Impact of surface defects and denaturation of capture surface proteins on nonspecific binding in immunoassays using antibody-coated polystyrene nanoparticle labels

Microtiter wells are commonly used for bioassays. The sensitivity of such an assay depends on several instrumental and biochemical parameters such as the signal-to-background ratio and nonspecific binding of the label molecules. In this study, we have investigated the possible effects of well surface defects, well edges and denaturation of capture antibodies on the assay sensitivity. We used internally dyed Eu(III) chelate polystyrene nanoparticles as high specific activity labels in a thyroid stimulating hormone (TSH) sandwich-type model assay. The nanoparticle labels provide a high signal-to-background ratio in assays but the major limiting factor of the assay sensitivity is nonspecific binding of the labels. In our model assay the capture monoclonal antibodies were immobilized on microtiter wells passively or through streptavidin (SA)–biotin linkage. At first, commercially manufactured microtiter well surfaces were probed with an atomic force microscopy and significant structural inhomogeneities were found. The nonspecific binding of the nanoparticle conjugates did not appear to follow any of the microtiter well surface defect patterns in a number of experiments. In addition, the microtiter well edges did not increase the nonspecific binding. Denaturation of capture antibodies on solid surfaces has been proposed to expose amino acid sequences promoting nonspecific binding. This was studied by intentionally denaturing the surface capture antibodies by heat, detergent or acid treatment prior to the assay. Although specific signal was almost entirely lost no significant effect on nonspecific binding was observed. The passively adsorbed antibodies denatured at lower temperatures than those captured through streptavidin–biotin linkage. Evidently, the additional protein (SA) layer protected the capture antibody from denaturation whereas the solid surface appeared to act as a “catalyst” making the passively adsorbed antibody more susceptible to denaturation.