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

This application note presents 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 simplifies the work flow without sacrificing sensitivity. Additionally, we compare the automation of the AlphaLISA® ADA assay with a solution ELISA ADA assay using liquid handling and dispensing instrumentation and a high performance multi-mode microplate reader which can be used to detect the presence of ADA activity in a model system3. The study will describe the assays, reagent preparation and automated methods used and will demonstrate several validation experiments comparing the performance of the two methods described above. Adaptation of the AlphaLISA ADA assay to be performed in a high-density 384-well format will also be described including serum screening data as well as additional analysis required for a full validation study using the AlphaLISA ADA assay technology with automated methods.

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 (Figure 1). 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 generation and as the beads are in close proximity, energy transfer to Acceptor beads is facile resulting in 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.


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