Development and characterization of Th17-related U-PLEX (R) assays

Abstract Th17-related cytokines mediate a host’s defense mechanisms against various infections and play a crucial role in crosstalk between the immune system and affected tissues. Here we describe the development and characterization of multiplexed Th17-related immunoassays on MSD’s flexible U-PLEX platform. Monoclonal and polyclonal antibodies were conjugated with biotin and/or SULFO-TAG™ and screened as capture and/or detection reagents. A number of analytical parameters were used to select antibody pairs, and assays were developed by optimizing antibody concentrations, calibrator curves, specificity, matrix tolerance, and assay robustness. Assay performance was verified to be compatible with other U-PLEX assays by running Th17-related multiplexes on the U-PLEX platform with controls and samples. Calibration curves showed expected signals, sensitivity, precision, and accuracy. Control samples for the assays had CVs < 10% within runs and < 25% between runs. Sensitivities were < 1 pg/mL for the majority of the assays. Dilution linearity and spike recovery studies demonstrated acceptable matrix tolerance and accurate quantification for most of the assays tested across all matrices (typically between 75% – 125%). Cross-reactivity between assays was shown to be typically < 0.5%. Results demonstrated a strong correlation between samples measured on U-PLEX multiplex and streptavidin singleplex panels with r2 values > 0.95 and slopes between 0.8–1.2. Over 20 Th17-related assays for human and mouse were developed for the U-PLEX platform. These assays enable the measurement of multiple proteins on biological matrices that are relevant to a wide range of life science applications and pre-clinical or clinical studies.