CHARACTERIZATION OF ANTIBODY-444 USING CHROMATOGRAPHICALLY PURIFIED ENANTIOMERS OF JUVENILE HORMONE-I, HORMONE-II, AND HORMONE-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 ch iral HPLC column using hexane:2-propanol (99.5:0.5) as the mobile phas e. The enantiomers of JH III were the best resolved (R = 4.26), follow ed by those of JH II (R = 2.29) and JH I (R = 1.47). These purified na tural and unnatural enantiomers were used to further characterize an a ntiserum (444) developed for JH radioimmunoassays (RIAs). Based on ED5 0 values generated using optically pure [methyl-H-3]-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 iso mers. When compared with the racemates, the natural isomers were appro ximately twice as immunoreactive. In competitive displacement studies where the natural enantiomers of the three JHs were compared, immunore activities were in the order JH II > JH I > JH III (ED50 = 109, 198, a nd 300, respectively). Availability of pure natural enantiomers of JH, both as tracers and competitors, should improve the sensitivity and a ccuracy of JH titer determinations made by RIA and facilitate various enzyme, binding protein, and receptor studies. (C) 1997 Academic Press .