A universal multiplex assay format for ultra-sensitive cytokine quantification in human serum and plasma

Abstract

Abstract Quantifying prospective biomarkers of immune signaling in biological samples requires high assay sensitivity. The Simoa Planar Array provides greater sensitivity than ELISA, but has a finite assay menu. This study aimed to develop a universal multiplex ‘homebrew’ planar immunoassay format for development of high sensitivity assays for any analyte. Anchor antibodies specific for a peptide tag were microprinted in microwell plates. The peptide tags were conjugated to analyte-specific capture antibodies via maleimide chemistry, enabling high affinity binding of capture to anchor antibody. Bound sample antigen was sandwiched between the peptide-tagged capture and biotinylated detector antibodies; chemiluminescent detection was used to quantify signal. This ‘homebrew’ approach successfully detected sub picomolar levels of cytokines and chemokines in human serum and plasma in a multiplex assay measuring IL-5, IL-6, IL-10, IL-22, TNFa. When compared to commercially available planar assays the universal planar assay format provided comparable LLOQ and LOD with sample correlation (R2) >0.97 for all analytes tested. The universal planar assay was used to successfully screen multiple antibody pairs for human and mouse biomarkers and to develop robust assays using mouse, rabbit or goat monoclonal and polyclonal antibodies. This universal multiplex planar homebrew assay format combines the accuracy, reproducibility and high sensitivity of Simoa Planar Array technology with total analyte flexibility, and provides assay developers a simple and versatile method to rapidly develop robust high-sensitivity multiplex assays for any analyte of their choice.