Rapid, Sensitive Detection of Protein Biomarkers in Minimally‐Processed Blood Products with a Monolithic Sandwich Immunoassay Reagent

Abstract For more than fifty years, the enzyme‐linked immunosorbent assay (ELISA) serves as the gold standard for protein biomarker detection. However, conventional ELISA requires considerable sample preparation including reagent addition, incubation, and washing steps, limiting its usefulness at the point‐of‐care. In this work, the “instant ELISA” (fluorophore‐linked immunosorbent assay) biosensor that can measure protein biomarkers in the picomolar range within 15 min in undiluted plasma or serum with no sample preparation is described. The sensor leverages a synthetic reagent termed the “monolithic dual‐antibody clamp” (MDAC) which preserves the specificity, sensitivity, and generalizability of an ELISA, but produces a fluorescence signal as two surface‐tethered antibodies form a “sandwich” by binding to two distinct epitopes on the target. As exemplars, picomolar quantification of tumor necrosis factor alpha (TNFα) and monocyte chemotactic protein (MCP)‐1, the latter of which is a useful prognostic indicator of cytokine release syndrome in patient plasma samples during chimeric antigen receptor T cell therapy are demonstrated.