Choosing the Best HuCAL® Antibody Format

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A critical step in any Human Combinatorial Antibody Library (HuCAL) generation project is selection of the optimal
antibody format.
Options include a monovalent Fab format or a bivalent version, which is functionally equivalent to a typical F(ab´)2
fragment, and choice of epitope or peptide tags which are best-suited to the intended applications.
Many assays benefit from the use of monovalent or bivalent Fab antibodies; certain applications however, requireconversion of the Fab antibodies into an immunoglobulin (Ig) format. The epitope tags are an additional feature of the Fabantibodies that can be used for immune-detection in an assay or for immobilization to a matrix. We offer a wide range ofcommon epitope tag Fab antibody formats, all available purified or conjugated to common enzyme and fluorescent labels.
This guide provides a compilation of recommended Fab antibody formats, for use in a range of applications, and details
on the composition and molecular weights of the various epitope tags. Please note that Ig formats do not contain anyepitope tags.
Contents:
1.
2.
3.
4.

Recommended Fab antibody formats by application
Fab antibody formats and epitope tag combinations
Tag sequences and homodimerization domains
Full-length immunoglobulin formats

Fab Antibody Formats

Ig Conversion

Examples of HuCAL antibody formats:
Fab-FH, monovalent Fab antibody with two epitope tags
Fab-dHLX-FH, bivalent Fab antibody formed via
dimerization of dHLX domains (blue cylinders) with twoepitope tags

Fab-FHFab-dHLX-FH

Fab-A-V5Sx2

IgG

Fab-A-V5Sx2, bivalent Fab antibody formed via
dimerization of bacterial alkaline phosphatase (bluemodules) with two epitope tags
IgG, a typical full-length immunoglobulin with no epitopetags

Suggested protocols, reagents tested for use with HuCAL antibodies, application examples, and a detailed discussion ofour recombinant antibody technology can be found in our HuCAL Antibodies Technical Manual or by visiting
bio-rad-antibodies.com/HuCAL.

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1. Recommended Fab antibody formats by application
Application

Monovalent

Bivalent

Western Blot√ELISA√

Immunoprecipitation√Immunohistochemistry√

Flow Cytometry√

Immunofluorescence√

Affinity Determination√

Affinity
Chromatography√

Co-crystalization√

Recommended Secondary
Antibodies
Anti-human FabAnti-Strep-tag
Anti-His-6
Anti-V5
Anti-BAP
Anti-DYKDDDDK
Anti-c-myc
Anti-human FabAnti-Strep-tag
Anti-His-6
Anti-V5
Anti-BAP
Anti-DYKDDDDK
Anti-c-myc
Anti-human FabAnti-Strep-tag
Anti-His-6
Anti-V5
Anti-DYKDDDDK
Anti-human Fab(for non-human tissue)
Anti-Strep-tag
Anti-His-6
Anti-DYKDDDDK
Anti-human Fab(for non-human tissue)
Anti-Strep-tag
Anti-DYKDDDDK
Anti-His-6
Anti-human Fab(for non-human tissue)
Anti-Strep-tag
Anti-His-6
Anti-V5
Anti-BAP se a Fab-ds or IgG format to avoid
Ucolumn leakage of the light or heavychain
Use Fab-H or Fab TC-MH: use
Thrombin to cleave off tags

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2. Fab antibody formats and epitope tag combinations
Description

 pprox
A
MW(kDa)FabClone
Code*

Fab-FHFab antibody (DYKDDDDK- and His-6-tags)

52ca

Fab-V5H

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

53

cd

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

52

cb

Fab-V5Sx2

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

56dc

Fab-FSx2

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

54

daFab-MSx2

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

55

db

Fab-H

Fab antibody (His-6-tag)

51cc

Fab-Sx2

Fab antibody (StrepX-StrepX-tags)

53

deFab-CysH

Fab antibody (Cys with His-6-tag)

51

cf

Fab-Cys3H

Fab antibody (three Cys with His-6-tag)

51

ce

Fab-k-ds-HDisulfide-linked Fab antibody (His-6-tag)

51

chFab-I-ds-H

Disulfide-linked Fab antibody (His-6-tag)

51

ci

Fab-Tc-MH

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

53

ck

Fab-dHLX-FH

Mini-antibody (DYKDDDDK- and His-6-tags)

115

ei

Fab-dHLX-MH

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

115

el

Fab-dHLX-H

Mini-antibody (His-6-tag)

112

ek

Fab-dHLX-FSx2

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

119

fe

Fab-dHLX-MSx2

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

120

ff

Fab-A-FH

Fab bacterial alkaline phosphatase (BAP) fusion antibody (DYKDDDDK- and His-6-tags)

198

ea

Fab-A-V5H

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

200

eb

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

199

ed

Fab-A-Cys3H

Fab BAP antibody (three Cys with His-6-tag)

197

ef

Fab-A-FSx2

Fab BAP fusion antibody (DYKDDDDK- and StrepX-StrepX-tags)

203

fa

Fab-A-V5Sx2

Fab BAP fusion antibody (V5- and StrepX-StrepX-tags)

205

fb

Fab-A-MSx2

Fab BAP fusion antibody (c-myc- and StrepX-StrepX-tags)

203

fc

Fab-A-HFab BAP fusion antibody (His-6-tag)

196

ec

Fab-Max-FH

Fab modified BAP fusion antibody with inactivated enzymatic activity (DYKDDDDK- and His-6-tags)

198

eg

Fab-Max-V5Sx2

Fab modified BAP fusion antibody with inactivated enzymatic activity (V5- and StrepX-StrepX-tags)

205

fd

Short Name
Monovalent

Bivalent

Note: the HuCAL Fab antibody format does not contain a disulfide bond between light chain and Fd chain; the exception is the Fab-ds-H format.
* From July 1, 2019, HuCAL Fab clone number designation includes a 2- or 3-letter code suffix, denoting the antibody format. Therefore, the same antibody in different
formats will have the same numerical number, but different suffixes. Clones generated prior to July 1, 2019 will keep their original clone numbers without the suffix.

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3. Tag sequences and homodimerization domains
Description

 pprox MWA(kDa)

FabHeavy chain variable and first constant domain, and complete light chain

50

dHLX

Synthetic double helix loop helix motif (dimer)

5.2

A

Bacterial alkaline phosphatase (dimer)

47

Max

Modified bacterial alkaline phosphatase with inactivated enzymatic activity (dimer)

47

p53

Domain derived from human p53 (tetramer)

5.8

H

HHHHHH

0.9

FH

DYKDDDDKGAPHHHHHH

2.1

V5H

GKPIPNPLLGLDSTDAPHHHHHH

2.9

MH

EQKLISEEDLNGAPHHHHHH

2.4

CysH

CHHHHHH

1.1

Cys3H

CCCHHHHHH

1.3

Tc-MH

 VPR↓GSGAPEQKLISEEDLNDAPHHHHHHL↓: Indicates Thrombin cleavage (Tc) site

3.3

S

WSHPQFEK

1.2FS

DYKDDDDKGAPWSHPQFEK

2.3

FSx2

DYKDDDDKGAPSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK

4.3

V5Sx2

GKPIPNPLLGLDSTDAPSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK

5.6

MSx2

EQKLISEEDLNDAPSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK

4.7

Short Name
Domains

His-tag Combinations

Strep-tag Combinations

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4. Full-length immunoglobulin formats
Fab antibodies can be converted to full length human and chimeric human-mouse, human-rat or human-rabbit antibodies
when an Fc region is required for the application. The variable heavy and light chain genes are cloned into vectors withthe desired constant regions and co-transfected for expression in mammalian cells. Additional isotypes and allotypes areavailable on request.
•Use Fc region for binding or agglutination reactions•Evaluate Fc receptor-mediated effects•
Use as a fully human standard e.g. as calibrator and/or control•
In vivo validation of therapeutic antibody interactions in animal models
Description

Ig Clone Code*Human IgG1 allele G1m3ia

Human IgG1 allele G1m17, isoallotype nG1m1im

Human IgG1 allele G1m17,1il

Human IgG1 allele G1m3,1in

Human IgG2ib

Human IgG3ic

Human IgG4id

Human IgG4-Pro**ie

Human IgA1if

Human IgEih

Human IgM

ii

Human/Cynomolgus monkey IgG1 chimera (human VH and VL)

khHuman/Mouse IgG1 chimera (human VH and VL)

kaHuman/Mouse IgG2a chimera (human VH and VL)

kbHuman/Rat IgG1 chimera (human VH and VL)

kc

Human/Rat IgG2a chimera (human VH and VL)

kd

Human/Rat IgG2b chimera (human VH and VL)

keHuman/Rat IgG2c chimera (human VH and VL)

kf

Human/Rabbit IgG chimera (rabbit CH2 and CH3)kg

* From June 2019, HuCAL Ig clone number designation includes a 2 or 3 letter code suffix, denoting the antibody format. Therefore, the same antibody in different formats
will start with “AbD” followed by the same 5 digit unique identifying number, but will have different suffixes denoting the different formats such as species, isotype andsubclass. New productions of clones generated prior to June 2019 will keep the unique identifying AbD number, and be assigned the new 2 or 3 letter suffix, replacing theold format information.
** This antibody format has a mutation in the core hinge region that prevents the formation of IgG4 half molecules.

If you have questions or require additional information on HuCAL antibody formats or would like a complete list of qualified
reagents for HuCAL assay development, please contact us. bio-rad-antibodies.com/HuCAL
Bio-Rad is a trademark of Bio-Rad Laboratories, Inc. in certain jurisdictions. HuCAL is a trademark of MorphoSys AG.
Streptag technology for western blot detection is covered by US patent 5,506,121 and by UK patent 2,272,698. All trademarks
used herein are the property of their respective owner.

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