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Western Blotting

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Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

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Exploring blocking assays using Octet, ProteOn, and Biacore biosensors.

Yasmina N Abdiche¹, Dan S Malashock, Alanna Pinkerton

¹Rinat Laboratories, Biotherapeutics and Bioinnovation Center (BBC), Pfizer Inc., South San Francisco, CA 94080, USA. yasmina.abdiche@pfizer.com

Analytical Biochemistry

|December 30, 2008

Summary

Label-free optical biosensors enable epitope binning of antibodies using competitive binding assays. This method groups antibodies with similar epitopes, aiding in the characterization of antibody panels and protein complexes.

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Area of Science:

Biochemistry
Immunology
Biotechnology

Background:

Antibody characterization is crucial for therapeutic development and biological research.
Epitope mapping helps understand antibody-antigen interactions and identify distinct antibody populations.
Label-free biosensing offers real-time, sensitive detection without the need for labels.

Purpose of the Study:

To demonstrate the utility of label-free real-time optical biosensors for epitope binning of antibody panels.
To compare three different assay orientations (in tandem, premix, classical sandwich blocking) across three biosensor platforms.
To establish a blocking profile for antibodies to group them into bins based on epitope similarity.

Main Methods:

Utilized competitive binding assays on three optical biosensor platforms (Octet QK, ProteOn XPR36, Biacore 3000).
Employed three distinct assay orientations: in tandem, premix, and classical sandwich blocking.
Performed pairwise antibody blocking experiments to determine epitope relationships.

Main Results:

Successfully generated blocking profiles for antibody panels, enabling epitope binning.
Demonstrated excellent agreement between data from different assay orientations and biosensor platforms.
Identified advantages and disadvantages of each biosensor and assay orientation for specific applications.

Conclusions:

Label-free optical biosensors are valuable tools for characterizing antibody epitopes and multiprotein complexes.
The described assay orientations and platforms provide a robust framework for epitope binning.
This approach facilitates the efficient grouping of antibodies with similar binding sites, aiding in antibody selection and development.