Monoclonal antibodies to complement components without the need of their prior purification. II. Antibodies to mouse C3 and C4.

Rat monoclonal antibodies (MAbs) to mouse complement components C3 and C4 were produced by immunizing rats with cell-bound C3 and C4. This principle involves: a) using mouse thymocytes coated with syngeneic rat antibody isotypes that show high affinity to C1q, b) the intercalation of C1q from serum and c) the subsequent activation of the classical complement pathway leading to deposition of cell-bound complement components. Screening for anti-complement antibodies was performed on antibody coating microtiter plates with mouse serum as source of complement. The reactivity of the MAbs was determined by variations of the ELISA screening system using EDTA-serum to inhibit complement activation by C1 dissociation, serum rendered deficient of functionally active C3 by treatment with cobra venom factor (CVF) or serum of genetically C5-deficient mice. The specificity of the MAbs was confirmed by affinity chromatography followed by SDS-PAGE and immunoblotting. We were able to establish a panel of anti-C3 and anti-C4 MAbs of various isotypes