A calcium-binding monoclonal antibody that recognizes a non-calcium-binding epitope in the short consensus repeat units (SCRs) of complement Clr

Abstract Clr is a Ca 2+ -binding serine protease that interacts with two other plasma proteins, Clq and Cls, to form Cl, the first component of the complement cascade. A monoclonal antibody, BG6, has been produced which binds to Clr only in the presence of Ca 2+ , requiring 3–5 μM Ca 2+ for half-maximal binding. The antibody reacts with native and heat-denatured C1r, and with zymogen Clr, but does not cross-react with Cls or Clq. BG6 did not significantly affect the esterolytic activity of Clr toward a synthetic thioester substrate nor the hemolytic activity of Cl reconstituted from subcomponents in the presence of the antibody. A tryptic fragment of Clr which consists of the C-terminal γ region of the A chain disulfide-linked to the B chain (γB) binds in a Ca 2+ -dependent manner to BG6-Sepharose. Western blotting experiments have further localized the epitope to the γ region of the A chain, which is composed of two short consensus repeat (SCR) units. The N -terminal α region contains the only previously determined Ca 2+ -binding site in the Clr molecule. Equilibrium dialysis experiments confirmed that Clr-γ B does not bind Ca 2+ , and showed that antibody BG6 and the γB/BG6 complex do bind Ca 2+ . Thus, the Ca 2+ -dependent nature of this interaction is due exclusively to binding of the metal ion to the antibody. Equilibrium dialysis and immunoblotting have further localized the Ca 2+ -binding site to the Fab fragment of BG6, indicating that the metal-induced conformational change resides in or near the variable region of the IgG. BG6 may set a precedent for the preparation of Ca 2+ -dependent antibodies to non-Ca 2+ -binding epitopes in other proteins.