Characterization of antibody 444 using chromatographically purified enantiomers of juvenile hormones I, II, and III: implications for radioimmunoassays.

Optically pure (> 99.5%) enantiomers of insect juvenile hormones (JH) I, II, and III were obtained by injection of racemic mixtures onto a chiral HPLC column using hexane:2-propanol (99.5:0.5) as the mobile phase. The enantiomers of JH III were the best resolved (R = 4.26), followed by those of JH II (R = 2.29) and JH I (R = 1.47). These purified natural and unnatural enantiomers were used to further characterize an antiserum (444) developed for JH radioimmunoassays (RIAs). Based on ED50 values generated using optically pure [methyl-3H]-10R,11S-JH II as a tracer, the natural isomers of JH I, JH II, and JH III were 30, 87, and 36 times more immunoreactive, respectively, than the unnatural isomers. When compared with the racemates, the natural isomers were approximately twice as immunoreactive. In competitive displacement studies where the natural enantiomers of the three JHs were compared, immunoreactivities were in the order JH II > JH I > JH III (ED50 = 109, 198, and 300, respectively). Availability of pure natural enantiomers of JH, both as tracers and competitors, should improve the sensitivity and accuracy of JH titer determinations made by RIA and facilitate various enzyme, binding protein, and receptor studies.