Application Note

May 1, 2022
Keywords or phrases:
Octet, ELISA, protein quantitation, assay development

Converting an ELISA Assay Intoan Octet® Quantitation Assay
Sae Choo, Sartorius, Fremont, CACorrespondence
Email: octet@sartorius.com

Abstract
Enzyme-linked immunosorbent assays (ELISA) are routinely used to quantitate molecules and despite their popularity,
these assays are labor- and time-intensive. Quantitation assays on the Octet® platform can be considered automatedforms of ELISA but allow a myriad of benefits compared to standard ELISA assays, notably the ability to detect lower affinity
interactions and a reduced hands-on time for the scientist. As with ELISA, the signal reported in quantiation assays is either
directly or inversely proportional to the amount of bound analyte.
The conversion of many existing ELISA to an Octet® assay typically requires simple re-optimization and validation of theconditions and configurations. This application note describes the key steps for ensuring successful conversion of anELISA-based assay to the Octet®, including defining the assay requirements, selecting the biosensor type and assay
format, minimizing non-specific binding, and optimizing the assay buffer.

Find out more: www.sartorius.com

Introduction
Quantitation assays on the Octet® platform have many
similarities to enzyme-linked immunosorbent assays(ELISA). Both are performed on a solid support on which
the capture molecule is immobilized and the analyte isbound from solution. Signal reported in the assay is either
directly or inversely proportional to the amount of boundanalyte. In fact, Octet® quantitation assays can beconsidered automated forms of ELISA. The conversion toan Octet® assay often involves simply re-optimizing and/orvalidating the conditions and configurations of the alreadyexisting ELISA assay. However, in some cases where theminimum essential requirements are stringent, moredevelopment work is required.
It is often beneficial to convert ELISA assays to the Octet®
platform as it allows the scientist to:
1. Choose from a number of assay formats (label-freedirect binding, sandwich, sandwich followed by signal
amplification, etc.) to suit detection limit requirements
2. Detect low-affinity analytes often missed by ELISA3. Minimize handling via automated and wash-free steps
4. Fully recover and re-use samples and reagents
5. Regenerate the assay surface and re-use for somebinding pairs (e.g., Protein A/human IgG).
This article provides guidance on converting an existing
ELISA-based assay to an Octet® assay. The process can be
broken down into five steps:
1. Defining assay requirements
2. Selecting a biosensor type
3. Selecting assay format
4. Minimize non-specific binding (NSB)
5. Optimizing assay buffer
Details on each of these steps are presented in thefollowing sections.

Defining Assay Requirements
Conversion activity can start with defining the minimum
assay requirements. Sensitivity and throughput
requirements often become critical factors in determining
the Octet® instrument, biosensor type and assay format,
while the sample matrix has the biggest impact on reagent
formulation and the blocking protocol. Sample volume
requirements also influence the choice of instrument(Octet® QKe, RED, and R8 require 80–200 μL while Octet®
RH16 and RH96 require 40–80 μL). Sample volume also2

indirectly influences the sensitivity of detection whendilution is required to compensate for limited availability ofsample. Physical characteristics (pI, size, polymeric status,hydrophobicity, stability, etc.) of the analyte should beconsidered when selecting the surface and theimmobilization protocol.

Selecting a Biosensor Type
There are four types of biosensors to be considered inbuilding quantitation assays on the Octet® platform. Theyare Streptavidin (SA), Aminopropylsilane (APS), AmineReactive (AR) and analyte-specific Biosensors.
SA, APS, and AR Biosensors can be used to build customquantitation assays for analytes that are process-specific.
SA Biosensors are most often preferred for their flexibility
to accommodate a variety of capture molecules (ligand)through streptavidin/biotin interaction and its superior
surface capacity derived from the use of specially designed
cross-linked streptavidin conjugates. There are several
advantages of immobilizing the ligand that is labeled with along-chain biotin at a low molar coupling ratio(biotin : ligand):1. Loss of binding capacity due to cross-linking and sterichindrance are reduced.
2. Biosensors can be prepared in batch mode and storedfor later use.
3. The biotin-ligand solution may be used to prepare
multiple batches of biosensors.
4. The strong, nearly irreversible binding between thebiotinylated ligand and the Streptavidin Biosensor
allows easy regeneration of the ligand-loadedbiosensor.
APS Biosensors are ideal for immobilizing ligands that are
not suitable for covalent linking chemistry. The mode ofimmobilization is a combination of hydrophobic and/orelectrostatic interactions. The APS Biosensor surface issomewhat more hydrophobic in comparison to the surfaceof ELISA microtiter plates , so the stability of the ligand on
the biosensor surface should be tested prior to loading toavoid denaturation.
AR Biosensors can be used to covalently immobilize aminegroup containing ligands onto carboxylate groups on thebiosensors. Batch mode preparation and regeneration of
the biosensor are possible.

Table 1: Octet® assay formats and features.
Assay features

1-Step2-Step3-StepPictorial
representation

HRP HRP HRP HRPHRP
HRP HRPHRP
HRP HRP HRP

Assay steps

1 Bind analyte

1 Bind analyte
2 Bind secondary reagent

Typical assay time
Typicalconcentration
range
Advantages-----

30 min (Octet® QKe, R8)
15 min (Octet® RH16, RH96)
Low mg/mL to low ng/mL

Single incubation step — fast, easy,
reduces reagent expenses
Low affinity analytes detected — eventhose missed by ELISANO labeled reagents
Kinetic parameters can be measured
Allows regeneration and re-use ofbiosensor in most cases---

1 Bind analyte – secondary antibody
complex
2 Bind HRP-loaded antibody---

3 Incubate in precipitating substratefor HRP
1 hr 30 min (Octet® QKe, R8)
1 hr 15 min (Octet® RH16, RH96)
Low ng/mL to low pg/mL

Two incubation steps — still fast, easy,
reduces reagent expenses incomparison to ELISA
Higher sensitivity of detection, down
to low pg/mL, depending on assay
NO labeled reagents
Automated and no-wash assay
minimizes handling

Analyte-specific biosensors are pre-immobilized withspecific capture proteins such as anti-human IgG Fc,
anti-murine IgG (Fab’)2, Protein A, Protein G, and antipenta HIS. These ready-to-use biosensors are suited forquantitative analysis of human IgG, mouse IgG, rat IgG,
all proteins that bind Protein A, those that bind Protein G,
and penta-HIS-tagged proteins, respectively.

Selecting Assay Format
The choice of an assay format is dependent on theconcentration range of analyte to be quantified. Octet®
systems offer the advantage of a direct binding assay(also called 1-step), that generally affords a dynamic rangeof detection from low ng/mL to low mg/mL, depending on
the analyte. The direct binding assay is fast, easy andeliminates the need for secondary reagents and steps. It
also allows regeneration of the biosensor in some cases.

2 hr (Octet® QKe, R8)
1 hr 30 min (Octet® RH16, RH96)
Low ng/mL to low pg/mL

Similar to most ELISA assays informat — but faster and easier
Excellent sensitivity — down to
low pg/mL, depending on assay
Automated and no-wash assay
minimizes handlingMulti-step assays provide enhanced sensitivity down tolow pg/mL, depending on the analyte. Octet® systems
measure signal as a function of the thickness and densityof the analyte binding layer, so increasing analyte bindingtranslates to bigger signals. When analyte is present in low
concentrations and the binding signal is low even after along incubation, building additional layers of secondary
reagents over the analyte binding layer enhances signal.
Such multi-layer assays include sandwich-style assays(also called 2-step assays), which use the biosensor tocapture analyte in the first step, followed by use of a second
antibody to sandwich the analyte in the second step. For
even more signal amplification, an enzyme-linked sandwich
assay (also called a 3-step assay) captures analyte bound by
two separately-labeled capture molecules to the biosensor
in the first step, binds an HRP-conjugated antibody to thecomplex in the second step, and precipitates a substrate
directly onto the biosensor surface in the third step.

3

Minimize Non-Specific Binding (NSB)40
200 ng/mL
75 ng/mL
25 ng/mL
8 ng/mLBinding (nm)32

2 ng/mL
1 ng/mL
0.5 ng/mL0 ng/mL

Managing NSB and matrix effects are critical parts of theassay development process that ensure acceptablespecificity and sensitivity. The major sources of NSB are
hydrophobic, electrostatic, and cross-reactive interactions
between the molecules on the biosensor surface and inthe solution. In addition to NSB, unrelated proteins andother components of the sample matrix often cause
assay interference.

2416800102030
Time (seconds)

40

5060

Figure 1: Raw data from the final amplification step of the CHO HCP
assay using Octet® R8 system.

The most effective way to minimize such effects is
passivation of the biosensor surface by including blocking
step(s) prior to sample incubation. Often, blocking buffer
formulation needs to be matched to the sample matrix (e.g.,
block with the serum for immunogenicity sample in serum).

Optimizing Assay Buffer

Binding Rate

1010
110Concentration (ng/mL)

100

Assay buffer formulation has a significant effect on NSB,
sample matrix interferences and signal over background.
The goal of optimizing reagent formulation is to maximize
specific signal and minimize NSB. A direct adaptation ofpreviously developed ELISA formulation may be sufficientin most cases, unless the formulation contains components
or conditions that are not compatible with Sartorius
biosensors. Examples of such incompatible reagentsinclude those with low pH (<4), high pH (>10), and certain
types of organic solvents. The characteristics of the analyte(pI, hydrophobicity and stability) and sample matrix
components are important to keep in mind whendeveloping an assay buffer.

Figure 2: Standard curve showing standards (n = 3 for each standard)
and unknown samples (n = 8 for each unknown). The lower limit ofdetection in this assay was less than 0.5 ng/mL.

On a related note, the rinsing step employed in Octet®
assays is often performed in physiological buffers such as
TBS and PBS in the presence of a detergent.

Table 2: Accuracy and robustnss of CHO host cell protein quantitation. In
this assay, CVs of less than 10% were achievable across the entire
dynamic range.

The following is a list of common assay buffer componentsthat are useful to keep in mind during optimization.
1. Salt: high salt concentrations slow down the reaction,
reduce stickiness of antigens with very high/low pI
values, reduce charge-induced NSB and denature
proteins in solution when needed (e.g., Protein A
contamination assay sample pre-treatment buffer).
2. Buffer capacity: higher buffer capacity minimizes pHchanges and stabilizes sample solutions.
3. Non-specific antibodies: reduce NSB from multi-species
cross-reactions.
4. Detergents: reduce sample aggregation, reducehydrophobic NSB and reduce sample coating out ofvery low concentration samples.

Spike conc.
(ng/mL)*

Calculatedconc.

%CV of calculatedconc.

% Recovery

200

201.6

4101

88.5

4

106

0.5

0.57

7114

* CHO Cell Protein, n = 8 for eac4

5. Bulk proteins (BSA, casein, antibodies): serve as blocking
agents, reduce sample coating out of solution, andstabilize analyte proteins in solution in other ways.
6. Sugars (trehalose, dextran, sucrose): stabilize some
proteins, enhance signal by increasing effective
concentration in solution.
7. PEG: helps reduce non-specific binding and canenhance signal in some cases by increasing the effective
concentration of proteins in solution.
8. pH: low pH may often reduce the affinity of competinginteractions and improves the proportion of ligandanalyte binding to the biosensor.
9. Dilution: to minimize matrix effects.

Germany
Sartorius Lab Instruments GmbH & Co. KG
Otto-Brenner-Strasse 2037079 Goettingen
Phone +49 551 308 0F
 or further contacts, visit
www.sartorius.com/octet-support

Specifications subject to change without notice.
Copyright Sartorius Lab Instruments GmbH & Co. KG.
For Research Use Only.
AN-4002 Rev DConclusion
ELISA-based assays and Octet® quantitation assays sharemany similarities. Therefore, conversion of a pre-configured
ELISA assay to the Octet® platform may only require transferof assay conditions. When assay conditions need
re-optimization on the Octet® platform, considerations areoften similar to those employed in ELISA.
The Octet® platform’s direct binding assay method is simple,
fast and accurate; the multi-step methods offer highsensitivity and expanded dynamic range. The automatedassay formats enhance time-to-results and walk-away timeand reduce operating expenses.

USA
Sartorius Corporation
565 Johnson Avenue
Bohemia, NY 11716
Phone +1 888 OCTET 75
Or +1 650 322 1360