Antibody binding characteristics of geometrical isomers of testosterone 3-(O-carboxymethyl)oxime

Geometrical isomers of testosterone 3-(O-carboxymethyl)oxime and their hist amine derivatives were purified on reverse-phase high pressure liquid chrom atography, and their antibody binding characteristics were studied. Using a competitive testosterone enzyme immunoassay, the unfractionated mixture of the oximes showed 75% cross-reactivity with respect to testosterone, where as the isolated 3Z- and 3E-isomers showed 124% and 26% cross-reactivity, re spectively. The cross-reactivity was increased in the histamine derivatives , but the difference in cross-reactivity of the two isomers was reduced. Su ppression of the ionization of the carboxyl group by lowering the pH of the incubation mixture in the antigen-antibody binding step raised the cross-r eactivity of the mixture of free oximes to 128%, at pH 4.0. Thus, the geome try and ionization state of the carboxymethyl oxime group has a profound ef fect on the affinity of the isomers for the antibody. (C) 1999 Elsevier Sci ence Inc. All rights reserved.