Developmental Biology Department Insect Gene Function Research Center, National Institute of Agrobiological Science
Yue-Jin Hua
Insect Gene Function Research Center, National Institute of Agrobiological Science Institute of Nuclear-Agricultural Science (INAS), Zhejiang University
Taneaki Tsugane
Insect Gene Function Research Center, National Institute of Agrobiological Science Chiba prefectural agriculture research center
Shirley Gee
Departments of Entomology and Environmental Toxicology, University of California
Bruce D. Hammock
Departments of Entomology and Environmental Toxicology, University of California
The practical conditions of an enzyme-linked immunosorbent assay (ELISA) for ecdysteroids were optimized. A complex of ecdysone and horseradish peroxidase (HRP) was prepared as an enzyme tracer, and a competition between the tracer and free ecdysteroids was employed for the ELISA system. The binding of rabbit antiserum against 20-hydroxyecdysone (20E) was better with the direct method than indirect method that used anti-rabbit IgG. Optimal blocking using 1% casein before the competition assay had lower background compared to no blocking or blocking using 1% BSA. The optimized method showed a determination range of 0.06 - 6ng equivalent of 20E. The system made it possible to determine ecdysteroids in both in vitro and in vivo samples. Changes in ecdysteroid titer in hemolymph of Agrius convolvuli between 4th molting and eclosion determined by the ELISA optimized in this study was similar to that reported concentrations in Manduca sexta.
