Development of a cross-reactive monoclonal antibody to sulfonamide antibiotics: Evidence for structural conformation-selective hapten recognition

A unique anti-sulfonamide antibody-secreting hybridoma that cross-reacts wi th several sulfonamides was isolated This was possible by using a N-sulfani lyl-4-aminobenzoic acid hapten-protein conjugate as the immunogen. Most of the antibodies that were detected in immunized mice did not recognize the f ree drug. However by screening a large number of antibody-secreting hybrido mas, cell lines were isolated that produced antibodies which recognized the free drug. The sensitivities of one such monoclonal antibody (MAb), referr ed to as Sulfa-1, for sulfanitran, sulfapyridine and sulfathiazole (express ed as IC50 values), were 1.41, 22.8, and 322 ng ml(-1), respectively. Molec ular modeling studies showed that the calculated minimum energy conformatio n of the hapten used as immunogen was different than those of the cross-rea ctive drugs. It was postulated that the MAb was derived from a cell line re sponsive to a form of the immunogen in which the structural conformation of the hapten was different than the molecular modeling calculated minimum en ergy conformation of the hapten. This was supported by further molecular mo deling studies, including the use of potential energy-conformational maps, and competitive ELISA experiments conducted at two different temperatures. The immunogen appeared to be in a structural conformation achieved at an en ergy of + 0.193 kcal mol(-1) with an energy barrier of 3.13 kcal above the minimum energy conformation; an energy easily found within the body tempera ture of the immunized mouse. Design of haptens for the purpose of generatin g cross-reactive antibodies should not just consider the two-dimensional st ructure, but also the three-dimensional conformation as well as the various structural combinations that can be easily attained within the body temper ature of the immunized animal.