A NEOEPITOPE-BASED ENZYME-IMMUNOASSAY FOR QUANTIFICATION OF C1-INHIBITOR IN COMPLEX WITH C1R AND C1S

Monoclonal antibodies (MoAb) recognizing neoepitopes exposed on activa tion products of complement proteins but hidden in the native componen ts have been used for quantification of activated complement. A previo usly produced and characterized mouse MoAb, recognizing a neoepitope o n the human plasma protein C1-inhibitor complexed with its substrates, was used to design an enzyme immunoassay for detection of C1-inhibito r complexed with C1r and C1s. These complexes are indicators of early classical complement pathway activation. The standard was serum activa ted with heat aggregated IgG defined to contain 1000 arbitrary units ( AU)/ml. The lower detection limit was approximate to 0.05 AU/ml corres ponding to 0.005% of fully activated serum. The reliability of the ass ay, including day-to-day variation, was tested. Intra-assay variation coefficients were 12% for low plasma control and 13% for high plasma c ontrol (n=12 for both). Inter-assay variation coefficients were 12% fo r low control (n=6), 19% for high control (n=6) and 15% for the normal plasma control (n=9). A 2.5-97.5 percentile reference range (normal b lood donors) was 16-33 AU/ml. Two patients with systemic lupus erythem atosus had considerably elevated plasma levels of the activation produ ct (56 and 62 AU/ml), and six patients with hereditary angioedema had normal plasma levels despite considerably reduced C1-inhibitor concent ration. We conclude that the present method is sensitive and reliable for detection of early classical pathway activation and superior to pr eviously published methods by utilizing neoepitope specificity and non -radiolabelled reagents.