Noise control in solid-phase immunoassays by use of a matrix coat

This paper describes the matrix coat noise control technique as applied in an ELISA to detect IgG antibody to the semen protein p30 (also known as prostate-specific antigen). Background noise due to non-specific binding of IgG in solid-phase immunoassays has recently been shown to be highly charge dependent. In this technique, the surface antigen (here, p30) is co-coated on the test surface with an anionic macromolecule, the noise reduction matrix component, to reduce non-specific IgG binding. A second matrix component, the noise balancing component, is added if necessary to balance non-specific IgG binding between a detecting well which contains matrix plus antigen and a control well which contains matrix alone. Sample noise can then be measured in the control well and subtracted to yield a noise-corrected signal. This approach both minimizes background noise and then precisely quantitates residual noise. In this study, human alpha 1-acid glycoprotein (AGP) served as the noise reduction component and bovine serum albumin (BSA) as the noise balancing component. Optimal coating concentrations of AGP and BSA for the matrix were determined by a new technique, the tetrad method of signal and noise analysis. A signal probe (immune serum), a noise probe (non-immune serum) and a set of four test wells are employed to analyze the noise control properties of a given combination of matrix components. For each such tetrad of wells, three ratios are calculated: the sensitivity, signal-to-noise and noise balance ratios, along with a composite index, the matrix index. These can be used to select the combination and concentrations of matrix components which optimize assay noise control and minimize losses of assay sensitivity. When an ELISA using matrix coat noise control (MCNC-ELISA) was compared with ELISAs using standard blocking techniques, the MCNC-ELISA was superior in its ability to control for false positive results due to non-specific IgG binding and binding of the polycation poly-L-lysine. Two subjects (14%) out of a clinic population of sexually active women were found to have significant serum levels of anti-p30 IgG (P less than or equal to 0.001) by MCNC-ELISA.