Simplified methods for the purification, quantitation, and functional estimation of human complement C-1-inhibitor (C-1-INH) with a monoclonal anti-C-1-INH antibody.
New methods have been developed for the isolation, quantitative detection, and functional measurement of human complement C-1-inhibitor (C-1-INH). The two-step purification procedure for C-1-INH from human plasma or serum employs affinity chromatography with a monoclonal anti-C-1-INH antibody coupled to CNBr-activated Sepharose 4B followed by fractionation on a FPLC Mono Q HR 5/5 column. It yields functionally active, homogeneous C-1-INH with about 40% recovery. For quantitative estimation of C-1-INH an ELISA was performed. ELISA plates were coated with a polyclonal anti-C-1-INH antibody, serum or plasma was added and bound C-1-INH was detected with the monoclonal anti-C-1-INH antibody. The method has a sensitivity of 0.4 ng C-1-INH per assay corresponding to 20 ng/ml. For the detection of functionally active C-1-INH an ELISA was developed using C1-s-coated microtiter plates. After incubation with serum or plasma, C1-s-bound C-1-INH was monitored with the monoclonal anti-C-1-INH antibody. With this method it is possible to measure as little as 0.3 ng of functionally active C-1-INH in 20 microliter of a biological sample. All methods described in the present paper are easy to perform, rapid, sensitive, and highly reproducible.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002846:Chromatography, Affinity; D003174:Complement C1 Inactivator Proteins / Q000276:immunology / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology
D000911:Antibodies, Monoclonal; D003174:Complement C1 Inactivator Proteins; D007074:Immunoglobulin G