PEGS Europe 2025 – Lisbon, Portugal

Poster session

Poster Title: Identification of Critical Reagent Leads Using High-Throughput SPR

Presenter: Andrew Goodhead, PhD, Carterra

Abstract:

Introduction
To identify lead critical reagents, we have implemented a high-throughput, biosensor-focused characterization methodology that is both time- and cost-efficient, provides a rich understanding of reagent performance characteristics, and permits identification of cooperative reagent pairs that could be overlooked using traditional plate-based screening approaches.

Method
Our workflow is initiated on the Carterra LSA, a high-throughput surface plasmon resonance (HT SPR) platform. The process involves establishing a covalently coupled array of up to 384 antibody reagents on a polycarboxylate biosensor chip, determining the binding kinetics of all coupled antibody ligands for their target analyte in parallel, serial epitope binning of the reagents on their target analyte to identify sandwiching (noncompetitive) reagent pairs, and measuring the dissociation rate of the sandwiched pairs to estimate the stability of the complete analytical complexes. All antibodies that are identified as potentially suitable as immunoassay reagents are subsequently evaluated by ELISA for matrix compatibility and finally for assay sensitivity and range as matched reagent pairs.

Results
In the case study presented, 94 anti-idiotype candidates were evaluated by the process described above, and from which 10 anti-IDs were identified as lead candidates to test as 8 sandwiching pairs. Two high-affinity blocking clones were also included as comparators in the confirmatory screens by ELISA. Per our predictions based on binding kinetics and analytical complex stability, the 6 lead sandwiching pairs produced the greatest assay sensitivity observed in our confirmatory ELISA, and generally did so in the expected capture/detect orientations. Strikingly, one of the two lead detection reagents identified has very low monovalent binding affinity for the analyte (> 1 uM), but appears to bind cooperatively with select capture reagents, and together these cooperative pairs form analytical complexes possessing enhanced stability and facilitate sensitive quantitation of the analyte.