Monoclonal antibodies against progesterone: effect of steroid-carrier coupling position on antibody specificity.

Monoclonal anti-progesterone antibodies were raised by immunizing mice with progesterone coupled through either the C3, C6 or C11 positions to protein carrier (bovine serum albumin, BSA). The specificity of four antibodies for a range of steroids related to progesterone, some carrying substitutions at various ring positions, was studied by competitive inhibition in an ELISA system. The results demonstrated that the ring coupling position has a determining effect on the cross-reactivity of the antibodies obtained. The patterns of cross-reaction were interpreted in the light of the structure of the combining site of an anti-progesterone antibody (DB3) recently determined by X-ray crystallography, and inferences drawn about the orientation of steroid in the combining sites of the antibodies studied. Specifically, in two antibodies raised against progesterone-11-BSA, the orientation of steroid resembled that of the progesterone-DB3 complex, with positions C11 and C3 exposed and C6 and C20 buried; an antibody raised against progesterone-6-BSA bound steroid in an apparently similar disposition, except that C6 was exposed and C11 buried; finally, in an antibody raised against progesterone-3-BSA, all steroid positions other than C3 were apparently buried in the steroid-antibody complex.