The effect of pH on the aggregation of biotinylated antibodies and on the signal-to-noise observed in immunoassays utilizing biotinylated antibodies

Abstract

During the development of immunoassays to detect gram-negative bacteria, an effect of pH on the aggregation of some murine monoclonal antibodies directed to Neisseria gonorrhoeae was observed. By reacting positively charged primary amines on these antibodies with the neutral NHS-biotin ( N-hydroxy-succinimidobiotin), the surface charge on the antibodies was altered and a concomitant change in the solubility of these antibodies noted. This derivatization produced not only a pH-dependent change in the solubility properties of the antibodies, but also affected the response of immunoassays in which these antibodies were used. Data presented suggests that the signal-to-noise (S/N) observed in these assays is maximized under conditions where the biotinylated antibody is introduced into the assay at a pH at least 2 U above its p I. Our hypothesis is that as the pH of the solution approaches the biotinylated antibodies' isoelectric point, they become ‘stickier’, perhaps by aggregation (which we have directly measured), leading to high non-specific binding and hence a lower S/N.