Epitope of 24G7 anti-bilirubin monoclonal antibody

We examined an antigenic epitope recognized by an anti-bilirubin monoclonal antibody designated 24G7 (Shimizu, S., Izumi, Y., Yamazaki, M., Shimizu, K., Yamaguchi, T., and Nakajima, H. (1988) Biochim. Biophys. Acta 967, 255-260). The reactivity of bilirubin-IX alpha, its analogues (III alpha, XIII alpha, and mesobilirubin-IX alpha) and related azo compounds, with 24G7 was compared by means of an enzyme-linked immunosorbent assay (ELISA). The order of reactivity was as follows: (a) aniline azopigment > ethyl anthranilate azopigment > sulfanilic acid azopigment, (b) bilirubin-XIII alpha > bilirubin-IX alpha > bilirubin-III alpha, (c) bilirubin-IX alpha = mesobilirubin-IX alpha. These findings indicated that the epitope is present in (a) the dipyrrolic moiety of the endovinyl (correspond to N-21 and N-22 of bilirubin), or exovinyl types (N-23 and N-24); (b) the dipyrrolic moiety of the endovinyl type, which contains (c) a methyl group (C-2) but not a vinyl group (C-3) in the dipyrrolic moiety of endovinyl type. Therefore, we concluded that the epitope was the region containing the oxo group at C-1, the methyl group at C-2, C-(4,5,6,9), and the N-21 and -22 of bilirubin-IX alpha.