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Automation of a Homogeneous Proximity Assay for Immunogenicity Testing of Biological Drug Products


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January 14, 2013

Authors: Peter Brescia and Peter Banks, BioTek Instruments, Inc., Winooski, Vermont, USA


Several challenges have surfaced during clinical evaluation of biological drug products due to a commonly associated immune response in patients. Anti-drug antibodies (ADA) are known to be frequently generated during administration of humanized monoclonal antibody therapeutics. These ADAs are nearly indistinguishable from antibody drug therapeutics thus requiring robust selective methods to determine the extent to which they impact safety and efficacy during treatment. A commonly used technology platform for assessment of immunogenicity relies on the bridging immunogenicity assay format typical of the Enzyme-Linked Immunosorbent Assay (ELISA). Other methods have been used to provide simpler workflows and higher sensitivity, such as Electrochemiluminescence (ECL) assays using streptavidin ECL plates to create the classic bridging assay. Here we present a homogeneous assay based on using a bridging assay format where all reagents and sample are in solution. This facilitates automation of reagent addition and simplifi es further the work flow without sacrificing sensitivity.

Here we compare the automation of an AlphaLISA® ADA assay with a solution ELISA ADA assay using liquid handling and dispensing instrumentation and a high-throughput screening (HTS) multi-mode reader which can be used to detect the presence of ADA activity in a model system. The AlphaLISA ADA assay utilizes bivalent binding of anti-drug antibodies to biotinylated drug which is then captured on streptavidin (SA)-coated Donor beads and drug antibody immobilized on Acceptor beads. The resulting complex is formed in the presence of ADAs resulting in the two beads coming into close proximity. Laser excitation of Donor beads at 680 nm results in singlet oxygen release and subsequent energy transfer to Acceptor beads and light emission at 615 nm. The formation of the complex in solution eliminates washing steps and secondary detection antibodies typically required with standard sandwich ELISA methods.