HuCAL Antibodies
Technical Manual®

Second Edition

By Francisco Ylera

Table of Contents
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ��������������������������������� 6
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ��������������������������������� 8
Chapter 1 Introduction to Recombinant Antibodies . . . . . . . . . .  ������������������������������� 10
What are Antibodies?  . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������ 10
Basic Antibody Structure  . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������ 11
Advances in Antibody Technology  . . . . . . . . . . . . . . . .  ������������������������������� 12
Recombinant Antibodies  . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 13
Advantages of Recombinant Antibodies . . . . . . . . . . . .  ������������������������������� 18
Considerations When Using Recombinant Antibodies  .  ������������������������������� 20
Chapter 2 HuCAL® Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 21
The HuCAL® Concept . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 21
The HuCAL PLATINUM® Platform . . . . . . . . . . . . . . . . .  ������������������������������� 25
The HuCAL® Antibody Generation Process . . . . . . . . . .  ������������������������������� 30
Antibody Generation Parameters . . . . . . . . . . . . . . . . .  ������������������������������� 33
Antibody Formats . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 33
Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 40
Guided Selection Strategies . . . . . . . . . . . . . . . . . . .  ������������������������������� 43
Screening Options  . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 46
Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 47
Antibody QC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 47
Additional Custom Services . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 49
Chapter 3 Applications: Western Blotting . . . . . . . . . . . . . . . . . . .  ������������������������������� 50
Use of HuCAL® Antibodies in Western Blotting  . . . . . .  ������������������������������� 50
General Tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 51
Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 52
Alternative Protocol Using the SNAP i.d.® Protein Detection System ����������� 53
Examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 54
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 56
Chapter 4 Applications: ELISA  . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 58
Use of HuCAL® Antibodies in ELISAs  . . . . . . . . . . . . . .  ������������������������������ 58
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3General Points  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 59
Indirect ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 60
Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 61
Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 62
Direct ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 62
Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 63
Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 64
Sandwich ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 64
Protocols  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 65
Examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 67
Competition (or Inhibition) ELISA . . . . . . . . . . . . . . . . .  ������������������������������� 70
Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 70
Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 71
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 72
Chapter 5 Applications: Immunoprecipitation . . . . . . . . . . . . . . .  ������������������������������� 74
Protocols  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 76
Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 78
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 79
Chapter 6 Applications: Immunohistochemistry  . . . . . . . . . . . . .  ������������������������������� 80
Use of HuCAL® Antibodies in IHC . . . . . . . . . . . . . . . . .  ������������������������������� 80
Paraffin Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 81
Cryosections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 81
Fixation  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 82
Mounting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 82
Staining  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 83
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 96
Chapter 7 Applications: FACS™ Analysis – Flow Cytometry  . . . .  ������������������������������� 97
Use of HuCAL® Antibodies in Flow Cytometry . . . . . . .  ������������������������������� 97
Protocols  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ������������������������������� 98
Examples  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ���������������������������� 100
Troubleshooting  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ���������������������������� 102

4Your first choice for antibodies!

Chapter 8 Applications: Immunofluorescence . . . . . . . . . . . . . . .  ����������������������������� 103
Protocol  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ���������������������������� 104
Examples  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ���������������������������� 105
Chapter 9 Applications: Bead-based Assays  . . . . . . . . . . . . . . . .  ���������������������������� 107
Luminex xMAP® Technology . . . . . . . . . . . . . . . . . . . . .  ����������������������������� 107
Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ����������������������������� 107
Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ����������������������������� 108
FMAT® FLISA assays . . . . . . . . . . . . . . . . . . . . . . . . . . .  ����������������������������� 109
Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ����������������������������� 111
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ����������������������������� 112
Chapter 10 Affinity Determinations  . . . . . . . . . . . . . . . . . . . . . . .  ����������������������������� 113
SPR on a Biacore Instrument  . . . . . . . . . . . . . . . . . . . .  ����������������������������� 114
QCM on an Attana Instrument . . . . . . . . . . . . . . . . . . .  ����������������������������� 116
SET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ����������������������������� 117
Chapter 11 Antibody Conjugation . . . . . . . . . . . . . . . . . . . . . . . . .  ����������������������������� 119
Conjugation Using LYNX Rapid Conjugation Kits® . . . .  ����������������������������� 119
Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ����������������������������� 121
Conjugation to FITC, Cy5, and Biotin Using Conventional Kits �������������������� 122
Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ����������������������������� 126
Examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ����������������������������� 127
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ����������������������������� 128
Chapter 12 Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ����������������������������� 129
Buffer Composition  . . . . . . . . . . . . . . . . . . . . . . . . . . .  ����������������������������� 129
Buffer Preparation  . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ����������������������������� 129
Source of Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ����������������������������� 130
Source of Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . ����������������������������� 132
Technical Assistance . . . . . . . . . . . . . . . . . . . . . . . . . . .  ����������������������������� 137
Confidentiality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ����������������������������� 137
Further Selected Publications . . . . . . . . . . . . . . . . . . . .  ����������������������������� 138

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5Abbreviations
ABC

Avidin biotin complex

ADA

Anti-drug antibody assay

AgX 

AbD Serotec antigen expression service®

AP

Alkaline phosphatase

BCIP

5-Bromo-4-chloro-3’-indolyphosphate p-toluidine salt

BSABovine serum albumin

CDR

Complementary determining region

CH1, 2, 3
CLConstant region of the antibody light chain

c-myc

c-myc epitope tag with the sequence: AEEQKLISEEDLL

CRAClosely related antigenDAB3,3’ Diaminobenzidine

DMSO

Dimethyl sulfoxide

DTT

Dithiothreitol

ECL

Enhanced chemiluminescence (Western blotting)

ECL

Electrochemiluminescence (SET)EDTA

Ethylenediaminetetraacetic acid

ELISA

Enzyme-linked immunosorbent assay™

FACS 

Fluorescence activated cell sorter

FDPS

Farnesyl diphosphate synthase

FITC

Fluorescein isothiocyanate®

FLAG 

Epitope tag with the sequence: DYKDDDDKFLISA

Fluorescence-linked immunosorbent assay

FMAT 

Fluorometric microvolume assay technology

®

HBS

Hepes-buffered saline

His-6

Epitope tag with the sequence: HHHHHH

HRP

Horseradish peroxidase

HuCAL ®

Human Combinatorial Antibody Library

IHC

Immunohistochemistrykon

Association rate, on rate

koff

Dissociation rate, off rate

KD

Dissociation constant

LOD
6Constant region 1, 2, or 3 of the antibody heavy chain

Limit of detection
Your first choice for antibodies!

LSAB

Labeled streptavidin biotin

MWCO

Molecular weight cut-off

MSD™

Meso Scale Discovery™

NBT
Ni-NTA

Nitro-blue tetrazolium chloride
Nickel-nitrilotriacetic acid

ORI

Origin of replication

PAGEPolyacrylamide gel electrophoresis

PBS

Phosphate-buffered saline

PBSTPhosphate-buffered saline-Tween® 20

PCR

Polymerase chain reaction

PE (or RPE)

R-Phycoerythrin

PK

Pharmacokinetics

PVDF
QC
QCM
RPE (or PE)Polyvinylidene fluoride
Quality control
Quartz crystal microbalance
R-Phycoerythrin

RT

Room temperature

scFv

Single-chain variable fragment

SDS

Sodium dodecyl sulfate

SET

Solution equilibrium titration

SPR

Surface plasmon resonance

ssDNA
Strep-tag®
Sx
TBST
Trf
TRIM

Single stranded DNA
Epitope tag with the sequence: WSHPQFEKExtended Strep-tag® with the sequence: SA-WSHPQFEK
Tris-buffered saline-Tween® 20
Human transferrin
Trinucleotide mutagenesis

VH

Variable region of the antibody heavy chain

VL

Variable region of the antibody light chain

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7Introduction
Over the last three decades, the use of antibodies has increased greatly, bothas tools for basic research and diagnostics, and as therapeutic agents. This has
been driven in part by advances in recombinant antibody technology. At theforefront of these advances is The Human Combinatorial Antibody Library(HuCAL®), one of the most powerful synthetic antibody libraries ever created.
At a glance, the HuCAL® library is simply a unique collection of billions ofhuman antibodies. However, a more detailed examination shows that it is ahighly sophisticated tool with features that make it the system of choice forbasic research, target validation, drug screening, and to study novel proteins
created by the proteomics revolution. It has proved to be a direct sourceof antibodies for diagnostic and therapeutic applications, and has been
integrated into the research and development processes of many leading
pharmaceutical and biotechnology companies.
With the production of more than 13,000 novel antibody specificities in recent
years, and the rapid 8-week production turnaround, this technology has also
succeeded in accelerating our clients´ research.
AbD Serotec is a Division of MorphoSys AG which provides for the custom
development of novel antibody specificities using HuCAL® technology on a
project by project basis for research, diagnostic, and other non-therapeuticcommercial applications as a service to individual customers.
Up-to-date information on AbD Serotec’s antibody generation services, andtheir catalog of over 14,000 antibodies and immunoreagents, can be foundonline at www.abdserotec.com.
For more information about MorphoSys AG, the development of therapeutic
antibodies, and access to MorphoSys’ HuCAL® technology, please visit
www.morphosys.com.
We cordially invite you to learn more about the benefits of our sophisticatedin vitro technology in this manual, which is divided into easy-to-use chaptersfull of key technical and application information.
The first two chapters outline the concept of the HuCAL® technology platform,and describe its many advantages over antibodies generated using traditional
animal-based technologies.
8

Your first choice for antibodies!

The next seven chapters explain the use of HuCAL® antibodies in applications
such as Western blotting, enzyme-linked immunosorbant assay (ELISA),
immunoprecipitation, immunohistochemistry (IHC), flow cytometry,immunofluorescence, and bead-based assays. These sections feature standard
protocols which have been optimized for HuCAL® monoclonals, as well as
actual examples, hints, and troubleshooting tips.
The manual also includes procedures for affinity determination and forlabeling antibodies with biotin, different fluorescent dyes, and enzymes, plus
several other useful appendices, making this publication a comprehensive
technical resource for HuCAL® recombinant antibody technology.

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9ChapterIntroduction to
Recombinant Antibodies

1

What are Antibodies?
Antibodies are glycoproteins that are naturally produced in response toinvading foreign particles (antigens) such as microorganisms and viruses.
They play a critical role in the immune system’s defense against infection anddisease.
Ideally, every antibody recognizes and binds to just one antigen1. Antigens canbe proteins (for instance, receptors expressed on cancerous cells), sugars onbacterial and viral cell surfaces, hormones, chemical compounds, nucleic acidstructures, and so on. The region of an antigen that interacts with an antibody
is termed the epitope. In the case of protein antigens, epitopes can be linear or
three dimensional configurations of amino acids, or individual amino acids
that have been modified (for example, by phosphorylation or oxidation).
Typically, only foreign substances elicit an immune response.
Normally, an animal’s immune system will generate a large group of
antibodies that recognize several epitopes from a particular antigen. Eachantibody is secreted by a different antibody-producing plasma cell, and may
be specific for a different epitope. Since the antibodies found in serum arecollectively produced by many plasma cells (clones), they are described aspolyclonal. While this is an advantage for fighting infections in nature, theheterogeneity of polyclonal antibodies limits their use as research tools.
A major breakthrough in the development of antibodies for research
applications was the production of monoclonal antibodies in 1975 by
Köhler and Milstein2. Their technique involved removing B-cells (plasma cell
precursors) from the spleen of an animal that had been challenged with an
antigen, and subsequent fusing with an immortal myeloma tumor cell line.
This resulted in a single-cell hybrid known as a hybridoma. The B-cells confer
antibody production capability, while the myeloma cells enable hybridomas1
In reality, most antibodies are not fully monospecific and will also bind to other substances, albeit withlower affinity.
2
Köhler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined
specificity. Nature 256:495–497

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CH A P T E R 1Your first choice for antibodies!

to divide indefinitely and to grow well in cell culture. Hybridomas secrete
only one antibody type, effectively ensuring an infinite supply of antibodies
selective for a single epitope, which are also known as monoclonals.

Basic Antibody Structure
Many of the key structural features of antibodies can be considered using
immunoglobulin G (IgG) as a model, since IgG is the most abundant antibody
in serum.
The classical representation of an antibody is as a Y-shaped moleculecomposed of four polypeptide subunits with two identical heavy and lightchains (Figure 1). The N-terminus of each heavy chain associates with one of
the light chains to create two antigen-binding regions; these are the armsof the Y shape and are termed Fab domains. The C-termini of the twoheavy chains combine to form the Fc domain; this is the tail of the Y. The
Fc domain is important for the antibody’s interaction with macrophages andfor activation of the complement cascade. The four polypeptide chains are held
together by covalent disulfide bridges and non-covalent bonds.

FIGURE 1

Schematic Structure of an IgG Molecule.

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CH A PTER 1

11

Sequence analysis of various light chains has revealed two distinct regions(Figure 2), a highly variable N-terminal half (VL) and a constant C-terminal half(CL). Similar studies on heavy chains have shown that they are also comprisedof variable and constant regions (VH and CH, respectively). However, IgG
heavy chains contain one variable region (found at the N-terminus) and threeconstant regions (Figure 2). Unsurprisingly, the variable regions of both chains
localize to the Fab domains, providing the structural basis for the antigen andepitope selectivity of antibodies.

FIGURE 2

Light and Heavy Chain Structure. Light chains are approximately 220 amino acids inlength and can be divided into two equal variable and constant regions. Heavy chainsare approximately 440 amino acids in length and are subdivided into four 110 aminoacid segments, with one variable and three constant regions.

The sequence heterogeneity within the variable regions is most pronounced
at three short segments (5–30 amino acids in length) within each chain
called the hypervariable regions. Since the residues in hypervariable regions
form the actual binding site for the antigen, they are also known as thecomplementarity determining regions (CDRs) (Figure 2).

Advances in Antibody Technology
Although the development of hybridoma technology was a landmark event,the methodology still has a number of limitations. Developing hybridomas
requires considerable time, expense, and expertise, as well as specialized
cell culture and animal facilities. Additionally, antigens can be toxic or poorly
immunogenic in mammals, and therefore, do not elicit an immune response.

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CH A P T E R 1Your first choice for antibodies!

Moreover, mouse monoclonal antibodies are unsuitable for use as therapeutic
agents because they are rejected by the human immune system. Various
approaches to overcome this therapeutic limitation have been tried, such as:
■■

■■

■■

Combining mouse monoclonal binding sites with human antibody
sequences to create chimeric or humanized antibodies
Using genetically engineered mice to produce antibodies with humansequences
Developing fully human antibodies directly with in vitro recombinant
antibody technology

Recombinant Antibodies
Recombinant antibody technology – such as HuCAL® – is a rapidly evolving
field with a number of major benefits over conventional antibody generationand production methods.
First, the availability of gene sequences enables antibodies to be geneticallyengineered, allowing for the following:
■■

■■

■■
■■

■■

Study of the structural and functional properties of antibodies (binding,folding, stability, catalysis, etc.)
Generation of smaller antibody fragments that retain the entire antigen
binding site, such as the Fab and single-chain variable fragment (scFv) seenin Figure 3
Enhancement of antibody affinity or specificity via mutagenesis
Production of novel antibody fusion proteins which couple the antibody
binding capacity to additional properties (enzymes, toxins, multimerization
modules, and epitope tags)
Direct creation of partially or fully human antibodies

Second, recombinant monoclonal antibodies can be produced in bacteria. Thisis easier, faster, and less expensive than using animals (ascites) or mammaliancell culture techniques.
Third, the technology enables the use of in vitro selection steps that facilitate
the isolation of antibodies with desired characteristics, e.g. antibodies that
distinguish closely related antigens.
Finally, the method does not rely on the use of laboratory animals and does
not require immunogenicity of the antigen. Antibodies can be produced from
non-animal derived materials and therefore are free from animal pathogens.

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CH A PTER 1

13

FIGURE 3

Commonly Used Recombinant Antibody Molecules. IgG: Full length molecules
exactly matching the natural antibody composition. Fab fragment: The variabledomain and the first constant domain of one heavy chain, plus one complete lightchain of an antibody. The VH and VL chains can be covalently linked by a disulfide bond,and are also linked by strong non-covalent interactions. scFv fragment: A single-chain
antibody consisting of one VH and one VL chains expressed as a single polypeptide
joined by a peptide linker. The polypeptide linker stabilizes the interaction between VH
and VL chains.

Methods for the Generation of Recombinant Antibodies
In the past, the initial step in generating recombinant antibodies was toisolate the relevant genetic material from a pre-selected hybridoma cell line
and clone the antibody genes into a bacterial expression vector. Anotheroption was to amplify the antibody repertoire of an immunized animal bypolymerase chain reaction (PCR), using degenerate primers, and to clone
the sequences into a bacterial selection system such as phage display (seepage 15).
Modern techniques to generate recombinant antibodies start instead fromvery large (>1 billion) libraries of antibody genes. These libraries usuallycontain human antibody gene sequences since they were originally designed
to produce human antibodies for therapeutic purposes. Such libraries canbe derived from B-cells taken from non-immunized donors, or they can begenerated de novo by gene synthesis (such as with HuCAL®). Therefore, they
are called unselected or naïve libraries. If they contain a sufficient number offunctional members, these libraries can be used to select specific antibodies
against virtually any antigen.

14

CH A P T E R 1Your first choice for antibodies!

Since large gene libraries cannot be screened for the binding properties ofindividual members, the antibodies that bind a given antigen are identified
instead by selection. All selection methods to date physically link the antibody
protein in the library (the phenotype) with the genetic information that encodes
the given antibody (the genotype). There are a number of techniques, of which
the most commonly used are phage display, ribosome display, and yeast display.
Phage display is the most popular and best established of the selection methods.

Phage Display Selection
Phage display is used to select E. coli host cells that express the desired
antibody fragment, i.e. the antibody that binds a given antigen3,4. Filamentous
E. coli phage, such as M13 (which is non-lytic), are most commonly used
(Figure 4). For phage display, the antibody library must be cloned into aphagemid vector with the following properties:■■

Has both a bacterial and a phage origin of replication (ORI)■■

Contains an antibiotic resistance marker■■

Allows expression of the encoded antibody■■

FIGURE 4

Links the expressed antibody to a phage surface protein (typically protein
III or protein VIII) by genetic fusion

Filamentous Phage M13. Left: Electron microscopy image of filamentous phage. Topright: Schematic drawing of M13 phage. Bottom right: Electron microscopy image ofthe tip of M13 phage with arrows marking the pIII surface protein.

Kretzschmar, T. and von Rüden, T. (2002) Antibody discovery: phage display. Curr Opin Biotechnol
13:598–602

3

Smith, G.P. and Scott, J.K. (1993) Libraries of peptides and proteins displayed on filamentous phage.
Methods Enzymol 217:228–257
4

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The process begins by using helper phage to infect E. coli host cells containing
phagemid vectors causing them to display antibody gene fragments. The
fragments are fused to the phage coat protein gene III (known as gIII), which
will later form part of the phage particle. The helper phage genome causes thecell to produce daughter phage. In these daughter phage, the cloned antibody
genes are transcribed and then translated as a fusion protein consisting of abacterial ‘leader sequence’, the antibody gene fragment, and the gIII phagecoat protein. The leader sequence directs the proteins to the periplasmic spaceof the bacterial cell, where the fused antibody fragment is incorporated into
viable phage particles via the protein coat. Since the helper phage contains
a defective ORI in its genome, which does not replicate, the daughter phage
incorporate the phagemid into their capsid. Finally, phage that contain the
antibody’s genetic information and simultaneously display the corresponding
antibody binding sites on their surfaces are secreted through the bacterial
outer membrane. About 1013 phage particles are harvested and subsequently
used for the antibody selection steps.
Phage displaying the desired antibodies are selected by ‘phage panning’,
which shares similarities with solid-phase immunoassays5 (Figure 10, page31). In this process, the antigen of interest is immobilized on a solid support,such as microplate wells or magnetic beads. The phage particles are thenadded to allow binding of phage that display appropriate antibodies. Afterextensive washing to remove all non-specifically bound material, the boundphage are eluted and amplified by replication in new host cells. The selection
procedure is repeated several times, resulting in a population that consists toa high degree of phage that express the desired antibodies (i.e. those thatbind the antigen of interest). After this selection step, the antibody genes are
isolated from the enriched pool, inserted into an expression vector, andintroduced in an expression host (usually E. coli).

Expression and Screening of Antibody Fragments
E. coli cells transformed with the pool of enriched antibody genes are grownon agar plates, forming colonies. Each colony contains the genetic informationfor one antibody (heavy and light chain) enriched by the previous phagedisplay selection. Therefore, each colony contains the genetic information for
one monoclonal antibody.

5
Barbas, C.F. and Lerner, R.A. (1991) Combinatorial immunoglobulin libraries on the surface of phage(phabs): Rapid selection of antigen-specific Fabs. METHODS: A Companion to Methods in Enzymology
2:119–124

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CH A P T E R 1Your first choice for antibodies!

At this stage, colonies are picked and grown, and antibody expression is
induced. Cells are subsequently lysed and the antibody-containing lysate is
tested using an ELISA for the presence of antigen-specific antibody material.
The plasmid DNA from ELISA-positive colonies is isolated and the antibody
gene is sequenced. At this point, the panning method typically results in
the isolation of a number of different antigen-specific monoclonal antibody
fragments from the original large library of antibody genes. The overall
procedure takes about 4–6 weeks, after which the antibodies can then be
expressed and purified. (See figure 9, page 30)

Purification of Antibody Fragments
Soluble antibody fragments (e.g. Fab or scFv) produced by bacterial cultures
are usually purified by one-step affinity chromatography using epitope tags
that have been genetically fused to the C-terminus of the antibody heavy
chain. Epitope tags commonly used for this purpose are the Strep-tag®, a short
peptide with 8–10 amino acids that binds to streptavidin6; or the Histidine tag(His-6), which is a series of six histidines that can be complexed by metalchelates such as nickel nitriloacetic acid (Ni-NTA). Since Strep and His-6
epitope tag antibodies are commercially available, the tags can also be used
for highly-specific immunodetection.
It should be noted that the antibody fragments which have been isolated will
include the complete antigen binding site, and as a result they usually havethe same intrinsic antigen-binding affinity as the complete antibody. However,full-length antibodies contain two binding sites per molecule, which increases
their avidity and apparent affinity in some assays. To match the affinity andavidity of natural Immunoglobulins (Igs), recombinant antibody fragments canbe dimerized or further multimerized by engineering.
Affinity is the strength of binding of an epitope to an antibody binding site.
Avidity is the overall stability of the antibody-antigen complex, which is dependent
on affinity, valency, and structural arrangements.

6

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Strep-Tactin®, a mutated Streptavidin with higher affinity to the Strep-tag is also used.
CH A PTER 1

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Advantages of Recombinant Antibodies
Recombinant antibodies offer many advantages over conventional
monoclonals as described in the following sections. These stem from several
properties inherent in the system:■■

All stages occur in vitro■■

Candidates can be engineered using readily available DNA sequences■■

Technology is based on a bacterial expression system

In Vitro Selection
The ability to select antibodies in vitro is especially valuable for generating
antibodies to non-immunogenic and toxic antigens which cannot be usedin animals. Furthermore, it is an open system which allows modification of
the selection conditions. Using guided selection, the process can be pushed
towards the required specificities to enable the generation of epitope-specificantibodies, e.g. those that are phospho-specific.
Once recombinant antibodies with desired specificities are found, they can befurther enhanced by generating new combinations of heavy and light chains,or by mutating individual CDRs7.

Engineering
Peptides and proteins with new functions can be linked by genetic fusion to
the antibody. Examples include:■■

Epitope tags for purification, immobilization, and detection■■

Enzymatic activities, such as alkaline phosphatase (AP), for direct detection■■

Multimerization modules which increase avidity■■

Heterodimerization modules that allow for the creation of bi-specific antibodies■■

Toxins for the elimination of tumor cells or enzymes that convert a pro-drug
into a drug at the site of a tumor (ADEPT)

E. coli Production
Antibody genes can be designed for high expression in E. coli 8, withdemonstrated production yields of more than 1 gram per liter E. coli culture.9
7Karu, A.E. et al. (1995) Recombinant antibody technology. ILAR Journal 37:132–141.

Ge, L. et al. (1995) Expressing antibodies in Escherichia coli. In: Borrebaeck, C.A.K., Antibody
Engineering, 2nd edition, 229–236. Oxford University Press

8

9
Carter P. et al. (1992) High level Escherichia coli expression and production of a bivalent humanizedantibody fragment. Biotechnology (N.Y.) 10:163–167

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CH A P T E R 1Your first choice for antibodies!

Bacterial production is also less expensive than mammalian cell culture andcan be performed without the use of animal-derived components. In addition,
the bacterial system is fully-defined, and free from animal-based viruses,
prions, and contaminating immunoglobulins. The high purity of antibodies
from E. coli is accomplished using one-step affinity chromatography.

Antibody Fragments
Antibody fragments, such as Fabs which lack the Fc domain, often show
benefits over full-size IgGs, including:
■■

■■

■■

■■

■■

■■

Elimination of non-specific binding to cellular Fc receptors leading to lower
background and increased sensitivity during cellular isolation and IHC
Avoidance of cytokine release in functional assays with immunocompetentcells, since complement cannot bind
Increased diffusion through tissue and cell membranes with smaller Fabfragments, leading to faster and more efficient staining
Creation of fewer artifacts in receptor studies because monovalent Fabs do
not cross-link receptors
Reduced non-specific binding to the solid phase in heterogeneous ELISA
experiments due to lower hydrophobicity
Improved stoichiometry with less interference from serum factors andmacromolecules (which mostly bind to the Fc region) in fluorescent or
enzyme conjugates■■

Simplified co-crystallization of target proteins with antibodies■■

Easy determination of monovalent affinities between antigen and antibody

Therapeutics
Antibodies selected from in vitro libraries of human antibody genes do not
elicit the same immune responses in patients that are seen with non-humanantibodies. Therefore, such antibodies can be used for therapeutic
development. More and more fully human antibodies obtained from antibody
libraries are entering clinical development and are reaching the market10.

Speed and High-throughput
Antibody selection using phage display of antibody libraries is a process thattakes around 3–4 weeks for antigen-specific members to be enriched andisolated. Further production and characterization of the monoclonal antibodies

10

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Weiner L.M. (2006) Fully human therapeutic monoclonal antibodies. J Immunother 29:1–9
CH A PTER 1

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is also rapid, due the fast growth rate of E. coli cultures. Nevertheless, there
are attempts to further accelerate the process with the goal being to perform
selections in just a few days11.
The phage display selection process has been automated to a large degree12
so that many target antigens can be processed in parallel, further increasing
the efficiency of the process and saving the end user significant time.

Considerations When Using Recombinant Antibodies
Some reagents commonly used for immunoassays are not appropriate for
protocols involving recombinant antibodies. Anti-mouse and anti-rabbit
Ig secondary reagents, for example, are unsuitable for detection because
recombinant antibodies are mostly of human origin and lack the Fc domain.
Instead, detection of recombinant antibody fragments is usually performed
using antibodies directed against epitope tags, such as: Strep-tag®, His-6,
V5, c-myc, and FLAG®. These antibodies are usually directly labeled with an
enzyme such as horseradish peroxidase (HRP) or a fluorophore. They canalso be used in combination with a labeled tertiary antibody. Alternatively,
detection of human recombinant Fabs can be achieved using anti-human Fabantisera labeled with AP, HRP, or fluorescent dyes.
In addition, immunoprecipitation using recombinant Fab cannot be performed
using Protein A Sepharose® (which relies on the presence of the Fc domain), but
can be performed by coupling the recombinant antibody to magnetic beads.

Hogan S. et al. (2005) URSA: ultra rapid selection of antibodies from an antibody phage display library.
Biotechniques 38:536–538
11

Krebs B. et al. (2001) High-throughput generation and engineering of recombinant human antibodies.
J Immunol Methods 254:67–84
12

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CH A P T E R 1Your first choice for antibodies!

Chapter

HuCAL® Technology

2

The HuCAL® Concept
HuCAL® technology is a unique and innovative concept for the in vitro
generation of highly-specific, fully human antibodies13. The technology sets thestandard for antibody production, taking the procedure out of the animal
house and onto the laboratory bench.
The concept behind HuCAL® technology14 is to represent the essential
features of the natural human antibody repertoire, defined by sub-families ofantibody germline gene segments (VH and VL), using the minimum numberof sequences that cover the structural diversity of the typical repertoire. This
aim to condense the repertoire into a few germline gene representatives isfacilitated by three features of antibody genes:
■■

■■

■■

There are relatively few antibody genes in the human germline as revealedby the human genome project
Many human antibody germline sequences are very rarely used during an
immune response
Germline genes can be grouped into families with high sequence andstructural similarity

Generation of the HuCAL® Library
Detailed analysis of the human antibody repertoire at MorphoSys revealed
that its structural diversity is best represented by seven heavy chain andsix light chain variable region genes, which give rise to 42 framework
combinations in the master library. The genes encoding these combinations
were chemically synthesized and unique restriction sites were incorporated
at the CDR boundaries (Figure 5). Highly variable genetic cassettes encoding
the diversity of CDRs, which were generated using trinucleotides15, were13
Ostendorp, R. et al. (2004) Generation, engineering and production of human antibodies using
HuCAL®. Subramanian, G. (Ed.). Antibodies Volume 2: Novel Technologies and Therapeutic Use. 13–52

Knappik, A. et al. (2000) Fully synthetic human combinatorial antibody libraries (HuCAL®) based on
modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol 296: 57–86

14

15
Virnekäs, B. et al. (1994) Trinucleotide phosphoramidites: Ideal reagents for the synthesis of mixed
oligonucleotides for random mutagenesis. Nucl Acid Res 22 5600–5607

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CH A PTER 2

21superimposed onto these frameworks to mimic the entire human antibody
repertoire. The resulting libraries contain billions of functional human antibody
specificities in a Fab format, which are selectable using in vitro methods, suchas phage display.

FIGURE 5

The HuCAL® Concept. The structural diversity of the human antibody repertoire isrepresented by seven heavy chain and six light chain variable region genes, which are
combined to produce 42 antibody frameworks in the master library. Superimposing
highly variable genetic CDR cassettes on these frameworks effectively mimics theentire human antibody repertoire.

Benefits of HuCAL® Technology
HuCAL® is one of the leading recombinant antibody libraries. It offers many
advantages when compared to conventional monoclonal antibody generationand production as summarized in Table 1, as well as benefits that extend
beyond both conventional methods and other recombinant libraries.
Since the antibody selection is performed in vitro in an open system, theprocess can be adjusted to the requirements of the project. Guided selection
approaches, such as applying blocking strategies or usage of more than one
antigen, lead to excellent antibody specificities (please refer to page 43).
Antibody generation against toxic antigens, immunosuppressants and nonimmunogenic antigens is also possible.

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CH A P T E R 2Your first choice for antibodies!

TABLE 1

Advantages of HuCAL® Over Conventional Monoclonal Antibodies.
Parameter

Conventional Monoclonals

HuCAL®

Selection

RandomGuidedProduction

ComplicatedEasy

Animal-based

Yes

No

Guaranteed Supply

No – source dies with thecell line

Yes – clones can be
resynthesized

Flexible Formats

No

Yes

Engineering andOptimization

No

Yes

HumannessNo

Yes

Throughput

Mostly manual

Automated

Success Rate

~75%

98%

Speed of Delivery

4–9 months

8 weeks

The production of the antibodies occurs in E. coli, thus the entire process
is free from laboratory animals. There is also no disease transmission risk
normally associated with mammalian cell culture and the antibodies can beproduced without the use of animal-derived components.
Furthermore, a secure supply is guaranteed by a double backup storage
system consisting of bacterial antibody clones and their plasmid DNA. Even
worst case scenarios will not impact the supply, since the antibody genesof selected candidates are sequenced so that every antibody produced canalways be resynthesized.
The unique modular structure of the HuCAL® antibodies allows for a rapidchange of format and engineering of selected antibodies, e.g. adding epitope
tags or dimerization domains. For more information on antibody formats, seepage 33. The modularity also allows for the optimization of antibodies withrespect to affinity by exchanging certain CDRs with CDR libraries followed bystringent selection (see affinity maturation on page 27).
Another unique feature, the complete humanness of the antibodies, becomes
an exceptional advantage when characterizing human immune responses indiagnostic and clinical settings, or when developing therapeutically active
compounds.

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CH A PTER 2

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Finally, the highly automated process reduces the risk of failures and provides
excellent traceability of single steps. Clients benefit from fast production
timelines and unrestricted capacity. With a project timeline of 8 weeks, thistechnology is considerably faster in delivering purified and characterized
antibodies than any animal-based system.

Application of HuCAL® Technology
While HuCAL® technology was originally developed for the generation of fully
human therapeutic antibodies, it has also proved to be highly valuable for thegeneration of research and diagnostic antibodies.
For many applications, the versatility of HuCAL® selection protocols, thespecificities and affinities achieved, and the engineering flexibility have proved
to be critical. For other applications, the humanness of HuCAL® antibodies has
been decisive. HuCAL® offers a seamless transition from a rapidly generated
antibody fragment with outstanding binding characteristics to a completely
human full length antibody (IgG, IgM, IgA, IgE), immediately ready forassessment as human diagnostic standards in autoimmune testing16. They arealso suitable for use in many other immune assays that currently rely onhuman sera as a control. Finally, human Fab and Ig molecules developed fromHuCAL® are perfect tools for analyzing patient responses to a drug17.
AbD Serotec has successfully generated and licensed HuCAL® antibodies for awide range of purposes, including:
■■
■■

■■
■■

In vitro and in vivo research
Diagnostic immunoassay development (as detection reagents, sera
replacements, and fully human controls)

In vivo diagnostic assay development
Preclinical and clinical drug monitoring in pharmacokinetic (PK) and antidrug antibody assays (ADA)■■

Quality control (QC) release assays for drugs and vaccines■■

Purification of drugs■■

Food testing■■

Biodefense assays■■

Medical devices

16
Knappik, A. et al. (2009) Development of recombinant human IgA for anticardiolipin antibodies assay
standardization. Ann N Y Acad Sci 1173:190–198
17Tornetta, M. et al. (2007) Isolation of human anti-idiotypic antibodies by phage display for clinicalimmune response assays. J Immunol. Methods 328: 34–44

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CH A P T E R 2Your first choice for antibodies!

More than half of the top twenty pharmaceutical companies and manyleading biotechnology companies have integrated HuCAL® technology into
their R&D processes (see www.morphosys.com). As both an antibody-based
drug and technology development company, MorphoSys is committed tospearheading the latest advances in antibody development technology. With
this in mind, innovations in antibody sourcing technologies are constantly
being developed in-house, certain of which may be available for licensing in
the future.

The HuCAL PLATINUM® Platform
The HuCAL PLATINUM® antibody library is the latest and most powerfulantibody library developed by MorphoSys. It contains several improvements
over previous versions of HuCAL®, including a large increase in library
members and a completely new approach to reproducing the naturalcomposition of the key antigen recognition region (HCDR3). Both lead to a
substantial increase in the diversity of selected antibodies as compared tothe previous library. They also provide a wider range of promising antibody
candidates to choose from during an initial screening of the library, withrespect to affinity and specificity. Furthermore, expression rates in E. coli andmammalian cells were improved by gene optimization.

Features of HuCAL PLATINUM® Technology■■■■

45 billion fully human antibodies in the proven HuCAL® design – one of thelargest high-quality antibody libraries worldwide
Maximum variability for the antigen binding site through diversification of
all six CDRs■■

Enhanced functionality by length-dependent design of HCDR3■■

Optimized selection of frameworks■■

TRIM technology applied to generate a high-quality library with over 74%
functional clones■■

Optimized codons for E. coli and eukaryotic expression systems■■

Removed mRNA secondary structures that may interfere with expression■■

Avoided negative regulatory sequence motifs in prokaryotic and eukaryotic
systems■■

Significantly reduced number of potential glycosylation sites in CDRs■■

Minimized number of non-germline positions

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The high diversity of the library guarantees the successful generation ofhigh-quality antibodies.

The new library is wholly compatible with the well-established screening
and selection methods at AbD Serotec. As with the previous library, HuCALPLATINUM® combines the advantages of the Fab format with the superior
selection properties of the proprietary CysDisplay® screening technology thatis used to select individual antibodies from the library (Figure 6).

CysDisplay® Selection Technology
CysDisplay® technology is a novel and efficient display method for selecting
high-affinity binders from antibody libraries using filamentous phage18. It
combines the advantages of monovalent phage display technology, such as
phenotype–genotype linkage, with the additional feature of a cleavabledisulfide bond. Therefore, it allows the efficient elution of interacting partners
during the panning procedure (Figure 6).

FIGURE 6

CysDisplay® Screening Technology. Fab fragments are linked to phage particles by adisulfide bond rather than a peptide bond. This allows elution of phage with reducing
agents during antibody selection.

Rothe, C. et al. (2008) The human combinatorial antibody library HuCAL GOLD combines diversificationof all six CDRs according to the natural immune system with a novel display method for efficient
selection of high-affinity antibodies. J. Mol. Biol. 376:1182–1200

18

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CH A P T E R 2Your first choice for antibodies!

CysDisplay® is based on the simultaneous periplasmic expression ofengineered phage coat proteins and antibody fragments, each containing
an unpaired cysteine residue. Disulfide bonds form between these partners,
resulting in heterodimerization. The heterodimers are incorporated into phage
particles, leading to the display of monovalent functional antibody fragmentson the phage surface. Since the disulfide linkage of the antibody fragments
is sensitive to reducing agents, an efficient elution protocol can be used torecover phage displaying specific high-affinity antibody fragments.
This protocol can be used for any type of antigen and is, therefore, well-suitedfor high-throughput applications. The elution of phage particles from theantigens during the selection process is independent of the affinity of thebinder to the antigen. This ensures that all phage displaying specific binders
are eluted from the antigen, including the ones displaying very high-affinity
antibodies, which cannot be easily retrieved using traditional elution methods.

Affinity Maturation of HuCAL® Antibodies
In contrast to animal-derived antibodies from serum or hybridoma
supernatant, HuCAL® antibodies can be rapidly optimized both in affinity andbiological activity, since the antibody DNA sequence is known and available.
The specificity of the antibody usually remains unchanged during the antibody
maturation, while the affinity can be routinely improved. Indeed, several
thousand-fold increases in affinity have been achieved using this approach19,20.
Optimization is facilitated by several features of HuCAL® antibodies:
■■

■■

■■

Modular design enables replacement of CDRs by highly variable CDR
cassettes in a simple cloning step, thereby creating new antibody libraries
based on existing HuCAL® specificities
CDR cassettes are built using TRIM technology, ensuring high functional
quality maturation libraries
Highly diversified affinity maturation cassettes exist for all six CDRs and arecompatible with all HuCAL® frameworks

In most antibodies, HCDR3 contains the most binding contacts to the antigen
and, therefore, determines to a large extent the epitope bound by the
antibody. Consequently, HCDR3 has the highest diversity in the HuCAL® library.
Affinity maturation of selected antibodies concentrates on one or more of theremaining CDRs, typically LCDR3 or HCDR2.
Steidl, S. et al. (2008) In vitro affinity maturation of human GM-CSF antibodies by targeted CDRdiversification. Mol Immunol 46:135–144

19

20
Prassler, J et al. (2009) In vitro affinity maturation of HuCAL® antibodies: complementarity determining
region exchange and RapMAT technology. Immunotherapy 1:571–583

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CH A PTER 2

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Affinity maturation is an optional step that can be performed once HuCAL®
antibodies have been selected that bind a specific antigen. Binding
characteristics are optimized by inserting a pre-built CDR library cassette(usually LCDR3 or HCDR2 as in Figure 7), which has been diversified according
to the natural repertoire of CDR sequences at unique flanking restriction sites.

FIGURE 7

Optimization of a HuCAL® Antibody’s Binding Characteristics. Optimization
occurs via CDR library creation for specific binders in a simple cloning step.

This simple and efficient ‘mix and match’ process generates a new antibody
library. Subsequent highly stringent phage display selection then permits rapid
affinity maturation (Figure 8) of single antibodies (‘lead optimization’), or evenof antibody pools (‘pool maturation’ or RapMAT®).

28

FIGURE 8

HuCAL® Antibodies with Biological Function are Identified during Standard
Selection. New CDR sequences are inserted at unique flanking restriction sites to augment
binding characteristics. Antibodies with improved binding characteristics are identified infurther rounds of selection. Optimized antibody candidates can then be converted into
various immunoglobulin formats. This leads to antibodies that have both higher binding
affinities and improved biological function, such as blocking a rat protease inhibitor.

CH A P T E R 2Your first choice for antibodies!

Sequential exchange of CDRs may lead to improvements in affinity by a factor
of greater than a thousand-fold as compared with non-optimized parental
binders, generating antibodies with affinities below 10–12 M19.

TRIM Technology
TRIM technology is used to facilitate targeted diversification of CDRs.
Pre-assembled trinucleotides are used for the chemical synthesis of the CDR
sequences, which provides complete control over the amino acid composition
and avoids stop codons. Consequently, the resulting CDR libraries display
significantly higher quality than those made using conventional approaches.

Developing Therapeutic Antibodies and Accessing
MorphoSys Technologies
MorphoSys employs its powerful HuCAL® technology primarily for therapeutic
antibody development and grants certain licenses to its technology. The
development of therapeutic antibodies and access to our proprietary
technologies is handled by MorphoSys AG. Companies have the following
partnership options (visit www.morphosys.com for further information):
■■

■■

■■

■■

■■

19

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Technology Subscription Licensing: Transfer and installation of the
HuCAL® technologies at partner premises, with comprehensive training
included. Such transfers may occur for the purposes of in vitro and invivo research, development of diagnostic reagents and assays, as well as
preclinical and clinical drug monitoring. Subscription licenses in infectious
disease indications may also be extended to include therapeutic antibody
development and use.
Collaborations in Therapeutic Antibody Generation: Funding ofMorphoSys personnel to generate highly optimized antibodies against
targets of interest to the partner. This work typically includes fullmaturation of the antibody with a focus on the therapeutic benefits (highaffinity, low immunogenicity, solubility, and half-life).
Co-development of Therapeutic Antibodies: Joint MorphoSys / partner
funding and resource allocation throughout the therapeutic antibody
generation project in targets of mutual interest to both parties.
Product In-licensing: MorphoSys in-licensing of partner developed
preclinical or early clinical stage antibodies for mutually agreed upon terms.
Product Out-licensing: MorphoSys also has a pipeline of antibody drugcandidates. These will be available for out-licensing when they reachclinical proof of concept.
Steidl, S. et al. (2008). See page 27.
CH A PTER 2

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The HuCAL® Antibody Generation Process
Generation of antibodies using HuCAL® technology involves seven steps asshown in the figure below:

FIGURE 930

CH A P T E R 2

HuCAL® Antibody Generation Process Overview.

Your first choice for antibodies!

1. Antigen immobilization
The first step in antibody generation is the immobilization of the antigen on asolid support. The standard method uses covalent coupling to magnetic beads.
An alternative strategy is coating polystyrene ELISA microtiter plates. Antigen
presentation by passive adsorption may be beneficial for certain applications,and is the only option if the antigen is in a buffer incompatible with the beadcoupling chemistry.

2. Phage display selection – panning
The HuCAL® library presented on phage particles is incubated with theimmobilized antigen. Nonspecific antibodies are removed by extensive
washing and specific antibody phage are eluted by adding a reducing agent.
An E. coli culture is infected with eluted phage and helper phage to generate
an enriched antibody phage library for the next panning round. Typically, threerounds of panning are completed (Figure 10).

FIGURE 10

Phage Panning. Selection round of phage-displayed HuCAL® Fab fragments on
antigen-coated magnetic beads.

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CH A PTER 2

31

3. Subcloning into antibody expression vector
After panning, the enriched antibody DNA is isolated as a pool and subcloned
into a Fab expression vector. Different vector formats are available formonovalent or bivalent Fab fragments with a choice of epitope tags. E. coli aretransformed with the ligation mixture and plated on agar plates. Each growingcolony represents a monoclonal antibody at this stage.

4. Primary screening
Colonies are picked and grown in a 384-well microtiter plate. Antibody
expression is induced and the culture is lysed to release the antibody
molecules. Cultures are screened for specific antigen binding either by ELISA
or a bead-based homogenous fluorometric microvolume assay technology(FMAT®) assay (see page 120 for more information on FMAT® assays).

5. Sequencing
Hits from the primary screening experiment are sequenced to identify
unique antibodies. The sequences not only guarantee the uniqueness of any
HuCAL® antibody, they also serve as an ideal antibody storage backup, anda guarantee of reproducibility. Every sequence can be rebuilt by chemical
synthesis, if necessary.

6. Expression and purification
The unique antibodies are expressed and purified using one-step affinity
chromatography.

7. Antibody QC
Purified antibodies are tested by ELISA, or in a bead-based fluorescencelinked immunosorbent assay (FLISA – usually FMAT®), for required specificity.
Purity is assessed by Coomassie® staining of a sodium dodecyl sulfatepolyacrylamide gel (SDS-PAGE) and yield is measured by UV absorbance at
280 nm.
Purified antibodies are typically shipped 8 weeks after commencementof a project.

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CH A P T E R 2Your first choice for antibodies!

A Typical HuCAL® Project Includes:■

Three rounds of panning■

Subcloning into selected antibody format■

Screening of 368 clones■

Sequencing of up to 20 clones
Shipment of antibodies (250 µg each) and – if available – 75 µg of each antigen(if produced or modified by AbD Serotec)■ ■

No antibody –no charge guarantee21

Antibody Generation Parameters
For a successful HuCAL® antibody project, it is important to be aware of allavailable options that can be used to obtain the desired antibody specificities.
Over the course of the following pages, parameters and options are explainedin more detail. Our scientists will be happy to offer advice on the best project
strategy.

Antibody Formats
The HuCAL PLATINUM® library is based on the human IgG1 Fab format, which
consists of the first two domains of the antibody heavy chain (VH and CH1)
plus the complete light chain (from either the κ or λ family). The Fab format istruly monovalent (one heterodimeric molecule has one antigen binding site).
This format, together with the monovalent phage display selection method,
ensures that the in vitro selection of antibodies from the library is driven byintrinsic affinity and not by avidity effects.
Antigen-antibody interactions depend on the following characteristics:■■■■

Affinity – binding strength of a single antigen binding site to a single
antigen epitope
Avidity – binding strength of a potentially multimeric antibody (e.g. whole
IgG) to a potentially multimeric antigen

After phage display selection of candidate antibodies, the antibody Fab genes
are cloned into an E. coli expression vector. The choice of expression vector
determines:

21
If we don’t find an ELISA-positive antibody against your antigen, there is no charge for library
screening.

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CH A PTER 2

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■■

Whether the final antibody is monovalent or bivalent■■

Whether an enzyme such as AP is fused to the antibody■■

Which peptide tag will be attached to the C-terminus of the antibody heavy
chain

Antibody formats can be changed by subcloning the gene into a new
expression vector. It is also possible to convert the antibody into a full-length
Ig format by choosing an expression vector that adds the Fc region (CH2and CH3 domains). Full-length antibody constructs are usually expressed in
mammalian cells to achieve sufficient quantities of Ig in a functional format.

Choosing the Fab or IgG Format
Many applications and assays benefit from monovalent or bivalent Fabfragments over classical full-size antibodies, as discussed on page 19. For
certain types of experiments, however, it will be necessary to convert therecombinant antibody into an IgG format. This is necessary in the following
circumstances:
■■

■■

■■

In experiments that depend on desired effects of the Fc region of theantibody (such as for binding or coating)
When the class- and subclass-specific antigen determinants located in the
Fc region are required
Where the Fc region is essential to sustain precipitation or agglutination
reactions

It is also important to remember that:
■■

■■

■■

Fab preparations generated from E. coli expression systems containendotoxins and cannot be used in some cellular assays without anadditional purification step
Since most commercial secondary detection systems are directed against
the Fc region of the primary antibodies, HuCAL® Fabs use a secondary
detection system directed against epitopes on the Fab portion or against
the epitope tags
In IHC on human tissues, the detection of human Fab or F(ab’)2 requires an
epitope tag antibody, since endogenous Igs will also be recognized by antihuman Fab secondary reagents

Choosing the Best Fab Format
When starting a project, our scientists will help to decide which format is mostsuitable for the application. Options include the monovalent Fab format or thebivalent format of AbD Serotec’s mini-antibodies (functionally equivalent to a
34

CH A P T E R 2Your first choice for antibodies!

F(ab’)2 fragment) (Figure 11 and Table 2). Certain dimerization domains, likethe dHLX domain, when fused to the C-terminus of the heavy chain fragment
create non-covalently linked Fab-homodimers which are also called miniantibodies.

FIGURE 11

Schematic of the Main Antibody Formats Available. Fab-V5Sx2: Monovalent
Fab (shown with two tags). Fab-dHLX-MSx2: Bivalent Fab, dHLX domains shown as
blue cylinders (with two tags). Fab-A-V5Sx2: Bivalent Fab formed by dimerization ofbacterial AP (blue/green) with two tags. IgG: Complete IgG antibody.
V5= V5 tag; M= c-myc tag; Sx2= two extended Strep-tags.

Bivalent Fab fragments are preferred to monovalent Fab fragments for mostapplications that detect surface-bound antigen (Western blots, IHC, andfluorescence-activated cell sorting [FACS™]), because they possess two antigen
binding sites and display an avidity effect. Monovalent Fabs work better insome cellular assays because they avoid cross-linking of antigens, i.e. thesignal strength is a better match for the binding stoichiometry. Furthermore,monovalent Fabs are usually preferred for crystallization experiments.
Some applications require special considerations. For example, AbD Serotec
recommends a monovalent Fab format when comparing affinities or IC50
values of different antibodies; or for use on an affinity chromatography
column, since elution of the Fab is prevented under conditions where theelution buffer disrupts the dHLX helix.
Since the bivalent format (Fab-dHLX) gives the best performance in mostassays, it is the standard antibody form used, with the heavy chain expressed
as a fusion with the dHLX domain22, 23. Two dHLX domains dimerize in a
22
Pack, P. et al. (1993) Improved bivalent miniantibodies, with identical avidity as whole antibodies,
produced by high cell density fermentation of Escherichia coli. Biotechnology (NY) 11:1271–1277
23
Plückthun, A. and Pack, P. (1997) New protein engineering approaches to multivalent and bispecificantibody fragments. Immunotech 3:83–105

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helix-turn-helix structure to form the bivalent mini-antibody. However,
AbD Serotec also offers bivalent Fab fragments in the Fab-A format, where theheavy chain is fused to bacterial alkaline phosphatase (AP). Since bacterial AP
forms a homodimer naturally, it creates a bivalent Fab fragment. This format
also allows direct detection via the AP activity and without the use of asecondary antibody. It has advantages for applications requiring
immobilization of the antibody by random chemical coupling, due to its large
size. Further information on the molecular weight of these domains can befound in Table 3 and Table 4.
With some applications, it is useful to have even higher valencies. In thoseinstances, AbD Serotec offers a tetravalent antibody format in which the Fab
is fused to the p53-tetramerization domain. Note that hetero-oligomerization
with native human p53 protein has been observed after incubation withhuman p53 containing lysates.
For applications where the Fc part of the antibody is required, we offer
subcloning of the antibodies into the human IgG1, IgG2, IgG4, IgA, IgM, andIgE formats, followed by transient expression in mammalian cells. If mouse
antibodies are required, AbD Serotec also offers production of chimerichuman/mouse IgG2a with human variable regions and mouse constantregions. For rat, human/rat IgG1, Ig2a, and Ig2b chimeras are available.

Choosing the Matching Epitope Tag
It is important to select the tag best-suited for the application. The tag is anadditional feature that can be used for immunodetection of the Fab in theassay or for immobilization on a matrix. Refer to Table 2 and Table 3 to identify
the most suitable tag. AbD Serotec offers a wide selection of common epitope
tag antibodies, either unconjugated or labeled with enzymes and fluorescentdyes (see appendix).

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CH A P T E R 2Your first choice for antibodies!

TABLE 2

Recommended Fab Antibody Formats by Application.
Applicationwww.abdserotec.com

Monovalent

Bivalent

Recommended
Secondaries

Comments

Western Blot✔

Anti-human FabAnti-Strep-tag®
Anti-His-6
Anti-V5
Anti-BAP
Anti-FLAG®
Anti-c-myc

Recommended
protocols
found in chapter 3

ELISA

✔

Anti-human FabAnti-Strep-tag®
Anti-His-6
Anti-V5
Anti-BAP
Anti-FLAG®
Anti-c-myc

Recommended
protocols
found in chapter 4

Immunoprecipitation✔

Anti-human FabAnti-Strep-tag®
Anti-His-6
Anti-V5
Anti-FLAG®

Recommended
protocols
found in chapter 5

IHC✔

Anti-human Fab (for nonhuman tissue)
Anti-Strep-tag®
Anti-His-6
Anti-FLAG®
Anti-BAP
Anti-V5

Recommended
protocols
found in chapter 6

FACS

✔

Anti-human Fab (for nonhuman tissue)
Anti-Strep-tag®
Anti-FLAG®
Anti-His-6

Recommended
protocols
found in chapter 7

Immunofluorescence✔

Anti-human Fab (for nonhuman tissue)
Anti-Strep-tag®
Anti-His-6
Anti-FLAG®
Anti-BAP
Anti-V5

Recommended
protocols
found in chapter 8

Affinity
Determination✔

Recommended
protocols
found in chapter 10

Chromatography✔

Use a Fab-ds or IgG
format to avoid column
leakage of the light or
heavy chain

Co-crystallization✔

Use Fab-H or Fab TCMH: use Thrombin tocleave off tags

CH A PTER 2

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All available Fab formats contain either a Strep-tag®, an extended Strep-tag®
(Strep II-tag with a Ser-Ala linker), or a His-6 tag, which is used for purificationof the antibody. A second tag is added in many formats to provide additional
options for detection (Table 3). Please note that Ig formats do not contain any
epitope tags.
His-6 is a widely used epitope tag that can be used for protein purification
on a Ni-NTA matrix. Several antibodies against His-6 are commerciallyavailable with different labels. For example, AbD Serotec offers MCA1396
in the following formats: Alexa Fluor® 488, Alexa Fluor® 647, AP, biotin,
DyLight® 549, fluorescein isothiocyanate (FITC), HRP, and R-phycoerythrin(RPE).
The Strep-tag® is an artificial epitope tag with 8 amino acids that was
originally generated to bind streptavidin. However, Strep-Tactin®, anengineered streptavidin, should be used instead due to its higher affinity.
The extended Strep-tag® has two additional amino acids which allow
capture of the tag on a solid support by the anti-Strep-tag® Immo antibody(MCA2488), which has a very high affinity. Both tags can be detected withgood sensitivity and specificity using the anti-Strep-tag® Classic antibody(MCA2489).
The extended Strep-tag® is generally preferred over His-6 because it yields
superior results in affinity purification.
Our standard format is the bivalent mini-antibody with a FLAG® and two
extended Strep-tags, referred to as Fab-dHLX-FSx2, which works well in mostapplications. The molecular weight of these mini-antibodies is approximately
115 kDa. Each Fab is genetically fused to a small homodimerization domain(termed dHLX; molecular weight 5 kDa), a FLAG® tag, and two extended
Strep-tags at the C-terminus of the heavy chain (see Table 4 for tag size andsequences).

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CH A P T E R 2Your first choice for antibodies!

TABLE 3

Fab Antibody Formats and Epitope Tag Combinations.
Short Name

Description

Approx. MW (kDa)

Monovalent
Fab-MSx2

Fab antibody (c-myc- and StrepX-StrepX-tags)

55

Fab-FSx2

Fab antibody (FLAG®- and StrepX-StrepX-tags)

55

Fab-V5Sx2

Fab antibody (V5- and StrepX-StrepX-tags)

55

Fab-MHFab antibody (c-myc- and His-6-tags)

53

Fab-FHFab antibody (FLAG®- and His-6-tags)

52

Fab-V5H

Fab antibody (V5- and His-6-tags)

53

Fab-S

Fab antibody (Strep-tag®)

51

Fab-H

Fab antibody (His-6-tag)

51

Fab-H-Cys

Fab antibody (His-6-tag with Cys)

51

Fab-MH-Cys

Fab antibody (c-myc- and His-6-tags with Cys)

53

Fab-MS-Cys

Fab antibody (c-myc- and Strep-tag® with Cys)

53

Fab-ds-H

Disulfide linked Fab antibody (His-6-tag)

51

Fab-ds-FS

Disulfide-linked Fab antibody (FLAG® and Strep-tag®)

52

Fab-Tc-MH

Fab antibody (Thrombin cleavable, c-myc- and His-6-tags)

53

Bivalent
Fab-dHLX-MSx2

Mini-antibody (c-myc- and StrepX-StrepX-tags)

115

Fab-dHLX-FSx2

Mini-antibody (FLAG®- and StrepX-StrepX-tags)

115

Fab-dHLX-MH

Mini-antibody (c-myc- and His-6-tags)

112

Fab-dHLX-FH

Mini-antibody (FLAG®- and His-6-tags)

112

Fab-dHLX-S

Mini-antibody (Strep-tag®)

111

Fab-dHLX-H

Mini-antibody (His-6-tag)

111

Fab-A-MSx2

Fab bacterial alkaline phosphatase fusion antibody(c-myc- and StrepX-StrepX-tags)

205

Fab-A-FSx2

Fab bacterial alkaline phosphatase fusion antibody(FLAG®- and StrepX-StrepX-tags)

205

Fab-A-V5Sx2

Fab bacterial alkaline phosphatase fusion antibody(V5- and StrepX-StrepX-tags)

205

Fab-A-SFab bacterial alkaline phosphatase fusion antibody (Strep-tag®)

201

Fab-A-FH

Fab bacterial alkaline phosphatase fusion antibody(FLAG®- and His-6-tags)

202

Fab-ds-A-FS

Disulfide-linked Fab bacterial alkaline phosphatase fusion antibody(FLAG®- and Strep-tag®)

202

Tetravalent

www.abdserotec.com

Fab-p53-H

Mini-antibody (His-6-tag)

231

Fab-p53-S

Mini-antibody (Strep-tag®)

231

Fab-p53-V5Sx2

Mini-antibody (V5- and StrepX-StrepX-tags)

235

CH A PTER 2

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TABLE 4

Tag Sequences and Homodimerization Domains.
Short Name

Description

Approx. MW (kDa)

DomainsFabHeavy chain variable and first constant domain, and complete light chaindHLX

Synthetic double helix loop helix motif (dimer)

5A

Bacterial alkaline phosphatase (dimer)

47

Domain derived from human p53 (tetramer)

5.8

p53

50

Strep-Tag Versions®

S

WSHPQFEKFSDYKDDDDKGAPWSHPQFEK

2.3

FSx2

DYKDDDDKGAPSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK

4.3

MS-CysEQKLISEEDLNGAPWSHPQFEKC

2.7

MSx2

EQKLISEEDLNDAPSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK

4.7

V5Sx2

GKPIPNPLLGLDSTDAPSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK

1

5

His-Tag Versions
H

HHHHHH

H-Cys

HHHHHHCFH

DYKDDDDKGAPHHHHHH

2.1

MH

EQKLISEEDLNGAPHHHHHH

2.4

MH-CysEQKLISEEDLNGAPHHHHHHC

2.5

Tc-MH

LVPR↓GSGAPEQKLISEEDLNDAPHHHHHH↓: Indicates Thrombin cleavage (Tc) site

3.3

V5H

GKPIPNPLLGLDSTDAPHHHHHH

2.9

1
1

Besides the standard purification (His-6 or Strep) and detection tags (c-myc,
FLAG®, or V5), others are also available to provide additional functionality. For
example, we offer a single cysteine or a thrombin cleavage site (Fab-Tc), whichenables the removal of the tags after purification.
Introduction of new sequences and tags for special applications is possible
via a simple cloning step. If the preferred tag or a combination of tags is
not listed in the tables above, please contact your account manager atsales.muc@abdserotec.com or visit www.abdserotec.com/HuCAL for
more information. For further guidance regarding specific applications, refer tothe relevant chapter of this manual, or consult the technical support scientists
at AbD Serotec.

Antigens
The majority of substances used as antigens for antibody development areproteins. Since HuCAL® technology does not involve immunization of animals,
only 0.5 mg of protein antigen is required for the entire antibody generation
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CH A P T E R 2Your first choice for antibodies!

project (under certain conditions even as little as 100 µg of protein may besufficient). If purified protein is not available, it is possible to generate antigen
by using our AgX® antigen expression technology, or to express the antigen as
an Fc fusion protein in a mammalian expression system. Alternatively, peptides
or other antigens, such as small molecules (haptens), can also be used for
HuCAL® antibody development.

Protein Antigens
Using the whole protein as antigen is usually the method of choice, providingthat a sufficient amount of purified protein is available. Proteins should be
at least 80% pure, as judged by Coomassie®-stained reducing SDS-PAGE ofa 3 µg sample. The sample buffer must not contain primary amines such astris or glycine, additives such as bovine serum albumin (BSA), or detergents,
since these reagents interfere with the immobilization reaction (covalent
coupling to magnetic beads). 4 M guanidine hydrochloride or low amountsof free sulfhydryl groups (e.g. 1 mM Dithiothreitol [DTT]) are compatible
with the coupling chemistry. The scientists at AbD Serotec are available toprovide advice on buffer compatibility. If it is not possible to change buffer
composition, the antigen would be included in a procedure called ‘solidphase panning’, where it is passively adsorbed onto an ELISA plate, andsubsequently handled with PBS.
If the protein contains a linker or purification tag, then 0.25 mg to 0.5 mg ofan unrelated protein with the same linker or tag should also be provided. Thisis used for counter-selection and screening to make sure that the antibodies
delivered will be against the protein of interest, and are not binding to thelinker or tag.

The AgX® Antigen Expression Service
When the protein is not available in purified form, the proprietary MorphoSys
AgX® antigen expression system is an ideal starting point for generating
antigens from cDNA or PCR fragments24. If purified DNA is unavailable, thesequence can be used by AbD Serotec for gene synthesis. This will add 2 weeks
to the timeline of AgX® antigen production. We use a bioinformatics toolbox todetermine the optimal protein domain for expression, and then subclone the
DNA fragment into a proprietary E. coli N1-fusion protein expression vector.

24
Frisch, C. et al. (2003) From EST to IHC: human antibody pipeline for target research. J Immunol
Methods 275:203–212

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After subcloning and sequence verification, the antigens are expressed asinclusion bodies in E. coli, and purified under denaturing conditions, via theHis-6 epitope tag, by one-step metal-affinity chromatography (Figure 12).
Next, the antigen is refolded. The AgX® approach has a high success rate, withmore than 90% of all antigens expressed, and it adds just 3 weeks to theantibody generation procedure. This system has two advantages over peptide
antigens:■■■■

Antibodies against protein domains are more likely to recognize theparental protein than anti-peptide antibodies
Many more epitopes are available for antibody recognition; hence, thediversity of antibodies selected is usually higher

For antigens that require a mammalian system for solubility or proper folding,
AbD Serotec offers the production of antigens as Fc-fusion proteins.

FIGURE 12

Antigens Expressed as AgX® Fusion Proteins. 15% SDS-PAGE (reducing conditions)of N1-fusions of nine protein fragments ranging in length from 100 to 700 amino acids.
N-terminal fusion of the AgX® to the N1 domain of the pIII protein results in strong
expression of protein fragments, while the C-terminal His-6 enables purification ofprotein samples under denaturing conditions.

Peptide Antigens and Haptens
Peptides have limitations as antigens, due both to the reduced number of
epitopes available and to their flexible structure. Antibodies against peptides
are often of lower affinity than antibodies against proteins, and there is no
guarantee that the anti-peptide antibody will recognize the natively folded
parent protein. However, in some cases they are the only option. Furthermore,
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CH A P T E R 2Your first choice for antibodies!

selection of certain epitope-specific antibodies (e.g. phospho-specific) often
requires a peptide antigen. AbD Serotec has a very good success rate ingenerating antibodies against peptides, and our technical specialists can helpchoose the best peptide sequence for antibody generation.
AbD Serotec routinely couples peptides to two carrier proteins (using different
linkers) for panning against the library. The standard carrier proteins are BSA
and human transferrin (Trf), with coupling via an N- or C-terminal cysteine, butalternative coupling strategies can be used where necessary. We can provide
peptide synthesis and coupling services, or just coupling for existing peptides.
Contact your account manager at sales.muc@abdserotec.com for more
information.
Using guided selection (see below), HuCAL® technology offers great
advantages for the selection of anti-hapten antibodies with demanding
specificities. Similar to peptide antigens, the first step involves coupling of
the hapten, or a homolog, to two different carriers. Contact us to discuss theoptions available for hapten antigens.

Guided Selection Strategies
Selection of antibodies using HuCAL® technology is performed entirely in vitro(Figure 9). This enables greater flexibility for antibody generation than isavailable with conventional methods based on the immunization of animals.
All selection strategies used for the identification of antibodies to certain
epitopes, or epitope areas, are termed guided selection.

Selection of Epitope-specific Antibodies
The HuCAL PLATINUM® library is highly suited for the selection of antibodies
against specific epitopes, such as phosphorylated sites. Chemically synthesizedpeptides are often used to ensure the purity and homogeneity of the antigen(which is phosphorylated) and the counter-antigen (a closely related antigen[CRA], which is non-phosphorylated). All antibodies in the library that bindthe non-phosphorylated peptide are efficiently blocked by incubation with anexcess of the non-phosphorylated peptide. The cleared library is then used forthe selection of epitope-specific antibodies (Figure 13).

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CH A PTER 2

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FIGURE 13

Guided Selection with Blocking. A closely related antigen is used to remove crossreactive antibodies (counter-selection).

Antibodies Lacking Cross-reactivity to Closely Related
Proteins
A subtraction strategy is used to eliminate cross-reactive antibodies fromthe selection process. Pre-adsorption steps and intelligent counter-selection
blocking drive the selection to unique epitopes on the antigen. This strategy
is highly suitable for various procedures, including the generation of antiidiotype antibodies.

Antibodies that Bind Homologous Proteins
Alternate selection using two homologous antigens allows identification of
antibodies that react with both antigens (Figure 14). This strategy is usefulfor generating cross-species antibodies or for recognizing two isoforms of aprotein.
This strategy is also used to select antibodies that bind peptides. The peptide is
first coupled to two carrier proteins using two different linkers. By alternating
the peptide carrier conjugate during the panning rounds, only antibodies thatbind the peptide, and not the linker or the carrier are selected from the library.

Antibodies that Bind Antigens under Special Conditions
AbD Serotec can alter the selection conditions to ensure that the antigen ispresented to the library in the form in which it will be assayed – denatured,captured, soluble, or masked. It is even possible to alter the buffer conditions
during binding to simulate the assay buffer of the relevant experimental
system.

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CH A P T E R 2Your first choice for antibodies!

FIGURE 14

Guided Selection with Alternating Antigens. The antigen is alternated during thethree rounds of selection, thereby enriching antibodies that bind to both antigens.
Primary screening on both antigens will confirm the desired cross-reactivity.

Generation of ELISA Sandwich Pair Antibodies
Two strategies are typically used for the generation of sandwich pairantibodies.
1. A standard panning against the antigen is followed by testing all possiblecombinations of the selected antibodies as capture and detection
antibodies in a sandwich ELISA using biotinylated detection antibodies.
The best combination of capture and detection antibodies is identified bythis method. However, this method depends on the number of selected
antibodies and their properties (which, in turn, depends on the antigenused), and does not always result in a good sandwich pair.
2. This method is used if the technique above is unsuccessful. Standard
panning against the antigen is followed by ELISA screening of the purified
antibodies for the best capture antibody. Labeled (e.g. biotinylated)
antigen is required for detection. The best capture antibody is then used
to capture the antigen and a subsequent panning is performed on the
antibody-antigen complex. The capture antibody is used to pre-clear the
library and an isotype-control antibody is used for blocking, ensuringwww.abdserotec.com

CH A PTER 2

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depletion of capture-antibody specific antibodies. Selected antibodies
will bind the captured antigen and therefore are matched detection
antibodies (Figure 15).

FIGURE 15

Selection with a Captured Antigen. The antigen is presented as an antigen-ligand
complex. The ligand is used as a CRA to pre-clear the library.

Screening Options
After panning, the enriched antibody gene pool is subcloned into an
expression vector to produce the final antibody format. For screening, E. coli
colonies containing single antibody genes are picked and transferred into
a 384-well microtiter plate. By default, one plate (368 colonies, because
one row is left blank for controls) is picked. It is possible to pick and screen
multiple plates if a maximum diversity of antibodies is required. At this stage,
antibodies with the required specificities will be identified and selected forfurther processing. Therefore, it is important to carefully choose all required
antigens for this primary screening experiment. For instance, for peptide
and AgX® antigen projects it is advisable to include the purified protein, todemonstrate binding of the resultant antibodies to the native protein, if at
least a small amount of purified native protein antigen is available.
The master plate with all 368 clones is stored for two years (as frozen glycerol
stocks) if at least one positive hit was found in the primary screening. This
allows additional screenings later, e.g. if new antigen material becomes
available.
The standard assay for primary screening is an indirect ELISA with coatedantigen, antibody-containing E. coli lysates, and labeled secondary antibody.
It is also possible to screen the antibodies in a capture setting. In that case,46

CH A P T E R 2Your first choice for antibodies!

the antibodies are captured from E. coli lysates by coated anti-human Fabantibody and then incubated with biotinylated antigen and detected via
streptavidin-HRP. For haptens, an inhibition ELISA (or competition ELISA)
is strongly recommended. Since the antibody selection is performed withconjugated hapten carrying a linker, an inhibition ELISA with free hapten(without linker) helps to identify only those antibodies which do not crossreact with the linker or the carrier.
If only very small amounts of antigen are available, or if the desired final
antibody application is a bead-based assay, the primary screening can be
performed as an FLISA assay on the FMAT® 8200 CDS instrument (see page120). With FMAT® screening, 100 µg of antigen may be sufficient for the entire
antibody generation project.

Sequencing
Sequencing of up to 20 primary screening hits is included in any standard
project. It identifies the truly unique antibodies and allows storage of theantibody data in an electronic format. To increase the chances of finding theperfect antibody for the application, it is possible to increase the number ofsequenced clones by multiples of 10 (providing sufficient primary screening
hits are available). Since the master plate with all 368 clones is stored for two
years, it is also possible to sequence additional clones later.

Antibody QC
The purity of all antibodies after one-step affinity purification is routinely
monitored by SDS-PAGE and subsequent Coomassie-staining of a 1.8 µg
sample. Concentration is determined by measuring UV absorbance at 280 nm.
Activity and specificity of the purified antibodies are tested by a QC ELISA
before shipment (Figure 16). All antibodies are tested on three non-specificstandard antigens and on all positive and negative control antigens, as
defined in the project plan. Since only small amounts of antigen are requiredat this step, more precious antigens can be reserved for this ELISA.

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CH A PTER 2

47

FIGURE 16

QC ELISA of Anti-peptide Antibodies Show Binding of Parental Protein. The
HuCAL® antibody library was panned against the M18 peptide and 11 binders wereexpressed as bivalent mini-antibodies. QC ELISA showed that all 11 antibodies
bound specifically to the M18 peptide but not to BSA and Trf carrier proteins, or toan unrelated peptide bound to Trf. The I-domain of the parental Mac1 protein is also
recognized by eight of the 11 antibodies. Differences in signal intensity between
peptide and protein antigens reflect coating densities of peptide vs. protein antigen(several peptides are conjugated to one BSA or Trf molecule), and do not represent
differences in affinity.

The QC ELISA is a single, one-point measurement and is not useful for ranking
the antibodies according to affinity. Consequently, AbD Serotec always
recommends testing all available antibodies in the final application(s). On
request we measure the antibody affinities or perform additional assays, suchas capture or inhibition ELISAs, as the final QC step. Alternatively, the assay
can be performed as a bead-based FLISA assay on the FMAT® instrument.

48

CH A P T E R 2Your first choice for antibodies!

Additional Custom Services
The laboratories at AbD Serotec are setup to run many additional assays andservices for a large number of antibodies in parallel. These assays and services
include:■■■■■■Antigen generation using proprietary AgX® technology, expression as Fc
fusion proteins, or with various other systems
Antibody testing in a range of typical assays (Chapters 3–8)
Small- and large-scale antibody conjugation to biotin, common reporter
enzymes (HRP and AP), or fluorescent dyes■■

Screening for antibody sandwich pairs (in 384-well format)■■

Bead-based assays on a Luminex® or FMAT® 8200 CDS instrument■■

Affinity ranking (koff ranking) and determination using several different
technologies (Chapter 10) including:–– Surface plasmon resonance (SPR) on a Biacore™ instrument from GE
Healthcare–– Quartz crystal microbalance technology (QCM) on an Attana A200®
instrument–– Solution equilibrium titration (SET) on a Meso Scale Discovery™ (MSD™)
instrument

For more information, please contact your account manager atsales.muc@abdserotec.com.

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CH A PTER 2

49

ChapterApplications: Western
Blotting

3

Western blotting is widely used for the detection and identification of protein
antigens in samples such as solubilized cell pellets, cell culture supernatants,
and tissue homogenates. The procedure involves separation of the proteins bysize using gel electrophoresis, followed by transfer of the proteins onto a solid
support membrane where they can be detected with antibodies.
Samples for Western blotting are denatured by boiling in a buffer containing
SDS and a reducing agent prior to loading, which helps to unfold the protein
and disrupt disulfide bonds. It is customary to measure the protein content ofeach sample before denaturation, so that equivalent amounts are loaded into
the wells of the gel.
After the gel electrophoresis, the proteins are transferred to a blotting
membrane using an electric field. The membrane is then blocked to prevent
non-specific binding of proteins, before incubation with a primary antibody
recognizing the antigen of interest. A labeled secondary antibody is thenapplied to detect the target antigen. The most common detection systemrelies upon the reporter enzyme HRP, which cleaves a substrate to generate achemiluminescent product that is detectible by an imager or on X-ray film.

Use of HuCAL® Antibodies in Western Blotting
HuCAL® antibodies can be used for Western blotting by following the standard
protocols developed for conventional antibodies. The only difference is thatthe secondary antibody cannot be directed against the Fc domain since this islacking in HuCAL® Fab antibodies. An anti-human Fab secondary antibody (e.g.
STAR126P) is recommended instead, due to the signal amplification achieved
with this polyclonal secondary.
Alternatively, antibodies against the epitope tags (which are typically part of
the HuCAL® Fab antibody), such as Strep-tag®, His-6, V5, FLAG®, and c-myc
are suitable. In addition, if the Fab antibody contains an AP domain, then an
antibody against bacterial AP can be used for detection. If a primary antibody
is used regularly, or if the secondary antibody causes background, thenthe primary antibody can be labeled directly with our easy-to-use

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CH A P T E R 3Your first choice for antibodies!

LYNX Rapid Conjugation kits® (see page 119). Antibodies conjugated withLYNX kits achieve comparable sensitivity to polyclonal antibody detection witha significantly reduced assay time.
The sensitivity of an antibody in a given application depends on its affinity andavidity. Antibodies with multiple binding sites have better binding properties
than monovalent antibodies when multiple epitopes are presented on asolid support. The affinities of HuCAL® antibodies are comparable to thoseof other monoclonal antibodies. Bivalent antibodies have higher sensitivity
than monovalent antibodies since their avidity is greater. The bivalent format
of HuCAL® antibodies is, therefore, recommended for most applications,
including Western blotting.
AbD Serotec is equipped to run a large number of Western blots in parallel,allowing us to offer the testing of all HuCAL® antibodies resulting from anantibody generation project as an additional service. Contact your accountmanager at sales.muc@abdserotec.com for more information.
AbD Serotec also offers a free, detailed guide dedicated to Western blotting,visit www.abdserotec.com/westernblot to learn more.

General Tips
We strongly recommend sonication of lysates using ultrasound. This serves to
disrupt genomic DNA and should reduce non-specific background signals.
Furthermore, it is recommended that both a positive (antigen used for theantibody generation) and negative control (e.g. lysate not containing theantigen of interest) be included to confirm the specificity of results.
It is also important to use an appropriately matching secondary antibody
for the HuCAL® monoclonal antibody being tested. It is best to refer to the
HuCAL® antibody product sheet for the antibody format and tags. A list ofrecommended HRP-conjugated secondary antibodies is given in Table 5. For
other conjugates, please consult www.abdserotec.com.
It is also possible to use colorimetric detection substrates instead ofchemiluminescent detection; this is described in Example 3, (page 55).
When working with antibodies that have been labeled with LYNX Rapid
Conjugation kits®, we strongly recommend HiSpec buffer (BUF049) for
antibody dilution. It is necessary to test for optimal antibody concentration toachieve sensitivities and background levels equal to detection with the mostfavorable secondary antibodies.

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TABLE 5

Selection of HRP-conjugated Secondaries for Western Blotting.
Secondary Antibody

Recommended Dilution

Product Code

Anti-human F(ab’)2:HRP

1:5000

STAR126P

Anti-Strep-tag® Classic:HRP

1:500–1:4000

MCA2489P

Anti-His-6:HRP

1:1000–1:5000

MCA1396P

Anti-V5:HRP

1:1000

MCA1360P

Anti-bacterial AP:HRP

1:8000–1:10000

AHP1108P

Anti-FLAG :HRP

MCA4764P®

Anti-FLAG :HRP

1:1000–1:5000

AHP1074P

Anti-c-myc:HRP

1:100–1:500

MCA2200P®

For other conjugates, visit www.abdserotec.com/HuCAL

Protocol
1. Run the samples on SDS-PAGE and transfer onto a polyvinylidene fluoride(PVDF) membrane. For initial testing, use 40 µg of cell lysate proteins or
100 ng of pure antigen.
2. Block the membrane with 5% non-fat dried milk in tris-buffered salineTween® 20 (TBST), for 1 hour on a shaker at room temperature (RT), or
overnight at 4°C.
3. Rinse the membrane with TBST.
4. Dilute the HuCAL® antibody (primary antibody) in TBST with 1%
non-fat dried milk and add to the membrane. The optimal amount of
HuCAL® antibody is typically between 1 and 10 µg/ml, but this must be
determined empirically for each antibody. A concentration of 5 µg/ml is
recommended as a starting point.
5. Incubate for 1 hour at RT on a shaker. Make sure that the membrane iscompletely immersed in the buffer.
6. Wash the membrane three times (5 minutes per wash) with a generous
amount of TBST.
7. Add secondary antibody to the membrane. Anti-human Fab:HRP(STAR126P) is recommended at a 1:5000 dilution, either in TBST with 1%
non-fat dried milk or in HiSpec buffer (BUF049).
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CH A P T E R 3Your first choice for antibodies!

8. Shake for 1 hour at RT.
9. Wash the membrane three times (5 minutes per wash) with a generous
amount of TBST.
10. Develop the membrane using Amersham™ ECL Plus or ECL Advance™ (GEHealthcare) according to the manufacturer’s instructions. Capture theimage with an imager or X-ray film.

Alternative Protocol Using the SNAP i.d.® Protein Detection
System
1. Run the samples on SDS-PAGE and transfer onto a PVDF membrane. For
initial testing, use 40 µg of cell lysate proteins or 100 ng of pure antigen.
2. Insert the blotting membrane into the SNAP i.d. ® system (Millipore)according to the manufacturer’s instructions.
3. Block the membrane using 0.05–0.1% non-fat dried milk in TBST. Use
30 ml, 20 ml, or 10 ml for the one, two, or three blot holder (Millipore),
respectively. Run the blocking solution through the membranes without
prior incubation.
4. Apply the primary antibody at a 3-fold higher concentration compared tothe conventional protocol above, in TBST with 0.05% non-fat dried milk(e.g. 15 µg/ml is recommended for initial experiments). Incubate for 10
minutes with the pump switched off. Then apply vacuum for ~ 20 seconds.
Use 10 ml, 6 ml, or 3 ml antibody solution for the one, two, or three blot
holder, respectively.
5. Wash the blot three times by applying 30 ml, 15 ml, or 10 ml TBST for theone, two, or three blot holder, respectively, with the pump switched on.
6. Apply the secondary antibody, also at a 3-fold higher concentrationcompared to our conventional protocols. An anti-human Fab: HRP(STAR126P) at a 1:1500 dilution in HiSpec buffer (BUF049) isrecommended. Incubate for 10 minutes with the pump switched off. Then
apply vacuum for ~ 20 seconds. Use 10 ml, 6 ml, or 3 ml of antibody
solution for the one, two, or three blot holder, respectively.
7. Wash the blot three times by applying 30 ml, 15 ml, or 10 ml TBST for theone, two, or three blot holder, respectively, with the pump switched on.
8. Develop the membrane using Amersham™ ECL Plus or ECL Advance™
according to the manufacturer’s instructions. Capture results with animager or X-ray film.
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Examples
Human cyclophilin A from HEK293 cell lysate was detected on a Western
blot using a HuCAL® bivalent mini-antibody (Fab-dHLX-MSx2) as the primary
antibody, and anti-human F(ab’)2: HRP (STAR126P) as the secondary antibody.
The blot was developed using Amersham™ ECL Plus (Figure 17).

FIGURE 17

Western Blot Analysis of HEK293 Lysates. HuCAL® Anti-cyclophilin A Fab-dHLXMSx2 Antibody (5 µg/ml) and Anti-F(ab’)2:HRP were used to probe the blot. Detection
was performed using Amersham™ ECL Plus. Molecular Weight Standard at left. Lanes
1–5: 1:1 serial dilutions of HEK293 lysate from 4000 cells to 250 cells.

It is possible to omit a secondary antibody by using a primary antibody labeledwith LYNX kits, thereby avoiding background issues associated with secondary
antibodies. The protocol is significantly faster and provides sensitivities thatare comparable to those achieved using optimal secondary antibodies. The
anti-cyclophilin A antibody was conjugated to HRP with a LYNX kit (e.g.
LNK002P) and used for immunodetection as described in Example 1 (without
a secondary antibody) (Figure 18).

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CH A P T E R 3Your first choice for antibodies!

FIGURE 18

Western Blot Analysis of HEK293 Lysate Using a Directly Labeled Antibody. The
blot was probed with HRP-conjugated (LYNX) HuCAL® Anti-cyclophilin A Fab-V5Sx2
antibody (5 µg/ml). Detection was performed using Amersham™ ECL Plus. Molecularweight markers at left. Lanes 1–5: 1:1 dilutions of HEK293 lysate from 4000 cells to
250 cells.

The sensitivity of the anti-cyclophilin A HuCAL® mini-antibody (HCA005)
was determined by dot blotting. Pure cyclophilin A was spotted onto a PVDF
membrane and detected using the HuCAL® anti-cyclophilin A bivalent miniantibody and an anti-human F(ab’)2:HRP (STAR126P) (Figure 19).

FIGURE 19

Cyclophilin A Dot Blot. Dot blot using HuCAL® anti-cyclophilin Fab-dHLX-MH as
the primary antibody, anti-F(ab’)2:HRP (STAR126P) as the secondary antibody, andAmersham™ ECL Plus for detection.

A second dot blot (Figure 20) was prepared using the antigen, farnesyl
diphosphate synthetase (FDPS), and detected with a HuCAL®-bacterial AP
fusion antibody in the Fab-A-FH format (AbD02012). This format offers theadvantages of a bivalent antibody (dimerization occurs via AP domain)www.abdserotec.com

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together with enzymatic activity for detection. The blot was developed with
5-bromo-4-chloro-3’ indolylphosphate p-toluidine salt/nitro-blue tetrazolium
chloride (BCIP/NBT) substrate (BUF045A), without the use of a secondary
antibody.

FIGURE 20

FDPS Dot Blot. Dot blot using HuCAL® anti-FDPS Fab-A-FH (AbD02012) without asecondary antibody and BCIP/NBT for detection.

Troubleshooting
ProblemPossible Cause and Course of Action

No bands on theWestern blot

1. Transfer did not work. Check protein by staining the gel and/or
the membrane.
2. Incorrect secondary antibody used. Try an anti-human Fab or asuitable epitope tag antibody.
3. Antibody stored at 4°C for several weeks or subjected to several
freeze–thaw cycles. Use a fresh aliquot of antibody that has been
stored at –20°C or below.
4. ECL detection reagents are contaminated. Make up fresh
detection reagents.
5. Antigen not expressed in the source used or insufficient antigen
loaded on the gel. Check source is appropriate or increase amountof source material.
6. Antibody not suitable for Western blotting. Run a Western or dot
blot with sufficient amounts of pure antigen.

Weak signal

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CH A P T E R 3

1. Low transfer efficiency. Perform transfer according to the
manufacturer’s protocol.

Your first choice for antibodies!

2. Insufficient sample loaded on the gel. Load more sample.
3. Primary or secondary antibody concentration was too low.
Optimize the protocol for the reagents.
4. Film exposure time (for ECL detection) was too short. Re-expose
the blot for a longer time.
White (negative)
bands on the film
after detection
with ECL system

1. Too much protein loaded or antibody concentrations too high,leading to depletion of substrate. Load less antigen and optimize
antibody concentrations.

High backgroundsignal

1. Concentration of primary and/or secondary antibody too high.
Optimize antibody concentrations.
2. Transfer and incubation buffers are contaminated. Use fresh
buffers.
3. Contaminated blocking agent. Use fresh blocking agent.
4. Incorrect concentration of blocking agent. Make sure the correct
concentration is used, as described in the protocol.
5. Membrane dried out during one or more steps of the procedure.
Repeat, making sure that the membrane is not allowed to dry at any
time during the procedure.
6. Washing time too short or insufficient buffer volume used for
washes. Increase length of washing steps and use larger volume ofwash buffer.
7. Insufficient amount of Tween® 20 in the buffers. Use TBS with
0.1% Tween® 20.
8. Film overexposed or blot developed for too long. Reduce exposure
and/or development times.
9. Antibodies stored at 4°C for several weeks or subjected to several
freeze–thaw cycles. Use a fresh aliquot of antibody that has been
stored at –20 °C or below.
10. Non-specific interaction with genomic DNA. Sonicate the lysateswith ultrasound or treat with DNases to disrupt/digest genomic
DNA.
11. Secondary antibody is cross-reactive. Perform Western blot
without primary antibody to confirm cross-reactivity of secondary.
Replace cross-reacting secondary with substitute that is suitable for
the HuCAL® antibody format in use as the primary.

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ChapterApplications: ELISA

4An ELISA is used to detect antigens or antibodies in samples, either
qualitatively or quantitatively. It is usually performed using two antibodies
for detection, one specific to the antigen, and the second specific to thefirst antibody. The second antibody is coupled to an enzyme that acts on achromogenic or fluorogenic substrate to generate a signal. The enzymatic step
amplifies the signal so that even low levels of protein can be detected.
There are several types of ELISA:
■■

■■

■■

■■

Indirect ELISA is used to confirm binding of the antibody to its antigen,but not to unrelated antigens. Since all HuCAL® antibodies are tested
for positive performance in ELISA, an indirect ELISA is a good control for
testing all the reagents in the assay set-up.
Direct ELISA is very similar, but avoids the use of a secondary antibody byusing a labeled primary antibody.
Sandwich ELISA is used to detect and quantify an antigen in a sample
by the use of two specific primary antibodies binding to non-overlapping
epitopes. One antibody captures the antigen on the plate and the second isused for detection.
Competition (or Inhibition) ELISA detects antigens and monitors
their concentration using competitive binding of an antibody to free orimmobilized antigen.

Use of HuCAL® Antibodies in ELISAsELISAs can be performed with HuCAL® antibodies in the same protocols
used for other polyclonal or monoclonal antibodies provided that a suitablesecondary antibody is employed. Since HuCAL® antibodies do not contain the
Fc domain, an anti-human Fab secondary antibody is recommended (the useof polyclonal antiserum amplifies the signal). Monoclonal antibodies against
the epitope tag (e.g. Strep-tag® or His-6) can also be used. The bivalent formatof the HuCAL® mini-antibodies (Fab-dHLX or Fab-A) is recommended for ELISA
assays with immobilized antigen because their avidity is higher and is similar
to that of full IgGs. A list of recommended secondary antibodies is given in
Table 6.

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CH A P T E R 4Your first choice for antibodies!

TABLE 6

Selection of Secondary Antibodies for ELISA.
Secondary Antibody

Recommended Dilution

Product Code

Anti-human F(ab’)2:AP

1:5000

STAR126A

Anti-human F(ab’)2:HRP

1:5000

STAR126P

Anti-human F(ab’)2:Biotin

1:5000

STAR126B

Anti-Strep-tag Classic:AP

MCA2489A®

Anti-Strep-tag Classic:HRP®

1:5000Anti-Strep-tag Immo
(for Fab immobilization)

MCA2489P
MCA2488®

Anti-His-6:HRP

1:20–1:1000

MCA1396P

Anti-V5:HRP

1:1000

MCA1360P

Anti-bacterial AP:HRP

1:3500

AHP1108P

Anti-FLAG :HRP

1:10000–1:50000

AHP1074P®

Anti-FLAG :HRP

MCA4764P®

Anti-c-myc:HRP

1:100–1:500

MCA2200P

For other conjugates, visit www.abdserotec.com/HuCALAll HuCAL® antibodies are routinely tested by indirect ELISA. The staff at
AbD Serotec are highly experienced and well equipped to run all types of
ELISAs (e.g. testing for sandwich pairs, inhibition ELISAs, etc.) at highthroughput. We offer additional ELISA services in conjunction with any HuCAL®
project. Contact your account manager at sales.muc@abdserotec.com for
more information.

General Points■■■■

www.abdserotec.com

96-well plates can be used instead of 384-well plates, (e.g. black, flatbottom MaxiSorp™ PS, NUNC, 437111). For the 96-well format, use 100 µl(instead of 20 µl) of antigen, antibodies, or substrate and 300 µl for theblocking step.
In the examples shown, an AP-conjugated antibody was used, together
with the AttoPhos® (Roche, 11681982) fluorescence detection system(excitation: 440 ± 25 nm, emission: 550 ± 35 nm). Alternatively, an HRPconjugated antibody can be used together with QuantaBlu® (Thermo Fisher
Scientific, 15169) fluorescence detection reagent (excitation: 320 ± 25 nm,
emission: 420 ± 35 nm). If detection is performed using a chromogenic
substrate (e.g.TMB Plus [BUF042A]), then transparent plates must be used.
CH A PTER 4

59

■■

■■

If the antibody is to be immobilized as a capture antibody, this can be done
either by direct coating to polystyrene plates or by using an antibody for
capture. The anti-Strep-tag® Immo antibody (MCA2488) offers an extremely
good affinity and is well-suited for this purpose.
HiSpec Buffer (BUF049) is recommended for antibody dilution. It inhibits
weak interactions resulting in lower background and higher reproducibility,
especially for secondaries and directly-labeled primary antibodies.

Indirect ELISA
Indirect ELISA is often recommended as a control assay to test theperformance of reagents. In an indirect ELISA, the antigen is immobilized ona surface, such as a well of a microtiter plate. The plate is blocked to preventthe non-specific binding of detection antibodies, and then a specific antibody
is added to each well. The plate is washed to remove unbound antibody,leaving only the specific antigen-antibody complexes of interest. A secondary
antibody is added next. This is typically conjugated to an enzyme such as
HRP or AP, and binds the antibody-antigen complexes. The plate is washedand the appropriate substrate is applied. Finally, the resulting chromogenic or
fluorescent signal is viewed using a spectrophotometer (Figure 21).

60

FIGURE 21

Indirect ELISA. The antigen (blue) is coated to a surface. The primary antibody (graymini-antibody) binds to the antigen and an enzyme-linked secondary anti-Fab antibody(gray full-size antibody) is used for detection. The star represents the detection
substrate.

CH A P T E R 4Your first choice for antibodies!

Protocol
Indirect ELISA with Fluorescence Readout
1. Coat the antigen by adding 20 µl of a 5 µg/ml antigen solution diluted inphosphate-buffered saline (PBS) to several wells of a 384-well microtiterplate, and incubate overnight at 4°C. Use a black, 384-well ELISA
microtiter plate with a square-well and flat-bottom, such as MaxiSorp™
PS (NUNC, 460518).
2. Wash the microtiter plate twice with phosphate-buffered salineTween® 20 (PBST).
3. Block the microtiter plate by adding 100 µl 5% non-fat dried milk in PBST
to each well. Incubate for 1–2 hours at RT.
4. Wash the microtiter plate twice with PBST.
5. Transfer 20 µl HuCAL® antibody to each well. A standard concentration of
2 µg/ml in PBST or HiSpec buffer (BUF049) is recommended. Incubate for
1 hour at room temperature.
Note: Optimize the concentrations of each HuCAL® antibody by titration.
6. Wash the microtiter plate five times with PBST.
7. Add 20 µl of the secondary antibody to each well and incubate for 1 hourat room temperature. Anti-human Fab: AP (STAR126A), diluted 1:5000 in
HiSpec buffer (BUF049) is recommended.
8. Wash the microtiter plate five times with PBST.
9. Add 20 µl AttoPhos® (Roche, 11681982) to each well. Measurefluorescence after 10 minutes (excitation: 440 ± 25 nm, emission: 550 ±35 nm).

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Example
The antigen (a peptide derived from the pKi-67 protein and coupled toa carrier protein) was detected by ELISA with different concentrations of
HuCAL® antibody (bivalent mini-antibody format Fab-dHLX-MH, (HCA006)(Figure 22).

FIGURE 22

Indirect ELISA. The experiment was carried out with a HuCAL® Anti-pKi-67 Fab-dHLXMH Antibody and Anti-F(ab’)2:AP, with AttoPhos® detection.

Direct ELISA
In a direct ELISA, the primary antibody is labeled, for example, by genetic
fusion with AP or with LYNX Rapid Conjugation kits® (see page 119). This
saves time and money because no secondary antibody is required, which
is valuable for frequently run assays. It also avoids the cross-reactivity andbackground issues sometimes introduced by secondary antibodies.
LYNX-conjugated antibodies show excellent sensitivity, often similar oreven better than those achieved in indirect ELISAs. However, the AP genetic
fusion system is usually less sensitive than indirect detection via secondary
antibodies (Figure 23).

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CH A P T E R 4Your first choice for antibodies!

FIGURE 23

Direct ELISA. The antigen (blue) is coated to a surface. An AP-fused primary antibody(gray/green) is used in combination with an AP substrate (yellow star) for detection.

ProtocolDirect ELISA with AP-conjugated Primary Antibody
1. Coat the antigen by adding 20 µl of a 5 µg/ml antigen solution diluted in
PBS to several wells of a 384-well microtiter plate, and incubate overnightat 4°C. Use a black, 384-well ELISA microtiter plate: square-well,flat-bottom, such as the MaxiSorp™ PS (NUNC, 460518).
2. Wash the microtiter plate twice with PBST.
3. Block the microtiter plate by adding 100 µl of 5% non-fat dried milk in
PBST to each well, and incubate for 1–2 hours at room temperature.
4. Wash the microtiter plate twice with PBST.
5. Transfer 20 µl of labeled HuCAL® antibody to each well. Use a standard
concentration of 2 µg/ml in PBST or HiSpec buffer (BUF049). Incubate for
1 hour at room temperature.
Note: Optimize the concentrations of each HuCAL® antibody by titration.
6. Wash the microtiter plate five times using PBST.
7. Add 20 µl AttoPhos® (Roche, 11681982) to each well and measure thefluorescence after 10 minutes (excitation: 440 ± 25 nm, emission: 550 ±35 nm).

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Example
The antigen (a peptide derived from the pKi-67 protein coupled to a carrier
protein) was detected by both direct and indirect ELISA. Direct ELISA was
performed using a HuCAL® Fab-A-FH anti-pKi-67 antibody fused with AP(AbD02815; this is the same antibody as HCA006, but cloned into Fab-A-FH
format). Indirect ELISA was performed using the same antibody in the
Fab-dHLX-MH format (HCA006), and anti-Fab:AP detection (STAR126A)(Figure 24).

FIGURE 24

Comparison of Direct and Indirect ELISA. Direct ELISA was performed using
a HuCAL® Fab-A-FH anti-pKi-67 antibody fused to AP. For the indirect ELISA, thesame antibody was used in the Fab-dHLX-MH format, and anti-Fab:AP was used for
detection. Note that the use of a secondary antibody provides an amplification effectthat increases the sensitivity of the assay compared to the AP fusion. Using LYNX
kits to conjugate the antibody achieves sensitivities similar to secondary antibody
detection.

Sandwich ELISA
Sandwich ELISAs are a highly sensitive and specific method of detecting
antigens, which provide fast and accurate determination of the antigen
concentration in a given sample. The technique uses two antibodies, both ofwhich are specific to the antigen of interest, but which bind the antigen at
non-overlapping epitopes.
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CH A P T E R 4Your first choice for antibodies!

In a sandwich ELISA, it is possible to use a HuCAL® antibody as the capture
or detection antibody together with an existing commercial antibody, or
to use two different HuCAL® antibodies as the sandwich pair (see page 45
for selection strategies for antibody sandwich pairs). The bivalent format of
HuCAL® mini-antibodies is highly suitable for both capture and detection.
When two HuCAL® antibodies are used, the detection antibody is typicallybiotinylated (see page 119) for detection via streptavidin. Alternatively, thetwo HuCAL® antibodies must be cloned in different formats with detection via
a tertiary antibody specific for the detection antibody (e.g. Fab-dHLX-MSx2
capture antibody, Fab-A-V5Sx2 detection antibody and HRP-conjugated antibacterial AP tertiary antibody (AHP1108P) (Figure 25).

FIGURE 25

Sandwich ELISA Schematic. Sandwich ELISAs are carried out using three detection
options. Panel A: Two HuCAL® antibodies with direct detection. Panel B: Two HuCAL®
antibodies with indirect detection (using anti-AP:HRP). Panel C: A HuCAL® capture
antibody and a mouse polyclonal detection antibody plus an HRP-conjugated antimouse secondary antibody. The capture antibody (gray bivalent mini-antibody) is
immobilized and binds the antigen (blue).

Protocols
Sandwich ELISA Using Two HuCAL® Antibodies
1. Coat the capture antibody by adding 20 µl of a 5 µg/ml solution diluted in
PBS to several wells of a 384-well microtiter plate, and incubate overnightat 4°C. Use a black, 384-well ELISA microtiter plate, which has squarewells and a flat-bottom, MaxiSorp™ PS (NUNC, 460518).
2. Wash the microtiter plate twice with PBST.
3. Block the microtiter plate by adding 100 µl 5% BSA in PBST to each well,and then incubate for 1 hour at RT.
4. Wash the microtiter plate twice with PBST.
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5. Add 20 µl of antigen to each well of the microtiter plate, and incubate for
1 hour at RT. Use a range of antigen concentrations, diluted in PBST with
1% BSA or in HiSpec buffer (BUF049).
6. Wash the microtiter plate five times with PBST.
7. Add 20 µl HuCAL® detection antibody (1–2 µg/ml concentration in HiSpec
buffer (BUF049) to each well, and incubate for 1 hour at RT. Use one of
the following options:A. Use a biotinylated detection antibody.
	B. Use an antibody in Fab-A format (e.g. Fab-A-V5Sx2) as the detection
antibody in combination with Fab-dHLX capture antibody.
	C. Use a detection antibody carrying different tags than the capture
antibody (e.g. His-6 tagged capture antibody and Strep-tag detection
antibody).
8. Wash the microtiter plate five times with PBST.
9. Transfer 20 µl of detection antibody to each well and incubate for 1 hourat RT. Choose option below to match step 7.
	A. Use either streptavidin HRP (STAR5B) or streptavidin AP (STAR6B) in
combination with matching substrate, diluted 1:1000 in HiSpec buffer(BUF049).
	B. Use rabbit anti-bacterial AP HRP (AHP1108P), diluted 1:3500 in HiSpec
buffer (BUF049).
	C. Use matching epitope tag antibodies, e.g. anti-Strep-tag® Classic HRP(MCA2489P) diluted 1:5000 in HiSpec buffer (BUF049).
10. Wash the microtiter plate five times with PBST.
11. Add 20 µl QuantaBlu® (Thermo Fisher Scientific, 15169) to each well for
HRP-conjugated secondaries or Attophos® (Roche, 11681982) for APconjugated secondaries, and measure the fluorescence directly.

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CH A P T E R 4Your first choice for antibodies!

Sandwich ELISA with HuCAL® and Polyclonal Mouse
Antibodies
1. Coat the plate with the HuCAL® capture antibody by adding 20 µl of a
5 µg/ml solution, diluted in PBS, to several wells of a 384-well MaxiSorp™
microtiter plate. Incubate overnight at 4°C.
2. Wash the microtiter plate twice with PBST.
3. Block the microtiter plate by adding 100 µl 5% BSA in PBST to each well,and incubate for 1 hour at RT.
4. Wash the microtiter plate twice with PBST.
5. Add 20 µl of antigen to each well of the microtiter plate, and incubate for
1 hour at RT. Use a range of antigen concentrations, diluted in PBST with
1% BSA or HiSpec buffer (BUF049).
6. Wash the microtiter plate five times with PBST.
7. Add 20 µl biotinylated polyclonal detection antibody (concentration as
specified by the manufacturer) in HiSpec buffer (BUF049) to each well,and incubate for 1 hour at RT.
8. Wash the microtiter plate five times with PBST.
9. Transfer 20 µl of AP-conjugated streptavidin (STAR6B) diluted
1:2000–1:10000 in HiSpec buffer (BUF049) and incubate for 1 hour at RT.
10. Wash the microtiter plate five times with PBST.
11. Add 20 µl of AttoPhos® (Roche; 11681982) to each well, incubate for10 minutes, and measure the fluorescence (excitation: 440 ± 25 nm,
emission: 550 ± 35 nm).

ExamplesA sandwich ELISA was performed using:■■■■

A HuCAL® sandwich pair
A HuCAL® capture antibody together with a commercial polyclonal
detection antibody

For the experiment with the HuCAL® sandwich pair, a bivalent mini-antibody
directed against IFNγ in the Fab-dHLX-MH format (AbD0676, HCA043)
was used for capture. A serial dilution of the antigen IFNγ (PHP050) was
detected with a bivalent mini-antibody fused to bacterial AP (AbD02503,
Fab-A-FH, HCA044). For direct detection (Figure 26), AttoPhos® AP substratewww.abdserotec.com

CH A PTER 4

67

(Roche; 11681982) was used for signal development. An HRP-conjugatedrabbit anti-bacteria AP antibody (AHP1108P) was used together with
QuantaBlu® HRP substrate (Thermo Fisher Scientific; 15169) for indirect
detection (Figure 27). The use of an anti-AP secondary antibody amplifies thesignal, leading to higher sensitivity for this assay.
The mixed sandwich ELISA was performed using a HuCAL® capture antibody
against IFNγ (AbD00068, Fab-MH) and a polyclonal anti-IFNγ detection
antibody (R&D Systems, BAF285). Since the polyclonal antibody is biotinylated,
streptavidin-AP (STAR6B) and AttoPhos® AP substrate can be used for signal
development (Figure 28).

Direct Sandwich ELISAFIGURE 26

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CH A P T E R 4

Direct Sandwich ELISA for IFNγ. The ELISA used HuCAL® capture (Fab-dHLX-MH)
and detection (Fab-A-FH) antibodies, with direct detection through the genetic fusion
with AP and AttoPhos®.

Your first choice for antibodies!

Indirect Sandwich ELISAFIGURE 27

Indirect Sandwich ELISA for IFNγ. This ELISA used HuCAL® capture (Fab-dHLXMH) and detection (Fab-A-FH) antibodies, and an HRP-conjugated anti-bacterial AP
antibody and QuantaBlu® for indirect detection. Use of a secondary antibody provides
an amplification effect that increases the sensitivity of the assay compared with thedirect detection experiment shown in Figure 26.

Indirect Sandwich ELISA with a Polyclonal Antibody

FIGURE 28

Indirect Sandwich ELISA for IFNγ. This ELISA used a HuCAL® capture antibody (FabMH) and a biotinylated polyclonal detection antibody. Results were visualized withstreptavidin-AP and AttoPhos®.

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Competition (or Inhibition) ELISA
A competition (or inhibition) ELISA is ideal when only one suitable antibody
is available for the target of interest, or when the antigen is too small tobe detected by two antibodies e.g. a hapten. The technique measures theconcentration of a substance by its ability to interfere with an established pretitrated system. The primary antibody is first incubated with the free antigen.
It is then added to an antigen-coated well, and the plate is washed to remove
unbound antibody. The amount of antibody that binds the immobilized antigen
is detected using a secondary antibody linked to a detection label such as
HRP. The appropriate substrate is applied and the resulting chromogenic or
fluorescent signal is viewed using a spectrophotometer. With higher amountsof free antigen in the sample, there are fewer antibody molecules available tobind the immobilized antigen, resulting in a weaker signal. Conversely, lower
amounts of free antigen in solution generate stronger signals. Other variationsof a competition ELISA are also possible.

FIGURE 29

Examples of Competition ELISAs. Panel A: Competition ELISA with low
concentration of free antigen (blue); HuCAL® antibodies (gray) bind to the coatedantigen. Panel B: Competition ELISA with high concentration of free antigen. MoreHuCAL® antibodies are kept in solution by binding to free antigen and are washedaway before the addition of the secondary antibody.

Protocol1. Coat the plate with antigen by adding 50 µl of a of 5 µg/ml antigen
solution diluted in PBS to several wells of a 384-well MaxiSorp™
microtiter plate, and incubate overnight at 4°C.
2. Wash the microtiter plate twice with PBST.
3. Block the microtiter plate by adding 100 µl of 5% non-fat dried milk in
PBST to each well, and incubate for 1–2 hours at RT.
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CH A P T E R 4Your first choice for antibodies!

4. Incubate 25 µl of antigen solution with 25 µl of HuCAL® antibody(final concentration 2 µg/ml) for 1 hour at RT. Use a range of antigen
concentrations diluted in PBS or HiSpec buffer (BUF049).
5. Wash the microtiter plate twice with PBST.
6. Transfer 50 µl of HuCAL® antibody/antigen mix to each well of the
microtiter plate, and incubate for 1 hour at RT.
7. Wash the microtiter plate five times with PBST.
8. Transfer 50 µl of the secondary antibody (anti-human Fab:AP, STAR126A),
diluted 1:5000 in HiSpec buffer (BUF049), and incubate for 1 hour at RT.
9. Wash the microtiter plate five times with PBST.
10. Add 20 µl AttoPhos® (Roche, 11681982) to each well. Incubate for10 minutes at RT and measure fluorescence (excitation: 440 ± 25nm,
emission: 550 ± 35nm).

ExampleThe concentration of the antigen, FITC, in solution was determined bycompetition ELISA. BSA-conjugated FITC was coated onto a microtiter plate
and a mix of primary HuCAL® antibody (AbD00756, HCA002) and varying
amounts of free FITC were transferred to the coated plate. Detection was
performed using anti-human F(ab’)2:AP (STAR126A).

FIGURE 30

Competition ELISA. An anti-FITC HuCAL® antibody (AbD00756) is added to coated
FITC-BSA. Binding of the antibody to FITC-BSA is inhibited by the addition of varying
amounts of soluble FITC.

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Troubleshooting
ProblemPossible Cause and Course of Action

No signal in ELISA

1. Assay incorrectly set up or incorrect reagents used. Include a
positive control.
2. Improper secondary antibody used. Use an anti-human Fab ora suitable epitope tag antibody.
3. Antibody stored at 4°C for several weeks or subjected toseveral freeze–thaw cycles. Use fresh aliquot of antibody that has
been stored at –20°C or below.
4. Detection reagent contaminated. Use freshly prepared
detection reagents.
5. Antigen not coated properly. Try longer coating times, different
buffers, or use avidin plates with biotinylated antigen.
6. Incorrect settings on plate reader for this detection system.
Check values (wavelength, filters, gain etc.).

Weak signal

1. Insufficient amount of antigen coated to microtiter plate. Use
more antigen for coating step.
2. Concentration of primary or secondary antibody too low.
Optimize the protocol for the reagents.
3. Detection reagent too old or contaminated. Use fresh
detection reagent.
4. Detection reagent diluted. Use a higher concentration of
detection reagent.
5. Incorrect settings on plate reader for this detection system.
Check values (wavelength, filters, gain etc.).

High backgroundsignal

1. Concentration of primary and/or secondary antibody too high.
Optimize antibody concentrations.
2. Too much detection reagent used. Repeat with more dilutedetection reagent.
3. Number of washing cycles too low. Increase number ofwashing cycles.
4. Contaminated blocking agent. Use fresh blocking agent.

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5. Incorrect concentration of blocking agent. Make sure that thecorrect concentration is used, as described in the protocol.
6. Incubation time of detection reagent too long. Start
measurement soon after addition of detection reagent.
7. Incorrect settings on the reader. Adjust the settings (e.g. gain).
8. Insufficient amount of Tween® 20 in the buffers. Use PBS with
0.05% Tween® 20.
9. Use HiSpec buffer (BUF049) for primary and secondary
antibody dilution.

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ChapterApplications:
Immunoprecipitation

5

Immunoprecipitation is used to isolate an antigen from cells or tissues with an
antigen-specific antibody attached to a sedimentable matrix. The method canalso help to identify members of protein complexes in solubilized cell or tissue
extracts by targeting any one of the proteins thought to be in the complex. The
antibody brings the complex out of solution so that it can be analyzed using
standard techniques such as Western blotting and mass spectrometry. HuCAL®
antibodies are ideal for immunoprecipitation.
The procedure consists of four stages:■■

Immobilization of the antibody onto a matrix■■

Solubilization of the antigen■■

Immunoprecipitation■■

Analysis of the precipitated protein(s)Basic procedures for each step are outlined in this chapter.
Protein A or G Sepharose® is often used as a matrix for sedimenting
conventional antibodies, since these compounds bind to the Fc portion of theantibody. However, since HuCAL® Fabs lack the Fc region, a different strategy
is required.
There are three procedures suitable for immobilizing HuCAL® Fabs:
■■
■■

■■

Covalent coupling on magnetic beads via primary amino groups
Immobilization on Ni-NTA magnetic beads via the His-6 tag or
immobilization on MagStrep Strep-Tactin® beads for Strep-tagged HuCAL®
antibodies
Immobilization of a capture antibody (see Table 7) on magnetic beads andincubation with the HuCAL® antibody

Magnetic beads are preferable to regular beads because they are easier towash. Ni-NTA beads should not be used with lysis buffer containing metalchelating compounds (e.g. ethylenediaminetetraacetic acid [EDTA]). Protocols
for the first two options are given below.

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TABLE 7

Methods to Pull Down the Immunocomplex.
Matrix

MethodAdvantages

Disadvantages

Primary antibody
coupled beads

Fab covalent coupled

Low background, little
nonspecific reaction
since secondary
antibodies are used

Coupling required
for every antibody;high primary antibody
consumption

Anti-F(ab’)2 (STAR126)
coupled beads

Capture FabGood for testing manydifferent antibodies

Pull-down of intrinsic
antibodies in thehuman samples

Anti-Strep®-tag Immo
(MCA2488) coupledbeads

Capture Fab via
StrepX tag

Excellent antibody
affinity, good fortesting many different
antibodies

None

Anti-Strep®-tag
Classic (MCA2489)
coupled beads

Capture Fab via Strep
tag

Good for testing manydifferent antibodies

None

Anti-His-6 tag(MCA1396) coupledbeads

Capture Fab via His-6
tag

Good for testing manydifferent antibodies

None

Anti-V5 tag(MCA1360GA)
coupled beads

Capture Fab via V5 tag

Good for testing manydifferent antibodies

None

Anti-FLAG® tag(MCA4764) coupledbeads

Capture Fab via
FLAG® tagGood for testing manydifferent antibodies

None

MagStrep type 2
Strep-Tactin® beads(IBA)

Capture Fab via Streptag®

Ready-to-use beads,
good for testing manydifferent antibodies

None

TALON™ beads(Ni-NTA beads;
Invitrogen)Capture Fab via His-6
tag

Ready-to-use beads,
good for testing manydifferent antibodies

Slightly higher
background, might
require extra washingsteps

Methods that use a bead coupling step were tested with Dynabeads® M-450 Epoxy(Invitrogen)

AbD Serotec offers testing of HuCAL® antibodies in immunoprecipitation
under customer specified conditions. Contact your account manager atsales.muc@abdserotec.com for more information.

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Protocols
Covalent immobilization on Dynabeads® M-450 Epoxy
1. Wash 300 µl magnetic Dynabeads® M-450 Epoxy (Invitrogen, 14011) twice
using 0.5 ml phosphate buffer, 0.1 M, pH 7.4.
2. Resuspend beads in phosphate buffer and add 100 µg HuCAL® Fab to givea final volume of approximately 350 µl.
3. Incubate on a rotator for 16–20 hours at RT.
4. Place the tube in a magnetic holder for 1 minute and remove the supernatant.
5. Wash the beads three times with 1 ml of PBS.
6. Resuspend beads in 700 µl blocking solution (3% BSA in PBS).
7. Block the beads on a rotator for 30 minutes at RT.
8. Wash the beads once with 700 µl PBS.
9. Resuspend the beads in 300 µl PBS.
Note: This protocol can also be used for the immobilization of capture
antibodies. In that case, capture 10 µg of HuCAL® antibody onto 100 µl
capture antibody coupled beads. Incubate for 1hour at RT on a shaker or
rotator and wash beads with PBST.

Immobilization on Dynabeads® TALON™ via the His-6 tag(Suitable for Fab formats with a His-6 tag, e.g. Fab-dHLX-MH)

1. Wash 50 µl Dynabeads® TALON™ (Invitrogen, 10101D) twice with 700 µl
PBS.
2. Resuspend the beads in 100 µl PBS and add 20 µg HuCAL® Fab. Add PBS toa final volume of 700 µl.
3. Incubate on a rotator for 10 minutes at RT.
4. Place the tube in a magnet holder for 1 minute and remove thesupernatant.
5. Wash the beads four times with 700 µl PBS.
6. Resuspend beads in 700 µl blocking solution (3% BSA in PBS).
7. Block the beads on a rotator for a further 30 minutes at RT.
8. Wash the beads once with 700 µl PBS.
9. Resuspend the beads in 100 µl PBS.
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Solubilization
Various techniques can be used for this step, depending on the origin andnature of the antigen. Below is a basic protocol suitable for extracting proteinsfrom mammalian cells.
1. Use either a fresh or frozen cell pellet (containing 0.5–2.0 × 107 cells)
thawed on ice.
2. Resuspend the cells in 1 ml ice-cold non-denaturing lysis buffer (Tris/NaCl/
NP 40/EDTA/protease inhibitor [TNEC]) by gentle agitation on a vortex
mixer for 30 seconds. Use medium speed to avoid foaming.
3. Store the suspension on ice for 15–30 minutes, then transfer to a
microfuge tube.
4. Clear the lysate by centrifugation for 15 minutes at maximum speed,
at 4°C.
5. Transfer the supernatant to a fresh microfuge tube. To avoid disturbing thepellet, leave 20–40 µl supernatant in the original tube.
6. Store the supernatant on ice until used for immunoprecipitation.

Immunoprecipitation
This step involves incubation of the immobilized antibodies with thesolubilized antigen, followed by extensive washing to remove unboundprotein.
1. Add 100 µl bead-coupled antibodies to 1 ml lysate.
2. Incubate on a rotator for 1–2 hours at 4°C.
3. Wash four times with 0.5 ml ice-cold TNEC buffer for a total wash time of
approximately 30 minutes. Store samples on ice for 3–5 minutes between
washes, if necessary.
4. Wash once with PBS.

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Analysis
This step is performed using standard techniques, such as: Coomassie®-stained
PAGE, Western blotting (see Chapter 3), or mass spectrometry.
1. Resuspend the pellet in 10 µl reducing SDS gel loading buffer (sample
buffer).
2. Heat to 95°C for 5 minutes, and then cool on ice.
3. Run the entire sample on SDS-PAGE and electrotransfer onto a PVDF
membrane, and then refer to chapter 3 for Western blot analysis steps.

ExampleGranta cells (~1.3 × 107 cells per immunoprecipitation) were used for
precipitation of vimentin with HuCAL® Fab mini-antibodies. Different formatsof the same clone were tested with appropriately matching precipitation
methods. Precipitated proteins were detected using Western blot analysis
with the same anti-vimentin antibody (Fab-dHLX-MSx2 format, HCA111)
conjugated to HRP (using the LNK 002P LYNX kit). Direct detection without asecondary antibody has the advantage that the Fab bands of the precipitating
antibody are not visible on the blot.

78

FIGURE 31

Immunoprecipitation of Vimentin. Western blot analysis using anti-Vimentin:HRP(Fab-dHLX-MSx2 [HCA111] conjugated to HRP with LNK002P kit) and ECL detection.
Different HuCAL® anti-Vimentin antibody formats and beads were used for the
immunoprecipitations. Lane 1: Fab-dHLX-MSx2 coupled to M450 Epoxy beads. Lane 2:
Fab-dHLX-MH on Talon™ beads. Lane 3: Fab-A-V5Sx2 on anti-V5 coupled beads, Lane
4: Fab-dHLX-MSx2 on anti-Strep-tag® Immo (MCA2488) beads. Lane 5: Fab-A-V5Sx2
on MagStrep Strep-Tactin® beads.

CH A P T E R 5Your first choice for antibodies!

Troubleshooting
ProblemPossible Cause and Course of Action

No bands on
Western blot

1. Insufficient antigen in sample. Increase amount of source
material used for immunoprecipitation.
2. Antigen not expressed in the source. Test for antigen expression
in source by Western blotting with concentrated lysate.
3. Insufficient antibody used. Use more immobilized antibody.
4. Antigen not properly solubilized. Try different lysis buffers.
5. Antibody not suitable for immunoprecipitation. Test
immunoprecipitation with pure antigen.
6. Antibody does not work in Western blotting. Test Western blot
with pure antigen (and lysate).
7. Antibody stored at 4°C for several weeks or subjected to several
freeze–thaw cycles. Use fresh aliquot of antibody that has been
stored at –20°C or below.

High backgroundsignal due to nonspecific binding of
unrelated protein

1. Use additional washing steps (e.g. 15 times with 0.5 ml for atleast 1 hour).
2. Optimize lysis buffer for your cells.
3. Use covalently immobilized antibody or other secondary
antibody for precipitation.
4. Try a different blocking agent for bead blocking step.
5. Increase number of washing steps.

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ChapterApplications:
Immunohistochemistry6

IHC is used to detect antigens directly in tissue sections. Experiments can be
performed on a wide range of biological specimens using different detection
methods. Therefore, it is important to optimize the IHC conditions for eachantigen under investigation.
Most samples for IHC are embedded in paraffin because this simplifies storage
and provides excellent morphological detail and resolution. Cryostat sections,or cryosections, are typically used if no paraffinized tissue is available. In
addition, they may be employed if the antigen cannot be detected after
formalin fixation and paraffin-embedding, due to protein cross-linking. This
chapter describes the standard procedures used with each type of tissue
section, followed by fixation methods and typical staining procedures.

Use of HuCAL® Antibodies in IHC
Monoclonal antibodies are generally preferred over polyclonal antibodies for IHC
because they usually have a higher specificity. HuCAL® antibodies offer a numberof additional advantages over conventional monoclonal antibodies, including rapid
generation time and pre-existing epitope tags to simplify detection.
TABLE 8

Recommended Secondary Antibodies for IHC.
Secondary Antibody

Recommended Dilution

Product Code

Anti-human F(ab’)2:HRP

1:50–1:500

STAR126P

Anti-Strep-tag Classic

1:50–1:200

MCA2489

Anti-His-6

1:50–1:200

MCA1396

Anti-His-6

1:60

Roche,1922416

1:50–1:100

AHP1108P®

Anti-AP:HRP
Anti-FLAG :HRP

MCA4764P®

Anti-V5
Anti-mouse IgG:Biotin

MCA1360
1:50–1:200

STAR11B

Visit www.abdserotec.com/HuCAL for more information. Note: Anti-c-myc
antibodies are not recommended for use in IHC.

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CH A P T E R 6Your first choice for antibodies!

Paraffin Sections
Paraffin tissue sections must be cleared of paraffin (deparaffinized) andrehydrated before use to ensure that the antibodies have full access to thetissue antigen. Samples frequently require pretreatment to unmask the
antigen. This is because treatment with formaldehyde fixative often induces
intramolecular and intermolecular cross-linking of proteins. As a result,conformational changes may occur that mask the domains of the antigen
bound by the antibody, leading to non-specific staining.

Procedure for Deparaffinization and Rehydration
1. Tissue sections are dried at 60°C for 1 hour. Drying increases adhesionof the tissue sections to the surface of the glass slide. Important: useadhesive slides.
2. Place the slides in a cuvette containing sufficient xylene to cover thetissue completely and incubate for 5 minutes with gentle shaking.
3. Transfer the slides to a cuvette containing fresh xylene and repeat step 2
for a further three washes, to give a total of four washes, each lasting 5
minutes. Important: Use fresh xylene for each wash.
4. Wash the slides twice in 100% ethanol (for 2 minutes per wash).
5. Wash the slides twice in 90% ethanol (for 2 minutes per wash).
6. Wash the slides twice in 80% ethanol (for 2 minutes per wash).
7. Wash the slides twice in 70% ethanol (for 2 minutes per wash).
8. Wash the slides twice in 60% ethanol (for 2 minutes per wash).
9. Wash the slides twice in 50% ethanol (for 2 minutes per wash).
10. Wash the slides twice in TBST (for 2 minutes per wash).

Cryosections
There are two types of cryosections:■■■■www.abdserotec.com

Fresh or unfixed sections: quickly frozen tissues are cut, and then airdried. Sometimes they are fixed before staining.
Fixed frozen tissue: the tissue is fixed first, then cryoprotected withsucrose or another stabilizer, before freezing and sectioning.

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Frozen sections offer a number of advantages: they allow excellent antigen
preservation; they are typically faster to perform; and they allow optimizationof the fixative for each antigen, since any fixative can be used.
Important notes:■■

Avoid thawing the tissue sample, since this may destroy the antigen■■

Use adhesive slides■■

Air-dry sections completely before fixation■■

All steps can be carried out at RT unless otherwise specified

Fixation
Methanol, acetone, or formaldehyde can be used for fixation. Methanol andacetone precipitate proteins, while formaldehyde cross-links them. The choiceof fixative is likely to affect the staining result, therefore the most suitablereagent should be determined by experimentation for each antigen and set of
conditions.

Procedure
1. Submerge slides in one of the following:
A. Methanol for 10 minutes.
B. Acetone for 10 minutes.
C. 1:1 mixture of methanol and acetone for 10 minutes.
D. 4% formaldehyde for 2 minutes.
2.	Wash sections several times in freshly made TBST to ensure that the
fixative is completely removed from the sample.

Mounting
Either aqueous or non-aqueous mounting media is suitable, depending on thechromogenic substrate used to detect the antigen. We recommend a nonaqueous medium for permanent mounting of 3,3’ diaminobenzidine (DAB)
slides, although the faster procedure using an aqueous medium can also be
used. Slides containing ethanol-soluble chromogens must be mounted inaqueous medium.

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Non-aqueous Mounting
1. Transfer slides to 50% ethanol and agitate gently for a few seconds.
2. Transfer slides to 70% ethanol and agitate gently for a few seconds.
3. Transfer slides to 80% ethanol and agitate gently for a few seconds.
4. Transfer slides to 90% ethanol and agitate gently for a few seconds.
5. Transfer slides to 100% ethanol and agitate gently for a few seconds.
6. Transfer slides to xylene for 2 minutes.
7. To each slide, apply one or two drops of non-aqueous mounting medium tothe uncovered tissue and apply the coverslip.
Important: apply the coverslip at an inclined angle to the slide and lower it
gently to avoid trapping air bubbles.
8. Leave the slides to air-dry overnight at RT, or dry in an incubator at 60°C for
15 minutes.

Aqueous Mounting
1. Add one or two drops of aqueous mounting medium to the stained
tissue and apply the coverslip. Important: apply the coverslip at anangle to the slide and lower it gently to avoid trapping air bubbles.
2. Observe slide immediately.

Staining
Cryosections and paraffin-embedded sections were stained with a HuCAL®
bivalent mini-antibody (Fab-dHLX-MH, AbD05054, HCA071) against
recombinant human desmin expressed in E. coli. Desmin belongs to theclass III intermediate filaments, which constitute part of the cytoskeleton.
It is the characteristic intermediate filament found in all three types of muscle
cells (skeletal, cardiac, and smooth muscle).
Structurally, desmin is a 52 kDa protein encoded by nine exons of a genelocated on chromosome 2q35. It forms cytoskeletal networks of muscle
fibers between the plasma and nuclear membranes, and is found in the
subplasmalemmal region and the Z-band. The anti-desmin antibody AbD05054
labels both smooth and striated muscle cells. It is useful for the identificationof rhabdomyosarcomas and leiomyomas.
This section describes six alternative staining protocols using a HuCAL®
antibody together with different amplification and detection systems.
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Staining of Tagged HuCAL® Fabs (HRP-labeled anti-His-6
antibody)
This is a fast, but relatively insensitive, method. It requires high concentrationsof primary antibody (10–25 µg/ml), which can lead to high backgroundsignals. The antigen is detected by a directly-labeled anti-tag antibody(Figure 32).

Procedure
1. Deparaffinize the slides according to the protocol on page 81.
2. Wash the slides a further three times (for 2 minutes per wash) in TBST.
3. Submerge the slides in TBST, place in a microwave oven (700–900 W), andheat until the buffer reaches boiling point.
4. Reduce the power to 250 W and boil the slides for 30 minutes.
5. Replace water evaporated during boiling with ddH2O and allow thesections to cool in the buffer for 15 minutes.
6. Wash the slides three times (for 2 minutes per wash) in TBST.
7. Incubate in 3% H2O2 in methanol for 10 minutes to quench peroxidase in
the tissue.
8. Wash the slides three times (for 2 minutes per wash) in TBST.
9. Transfer slides to a solution of 10% BSA in PBS and incubate for 15
minutes at room temperature to block proteins.
10. Tip excess blocking solutions off the sections, apply 10–25 µg/ml ofprimary HuCAL® antibody in antibody-diluent (BUF014), and incubate for
1 hour. The concentration of antibody should be determined empiricallyfor each antigen, staining system, and laboratory.
11. Wash the slides three times (for 2 minutes per wash) in TBST.
12. Apply mouse anti-His-6 antibody conjugated to HRP (MCA1396P), diluted
1:50–1:200 in antibody diluent (BUF014), and incubate for 30 minutes.
13. Wash the slides three times (for 2 minutes per wash) in TBST.
14. Add 1 drop (approximately 40 µl) of DAB chromogen per 1 ml of DAB
substrate buffer (BUF21A and BUF022), mix by swirling, and apply totissue. Incubate for 10 minutes.
15. Wash the slides three times (for 2 minutes per wash) in TBST.
16. Counterstain using hematoxylin (Sigma, MHS16) for 1 minute.
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CH A P T E R 6Your first choice for antibodies!

17. Rinse slide with tap water to enhance color development.
18. Rinse slides with ddH2O.
19. Dehydrate with graded alcohol (see mounting procedure on page 82) andapply a permanent coverslip to the slides.

FIGURE 32

Staining of Human Small Bowel. 400x magnification, using HRP-labeled HuCAL®
Fab AbD05054 at 10 µg/ml and DAB detection.

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Staining of Tagged HuCAL® Fabs without Amplification(ABC or LSAB Method)
This straightforward procedure is suitable only for tissues that do not containendogenous biotin. A major advantage is the low concentration of primary
antibody necessary (usually only 1–5 µg/ml), which reduces the risk ofbackground staining. It is the most common method found in commercial
detection kits.
The antigen is visualized using a primary antibody, an anti-tag secondary
antibody, and a biotinylated anti-species antibody, followed by an enzymelabeled streptavidin complex (Figure 33).

Procedure
1. Deparaffinize the slides according to the protocol on page 81.
2. Wash the slides three times (for 2 minutes per wash) in TBST.
3. Submerge the slides in TBST, place in a microwave oven (700–900 W), andheat until the buffer reaches boiling point.
4. Reduce the power to 250 W and boil the slides for 30 minutes.
5. Replace water evaporated during boiling with ddH2O and allow thesections to cool in the buffer for 15 minutes.
6. Wash the slides three times (for 2 minutes per wash) in TBST.
7. Incubate in 3% H2O2 in methanol for 10 minutes to quench peroxidase in
the tissue.
8. Wash the slides three times (for 2 minutes per wash) in TBST.
9. Transfer slides to a solution of 10% BSA in PBS and incubate for 15
minutes to block proteins.
10. Tip excess blocking solution off the sections, apply 1–5 µg/ml primary
HuCAL® antibody in antibody diluent (BUF014), and incubate for 1 hour.
The concentration of antibody should be determined empirically for eachantigen, staining system, and laboratory.
11. Wash the slides 3 times (for 2 minutes per wash) in TBST.
12. Apply mouse anti-His-6-tag antibody (MCA1396), diluted 1:50–1:200 in
antibody diluent (BUF014), and incubate for 30 minutes.
13. Wash the slides three times (for 2 minutes per wash) in TBST.

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CH A P T E R 6Your first choice for antibodies!

14. Apply biotinylated anti-mouse antibody (STAR11B), diluted to 1:50–1:200
in antibody diluent and incubate for 15 minutes.
15. Wash the slides three times (for 2 minutes per wash) in TBST.
16. Apply streptavidin-peroxidase complex (STAR5B), and incubate for 15
minutes.
17. Wash the slides three times (for 2 minutes per wash) in TBST.
18. Add 1 drop (approximately 40 µl) of DAB Chromogen per 1 ml of DAB
substrate (BUF021A and BUF022), mix by swirling, and apply to tissue.
Incubate for 10 minutes.
19. Wash with ddH2O.
20. Counterstain using hematoxylin (Sigma, MHS-16) for 1 minute.
21. Rinse slide with tap water to enhance color development.
22. Rinse slides with ddH2O.
23. Dehydrate with graded alcohol (see mounting protocol on page 82) andapply a permanent coverslip to the slides.

FIGURE 33

Staining of Human Small Bowel. 400x magnification, with HuCAL® Fab AbD05054
at 5 µg/ml, detected using the ABC or LSAB method.

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Staining of Tagged HuCAL® Fabs with Amplification Using
the EnVision™ System
The EnVision™ system is a fast and simple three-step amplification procedurethat does not use streptavidin. The antigen is detected using a HuCAL®
antibody, followed by a mouse anti-His-6 and an anti-mouse secondary
antibody bound to an HRP-labeled dextran backbone. This system also uses
low concentrations of primary antibody, which reduces background staining(Figure 34).
1. Deparaffinize the slides according to the protocol on page 81.
2. Wash the slides twice (for 2 minutes per wash) in TBST.
3. Submerge the slides in the buffer, place in a microwave oven
(700–900 W), and heat until the buffer reaches boiling point.
4. Reduce the power to 250 W and boil the slides for 30 minutes.
5. Replace water evaporated during boiling with ddH2O and allow thesections to cool in the buffer for 15 minutes.
6. Wash the slides three times (for 2 minutes per wash) in TBST.
7. Incubate in 3% H2O2 in methanol for 10 minutes to quench peroxidase in
the tissue.
8. Wash the slides twice (for 2 minutes per wash) in TBST.
9. Transfer slides to a solution of 10% BSA in PBS and incubate for 15
minutes to block proteins.
10. Tip excess blocking solution off the sections, apply 1–5 µg/ml primary
HuCAL® antibody in antibody diluent (BUF014), and incubate for 1 hour.
The concentration of antibody should be determined empirically for eachantigen, staining system, and laboratory.
11. Wash the slides three times (for 2 minutes per wash) in TBST.
12. Apply mouse anti-His-6-tag antibody (MCA1396), diluted 1:50–1:200 in
antibody diluent (BUF014), and incubate for 30 minutes.
13. Wash the slides three times (for 2 minutes per wash) in TBST.
14. Apply ready-to-use EnVision™ anti-mouse HRP (Dako, K4007), andincubate for 15 minutes.
15. Wash the slides three times (for 2 minutes per wash) in TBST.

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CH A P T E R 6Your first choice for antibodies!

16. Add 1 drop (approximately 40 µl) of DAB Chromogen per 1 ml of DAB
substrate (BUF021A and BUF022), mix by swirling and apply to tissue.
Incubate for 5 minutes.
17. Wash with ddH2O.
18. Counterstain using hematoxylin (Sigma, MHS-16) for 1 minute.
19. Rinse slide with tap water to enhance color development.
20. Rinse slides with ddH2O.
21. Dehydrate with graded alcohol (see mounting protocol on page 82) andapply a permanent coverslip to the slides.

FIGURE 34

Staining of Human Small Bowel. 400x magnification, with HuCAL® Fab AbD05054,
1 µg/ml, detected with the EnVision™ system.

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Staining of Tagged HuCAL® Fabs with Tyramide
Amplification
This procedure avoids the use of tertiary antibodies and is suitable for murine
tissues. It uses biotinylated tyramide to amplify the signal, resulting in
improved detection sensitivity and requiring only very low concentrations ofprimary antibody (0.1–1 µg/ml). Antigens are detected using an HRPconjugated secondary antibody against the tag of the HuCAL® Fab.
Biotinylated tyramide is activated by the peroxidase of the secondary antibody
to form highly-reactive, short-lived tyramide radicals, which bind to tyrosine
residues of proteins in the vicinity of the HRP target. The biotin on the boundtyramide is easily visualized using standard ABC techniques25 (Figure 35).
1. Deparaffinize the slides according to the protocol on page 81.
2. Wash the slides three times (for 2 minutes per wash) in TBST.
3. Submerge the slides in TBST, place in a microwave oven (700–900 W), andheat until the buffer reaches boiling point.
4. Reduce the power to 250 W and boil the slides for 30 minutes.
5. Replace water evaporated during boiling with ddH2O and allow thesections to cool in the buffer for 15 minutes.
6. Wash the slides three times (for 2 minutes per wash) in TBST.
7. Incubate in 3% H2O2 in methanol for 10 minutes to quench peroxidase in
the tissue.
8. Wash the slides three times (for 2 minutes per wash) in TBST.
9. Transfer slides to a solution of 10% BSA in PBS and incubate for 15
minutes to block proteins.
10. Tip excess blocking solution off the sections, apply 1–25 µg/ml primary
HuCAL® antibody in antibody diluent (BUF014), and incubate for 1 hour.
11. Wash the slides three times (for 2 minutes per wash) in TBST.
12. Apply HRP-labeled mouse anti-His-6-tag antibody, (MCA1396P), diluted
1:50–1:200 in antibody diluent (BUF014). Incubate for 30 minutes.
13. Wash the slides 3 times (for 2 minutes each time) in TBST.

von Wasielewski, R. et al. (1997) Tyramine amplification technique in routine immunohistochemistry.
J Histochem Cytochem 45:1455–1460
25

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CH A P T E R 6Your first choice for antibodies!

14. Prepare a working solution of activated biotinylated tyramide (see‘Composition of Buffers’, page 130) by adding 5 µl tyramide stock
solution (freshly thawed) and 2 µl 30% H2O2 per 1 ml of TBST.
15. Apply the activated biotinylated tyramide to the slides for 15 minutes.
16. Wash the slides three times (for 2 minutes per wash) in TBST.
17. Apply streptavidin-peroxidase complex (STAR5B), diluted 1:500, andincubate for 15 minutes.
18. Wash the slides three times (for 2 minutes per wash) in TBST.
19. Add 1 drop (approximately 40 µl) of DAB Chromogen per 1 ml of DAB
substrate (BUF021A and BUF022), mix by swirling and apply to tissue.
Incubate for 10 minutes.
20. Wash with ddH2O.
21. Counterstain using hematoxylin (Sigma, MHS-16) for 1 minute.
22. Rinse slide with tap water to enhance color development.
23. Rinse slides with ddH2O.
24. Dehydrate with graded alcohol (see mounting protocol on page 82) andapply a permanent coverslip to the slides.

FIGURE 35

Staining of Human Small Bowel. 400x magnification, labeled with HuCAL Fab
AbD05054 at 0.1 µg/ml using tyramide amplification.

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Staining of Tagged HuCAL® Fabs Using a Mouse-on-mouse
System
The mouse-on-mouse system is a simple method for staining mouse tissues.
A special blocking reagent eliminates background from endogenous mouse
Igs. Antigens are detected with a mouse anti-tag antibody and a biotinylated
anti-mouse secondary antibody, followed by an enzyme-labeled streptavidin
complex.
The protocol is performed using a customized detection kit from Vector
Laboratories (PK-2200) (Figure 36).
Important note: before starting the protocol, prepare working solutions
according to the manufacturer’s instructions.

Procedure
1. Deparaffinize the slides according to the protocol on page 81.
2. Wash the slides three times (for 2 minutes per wash) in TBST.
3. Submerge the slides TBST, place in a microwave oven (700–900 W), andheat until the buffer reaches boiling point.
4. Reduce the power to 250 W and boil the slides for 30 minutes.
5. Replace water evaporated during boiling with ddH2O and allow thesections to cool in the buffer for 15 minutes.
6. Wash the slides three times (for 2 minutes per wash) in TBST.
7. Incubate in 3% H2O2 in methanol for 10 minutes to quench peroxidase in
the tissue.
8. Wash the slides three times (for 2 minutes per wash) in TBST.
9. Incubate the tissue sections for 1 hour in a working solution of mouseon-mouse Ig blocking reagent, prepared according to the manufacturer’s
instructions.
10. Wash the slides three times (for 2 minutes per wash) in TBST.
11. Incubate tissue sections for 5 minutes in a working solution of M.O.M™
diluent, prepared according to the manufacturer’s instructions.
12. Tip excess M.O.M™ diluent off slides. Dilute the primary antibody in
M.O.M™ diluent to the appropriate concentration and incubate thesection with the diluted primary antibody for 1 hour.
13. Wash the slides three times (for 2 minutes per wash) in TBST.
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CH A P T E R 6Your first choice for antibodies!

14. Incubate the slides for 30 minutes in a working solution of mouse antiHis-6 antibody (MCA1396), diluted 1:50–1:200 in M.O.M™ diluent.
15. Wash the slides three times (for 2 minutes per wash) in TBST.
16. Apply a working solution of M.O.M™ biotinylated anti-mouse Ig reagent,
prepared as described in the manufacturer’s instructions Incubate thesections for 10 minutes.
17. Wash the slides 3 times (for 2 minutes each time) in TBST.
18. Apply Vectastain ABC Reagent, prepared according to the manufacturer’s
instructions, and incubate the sections for 5 minutes.
Note: the reagent must be prepared 30 minutes before use.
19. Wash the slides 3 times (for 2 minutes each time) in TBST.
20. Add 1 drop (approximately 40 µl) of DAB Chromogen per 1 ml of DAB
substrate (BUF021A and BUF022), mix by swirling and apply to tissue.
Incubate for 10 minutes.
21. Wash the slides three times (for 2 minutes per wash) in TBST.
22. Counterstain using hematoxylin (Sigma, MHS-16) for 1 minute.
23. Rinse slide with tap water to enhance color development.
24. Rinse slides with ddH2O.
25. Dehydrate with graded alcohol (see mounting protocol on page 82) andapply a permanent coverslip to the slides.

FIGURE 36

Staining of Mouse Small Bowel. 400x magnification, labeled with HuCAL® Fab
AbD05054 at 5 µg/ml, using mouse-on-mouse system.

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Staining of Tagged HuCAL® Fabs with an Anti-Strep-tag®
Antibody and HISTAR IHC Detection Kit
AbD Serotec’s HISTAR IHC Detection System reagents are designed specificallyto enhance the IHC technique with a simple kit format. This kit provides
linking and labeling reagents intended for use with species specific primary
antibodies for visualizing cellular antigens in tissue specimens. Visualizationis accomplished using an ultra-sensitive indirect labeling method that utilizes
novel polymer labeling technology. Secondary antibodies are linked directlywith HRP into compact polymers bearing a high ratio of enzyme to antibody.
This biotin-free system offers enhanced sensitivity and minimal backgroundstaining.
All labeling and blocking reagents are pre-diluted for immediate use, andchromogen is provided in concentrated format. The kit is suitable for use
with HuCAL® antibodies in combination with a wide range of secondary
antibodies. In the example shown in Figure 37, the anti-desmin antibody wasused as bivalent mini-antibody with a c-myc and double extended Strep-tag®
(Fab-dHLX-MSx2, AbD08860) in combination with an anti-Strep-tag® antibody.
The protocol has also been successfully used with the following: an anti-V5
tag antibody (MCA1360) or anti-bacterial AP antibody (AHP1108P) together
with anti-desmin in Fab-A-V5Sx2 format.

Procedure
1. Deparaffinize the slides according to the protocol on page 81.
2. Wash the slides twice (for 2 minutes per wash) in TBST.
3. Submerge the slides in TBST, place in a microwave oven (700–900 W), andheat until the buffer reaches boiling point.
4. Reduce the power to 250 W and boil the slides for 30 minutes.
5. Replace water evaporated during boiling with ddH2O and allow thesections to cool in the buffer for 15 minutes.
6. Wash the slides three times (for 2 minutes per wash) in TBST.
7. Incubate in Peroxide Buffer (BUF017A) for 15 minutes to quench
peroxidase in the tissue.
8. Wash the slides three times (for 2 minutes per wash) in TBST.
9. Transfer slides to Serum Block, vial 2 of HISTAR detection kit (STAR3000)
and incubate for 15 minutes.

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10. Tip excess blocking solution off the sections, apply 5–40 µg/ml primary
HuCAL® antibody in TBST or antibody diluent (BUF014) and incubate for
30 minutes. The concentration of antibody must be determined empiricallyfor each antigen, staining system, and laboratory.
11. Wash the slides three times (for 2 minutes per wash) in TBST.
12. Apply Special Block (vial 3; shake before use) and incubate for 20
minutes.
13. Wash the slides three times (for 2 minutes per wash) in TBST.
14. Apply anti-Strep-tag® Classic antibody (MCA2489), diluted 1:50–1:200 in
antibody-diluent (BUF014) and incubate for 30 minutes.
15. Wash the slides three times (for 2 minutes per wash) in TBST.
16. Apply Polymer Link (HRP) (vial 4; shake before use), and incubate for
30 minutes.
17. Wash the slides three times (for 2 minutes per wash) in TBST.
18. Add 1 drop (approximately 40 µl) of DAB Chromogen per 1 ml of DAB
substrate (BUF021A and BUF022), mix by swirling and apply to tissue.
Incubate for 5 minutes.
19. Wash with ddH2O for 3 minutes.
20. Counterstain using hematoxylin (Sigma, MHS-16) for 1 minute.
21. Rinse slide with tap water to enhance color development.
22. Rinse slides with ddH2O.
23. Dehydrate with graded alcohol (see mounting protocol on page 82) andapply a permanent coverslip to the slides.

FIGURE 37

Staining of Human Appendix. Left: Human appendix at 400x magnification, labeledwith HuCAL® Fab AbD08860, 10 µg/ml using an anti-Strep-tag® antibody and the
HISTAR Detection Kit. Right: Negative control staining without HuCAL® antibody.

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Troubleshooting
ProblemPossible Cause and Course of Action

No staining ofsamples, controls,
or background

1. Primary or secondary antibody not used. Repeat experimentaccording to the protocol.
2. Antibody stored at 4ºC for several weeks or subjected to several
freeze–thaw cycles. Repeat experiment using a fresh aliquot of
antibody that has been stored at –20ºC or below.
3. Slides prepared with alcohol-soluble chromogen were mounted inalcohol-based medium. Repeat the color reaction and use aqueousmounting medium.
4. Buffers for peroxidase staining contained azide. Repeat
experiment with buffers that do not contain azide.
5. Incompatible buffers used for the enzyme and substrate reagents.
Repeat the staining reaction with compatible buffers.

Weak or nonspecific staining

1. Concentration of primary antibody too low (or too high). Repeat
the experiment, adjusting the concentration of primary antibody(often by 2–5 fold), and increasing the incubation time if the
background staining was low.
2. Proteolytic digestion omitted. Some antigens require proteolytic
digestion, depending on the fixation protocol and the antibody.
3. Too much washing buffer left on slide before incubation withthe primary antibody, leading to dilution of the antibody. Repeat
the staining procedure and remove the buffer by tapping the slide
carefully on a paper towel.
4. Use of expired reagents. Repeat staining with fresh reagents.
5. Incubation times too short. Repeat experiment with longer
incubation times.

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CH A P T E R 6Your first choice for antibodies!

ChapterApplications: FACS™
Analysis – Flow Cytometry

7

Fluorescence activated cell sorting (FACS™) analysis or flow cytometry is atechnique that measures characteristics, such as surface markers, on single
cells. It is also used to detect antigen-expressing cells, to detect antigens
bound to cells, and to separate antigen-expressing from non-expressing cells.
As with many other immunoassays, cells are incubated with an antigenspecific antibody and a fluorescence-labeled secondary antibody. Detection
is performed by passing the cell suspension through a flow cytometer or
FACS™ instrument. The suspension is then scanned by a laser and the lightscattering characteristics and fluorescence are measured, allowing detectionof cells carrying fluorescence-labeled antibodies. If required, cells expressinga particular antigen can also be separated from those that do not by applying
an appropriate electric charge which deflects the cells into different sample
tubes.
Contact your account manager at sales.muc@abdserotec.com for more
information. AbD Serotec also offers a detailed application guide that can beaccessed at www.abdserotec.com/flow.

Use of HuCAL® Antibodies in Flow Cytometry
HuCAL® antibodies are well suited for use in flow cytometry. Their small
size and lack of an Fc region results in low background and avoids potential
interactions between the primary antibody and Fc receptors on cells.
In addition, HuCAL® antibodies routinely carry epitope tags that allow them
to be detected on antibody-expressing cells, such as B-cells. While the bivalentmini-antibody format (Fab-dHLX) is recommended, since it has higher avidity,
the monovalent format can also be used. Table 9 lists the recommended
secondary antibodies.
For the detection of HuCAL® antibodies with cells that do not produce human
IgG, AbD Serotec recommends the use of an anti-human IgG conjugate that is
specific for heavy and light chains (STAR97PE), allowing detection of any Fabformat, regardless of the tags present.
For cells which inherently display human IgG, detection via the tag on the
HuCAL® antibody is required. Check the format of the HuCAL® antibody anduse an appropriate secondary antibody from the list in Table 9. A conjugatedwww.abdserotec.com

CH A PTER 7

97

anti-tag antibody or an unconjugated secondary antibody followed by aconjugated anti-mouse antibody can be used. Use of an anti-c-myc antibody
is not recommended, since it can give higher background due to endogenous
c-myc in dead cells.
TABLE 9Secondary Antibodies Recommended for Flow Analysis.
Secondary Antibody

Recommended Dilution

Product Code

Anti-human IgG (H+L):PE
F(ab’)2 fragment

1:400

STAR97PE

Anti-Strep-tag® Immo

MCA2488

Anti-Strep-tag Classic

MCA2489®

Anti-V5:FITC

Neat-1:5

MCA2894F

Anti-V5:FITC

Neat-1:5

MCA2895F

Anti-FLAG M2

1:2000Sigma, F3165

Anti-His-6:FITC

1:10

MCA1396F

Anti-His-6

1:500

MCA1396

Anti-mouse IgG (H+L):PE
F(ab’)2 fragment

1:5

STAR105PE

Anti-mouse IgG:FITC
F(ab’)2 fragment

Undiluted – 1:500

STAR9B®

Protocols
Cells expressing the target antigen, and, where available, a negative control
cell line, are required. All buffers used during the staining procedure must
contain the appropriate supplements e.g. azide and ions, where necessary.

Recommended Controls
■■

■■

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CH A P T E R 7

Background control: Use PBS instead of the HuCAL® antibody, withflow buffer and detection antibody. Use two control samples if a tertiary
antibody is required (Sample 1: anti-tag + anti-mouse antibodies; Sample
2: anti-mouse antibody only).
Negative control: Use unrelated negative control HuCAL® antibody,with flow buffer and detection antibody. AbD Serotec recommends testing
several dilutions of purified Fab for flow cytometry and using a non-related
Fab in identical dilutions as a negative control.

Your first choice for antibodies!

■■Positive control (if available): Include with each flow cytometry
experiment to check whether the cells are expressing the selection antigen.

Preparation of Cells
1. Adherent cells: Detach cells from their support using Trypsin/EDTA(Versene), and collect in a 50 ml sterile plastic tube.
Suspension cells: Collect in a 50 ml sterile plastic tube.
2. Wash the cells once in ice-cold flow buffer, add buffer and invert to mix,
then centrifuge at 190 × g for 5 minutes at 4°C. Preparation of flow
buffer is described on page 129.
3. Discard the supernatant and resuspend the cell pellet in the remaining
drop. Add 5 ml ice-cold flow buffer and mix by inverting the tube.
Centrifuge at 190 × g for 5 minutes at 4°C.
4. Discard the supernatant and gently resuspend the cell pellet in theremaining drop. Add a small volume of ice-cold flow buffer.
5. Stain cells with Trypan Blue and count the cells. Add flow buffer andadjust the density of live cells to 0.5 × 106 cells/100 µl.
6. Add 100 µl of cell suspension (0.5 × 106 cells) to each well of a 96-wellround bottom tissue culture plate, and centrifuge the plate at 716 × g for
2 minutes at 4°C.
7. Carefully remove the supernatant and gently vortex the plate to bringthe cells into suspension in the remaining liquid. When using only a few
wells, aspirate the supernatant with a pipet. If using an entire 96-wellplate, invert the plate quickly but carefully over a tray to remove thesupernatant. Then, do not tap the plate on a paper towel, instead turnplate upwards and blot surface moisture from the top with a paper towel.

Procedure for Cell Staining
1. Prepare serial dilutions of HuCAL® antibodies, between 0.1 and 100 µg/ml
in flow buffer. (10 µg/ml is a standard concentration.) Preparation of flow
buffer is described on page 129.
2. Add 100 µl of diluted antibody and control solutions to the wells
containing antigen-expressing cells and negative control cells. Incubate
for 1 hour at 4°C with gentle agitation.
3. Centrifuge at 716 × g for 2 minutes at 4°C. Remove the supernatant andwash each well 2–3 times with 200 µl flow buffer. When using only a few
wells, aspirate the supernatant with a pipet. If using an entire 96-wellwww.abdserotec.com

CH A PTER 7

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plate, invert the plate quickly but carefully over a tray to remove thesupernatant. Then, do not tap the plate on a paper towel; instead blot
surface moisture from the top of the plate with a paper towel.
4. Add one of the following secondary antibodies:
A. Add 100 µl RPE-conjugated goat anti-human IgG (H+L) specific F(ab’)2
fragment (STAR97PE) diluted 1:400 in flow buffer to the wells (samples
and controls) containing antigen-expressing cells and negative control
cells.
B. Add 100 µl conjugated anti-tag secondary antibody (e.g. STAR1396F)
diluted in flow buffer to the wells (samples and controls) containing
antigen-expressing cells and the negative control cells.
C. Add 100 µl mouse anti-tag secondary antibody (see Table 9) diluted
in flow buffer to the wells (samples and controls) containing antigenexpressing cells and the negative control cells.
5. Incubate for 1 hour at 4°C with gentle agitation.
6. Centrifuge at 716 × g for 2 minutes at 4°C. Remove the supernatant andwash 2–3 times with 200 µl flow buffer, as described in step 3.
7. Tertiary antibody incubation (only for unconjugated secondary antibodies,step 4C). Add 100µl of RPE-conjugated goat anti-mouse IgG antibody(STAR105PE) to each well, diluted 1:5 in flow buffer. Incubate for 1 hourat 4°C with gentle agitation followed by centrifugation and wash, as
described in step 3.
8. After the final washing step, centrifuge for 2 minutes at 4°C. Resuspend
cells in 100–200 µl flow buffer and transfer to FACS™ tubes. For shortterm storage (up to 4 days), stained cells can be resuspended in 100 µl
of 4% paraformaldehyde in PBS after the final washing step. Cover cellswith aluminum foil and store at 4°C. Immediately before FACS™ analysis,
add flow buffer to the required final volume.

ExamplesTransiently transfected HEK293 cells were analyzed by flow cytometry using amonovalent HuCAL® antibody in Fab-FH format (with FLAG® and His-6 epitopetags) against a human transmembrane protein. Detection was performed
either with a PE-labeled anti-human IgG heavy and light chain specificsecondary antibody, or with an anti-FLAG® or an anti-His-6-tag antibody in
combination with a PE-conjugated anti-mouse antibody. A clear shift of thetransfected cells is visible for all three detection systems (Figure 38).
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FIGURE 38

Flow Cytometry of Transiently Transfected HEK293 Cells.
Left column: Unrelated antibody (control). Right column: Antigen-specific antibody.
Row 1: Detection with a PE-conjugated anti-human IgG antibody. Row 2: Detection
with a mouse anti-FLAG® M2 antibody and a PE-conjugated anti-mouse IgG. Row 3:
Detection with a mouse anti-His-6-tag antibody and a PE-conjugated anti-mouse IgG.

CD81-positive cells were detected by FACS™ analysis from washed humanblood using an anti-CD81 HuCAL® antibody (AbD06083, HCA086 and
AbD06084, HCA087 in Fab-dHLX-MH format or AbD08863, HCA112 and
AbD8864, HCA113 in Fab-dHLX-MSx2 format, respectively) together with antiStrep-tag® Classic (MCA2489) and FITC-conjugated anti-mouse IgG antibody(STAR9B) detection. A clear shift of the peak can be observed when comparedto the negative control sample.

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FIGURE 39

Flow Cytometry of CD81-positive Cells from Washed Human Blood.
Left: Anti-CD81 HuCAL® antibody (AbD08864, HCA113) and detection with anti-Streptag® Classic (MCA2489) and anti-mouse IgG FITC (STAR9B). Right: Negative control
without HuCAL® antibody.

Troubleshooting
ProblemPossible Cause and Course of Action

No staining

1. Antibody stored at 4°C for several weeks or subjected to several
freeze–thaw cycles. Repeat the experiment using a fresh aliquot of
antibody that has been stored at –20°C or below.
2. Incorrect secondary antibody used. Use a conjugated anti-humanFab specific, IgG (H + L), or a suitable anti-tag antibody with amatching tertiary antibody (see Table 9, page 98).
3. Failure of conjugation reaction (primary antibody to PE). Repeat
the experiment, following the protocols on antibody conjugation(Chapter 8).

Weak shift

1. Antibodies not stored properly. Repeat the experiment using fresh
antibodies.
2. Affinity of secondary antibody for the primary antibody too low. Trya different secondary antibody. If the recommended anti-human IgG(H + L) was used, try the anti-human Fab.
3. Antibody concentrations too low. Use a higher concentration ofprimary and/or secondary antibody.

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Chapter

Immunofluorescence

8

Immunofluorescence is a technique frequently used for the analysis of tissue orcells stained with fluorescence-labeled antibodies. Labeled samples are studied
under a fluorescence microscope or by confocal microscopy. Similar to otherimmunoassays, detection is usually accomplished with a labeled secondary
antibody, although direct labeling of the primary antibody may be an advantage
if the secondary antibody causes non-specific background signal.
HuCAL® antibodies can be used in immunofluorescence assays in the samemanner as conventional antibodies with appropriate secondary reagents.
Their exquisite specificity makes them ideal reagents for immunofluorescenceanalysis. The bivalent mini-antibody format is recommended due to the avidity
effect. Recommended secondary antibodies are listed in Table 10.
For a complete list of all available conjugates, contact your account manager
at sales.muc@abdserotec.com or visit www.abdserotec.com for more
information.
TABLE 10

Selection of Secondary Antibodies Recommended for Immunofluorescence.
Secondary Antibody

Recommended Dilution

Product Code

Anti-human F(ab’)2:FITC

1:50–1:200

STAR126F

Anti-human F(ab’)2:TRITC

1:50–1:200

STAR126TRC

Anti-Strep-tag Classic

1:50–1:200

MCA2489

1:50–1:200

MCA1396

Anti-His-6-tag:Alexa Fluor 488

Undiluted – 1:100

MCA1396A488

Anti-His-6-tag:Alexa Fluor 647

Undiluted – 1:100

MCA1396A647

Anti-His-6-tag®
®

Anti-His-6-tag: DyLight 549

1:50–1:200

MCA1396D549

Anti-AP

1:50–1:100

AHP1108P®

Anti-V5 tag

MCA1360

Anti-V5 tag:Alexa Fluor® 488

1:100

MCA1360A488

Anti-V5 tag: Alexa Fluor® 647

1:100

MCA1360A647

Anti-V5 tag:DyLight 549

1:100–1:500

MCA1360D549®

Anti-mouse IgG:Cy5

OBT1725C

Anti-mouse IgG (H + L):DyLight 649®

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1:500–1:1000

STAR117D649

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Protocol
Sample Preparation
Cultured cells
1. Grow cells in multi-well plates.
2. Rinse cells briefly with sterile PBS.
3. Aspirate PBS and fix cells with either:	A. 2–4% paraformaldehyde (PFA) for 10 min at 37°C.
	B. 100% methanol for 10 min at –20°C
Note: use sufficient solution to cover cells properly.
4. Aspirate fixative and wash cells with PBS, three times for 5 minutes per wash.
5. Optional: Permeabilization. Choose either option A or B.
	A. Add 0.2-0.5% Triton® X-100 for 5 min at 37°C, rinse with PBS for
5 minutes.
	B. Remove PBS and add ice-cold methanol to cells. Use a sufficient amountto cover cells completely. Cells must not dry out. Incubate cells for 10
minutes at –20°C, rinse with PBS for 5 minutes.
Paraffin sections
Refer to the protocol on page 81 for deparaffinization and rehydration. For
antigen retrieval, see steps 1–6 of the IHC protocol on page 84
Frozen sections
Refer to the protocol on page 82 for fixation of cells.

Immuostaining
1. Block sample with 3% BSA or fetal calf serum (FCS) in PBS for 1 hour.
Alternatively, use 3–5% serum from the same species as the secondary
antibody, in PBS.
2. Remove blocking solution and apply HuCAL® antibody at 1–10 µg/ml in
PBS. Incubate overnight at 4°C.
3. Wash sample three times with PBS (5 minutes per wash).
4. Apply secondary antibody in PBS with 1% BSA or FCS and incubate for 1–2
hours in the dark.
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CH A P T E R 8Your first choice for antibodies!

5. Wash sample three times with PBS (5 minutes per wash).
6. Mount and seal the slides. For best results, examine samples immediately.
Store slides at 4°C in the dark.

ExamplesMCF7 breast carcinoma cells were stained for Ki-67, a proliferation marker
localized in the nucleus, with a Ki-67 specific HuCAL® mini-antibody(AbD02531, HCA006). Detection was performed with an Alexa Fluor® 488
conjugated anti-His-6 secondary antibody (MCA1396A488). Cells were also
stained with Alexa Fluor® 660 Phalloidin (red), and the cell nucleus was
stained blue with Hoechst 33342 (Invitrogen, H1399). See Figure 40. The green
spots in Figure 40 indicate Ki-67 in growing cells.

FIGURE 40

Immunofluorescence Detection of Ki-67 in MCF-7 Breast Cancer Cells. Cells
were stained with HuCAL® Antibody AbD02532 (green spots) and images were taken
on an IN Cell Analyzer 1000 (GE Healthcare) at 40x magnification.

Contactin-associated protein, Caspr, was examined in mouse nerve cells using
a HuCAL® anti-Caspr mini-antibody (AbD06152). Detection was performed
with an Alexa Fluor® 647 conjugated anti-human IgG secondary antibody (red)on an Olympus FluoView® 1000 laserpoint scanning confocal microscope.
Ankyrin G delineates the nodes of Ranvier and was stained green with amouse anti-ankyrin G antibody to make the outline of the nerves visible(Figure 41). Caspr is found at the flanking paranodes.

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CH A PTER 8105

FIGURE 41

Immunofluorescence Analysis of Caspr and Ankyrin G in Mouse Cerebellum
Sections. Nerves were stained with an anti-ankyrin G antibody to mark the nodes ofRanvier (green). Anti-Caspr HuCAL® antibody (AbD06152) was used in combination
with an anti-human IgG antibody to detect Caspr at the flanking paranodes (red).
Images were captured with an Olympus FluoView® 1000 laserpoint scanning confocal
microscope26.

26

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Data courtesy of B. Ranscht, Burnham Institute for Medical Research, La Jolla, CA, USA.
Your first choice for antibodies!

ChapterApplications:
Bead-based Assays

9

Luminex xMAP® Technology
The Luminex xMAP® technology is popular for antibody assays and diagnostic
testing due to its large multiplexing capacity of up to 100 analytes per well.
xMAP® is based on flow cytometry technology using fluorescent microspheres(beads). The use of beads with different fluorescent dyes allows differentiation
between the types of beads and thus, between different samples that arecovalently coupled to the beads. With just one well, it is possible to test
the binding of an antibody to different immobilized antigens; or by using
immobilized antibodies, the presence of different antigens in one sample canbe detected (multiplex assay).
The technology has also proved to be very useful for the detection of
sandwich pairs. For example, if 10 different antibodies are to be screened fortheir suitability as sandwich pairs, it would require at least 100 wells in an
ELISA (10 capture antibodies × 10 detection antibodies). With the xMAP®
technology, the same assay can be performed in just 10 wells.
HuCAL® antibodies are well suited for this application due to their highspecificity. xMAP® technology is well established at AbD Serotec. For more
information about timelines and costs, contact your account manager atsales.muc@abdserotec.com.

ProtocolThis is a standard protocol for a Luminex® xMAP® sandwich assay using a
HuCAL® capture antibody together with a biotinylated HuCAL® detection
antibody.

Bead Coupling
For coupling to LiquiChip® Carboxy Beads (QIAGEN, 922402), follow the
manufacturer’s instructions.
To reduce the loss of beads during the coupling steps, AbD Serotec
recommends the use of pipet tips from Mettler Toledo (RT-250, RT-1000) andvials from USA Scientific (1415-2500).
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xMAP® Assay
1. Prewet filter microtiter plate (MultiScreenHTS-BV, 1.2 µm PVDF, Millipore,
MSBVN1210) with 200 µl assay buffer [PBST (0.05% Tween® 20) with
1% BSA] per well.
2. Transfer the plate to vacuum manifold base (e.g. Vac-Man® 96 Vacuum
Manifold, Promega, A2291) and apply low vacuum. Tap the plate briefly
onto a paper towel to remove remaining droplets.
3. Dilute capture antibody coupled beads in assay buffer to 1.5 × 105 beads/
ml and transfer 1–10 µl of each bead type to the well of a filter plate(150–1500 beads of each type per well).
Note: Starting with 1500 beads/well is recommended only if a few
different beads are used.
4. Add 50 µl antigen diluted in PBS. Use a concentration range of 0–5000
ng/ml. Resuspend beads on a shaker for 1 hour at RT.
5. Transfer the plate to a vacuum manifold, apply low vacuum and wash
three times with 200 µl assay buffer. Tap the plate briefly onto a paper
towel to remove remaining droplets.
6. Add 70 µl/well biotinylated detection antibody at 2 µg/ml in PBS.
Resuspend beads on a shaker for 1 hour at RT.
7. Wash the plate five times with 200 µl assay buffer, as described in step 5.
8. Add 70 µl/well streptavidin-RPE (STAR4B) diluted 1:5 in assay buffer.
Resuspend beads on a shaker for 1 hour at RT.
9. Wash plate five times with 200 µl assay buffer, as described in step 5.
10. Add 100 µl assay buffer and vortex the plate for 1 minute.
11. Analyze the samples on a Luminex® 100™ instrument as per the
manufacturer’s instructions.

ExampleA sandwich assay was performed with a HuCAL® capture and detection
antibody against human IFNγ. The capture antibody (AbD00676, HCA043)
was immobilized on beads and detection was performed using biotinylated
detection antibody (AbD02503, HCA044). Human IFNγ (PHP050) was used
in a concentration range from ~1 pg/ml to 10 ng/ml. The calculated limit ofdetection (LOD) was 2.9 pg/ml (Figure 42).

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CH A P T E R 9Your first choice for antibodies!

FIGURE 42

Luminex Sandwich Assay for IFNγ. The assay used two HuCAL® antibodies, one
for capture and one for detection. The detection antibody was biotinylated for further
detection by streptavidin-PE.

FMAT® FLISA assays
FMAT® is a fluorescence-linked immunosorbent assay (FLISA) which uses the8200 Cellular Detection System (8200 CDS; Applied Biosystems). The 8200
CDS is a macroconfocal imager that detects fluorescence on a particle (cellor bead). Speed and simplicity are the main advantages of this technique. In
contrast to ELISA, it is a homogenous mix and measure assay without any
washing steps. Though this technology was originally developed for cellular
assays, it has proved very useful for bead-based assays.
In this example, antigen-coated beads, primary antibody, and fluorescencelabeled secondary antibody are mixed in a 96- or 384-well microtiter plate.
A laser scans the bottom 100 µm layer of the well and detects fluorescenceof labeled secondary antibody bound to beads. The background from freesecondary antibody in solution is automatically subtracted (Figure 43).

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FIGURE 43

FMAT® Technology. Schematic drawing of one well of a FMAT® plate with beads (inred). A laser scans a square area and detects the fluorescence-labeled sample on thesurface of the beads. Background from sample in solution in the bottom layer issubtracted (red line in the diagram), resulting in images with white spots in front of a
black/gray background.27

One other advantage of this system is the low antigen consumption required
for screening a 384-well plate in comparison to an ELISA. AbD Serotec
recommends this assay for all antibody generation projects with limited
antigen availability.
Screening in a bead-based assay can also be superior to an ELISA dependingon the intended use of the antibodies. The conformation of antigens on beads
is believed to be closer to native conformation than is achieved in coating toan ELISA plate. It is known that differences in antigen presentation may leadto differing results with some antibodies. In some cases, an antibody may bepositive in one assay, but not in another.

27

110

CH A P T E R 9Image included courtesy of Applied Biosystems.
Your first choice for antibodies!

Protocol
Bead Coupling
1. Resuspend M-450 Epoxy Dynabeads® (Invitrogen, 14011) by vortexing for
1–2 minutes.
2. Immediately transfer 300 µl (~1.2 × 108 beads) into a 2.0 ml vial or
24-well microtiter plate.
3. Place the vial or plate on a magnet (DynaMag™-2, Invitrogen, 123-21D)
for 1 minute and carefully remove the supernatant.
4. Remove vial or plate from the magnet and resuspend beads in 1 ml of
100 mM sodium phosphate buffer, pH 8.0. Mix by inversion with gentle
tilting and rotation.
5. Repeat steps 3 and 4, and then remove supernatant.
6. Calculate the required volumes of antigen (60 µg) and buffer to yield afinal volume of 400 µl.
7. Resuspend the beads in 100 mM sodium phosphate buffer, pH 8.0, andadd the antigen solution as in step 6.
8. Incubate for 16–20 hours on an overhead rotator/shaker at RT. Ensurethat the beads do not settle during incubation, and are mixed by inversion
with gentle tilting and rotation.
9. Add 20 µl 1M Tris-HCl, pH 7.4, and incubate on a rotator/shaker for 2
hours.
10. Wash beads three times with 500 µl PBS.
11. Resuspend the beads in 500 µl PBS and store at 2–8°C until needed.
Notes: Amount of antigen is calculated for a~30 kDa protein and can be
adjusted. Antigen buffer must not contain primary amines (e.g. Tris) or
detergents (e.g. Tween® 20, Triton® X-100).

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FMAT® Assay
1. Resuspend antigen-coated beads in PBST (0.05% Tween® 20) with 3% BSA
and secondary antibody to a final concentration of ~1.3 × 105 – 5.0 × 105
beads/ml. AbD Serotec recommends using a Cy5 conjugated anti-humanFab secondary antibody (Jackson ImunoResearch, 109-175-097 at a 1:5000
dilution).
2. Transfer 40 µl into each well of a 384-well plate.
3. Add 5 µl of the HuCAL® antibody at 45 µg/ml (final concentration of
5 µg/ml).
4. Incubate for 1 hour at RT in the dark. Measure plate with the 8200 CDS, as
per the manufacturer’s instructions.

Troubleshooting
ProblemPossible Cause and Course of Action

No signal

1. Insufficient beads per well. Prepare a fresh dilution of beads.
Resuspend beads before each pipetting step.
2. Antibody stored at 4°C for several weeks or subjected to several
freeze–thaw cycles. Repeat the experiment using a fresh aliquot of
antibody that has been stored at –20°C or below.
3. Incorrect secondary antibody used. Use a Cy5 conjugated antihuman Fab specific secondary antibody, or a suitable anti-tag
antibody.
4. Failure of bead-coupling reaction. Make sure the antigen buffer
does not contain primary amines or detergents, or dialyze the antigen
against PBS and repeat bead coupling reaction.
5. Insufficient secondary antibody used. Increase the amount of
secondary antibody.

Weak signal

1. Antibodies not stored correctly. Repeat the experiment using fresh
antibodies.
2. Antibody concentration too low. Use a higher concentration ofprimary and/or secondary antibody.

Highbackground112

CH A P T E R 9

1. Reduce the amount of Cy5 conjugated secondary antibody.

Your first choice for antibodies!

ChapterAffinity Determinations

10

Measuring antibody affinity is a useful tool for characterizing, evaluating, andranking antibodies. Although there are many different technologies available,
there are essentially two different principles applied to determine affinities:■■■■

Kinetic data with association on and off rates (kon and koff), which is
acquired by using instruments with a flow cell (e.g. Biacore and Attana)Endpoint analysis, which measures concentrations after equilibrium isreached between antigen-bound and free antibody (e.g. ELISA titration)

Flow-cells are often used in label-free technologies where none of thereagents are required to carry a label or dye and no secondary reagents arenecessary. For most kinetic measurements, one binding partner (the antigen
or antibody) is immobilized on a solid support. Measured affinities for one
antibody–antigen pair may vary depending on the method used due to thedifferences in set-up, antigen presentation, or steric hindrance.
To get true monomeric affinity values, it is important to avoid avidity effects
since these may be an issue if the antigen or antibody forms oligomers orshows a tendency to aggregate. Typical examples of oligomeric antigens are
Fc-fusion proteins, homodimers, or conjugated peptides. Full IgGs, HuCAL®
mini-antibodies, and AP-fusion antibodies are examples of bivalent antibodies.
Whenever one of the two partners (or both) is oligomeric, the setup must
be chosen with care to avoid avidity influencing the measurement. This isalways the case when one molecule can bind the immobilized partner withtwo binding sites at the same time (e.g. IgG binds with both arms to coatedantigen).
Although there are many technologies available for KD determination, the only
ones covered in this book are those that are offered by AbD Serotec for the
ranking or affinity determination of HuCAL® antibodies. Contact your accountmanager at sales.muc@ abdserotec.com for more information about these
services. The scientists at AbD Serotec can advise which technology (QCM on
Attana instruments, SPR on Biacore, or SET) is best suited for the project.

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SPR on a Biacore Instrument
For Biacore28 analysis of HuCAL® Fabs, the antigen (ligand) is usuallyimmobilized on the surface and the Fab (analyte) is in the mobile phase. For
most applications, hepes buffered saline (HBS) or PBS is suitable as therunning buffer, to which 0.05% Tween® 20 may be added to reduce nonspecific binding (if necessary). For affinity determinations, the use ofmonomeric (i.e. not aggregated) fractions of purified Fab preparations inrunning buffer are recommended. The affinity determination must be
performed with at least five different concentrations of the purified Fab in HBS
or PBS, in order to ensure that a measurement with an optimal affinity fit canbe obtained.

Immobilization of the Ligand
The CM5 sensor chips (GE Healthcare, BR-1000-14) and the Amine Coupling
Kit (GE Healthcare, BR-1000-50) are used for the immobilization of the ligand
according to the manufacturer’s instructions (BIAapplication Handbook).
The amount of immobilized antigen has to be adapted to its mass and theexperiment that will be performed. Immobilization levels of antigens typicallyrange from ~5–3000 RU. A low-density immobilization of the antigen isappropriate for affinity measurements, whereas koff ranking studies may
require a higher density of immobilized antigen. Absolute immobilizationlevels also depend on the activity of the antigen (including loss of activity
during immobilization) and stability during regeneration among other factors.
The following values may serve as a starting point, but may have to be
optimized in the corresponding assay. For example:
■■

■■

■■

28

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CH A P T E R 1 0

For a 30 kDa protein, immobilize ~300 RU for affinity measurements and~3000 RU for koff ranking
For a peptide coupled to BSA or transferrin (with a coupling rate of 9–15
peptides per carrier), immobilize ~300 RU for affinity measurements and~3000 RU for koff ranking
For an uncoupled peptide, immobilize ~5 RU for affinity measurements and~50 RU for koff ranking

Biacore 3000 with BIAevaluation software, version 3.2, from GE Healthcare.
Your first choice for antibodies!

Procedure
1. Set the flow rate to 5 µl/min.
2. Inject 10–20 µl NHS/EDC (Biacore, BR-1000-50) for low-density
immobilization and 20–30 µl NHS/EDC for high-density immobilization,
depending on the amount of antigen that has to be immobilized.
3. Dilute the antigen to a concentration of ~20 µg/ml (proteins and peptides
coupled to proteins) or to a concentration of ~1 µg/ml (uncoupled
peptides), using, for example, 10 mM acetic acid. The pH of the acetic acidbuffer used should be ~2 pH units below the pI of the antigen. Inject thesolution until the desired amount of antigen is immobilized.
4. Deactivation of the surface is achieved by injecting 35 µl of 1 M
ethanolamine.

Affinity Determination
Use purified Fab in running buffer and a low-density sensor chip.
1. Dilute the analyte (Fab) to six different concentrations with running buffer,e.g. 1:2 dilutions, starting with 500 nM.
2. Set the flow rate to 20 µl/minute.
3. Using the injection mode ’kinject’, inject 20 µl of the analyte (Fab) diluted
to the lowest concentration.
4. Measure the binding of the Fab to the low-density chip and allow theanalyte to dissociate for at least 100 seconds.
5. Regenerate the chip by injection of 5 µl 10 mM glycine/HCl, pH 1.5, or
5 mM NaOH (for example, depending on the antigen). Several 5 µl pulses
may be necessary for complete regeneration.
6. After the regeneration, repeat steps 3–5 with the next concentration of theanalyte. Include buffer injections, as well as positive and negative controls,
if available.
7. Repeat steps 3–5 for all dilutions.
8. Analyze the data set using the BIAevaluation software, version 3.2, byselecting ’simultaneous fit’ and entering the concentrations of the analyte.
Notes:
1. Include a control with a defined affinity as the first and last sample to be measured
in order to monitor the quality of the antigen after each regeneration cycle.

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2. Include several measurements with buffer as a negative control (at least before
and after analysis of the Fabs) for double referencing (subtraction of blank signal tocompensate for flowcell-specific artifacts).
3. If the antibody must be captured on the chip, no affinities greater than that of thecapture antibody can be measured. AbD Serotec recommends the anti-human Fabsecondary antibody (STAR126), although suitable anti-tag antibodies can also be used.

QCM on an Attana Instrument
Affinity determination on an Attana A200® instrument is very similar tomeasurements on SPR (e.g. Biacore) instruments, even though the physicalprinciples behind the technologies are different. In quartz crystal microbalance(QCM), the frequency of a quartz crystal, which is mass dependent, ismeasured. When an antibody binds to an antigen immobilized on a quartz
chip, the mass on the chip increases and the frequency of the quartz changes.
The setup with a flow cell, immobilized sample, and analyte flowing over thechip, as well as the output curves, are very similar to SPR. Thus, the technology
allows determination of kinetic parameters (kon and koff) and affinities.
As with SPR, the antigen is usually immobilized on a chip and the antibody
in the mobile phase. HBS or PBS with 0.05% Tween® 20 is used as runningbuffer. For affinity determinations, the use of monomeric, i.e. non-aggregatedfractions, of purified Fab preparations in PBS are recommended. The affinity
determination has to be performed with at least five different concentrationsof the purified Fab in HBS or PBS to ensure that a measurement with an
optimal affinity fit can be obtained.

Immobilization of the Ligand
Different chips are available for the Attana instruments. The most commonlyused chip is a Carboxyl chip (Attana, 3616-3033) on which the ligand iscovalently immobilized. Other available chips include a polystyrene surface
similar to an ELISA microtiter plate and a biotin surface for immobilization of
biotinylated samples via streptavidin.
Immobilization on Carboxyl sensor chips together with the amine coupling kit(Attana, 3501-3001) can be easily performed by following the manufacturer’s
instructions.

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CH A P T E R 1 0

Your first choice for antibodies!

Affinity Determination
Use purified Fab in HBST or PBST running buffer and a low-density sensor chip.
1. Dilute the analyte (Fab) to six different concentrations with running buffer;e.g. 1:2 dilutions, starting with 500 nM.
2. Set the flow rate to 25 µl/minute.
3. Inject sample (80 seconds) of the analyte (Fab) diluted to the lowest
concentration.
4. Measure the binding of the Fab to the antigen on the sensor and allow theanalyte to dissociate for 100 seconds.
5. Regenerate by 1–3 injection pulses of 10 seconds of an appropriate
regeneration solution. In many cases 10 mM glycine/HCl, pH 1.5, will beappropriate. However, depending on the antigen, other conditions mayneed to be tested. Alternatives include: 10 mM HCl; 10 mM glycine/HCl,
pH 2–3; and 10 mM NaOH.
6. After the regeneration, repeat steps 3–5 with the next concentration of theanalyte.
7. Repeat steps 3–5 for all dilutions.
8. Analyze the data set using the Attester Evaluation software and ClampXP
fitting software.

SET
Determination of affinities in the low picomolar (pM) range is difficult,since routinely applied methods such as SPR or QCM reach their limit when
determining very slow dissociation rate constants (koff). MorphoSys has
established an alternate method to overcome this limitation and also offers away to carry out the affinity determination in a medium throughput fashion.
Solution Equilibrium Titration (SET)29 in combination with a highly sensitive
detection method such as electrochemiluminescence (ECL) on a BioVeris(Roche Group) or MSD™ instrument (Meso Scale Discovery™, SECTOR Imager6000) allows for the determination of high-affinity antibody to antigen
interactions. In contrast to kinetic assays using sensor surfaces, such as SPR or
QCM, SET is a method which determines affinities in solution. It is anequilibrium measurement that does not deliver kinetic data.
Haenel, C. et al. (2005) Characterization of high-affinity antibodies by electrochemiluminescencebased equilibrium titration. Anal Biocemistry 339:182–184

29

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In SET, a constant amount of antibody is incubated with different
concentrations of antigen until equilibrium is reached. The concentrationof free antibody in the equilibrated solution is determined by applying
the solution on an antigen coated MSD™ plate (Meso Scale Discovery™,
L11XA3), followed by incubation with an ECL-labeled secondary antibody
and measurement of signal intensity. At low antigen concentrations, a strong
signal is achieved (high concentration of free antibody which binds to theantigen on the plate) whereas for high antigen concentration, the antibody iscompletely antigen-captured, resulting in a low signal.
If a sufficient number of antigen concentrations in a matching range areavailable, the titration curve allows for a reasonable determination of theaffinity, using the appropriate fit model. For a complete titration, antigen
concentrations of at least 10-fold higher than the anticipated KD have to be
applied. The constant concentration of antibody applied in the assay should bein the range of, or below, the KD.

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CH A P T E R 1 0

Your first choice for antibodies!

Chapter

Antibody Conjugation

11

There are two methods for the direct detection of HuCAL® antibodies without
the need for secondary antibodies. One is the use of AP-fusion antibodies (e.g.
the Fab-A-FH format) together with an AP detection substrate, and the other isby directly-labeling the HuCAL® primary antibody by chemical coupling.
The advantage of using a secondary antibody is that it works with manydifferent antibodies and can enhance sensitivity. However, working with only a
primary antibody is often preferred because it reduces the risk of non-specificbackground staining from cross-reactive secondary antibodies. It also reduces
incubation times, washing steps, and costs by avoiding secondary reagents.
Conjugation of HuCAL® antibodies with biotin and various fluorescence dyes
works the same as for complete antibodies. To date, in house testing has
shown that conjugation with LYNX kits delivers antibodies with excellent
sensitivities, which are similar to those achieved with the best secondary
antibodies.
For more information on receiving conjugated HuCAL® antibodies, contact
your account manager at sales.muc@abdserotec.com.

Conjugation Using LYNX Rapid Conjugation Kits®
LYNX kits are based on a revolutionary new technology that offers extremely
fast and simple conjugation of antibodies and other proteins to biotin,
fluorescent, or enzymatic labels. LYNX kits offer the following advantages:■■

Easy protocol with just 30 seconds hands-on time■■

No purification required, so there is no loss of antibody■■

High-quality conjugates are ready to use■■

Assay sensitivities are equal to those achieved with secondary antibodies

LYNX kits are available in a range of sizes for easy, scaleable conjugations
from microgram to milligram quantities. In addition, they are available forall commonly used reporter enzymes, fluorescent dyes, and biotin as listed inTable 11.

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LYNX Rapid Conjugation Kits®.

Tandem Dyes

Single Dyes

Enzymes

TABLE 11

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CH A P T E R 1 1

Label

Reaction Size

Product Code

Biotin3 × 200 µg
1 mg

LNK041B
LNK042BAP

100 µg3 × 100 µg
1 mg

LNK011AP
LNK012AP
LNK013AP

HRP

400 µg3 × 400 µg
4 mg
5 × 4 mg
20 mg

LNK001P
LNK002P
LNK003P
LNK004P
LNK005P

Glucose oxidase (GOx)

3 × 100 µg
1 mg

LNK051GOX
LNK052GOX

Allophycocyanin (APC)

100 µg3 × 100 µg
1 mg

LNK031APC
LNK032APC
LNK033APC

Cy5200 µg
2 mg

LNK101CY5
LNK102CY5

Fluorescein

200 µg
2 mg

LNK061F
LNK062F

PerCP

100 µg3 × 100 µg
1 mg

lNK071PERCPlNK072PERCPlNK073PERCP

RPE

100 µg3 × 100 µg
1 mg

LNK021RPE
LNK022RPE
LNK023RPE

APC-Cy5.5

150 µg3 × 150 µg
1.5 mg

LNK121APCCY5.5
LNK122APCCY5.5
LNK123APCCY5.5

APC-Cy7

150 µg3 × 150 µg
1.5 mg

LNK131APCCY7
LNK132APCCY7
LNK133APCCY7PerCP-Cy5.5

100 µg3 × 100 µg
1 mg

LNK141PERCPCY5.5
LNK142PERCPCY5.5
LNK143PERCPCY5.5

RPE-Cy5

60 µg
3 × 60 µg
600 µg

LNK081C
LNK082C
LNK083C

RPE-Cy5.5

60 µg
3 × 60 µg
600 µg

LNK091PECY5.5
LNK092PECY5.5
LNK093PECY5.5

RPE-Cy7

60 µg
3 × 60 µg
600 µg

LNK111PECY7
LNK112PECY7
LNK113PECY7Your first choice for antibodies!

In the LYNX procedure, the antibody is mixed with the labeling reagents
followed by 3-hour, or overnight, incubation. Next, the reaction is stopped andthe antibody is now ready to use (Figure 44). All materials and reagents areprovided with the kit and no purification of the antibody is required after thelabeling reaction.

FIGURE 44

LYNX Kit Procedure.

Antibodies conjugated with LYNX kits offer excellent sensitivities and,
therefore, can often be used at lower concentrations than needed with typicalsecondary reagents. Conditions that yield the best signal with low backgroundneed to be optimized for each antibody. For many assays, we recommend
diluting the conjugated antibody in HiSpec buffer (BUF049) in order to obtainsuperior results.

ExampleWestern Blotting
An anti-cyclophilin A antibody in Fab-A-V5Sx2 format (AbD08966) was used inWestern blot analysis of HEK293 cell lysate, either after direct HRP conjugation
with a LYNX kit (LNK001P) or in combination with a HRP-conjugated anti-V5 tag
secondary antibody (MCA1360P). The labeled antibody was diluted in HiSpec
buffer (BUF049) and the blot was developed with Amersham™ ECL Plus.
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FIGURE 45

Western Blot Comparison of LYNX-HRP Conjugate Antibody vs. Typical
Secondary Antibody. Lanes 1–5: Serial dilutions of HEK293 cell lysate detected withLYNX HRP-conjugated anti-cyclophilin A. Lanes 7–10: The same samples detected withunconjugated anti-cyclophilin A and an anti-V5:HRP secondary antibody.

Conjugation to FITC, Cy5, and Biotin Using Conventional
Kits
Important points before starting
■■

■■

■■

■■

■■

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CH A P T E R 1 1

The appropriate quantity of reagent dye depends on antibody
concentrations and on desired levels of labeling
For most situations, a 20-fold molar excess of the reagent dye isrecommended; this will label each antibody with an average of 2–4 dye
molecules
For antibody concentrations below 0.5 mg/ml, an approximately 30-foldmolar excess of reagent dye is recommended
If lower levels of labeling are required, use a 5–10 fold molar excess ofreagent dye
Sample calculations for the required molar excess of reagent dye areprovided for each procedureYour first choice for antibodies!

Conjugation with Fluorescein Isothiocyanate (FITC)
FITC-labeling of antibodies can be performed using commercially available
kits. The protocol described below uses the kit supplied by Thermo Fisher
Scientific, and follows the manufacturer’s instructions. It describes the
conjugation of 1 mg of a bivalent and monovalent antibody, and can be scaled
down to provide labeling of smaller amounts.
TABLE 12Calculation of Reagent Dye Volumes.
CompoundMolecular
weightnmol in 1 mg
reagent

nmol in 20-foldmolar excess ofreagent

Requiredvolume ofreagent

FITC

389 g/mol

2500 nmol/ml(solution of 1
mg/ml)––

Fab~50 kDa20 nmol

20 × 20 nmol =400 nmol

400 nmol of a
FITC reagent
solution at 2500
nmol/ml = 160 µl

Fab-dHLX~120 kDa

8 nmol20 × 8 nmol =160 nmol

160 nmol of a
FITC reagent
solution at 2500
nmol/ml = 64 µl

Procedure
1. Prepare a fresh solution with 1 mg of FITC in 10 ml PBS.
2. The solution is stable for several hours. Higher temperatures, traces ofamines (e.g. Tris), and non-neutral pH greatly reduce the stability. Minimize
exposure to light, as FITC is light sensitive.
3. For bivalent Fab labeling (Fab-dHLX format), add 64 µl reagent solution to
1 mg antibody.
For monovalent Fab labeling (Fab format), add 160 µl reagent solution to
1 mg antibody.
Antibody concentration should be between 0.5 and 2 mg/ml. Lower
concentrations of antibody require a higher reagent excess (see ‘Important
points before starting’).
4. Incubate for 30 minutes at RT, or on ice for 2 hours.
5. Follow the purification protocol described on page 126.

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Conjugation with Cy5
TABLE 13

Calculation of Reagent Dye Volumes.
CompoundMolecular
weight

Cy5-NHSnmol in 1 mg
reagent

nmol in 20-foldmolar excess ofreagent

Requiredvolume ofreagent

6300 nmol/ml(solution of 5
mg/ml)––

Fab~50 kDa20 nmol

20 × 20 nmol =400 nmol

400 nmol of a
Cy5 reagent
solution at 6300
nmol/ml = 63 µl

Fab-dHLX~120 kDa

8 nmol20 × 8 nmol =160 nmol

160 nmol of a
Cy5 reagent
solution at 6300
nmol/ml = 25 µl

Procedure
1. Prepare a fresh solution of 5 mg of Cy5 mono NHS ester (Cy5-NHS, GE
Healthcare) in 1 ml dimethylsulfoxide (DMSO).
The solution is stable for several hours. A fresh solution should be preparedfor each experiment since DMSO is hygroscopic and traces of water will
greatly reduce the stability of the solution. Minimize exposure to light,
since Cy5-NHS is light sensitive.
2. For bivalent Fab labeling (Fab-dHLX format), add 25 µl reagent solution to
1 mg antibody.
For monovalent Fab labeling (Fab format), add 63 µl reagent solution to
1 mg antibody.
Antibody concentration should be between 0.5 and 2 mg/ml. Lower
concentrations of antibody require a higher reagent excess (see ‘Important
points before starting’).
3. Incubate for 30 minutes at RT, or on ice for 2 hours.
4. Follow the purification protocol described on page 126.

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Your first choice for antibodies!

Conjugation with Biotin
Conjugation with biotin can be performed using the EZ-link Sulfo-NHS-LCBiotin (Thermo Fisher Scientific, 21335).
Table 14

Calculation of Reagent Dye Volumes.
CompoundMolecular
weightBiotin-NHS

nmol in 1 mg
reagent

nmol in 20-foldmolar excess ofreagent

Requiredvolume ofreagent

1800 nmol/ml(solution of 1
mg/ml)––

Fab~50 kDa20 nmol

20 × 20 nmol =400 nmol

400 nmol of abiotin reagent
solution at 1800
nmol/ml = 222 µl

Fab-dHLX~120 kDa

8 nmol20 × 8 nmol =160 nmol

160 nmol of abiotin reagent
solution at 1800
nmol/ml = 89 µl

Procedure
1. Prepare a fresh solution with 1 mg of EZ-link Sulfo-NHS-LC-Biotin (BiotinNHS) in 1 ml PBS.
The solution is stable for several hours. High temperatures, traces of amines(e.g. Tris), or non-neutral pH greatly reduce the stability of the solution.
2. For bivalent Fab labeling (Fab-dHLX format), add 89 µl reagent solution to
1 mg of antibody.
For monovalent Fab labeling (Fab format), add 222 µl reagent solution to
1 mg antibody.
Antibody concentration should be between 0.5 and 2 mg/ml. Lower
concentrations of antibody require a higher reagent excess (see ‘Important
points before starting’).
3. Incubate for 30 minutes at RT, or on ice for 2 hours.
4. Follow the purification protocol on page 126.

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CH A PTER 11

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Purification
For each type of labeling, there are three options for separating the antibody
from the unreacted reagent.

Purification on a PD10-column (GE Healthcare) or
Comparable SEC Column
1. Add 50 µl 1 M Tris, pH 7.4, to stop the reaction (this inactivates NHS esters),
and use the resulting solution in experiments.
2. Equilibrate the column twice with 8 ml 3x PBS.
3. Adjust the reaction volume to 2 ml using 3x PBS.
4. Apply 2 ml reaction mix to the column and discard the flow-through.
5. Wash the column using 0.5 ml 3x PBS and discard the flow-through.
6. Elute with 2.5 ml 3x PBS.
Note: The antibody yield is approximately 80% of the starting amount.

Dialysis Using 3x PBS
1. Add 50 µl 1 M Tris, pH 7.4, to stop the reaction (this inactivates NHS esters).
2. Perform two dialysis steps using at least a 50-fold excess of dialysis
volume. ‘Slide-A-Lyzer’ cassettes (0.5–3 ml; Thermo Fisher Scientific) with amolecular weight cut-off (MWCO) of 10,000 are recommended.

Dialysis for Small-scale Reactions
1. Add 50 µl 1 M Tris, pH 7.4, to stop the reaction (this inactivates NHS esters).
Perform one dialysis step using 3x PBS using at least a 200-fold excess of
dialysis volume. The ’Mini Slide-A-Lyzer’ with a MWCO of 3,500 (Thermo Fisher
Scientific) is recommended.
Note: For monovalent Fab fragments, 1x PBS can also be used as dialysis
buffer.

126

CH A P T E R 1 1

Your first choice for antibodies!

Examples
FITC conjugation
The successful FITC conjugation of a monovalent antibody, AbD00178 in
Fab-MH format, and a bivalent antibody, AbD00908 in Fab-dHLX-MH format,
was demonstrated by direct ELISA. The antibodies in this example bind human
NFκB (Figure 46).

FIGURE 46

Direct ELISA. Left: FITC-labeled anti-NfkB HuCAL® bivalent mini-antibody AbD00908(Fab-dHLX-MH). Right: FITC-labeled anti-NfkB HuCAL® monovalent Fab fragment
AbD00178 (Fab-MH).

Biotinylation
Successful biotinylation of a mono and bivalent HuCAL® antibody against
NFκB was shown by ELISA. The biotinylated antibody was immobilized on aneutravidin plate and detected with an anti-Fab antibody conjugated to AP
and AttoPhos® substrate (Figure 47).

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FIGURE 47

ELISA Detects Biotinylated HuCAL® Fab. Detection was performed with ananti-Fab polyclonal antibody. The negative control is the same HuCAL® Fab without
biotinylation.

Troubleshooting
ProblemPossible Cause and Course of Action

Failed labelingreaction

1. Incompatible buffer used. Use a buffer that does not contain
primary amines or SH-groups.
2. Reagent hydrolyzed and non-reactive. Prepare labeling reagent
immediately before use, and do not store the reagent in aqueous
solution.
3. Reagents expired or not stored correctly. Use fresh reagents andstore according to the manufacturer’s instructions.

Fluorescencesignal too low

128

CH A P T E R 1 1

1. Too few or too many dye molecules coupled to the antibody. Use ahigher or lower ratio of antibody to dye (see ‘Important points before
starting’ page 122).

Your first choice for antibodies!

ChapterAppendices12

Buffer Composition
BufferComposition

StorageHBS

20 mM Hepes, pH 7.4
150 mM NaCl

4°C

PBS

136 mM NaCl

RT

2.68 mM KCl
8.1 mM Na2HPO4
1.46 mM KH2PO4
PBST

PBS with 0.05% Tween®-20RT

10x TBS

500 mM Tris-HCl, pH 7.4

RT

1.5 M NaCl
TBST

50 mM Tris-HCl, pH 7.4

RT0.15 M NaCl
0.1% Tween® 20
TNEC buffer

50 mM Tris-HCl, pH 8.0

4°C

150 mM NaCl
1% Nonidet P40
2 mM EDTA
1 tablet/10 ml buffer of complete mini proteaseinhibitor (Roche, 1332473)

Buffer Preparation
Flow Buffer
1. Mix ice-cold PBS, pH 7.4, 3% FCS, and 0.02% sodium azide.
2. Centrifuge at high speed until the suspended matter is pelleted(~ 30 minutes at 17700 × g), at 4°C.
3. Transfer supernatant to a new tube.
4. Store at 4°C for up to one week.
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CH A PTER 12

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Activated Biotinylated Tyramide Buffer for IHC
1. Add 100 mg EZ-link Sulfo-NHS-LC-biotin (Thermo Fisher Scientific, 21335)
to 40 ml of 25 mM borate buffer.
2. Add 30 mg tyramine-HCl (Sigma, T-2879).
3. Shake gently at room temperature for 12 hours.
4. Sterilize by filtration through a 20 µm filter.
5. Divide into single-use aliquots of 50 µl and store at –20°C.

Source of Reagents

130

CH A P T E R 1 2

Reagent

Supplier

Product Code

4% Paraformaldehyde in PBS

Sigma

P6148

96-well round bottom microtiter plates, sterile

NUNC

16332096-well, flat-bottom, black MaxiSorp™ PS
microtiter plates

NUNC

437111384-well, flat-bottom, black MaxiSorp™ PS
microtiter plates

NUNC

460518Antibody diluents

AbD Serotec

BUF014

Attana Carboxyl Sensor Chip

Attana3616-3033

Attana Amine Coupling Kit

Attana3501-3001

AttoPhos®

Roche

11681982

BCIP/NBTAbD Serotec

BUF045A

Biacore Sensor Chip CM5

GE Healthcare

BR-1000-14

Biacore Amine Coupling Kit

GE Healthcare

BR-1000-50

BM Blue Soluble Peroxidase Substrate

Roche

1484281

BSA

Sigma

A7960

Cy5 Mono NHS Ester

GE Healthcare

PA15100

DAB substrate

AbD Serotec

BUF021ADAB substrate buffer

AbD Serotec

BUF022Dynabeads® TALON™

Invitrogen

10101D

DynaMag™-2

Invitrogen

123-21DECL Plus™

GE Healthcare

RPN2132

ECL Advance™ Western Blotting Detection Kit

GE Healthcare

RPN2135

EZ-link sulfo-NHS-LC-Biotin

Thermo Fisher Scientific

21335

Your first choice for antibodies!

Reagent

Supplier

Product Code

FCS

PAN

3302-P241105

FITC labeling reagents

Thermo Fisher Scientific

46110

Hematoxylin

Sigma

MHS16

HiSpec Buffer

AbD Serotec

BUF049HISTAR Detection System

AbD Serotec

STAR3000IFNγ

AbD Serotec

PHP050LiquiChip Carboxy Beads Set A

QIAGEN

922402LiquiChip Carboxy Beads Set B

QIAGEN

922404

LiquiChip Carboxy Beads Set C

QIAGEN

922406LYNX Rapid Conjugation Kit for HRP

AbD Serotec

LNK001P

Dynabeads M-450 Epoxy

Invitrogen

14011

MagStrep type 2 Strep-Tactin® coatedmagnetic beads

IBA

2-1611-002

Microcentrifuge tubes for Luminex assays

USA Scientific1415-2500

Mini Slide-A-Lyzer, MWCO 3,500

Thermo Fisher Scientific

69550

®
®
®

®

®

Mouse Envision Kit

Dako

K4007

Mouse-on-Mouse (M.O.M™) Kit

Vector Laboratories

PK-2200

MSD Sector Imager 6000 Reader 96-well
PlatesMeso Scale Discovery

L11XA3™

MultiScreenHTS-BV, 1.2 µm PVDF Filter Plate

Millipore

MSBVN1210

PBS

Gibco

14190-094

PD10 SEC columns

GE Healthcare

17-0851-01

Peroxide Blocking Reagent

AbD Serotec

BUF017A

QuantaBlu® Fluorogenic Peroxidase Substrate

Thermo Fisher Scientific15169

Slide-A-Lyzer, 0.5–3 ml, MWCO 10,000

Thermo Fisher Scientific

66380

Streptavidin-AP

AbD Serotec

STAR6B

Streptavidin-RPE

AbD Serotec

STAR4B

Streptavidin-HRP

AbD Serotec

STAR5B®

Tips for Luminex assays, 250 µl

Mettler Toledo

RT-250Tips for Luminex assays, 1000 µl

Mettler Toledo

RT-1000

Trypan Blue

Sigma

T-B154Trypsin/EDTA

Gibco BRL

25300-054

Tyramide

Sigma

T-2879

Vac-Man 96 Vacuum Manifold

Promega

A2291

®

®

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CH A PTER 12

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Source of Antibodies
Visit www.abdserotec.com for additional conjugates or to download
detailed datasheets.

HuCAL® Antibodies
Antibody

Clone (Product Code)FormatAnti-Caspr

AbD06152

Fab-dHLX-MH

Anti-CD81

AbD06083 (HCA086)

Fab-dHLX-MH

AbD06084 (HCA087)

Fab-dHLX-MH

AbD08863 (HCA112)

Fab-dHLX-MH

AbD08864 (HCA113)

Fab-dHLX-MH

AbD00794 (HCA005)

Fab-dHLX-MH

AbD08865

Fab-dHLX-MSx2

AbD08966

Fab-A-V5Sx2

AbD05054 (HCA071)

Fab-dHLX-MH

AbD08860 (HCA114)

Fab-dHLX-MSx2

AbD08961

Fab-A-V5Sx2

AbD02012

Fab-A-FH

AbD02122 (HCA012)

Fab-dHLX-MH

Anti-FITC

AbD00756 (HCA002)

Fab-FS

Anti-IFNγ

AbD00068

Fab-MH

AbD00676 (HCA043)

Fab-dHLX-MH

AbD02503 (HCA044)

Fab-A-FH

AbD02531 (HCA006)

Fab-dHLX-MH

AbD02815

Fab-A-FH

AbD00908

Fab-dHLX-MH

AbD00178

Fab-MH

Anti-Rootletin

AbD02085 (HCA009)

Fab-A-FH

Anti-Vimentin

AbD02701 (HCA032)

Fab-dHLX-MH

AbD08866 (HCA111)

Fab-dHLX-MSx2

AbD08962

Fab-A-V5Sx2

Anti-Cyclophilin A

Anti-Desmin

Anti-FDPS

Anti-Ki-67

Anti-NFκB

132

CH A P T E R 1 2

Your first choice for antibodies!

HuCAL® Negative Control Antibodies (anti-GFP,
MOR06391)

www.abdserotec.com

FormatProduct CodeFab-MH

HCA051

Fab-V5H

HCA098Fab-FH

HCA045Fab-H

HCA124

Fab-V5Sx2

HCA125

Fab-MSx2

HCA100Fab-FSx2

HCA101Fab-FS

HCA046

Fab-dHLX-MH

HCA052Fab-dHLX-FH

HCA097

Fab-dHLX-MSx2

HCA102Fab-dHLX-FSx2

HCA104Fab-dHLX-MS

HCA048Fab-dHLX-FS

HCA047Fab-A-FH

HCA096

Fab-A-V5Sx2

HCA123Fab-A-MSx2

HCA106

Fab-A-FSx2

HCA107

Fab-p53-H

HCA099Fab-p53-V5Sx2

HCA139hIgG1

HCA049hIgG2

HCA108hIgG4

HCA050h/mIgG2a

HCA110

CH A PTER 12

133

Secondary Antibodies

134

CH A P T E R 1 2

Anti-human IgG Antibodies

Supplier

Product CodeApplication

Goat anti-human F(ab’)2

AbD Serotec

STAR126

E, IP

Goat anti-human F(ab’)2:AP

AbD Serotec

STAR126A

E, WB

Goat anti-human F(ab’)2:HRP

AbD Serotec

STAR126P

C, E, P, WB

Goat anti-human F(ab’)2:Biotin

AbD Serotec

STAR126B

C,E, IP, P, WB

Goat anti-human F(ab’)2:FITC

AbD Serotec

STAR126F

C, F, IF, P

Goat anti-human F(ab’)2:TRITC

AbD Serotec

STAR126TRC

C, F, IF, P

Goat anti-human F(ab’)2:Cy5

JacksonImmunoResearch

109-175-097

C, F, IF, P

Goat anti-human IgG (H + L), F(ab’)2
fragment:R-PE

AbD Serotec

STAR97PE

F

Goat anti-human kappa light chain

AbD Serotec

STAR127

E

Goat anti-human kappa light chain:HRP

AbD Serotec

STAR127P

C, E, P, WB

Goat anti-human kappa light chain:FITC

AbD Serotec

STAR127F

C, F, IF

Goat anti-human kappa light chain

AbD Serotec

STAR128

E

Goat anti-human kappa light chain:Biotin

AbD Serotec

STAR128B

E, IF, WB

Goat anti-human kappa light chain:HRP

AbD Serotec

STAR128P

C, E, P, WB

Goat anti-human kappa light chain:R-PE

AbD Serotec

STAR128PE

F

Goat anti-human lambda light chain

AbD Serotec

STAR129E

Goat anti-human lambda light chain:HRP

AbD Serotec

STAR129P

C, E, P, WB

Goat anti-human lambda light chain:FITC

AbD Serotec

STAR129F

C, F, IF

Anti-mouse IgG Antibodies

Supplier

Product CodeApplication

Rabbit anti-mouse IgG, F(ab’)2
fragment:Biotin

AbD Serotec

STAR11B

C, E, F, P

Goat anti-mouse IgG (H+L), F(ab’)2
fragment

AbD Serotec

STAR105

C, E

Goat anti-mouse IgG (H+L), F(ab’)2
fragment:HRP

AbD Serotec

STAR105P

C, E, P

Goat anti-mouse IgG (H+L), F(ab’)2
fragment:FITC

AbD Serotec

STAR105F

F

Goat anti-mouse IgG (H+L), F(ab’)2
fragment:R-PE

AbD Serotec

STAR105PE

F

Rabbit anti-mouse IgG (H+L), F(ab’)2
fragment:FITC

AbD Serotec

STAR9BF

Goat anti-mouse IgG (H+L):Dylight®649

AbD Serotec

STAR117D649

F, IF

Rabbit anti-mouse IgG:Cy5

AbD Serotec

OBT1725C

IF

Your first choice for antibodies!

Anti-tag Antibodies

Supplier

Product CodeApplication

Mouse anti-Strep-tag® Classic

AbD Serotec

MCA2489

C, E, F, IF, IP, P, WB

Mouse anti-Strep-tag Classic:AP

AbD Serotec

MCA2489A

E, WB

Mouse anti-Strep-tag Classic:HRP

AbD Serotec

MCA2489P

C, E, WB

Mouse anti-Strep-tag Immo (capture
antibody)

AbD Serotec

MCA2488

E, IP

Mouse anti-His-6

AbD Serotec

MCA1396

C, E, F, IF, IP, P, WB

Mouse anti-His-6:Biotin

AbD Serotec

MCA1396B

C, E, F, IF, IP, P, WB

Mouse anti-His-6:HRP

AbD Serotec

MCA1396P

C, E, F, P, WB

AbD Serotec

MCA1396F

F, IF

Mouse anti-His-6:Alexa Fluor 488

AbD Serotec

MCA1396A488

F, IF

®

Mouse anti-His-6:Alexa Fluor 647

AbD Serotec

MCA1396A647

F, IF

Mouse anti-His-6:DyLight® 549

AbD Serotec

MCA1396D549

F, IF

Mouse anti-His-6

Roche

1922416

C, E, F, IF, IP, P, WB

Mouse anti-V5

AbD Serotec

MCA1360

C, E, IF, IP, WB

Mouse anti-V5:AP

AbD Serotec

MCA1360A

E, WB

Mouse anti-V5:HRP

AbD Serotec

MCA1360P

E, WB

Mouse anti-V5:Biotin

AbD Serotec

MCA1360B

C, E, IF, IP, WB

AbD Serotec

MCA1360F

IF

Mouse anti-V5: Alexa Fluor 488

AbD Serotec

MCA1360A488

IF®

Mouse anti-V5: Alexa Fluor 647

AbD Serotec

MCA1360A647

IF

Mouse anti-V5:DyLight 549

AbD Serotec

MCA1360D549

IF

Mouse Anti-V5

AbD Serotec

MCA2894

E, IF, IP, WB

Mouse Anti-V5:FITC

AbD Serotec

MCA2894F

F, IF

Mouse Anti-V5:DyLight® 549

AbD Serotec

MCA2894D549

F, IF

Mouse Anti-V5:DyLight® 649

AbD Serotec

MCA2894D649

F, IF

Mouse Anti-V5

AbD Serotec

MCA2895

E, IF, IP, WB

AbD Serotec

MCA2895F

F, IF

Mouse Anti-V5:DyLight 549

AbD Serotec

MCA2895D549

F, IF

Mouse Anti-V5:DyLight 649

AbD Serotec

MCA2895D649

F, IF

Rat anti-FLAG

AbD Serotec

MCA4764

E, IF, IHC, WB

Rat anti-FLAG :HRP

AbD Serotec

MCA4764P

E, IF, IHC, WB

Rat anti-FLAG :Biotin

AbD Serotec

MCA4764B

E, IF, IHC, WB®
®
®

Mouse anti-His-6:FITC®

Mouse anti-V5:FITC®

®

Mouse Anti-V5:FITC
®
®

®
®
®

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CH A PTER 12

135

Anti-tag Antibodies

Supplier

Product CodeApplication

Rabbit anti-FLAG®

AbD Serotec

AHP1074

E, WB

Rabbit anti-FLAG :HRP

AbD Serotec

AHP1074P

E, WB

Mouse anti-FLAG M2

Sigma

F3165

E, F, IF, WB

Rabbit anti-bacterial alkaline
phosphatase:HRP

AbD Serotec

AHP1108P

C, E, IF, WB

Mouse anti-c-myc (9E10)AbD Serotec

MCA2200

C, E, F, P, WB

Mouse anti-c-myc (9E10):HRP

AbD Serotec

MCA2200P

C, E, P, WB

Mouse anti-c-myc (9E10):AP

AbD Serotec

MCA2200A

C, E, P, WB

Mouse anti-c-myc (9E10):Biotin

AbD Serotec

MCA2200B

C, E, F, P

Mouse anti-c-myc (9E10):FITC

AbD Serotec

MCA2200F

F

Mouse anti-c-myc (9E10):R-PE

AbD Serotec®

®

MCA2200PE

F

Mouse anti-c-myc (9E10): Alexa Fluor® 488 AbD Serotec

MCA2200A488

F

Mouse anti-c-myc (9E10): Alexa Fluor® 647 AbD Serotec

MCA2200A647

F

Mouse anti-c-myc (9E10):DyLight® 549

MCA2200D549

F, IF

AbD Serotec

Applications: C: IHC – frozen; E: ELISA; F: Flow cytometry; IF: Immunofluorescence;
IP: Immunoprecipitation; P: IHC – paraffin; WB: Western blotting.

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Your first choice for antibodies!

Technical AssistanceA group of experienced technical advisors is available at AbD Serotec to help
with any questions regarding HuCAL® antibodies and their applications, both
before project start and after antibody delivery. Since HuCAL® technology
provides many opportunities for optimizing the antibody generation processaccording to customer needs, AbD Serotec encourages the discussion ofyour antibody project with one of our scientists at an early stage. They canhelp with project design, antigen preparation, and provide information
about the many options that are simply not available with typical animalbased technologies. Once the antibodies are delivered, they can also provide
valuable advice about protocols and detection systems.
Contact your account manager at sales.muc@abdserotec.com or visit
www.abdserotec.com/HuCAL for more information. General technicalsupport requests related to catalog or accessory products can also be
addressed to tech.uk@abdserotec.com.

Confidentiality
AbD Serotec treats all data and material provided to us by customers ascompletely confidential, including customer name and affiliation. Informationfrom customers is used only as necessary for the successful and safe
performance of the antibody generation project. We are pleased to provide asigned Confidential Disclosure Agreement (CDA) upon request.

www.abdserotec.com

CH A PTER 12

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Further Selected Publications
For a complete HuCAL antibodies publication list, please visit our website
www.abdserotec.com/HuCAL.
Gustchina, E. et al. (2009) Affinity maturation by targeted diversification of
the CDR-H2 loop of a monoclonal Fab derived from a synthetic naïve humanantibody library and directed against the internal trimeric coiled-coil of gp41
yields set of Fabs with improved HIV-1 neutralization potency and breadth.
Virology 39:112–119
Knappik, A. et al. (2009) Development of recombinant human IgA for
anticardiolipin antibodies assay standardization. Ann N Y Acad Sci
1173:190–198
Lochmatter, C. et al. (2009) Schistosoma mansoni TOR is a tetraspanningorphan receptor on the parasite surface. Parasitology 136:487–498
Chernov, A.V. et al. (2008) The two-component NS2B-NS3 proteinase represses
DNA unwinding activity of the West Nile virus NS3 helicase. J Mol Biol
283:17270–17278
Hillig, R.C. et al. (2008) Fab MOR03268 triggers absorption shift of adiagnostic dye via packaging in a solvent-shielded Fab dimer interface.
J Mol Biol 377:206–219
Gustchina, E. et al. (2007) A monoclonal Fab derived from a human nonimmune phage library reveals a new epitope on gp41 and neutralizes diverse
HIV-1 strains. J Virol 81: 12946–12953
Jarutat, T. et al. (2007) Selection of vimentin-specific antibodies from the
HuCAL® phage display library by subtractive panning on formalin-fixed,
paraffin-embedded tissue. Biol Chem 388:651–58
Zimmerman, T. et al. (2006) Simultaneous metal chelate affinity
purification and endotoxin clearance of recombinant antibody fragments.
J Immunol Methods 314:67–73
Jarutat, T. et al. (2006) Isolation and comparative characterization of Ki67 equivalent antibodies from the HuCAL phage display library. Biol Chem
387:995–1003
Ooe, H. et al. (2006). Establishment of specific antibodies that recognize C106oxidized DJ-1. Neurosci Lett 404:166–169
Ohara, R. et al. (2006) Antibodies for proteomic research: comparison of
traditional immunization with recombinant antibody technology. Proteomics
6:2638–2646
138

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Your first choice for antibodies!

Rauchenberger, R. et al. (2003) Human combinatorial Fab library yielding
specific and functional antibodies against the human fibroblast growth factor
receptor 3. J Biol Chem 278:38194–38205
Nagy, Z.A. et al. (2002) Fully human, HLA-DR-specific monoclonal antibodies
efficiently induce programmed death of malignant lymphoid cells. Nat Med
8:801–807

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Acknowledgements
The author gratefully recognizes Inge Altinger-Ralle, Dr. Karin Jury, Dr. Achim
Knappik, and Claire Moore, for their key contributions and valuable assistance.
Thanks to the following for images and data:
Figure 37: Joanna Rowe, PhD., AbD Serotec, Oxford, UK
Figure 39: Joanna Rowe, PhD., AbD Serotec, Oxford, UK
Figure 40: Carolin Zehetmeier, MorphoSys AG, Martinsried, Germany
Figure 41: Barbara Ranscht, PhD., The Burnham Institute for Medical Research,
La Jolla, CA, USA
Figure 43: Scott Cribbes, PhD., Applied Biosystems, Warrington, UK

140

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Your first choice for antibodies!