Universal planar assay format for high sensitivity cytokine quantification in human serum and plasma | Poster


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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 ‘homebrew’ planar immunoassay format for development of high sensitivity assays for any analyte, by using an anchor antibody/peptide tag system to develop a universal capture plate. Anchor antibody specific for a peptide tag was microprinted in microwell plates. The peptide tag was 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 or mouse serum and plasma, including IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-22, ITAC, MIP1a and MIP3b. 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 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 assays for any analyte of their choice.