The immunochemistry of sandwich ELISAs—V. The capture antibody performance of polyclonal antibody-enriched fractions prepared by various methods

Abstract

Studies compare the performance of antibody-enriched serum fractions prepared by various methods, when adsorbed on polystyrene microtiter wells as capture antibodies (CAbs) and tested against multivalent antigens. The criteria of performance in the RIA used included antigen capture capacity (AgCC) and the nmol of functional capture sites per microtiter well (CAb t). Affinity purified polyclonal (pAb) and monoclonal antibodies (mAb) were employed as reference CAbs. AgCC was highest for enriched fractions prepared using caprylic acid and a high-pressure SpG affinity column. The performance of capture antibodies is expressed by an equation which was empirically derived and experimentally tested; CAb t × AgCC/ng adsorbed IgG. In terms of this parameter, CAb-enriched fractions prepared with caprylic acid performed best. The data reported also provide insight into solid-phase ligand immunochemistry. Adsorbed polyclonal CAb performed with remarkable homogeneity in percent bound and in Scatchard plots. Values obtained for CAb t from Steward-Petty plots were directly correlated with the length of the LBR of log-log percent bound plots but indicated that < 10% of the potential capture sites of polyclonal CAbs remained functional after adsorption; mAb showed a more serious loss of activity. The loss of CAb t was a general phenomenon for all preparations tested although relative to their antibody content, certain antibody-enriched fractions retained a higher proportion of CAb t than their affinity-purified counterparts. Comparative studies in which the activity of adsorbed mAb and pAb was compared to the same antibodies immobilized by a non-adsorptive procedure, indicated that adsorbed CAbs also express lower affinity. The studies we report offer a single parameter critérium for comparatively evaluating CAb performance while simultaneously revealing the need to develop immobilization procedures that can preserve CAb t and antibody affinity so that immunoassays with wide dynamic ranges and high AgCC can be developed without waste of antibody.