Anti-Idiotypic Antibodies Overview

All antibodies display a feature on their variable portion that is a unique set of antigenic determinants (epitopes), known as the idiotope. An antibody that is anti-idiotypic (Anti-ID) will bind to the idiotope of another antibody, usually an antibody drug, which makes it an incredibly powerful tool in the development of antibody drugs, especially for immunogenicity and PK/PD analysis.

ACROBiosystems has recently developed a series of high-affinity anti-idiotypic antibodies to assist in preclinical/clinical immunogenicity and PK analysis. Their pipeline covers five hot targets including adalimumab, rituximab, cetuximab, trastuzumab, and bevacizumab. They also provide assay protocols which can be applied to different application scenarios, to help along the drug development process.

Product List

Cat.No. Antigen Neutralizing Activity Affinity KD, nM Application
TRB-Y1 Trastuzumab F(ab')2 Neutralizing Antibody 0.296 PK bridging ELISA with TRB-BY5
Neutralizing assay
Indirect ELISA
TRB-Y7 Trastuzumab F(ab')2 Neutralizing Antibody 0.452 ADA assay
Neutralizing assay
Indirect ELISA
ADB-Y19 Adalimumab F(ab’)2 Neutralizing Antibody 0.0013 ADA assay
Neutralizing assay
Indirect ELISA
CEB-Y27 Cetuximab F(ab’)2 Neutralizing Antibody 0.007 ADA assay
Neutralizing assay
Indirect ELISA
CEB-Y28 Cetuximab F(ab’)2 Neutralizing Antibody 0.0015 ADA assay
Neutralizing assay
Indirect ELISA
CEB-Y31 Cetuximab F(ab')2 Non-Neutralizing Antibody 0.421 ADA assay
Indirect ELISA
RIB-Y35 Rituximab F(ab’)2 Neutralizing Antibody 0.03 ADA assay
Neutralizing assay
Indirect ELISA
RIB-Y36 Rituximab F(ab')2 Neutralizing Antibody 0.01 ADA assay
Neutralizing assay
Indirect ELISA
RIB-Y37 Rituximab F(ab')2 Neutralizing Antibody / PK bridging ELISA with RIB-BY35
Neutralizing assay
Indirect ELISA
BEB-Y12 Bevacizumab F(ab’)2 Neutralizing Antibody 0.0828 Neutralizing assay
Indirect ELISA
BEB-Y9 Bevacizumab F(ab’)2 Neutralizing Antibody 1.92 ADA assay
Indirect ELISA

ADA Assay - Perfect as CalibratorApplication

Therapeutic proteins such as monoclonal antibodies are currently vital for treating cancer, autoimmune disease, and other diseases. The structure of protein contains potential B-cell and T-cell epitopes, making it intrinsically immunogenic. Therefore, therapeutic proteins have the potential to induce Anti-Drug Antibodies (ADA) even if the protein has the same amino acid sequence as endogenous human proteins.

If ADA starts to arise in patients it can potentially lead to loss of efficacy and/or adverse events. This means that throughout the development of therapeutic protein products, researchers must carry out immunogenicity risk assessment and risk-mitigating strategies.

At the moment it is extremely time-consuming to develop a mono/multi-clonal antibody in-house as a positive control for ADA assay. To solve this problem, ACROBiosystems developed a series of anti-drug antibody standards for ADA assays.

Anti-Rituximab Antibodies bridging ELISA for Anti-Drug Antibody (ADA) assay development. Immobilized rituximab at 5 µg/ml, add increasing concentrations of Anti-Rituximab Antibodies (Cat. No. RIB-Y35, 10% human serum) and then add biotinylated rituximab at 5 µg/ml. Detection was performed using HRP-conjugated streptavidin with a sensitivity of 20 ng/mL.

Anti-Rituximab Antibodies bridging ELISA for Anti-Drug Antibody (ADA) assay development. Immobilized rituximab at 5 µg/ml, add increasing concentrations of Anti-Rituximab Antibodies (Cat. No. RIB-Y35, 10% human serum) and then add biotinylated rituximab at 5 µg/ml. Detection was performed using HRP-conjugated streptavidin with a sensitivity of 20 ng/mL.

Measured by its neutralizing ability in FACS. The data shows that the binding of rituximab to 293F overexpressing CD20 was inhibited by increasing concentrations of Anti-Rituximab Antibodies (Cat. No. RIB-Y35). The concentration of rituximab used is 10 ng/ml. The IC50 is 0.013 μg/ml.

Measured by its neutralizing ability in FACS. The data shows that the binding of rituximab to 293F overexpressing CD20 was inhibited by increasing concentrations of Anti-Rituximab Antibodies (Cat. No. RIB-Y35). The concentration of rituximab used is 10 ng/ml. The IC50 is 0.013 μg/ml.

PK Assay – Quantitative Analysis of Therapeutic Antibody in Matrix

An enzyme-linked immunosorbent assay, or ELISA, is the most commonly used technology for quantitative analysis of therapeutic antibody in matrix. There are several ways to carry such an assay out including: indirect ELISA, competitive ELISA, sandwich ELISA or bridging ELISA. Out of this set, bridging ELISA is usually the preferred option, due to its low non-specific background and high sensitivity. As seen from the data, using goat anti-human IgG as secondary antibody leads to high background due to unspecific binding, and therefore makes it necessary to perform pre-dilution before analyses.

On the other hand, the bridging ELISA by anti-idiotypic antibodies minimizes the background interference by using HRP-conjugated Streptavidin for secondary detection. ACROBiosystems has developed a series of anti-idiotypic antibodies that are used for bridging ELISA.

Comparison between anti-idiotypic capture ELISA and anti-idiotypic bridging ELISA for rituximab detection in patient samples. Left: anti-idiotypic capture ELISA; Right: anti-idiotypic bridging ELISA.

Comparison between anti-idiotypic capture ELISA and anti-idiotypic bridging ELISA for rituximab detection in patient samples. Left: anti-idiotypic capture ELISA; Right: anti-idiotypic bridging ELISA.

Detection of rituximab by bridging ELISA in serum. Immobilized Anti-Rituximab Antibodies (Cat. No. RIB-Y37) at 2 μg/ml, added increasing concentrations of Rituximab (10% human serum) and then added biotinylated Anti-Rituximab Antibodies (Cat. No. RIB-BY35) at 1 μg/ml. Detection was performed using HRP-conjugated streptavidin with a sensitivity of 1 ng/ml.

Detection of rituximab by bridging ELISA in serum. Immobilized Anti-Rituximab Antibodies (Cat. No. RIB-Y37) at 2 μg/ml, added increasing concentrations of Rituximab (10% human serum) and then added biotinylated Anti-Rituximab Antibodies (Cat. No. RIB-BY35) at 1 μg/ml. Detection was performed using HRP-conjugated streptavidin with a sensitivity of 1 ng/ml.

High Affinity

The measure of the strength of interaction between an antigen and an antibody is known as the affinity, which can be described as KD. The smaller KD represents a stronger affinity. ACROBiosystems measured the affinity of all the anti-idiotype antibodies using the SPR method. This piece of data helps to predict the sensitivity in assay development.

Anti-Adalimumab Antibodies (mouse IgG1, Cat. No. ADB-Y19) captured on CM5 chip via anti-mouse antibodies surface, can bind human adalimumab with an affinity constant of 1.36 pM.

Anti-Adalimumab Antibodies (mouse IgG1, Cat. No. ADB-Y19) captured on CM5 chip via anti-mouse antibodies surface, can bind human adalimumab with an affinity constant of 1.36 pM.

High Specificity

As well as the affinity, AcroBiosystems ensures the specificity of each anti-idiotype antibody product before it goes on the market.

Demonstration of the specificity of Anti-Cetuximab Antibodies (Cat. No. CEB-Y28) to the cetuximab.

Demonstration of the specificity of Anti-Cetuximab Antibodies (Cat. No. CEB-Y28) to the cetuximab.

High Stability

To ensure the stabilities of their products before they go on the market, ACROBiosystems performs accelerated stability tests and freeze-thaw tests.

Reconstituted Anti-Trastuzumab Antibodies were diluted to 0.4 mg/ml, aliquoted and placed at 37°C. Aliquots were removed from 37°C at every time point and placed at 4°C along with the control. No significant loss of activity was observed.

Reconstituted Anti-Trastuzumab Antibodies were diluted to 0.4 mg/ml, aliquoted and placed at 37°C. Aliquots were removed from 37°C at every time point and placed at 4°C along with the control. No significant loss of activity was observed.

Anti-Trastuzumab Antibodies were subjected to the indicated number of freeze-thaw cycles (FT). No  significant loss of activity was observed.

Anti-Trastuzumab Antibodies were subjected to the indicated number of freeze-thaw cycles (FT). No  significant loss of activity was observed.

About ACROBiosystems

ACROBiosystems is an internationally recognized manufacturer of recombinant proteins committed to supporting cancer immunotherapy. We specialize in mammalian cell-based recombinant protein production and process development.

Our goal is to support professionals from pharmaceutical companies, CROs and research institutes who are working on the cancer immunotherapy area by providing high-quality proteins, antibodies and assay kits.

We have multiple offices and branches in North America, Europe, and Asia, and we are proud of serving customers from over 50 countries.


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Last updated: Apr 30, 2019 at 6:19 AM

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