A C3 convertase assay for nephritic factor functional activity

C3 nephritic factor (C3NeF) is an autoantibody against the C3 convertase wh ich stabilizes this otherwise inherently labile neoenzyme and induces a con tinuous activation of the alternative pathway with C3 depletion. NeF is fou nd in patients with membranoproliferative glomerulonephritis and/or partial lipodystrpohy. NeF activity is usually detected in plasma by hemolytic tes ts. In order to obtain reproducible data for the functional activity of pur ified C3NeF IgG a solid phase assay was developed. C3 convertase was genera ted on immobilized C3b by incubation with factors B and D in the presence o f Ni2+. Convertase sites were left to decay in the presence of normal IgG o r NeF IgG. Residual convertase activity was measured by adding I-125-C3 and capturing nascent I-125-C3b on the plate surface via covalently coupled NH 2-Glu-Tyr dipeptide, In the presence of factor H during C3 convertase decay , a dose dependent stabilizing activity was shown for NeF IgG including NeF IgG purified from urine. A second format of the assay was developed in whi ch C3 convertase was assembled on C3b(2)-IgG complexes in the presence of M g2+. Since these complexes are more efficient as convertase precursors the signal was five-fold higher than with C3b. Convertase decay, on the other h and, was not influenced by the nature of the precursor and in both systems the stabilizing activity of NeF IgG was similar. (C) 2001 Elsevier Science B.V. All rights reserved.