Non-fouling hyaluronic acid coatings for improved sandwich ELISA measurements in plasma

Abstract

Nonspecific protein adsorption can interfere with blood diagnostics, especially in point-of-care tests for which minimal sample processing is required. Here we report a non-fouling coating material for presenting capture antibodies in ELISA that significantly increased precision and accuracy compared to a commercial ELISA microplates in human plasma and model solutions containing plasma proteins. The coating was prepared by functionalizing the hydrophilic polysaccharide hyaluronic acid (HA) with a thermoresponsive polymer, poly(di(ethylene glycol)) methyl ether methacrylate (PMEO₂MA). Previous studies demonstrated that these coatings were resistant to adsorption of major blood proteins, and we demonstrate that functionalization of the coatings with a monoclonal antibody against tumor necrosis factor-α (TNF-α) provided enhanced detection accuracy for this pro-inflammatory cytokine in ELISA. Three plasma-type solutions were explored in this work based either on buffer containing known concentrations of albumin, fibrinogen and immunoglobulin at sub-physiological and physiological concentrations, and non-diluted human plasma. In model solutions of plasma, even sub-physiological concentrations of plasma proteins resulted in a 20% overestimate of TNF-α concentration in the commercial ELISA kit but a 4% overestimate in HA-coated microwells. However, in human plasma, the commercial ELISA kit underestimated the analyte concentration by up to 95% while the HA-coated microwells did so by only 15% at an analyte concentration of 12.5 pg mL⁻¹. The improvements in precision and accuracy provided by HA coatings suggests they could be used to enhance ELISA measurements in a broad range of complex biological media.