Rapid Immunoassay Development using the U-PLEX (R) Assay Platform.

Abstract In the expanding field of biomarker research, methods to identify immunoassay reagents and optimize their use can be rate limiting for product development programs. Flexible tools that accelerate assay development, from antibody screening and selection to assay feasibility, can accelerate these programs. Here we demonstrate the use of the MSD® U-PLEX platform to conduct early assay development steps in parallel in a multiplex format. This resulted in rapid identification of a multiplexed panel of compatible assays. Unbiased pairwise screening of 59 antibodies was performed on MULTI-SPOT® U-PLEX plates using biotinylated capture antibodies and detection antibodies conjugated with SULFO-TAG™ label. Feasible antibody pairs were identified based on high signal and low background, then ranked using parameters such as dynamic range, sensitivity, specificity, sample recognition, and matrix tolerance. Within four weeks, two prototype immunoassays were developed and characterized. This screening time frame can range from days to weeks depending on the number of starting antibodies (typically 10–50) and the level of characterization required. Due to the flexible nature of the platform and the ability to mix and match reagents, the same conjugated reagents identified in screening were used to create and characterize multiplex assay panels. Parameters such as calibration curve, assay protocol, detection antibody concentration, and non-specific binding were characterized with up to ten assays simultaneously in a multiplex. In conclusion, we demonstrate the utility of a flexible assay development system that allows rapid identification and optimization of assays for stand-alone use or in combination with existing MSD assays.