Monoclonal antibody has been obtained to the human complement control protein C3b inactivator after immunization of mice with the enzyme prepared by conventional methods. Antibody from ascitic fluid was purified and coupled to Sepharose-CL-4B to give a specific affinity column, which was used to isolate C3b inactivator from human serum in 70% yield. The product was characterized by size, chain structure, amino acid analysis and proteolytic activity.

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The light chain and heavy chain of reduced and alkylated human complement Factor I were purified by high-pressure gel-permeation chromatography. CNBr cleavage of Factor I light chain yielded four major fragments, which were purified by gel filtration. N-Terminal sequence analysis of the CNBr-cleavage fragments allowed identification of 200 of the approx. 240 amino acid residues of the light chain. An alignment is proposed, based on sequence analysis of peptides obtained after cleavage at arginine residues of the light chain and on homology of the sequence determined with that of other serine proteinases. The sequence around the active-site serine residue was established and three potential attachment sites for carbohydrate moieties were identified.

A monoclonal antibody (SB-4) to human Clq was prepared. The equilibrium constant of the antibody for CI q was found to be greater than 10 ~° M -1. It has been shown that the antibody binds to the A-B chain dimer, probably via the B chain of Clq. Pepsin digestion of Clq at pH 4.5, which fragments the globular regions but leaves the collagenous region intact, allowed the demonstration that the antigenic site is located in the collagenous region of the molecule. The effect of the antibody on haemolytic activity has shown that it is capable of inhibiting the formation of EAC1 cells from EAClq cells plus Clr and Cls but is incapable of inhibiting the CI activity of preformed EAC1 cells. This indicates that the binding of the antibody to the collagenous portion of the B chain of Clq probably prevents interaction between Clq and the Clr2-Cls z complex.

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