Simple determination of polysaccharide specific antibodies by means of chemically modified ELISA plates

Abstract

A new ELISA technique using Nunc CovaLink NH microtiter plates has been developed to measure anticapsular polysaccharide specific antibodies. Capsular polysaccharide (PS) of Haemophilus influenzae type b (PRP) and pneumococcal antigens types 3, 6, 8, 14, 19, 23 were immobilized on CovaLink NH. These are modified plates with secondary amino groups bound to their surface which, in the presence of a water-soluble carbodiimide as coupling reagent, facilitate the direct binding of polysaccharides. We compared the binding characteristics of PS antigens to CovaLink NH and a conventional polystyrene ELISA plate. Checkerboard titration of PS antigens between 0.04–30 μg/ml clearly demonstrated that with Covalink NH optimal binding of a pooled serum from immunized donors was achieved for all PS antigens tested at a concentration of 1 μg/ml, while binding of PS to the conventional plate was rather poor even at concentrations of 30 μg/ml. The CVs for the ELISA ranged from 1.1 to 2.8% for intra-assay comparisons and from 3.6 to 7.3% for inter-assay comparisons. In addition, when PRP-IgG antibodies were determined with the CovaLink NH ELISA and compared with the Farr assay an acceptable correlation ( r = 0.89, p < 0.0001) was obtained. The technique described provides a simple and sensitive tool for evaluating specific immunity to PS antigens.