Critical role of the sample matrix in a point-of-care protein chip for sepsis

Abstract

Both highly specific antibodies and appropriate assay buffers are key elements in the development of sensitive multi-analyte diagnostic tests and essential assay components to minimize interferences from the sample matrix. Herein, we investigate the influence of 0.1 M Tris (pH 7.4)/0.1 M NaCl/10 mM CaCl(2)/0.1% Tween-20 used as assay buffer and diluent for serum, plasma and saliva samples in a protein biomarker chip for the diagnosis of sepsis. In detail, on-chip sandwich assays for detection of IL-6 and PCT are established using pure assay buffer and serum, plasma, and saliva, each diluted by a factor of 10 and 100 with assay buffer. The dilution linearity as well as the cross-reactivity to immobilized IL-8, IL-10 and TNF-α antibodies (<1.8% in plasma and serum) is investigated; furthermore the influence of immunoglobulin G, fibrinogen and lysozyme, highly abundant proteins in serum, plasma and saliva. This effect is two times more pronounced in serum than in plasma and saliva and strongly decreases with increasing analyte concentration. Though the matrix proteins bind unspecifically to the immobilized receptors, they do not prevent the analyte binding; on the contrary, the analyte is reliably detected with high sensitivity, featuring limits of detection of 16 ng/L and 0.31 μg/L, and coefficients of variation of 18% and 29% for IL-6 and PCT in 10% serum.