Development and analytical validation of a flexible multiplexing platform for cytokine assays.

Abstract

Abstract Multiplex assays have advantages in being able to reduce time and sample volumes; however, multiplexing offers additional challenges, which include developing multiple assays that can be run with the same protocol, reagents, and sample dilution. Moreover, transferring reagents that work in one assay format to another without compromising performance and the integrity of sample measurement is difficult. The U-PLEX® platform enables flexible multiplexing of immunoassays using MSD’s MULTI-ARRAY® technology. Antibodies used in V-PLEX® kits were transferred to the U-PLEX platform by biotinylating the capture antibodies. The assays represented a variety of analyte classes (chemokine, interleukin, interferon), antibody types (monoclonal, polyclonal), and analytical properties (sensitivity, dynamic range, concentration-response slope). During development, antibody concentration, biotin and MSD SULFO-TAG™ label ratios, and calibrator concentrations were evaluated and optimized. Assays were readily transferred to the U-PLEX platform with calibration curves showing expected signals, sensitivity, precision, and accuracy. Controls for the assays showed CVs of <10% within runs. Sensitivities were <1 pg/mL for many assays. All assays used the same assay diluents. Non-specific binding between assays was typically <0.1%. We measured 40 human serum and 40 EDTA plasma samples, and demonstrated good correlation with V-PLEX assays (r2 > 0.9; slopes 0.8 – 1.2). The utility and convenience of the U-PLEX platform was demonstrated by easily transferring over 40 human cytokine assays onto the platform without compromising performance. When multiplexed, these assays enable multiple measurements from small amounts of human samples.