Development and Characterization of V-PLEX® TH17 Cytokine. Assays

Abstract

Abstract Th17 cytokines are important mediators of the host defense against infection and are increasingly investigated for their role in autoimmune disorders and immune regulation at mucosal surfaces. Here we describe the development, characterization, and analytical validation of a multiplexed immunoassay panel for seven Th17 cytokines, IL-17A, IL-21, IL-22, IL-23, IL-27, IL-31, and MIP-3alpha, on MSD’s V-PLEX platform. To efficiently and rapidly identify potential antibody pairs, biotinylated capture antibodies and detection antibodies conjugated with SULFO-TAG™ label were screened on MSD’s U-PLEX® platform, which enables the solution phase assembly of capture antibody arrays. Subsequent development used printed arrays of capture antibodies. Antibody concentrations, calibrator curve linearity, dynamic range, specificity, matrix tolerance, and assay robustness were analyzed for each assay during development. Calibration curves demonstrated a three-log dynamic range while achieving a lower limit of quantitation (LLOQ) of less than 1 pg/mL for most assays. Control samples for the assays had CVs of less than 10%. Dilution linearity and spike recovery studies in serum, plasma, urine, and cell culture media were conducted to demonstrate matrix compatibility. Spiked matrix samples were typically found to recover between 80%–120% of the expected value. Cross-reactivity was shown to be less than 0.3% between assays within the panel and less than 0.5% when panned against more than 30 other blood-related biomarkers. These validated, multiplexed assays provide sensitive measurement of Th17 cytokines in a variety of matrices and can be used as part of a researcher’s pre-clinical and clinical studies.