Fluorescent probes for antibody active sites. I. Production of antibodies specific to the N-methyl-2-anilinonaphthalene-6-sulfonyl group in rabbits and some fluorescent properties of the hapten bound to the antibodies.

1. Rabbits were immunized with N-methyl-2-anilinonaphthalene-6-sulfonyl (MANS) bovine serum albumin (MANS-BSA) prepared by the reaction of bovine serum albumin with N-methyl-2-anilinonaphthalene-6-sulfonyl chloride to obtain IgG fraction containing anti-MANS antibodies. 2. Both N-methyl-2-anilinonaphthalene-6-sulfonate (MANSate) and N-methyl-2-anilinonaphthalene-6-sulfonamide (MANSamide), which are virtually non-fluorescent in aqueous solution, became strongly fluorescent, with a blue shift of about 100 nm, when they were specifically bound to the antibodies against the hapten group contained in the IgG fractions. 3. IgG fractions were obtained from five immunized rabbits at intervals after the primary immunization. The fluorescence characteristics of the hepten were carefully examined with preparations of IgG fraction differing in source and time of bleeding. One of the rabbits showed a very interesting immune response. The rabbit produced two groups of anti-MANS antibodies which were significantly different in their effect on the fluorescence properties of the hapten group. One of the groups was exclusively produced at the early stage in the immune response. The other group appeared at the late stage. It is suggested that the two groups differ as regards the structure of the hepten combining sites. 4. The above findings indicate that the MANS group can be successfully used as a hapten with useful properties as a fluorescent probe.