Development of a Cross-Reactive Monoclonal Antibody to Sulfonamide Antibiotics: Evidence for Structural Conformation-Selective Hapten Recognition

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Abstract
A unique anti-sulfonamide antibody-secreting hybridoma that cross-reacts with several sulfonamides was isolated. This was possible by using a N-sulfanilyl-4-aminobenzoic acid hapten-protein conjugate as the immunogen. Most of the antibodies that were detected in immunized mice did not recognize the free drug. However, by screening a large number of antibody-secreting hybridomas, cell lines were isolated that produced antibodies which recognized the free drug. The sensitivities of one such monoclonal antibody (MAb), referred to as Sulfa-1, for sulfanitran, sulfapyridine and sulfathiazole (expressed as IC50 values), were 1.41, 22.8, and 322 ng ml−1, respectively. Molecular modeling studies showed that the calculated minimum energy conformation of the hapten used as immunogen was different than those of the cross-reactive drugs. It was postulated that the MAb was derived from a cell line responsive to a form of the immunogen in which the structural conformation of the hapten was different than the molecular modeling calculated minimum energy conformation of the hapten. This was supported by further molecular modeling 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 energy 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 temperature of the immunized mouse. Design of haptens for the purpose of generating cross-reactive antibodies should not just consider the two-dimensional structure, but also the three-dimensional conformation as well as the various structural combinations that can be easily attained within the body temperature of the immunized animal.