Optimization of an enzyme-linked immunosorbent assay for ecdysteroids

Takahiro Shiotsuki, Yue Jin Hua, Taneaki Tsugane, Shirley Gee, Bruce D. Hammock

Research output: Contribution to journalArticlepeer-review

9 Scopus citations


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 - 6 ng 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.

Original languageEnglish (US)
Pages (from-to)1-4
Number of pages4
JournalJournal of Insect Biotechnology and Sericology
Issue number1
StatePublished - Feb 2005


  • Agrius convolvuli
  • Bombyx mori
  • Ecdysteroid titer
  • Enzyme-linked immunosorbent assay (ELISA)

ASJC Scopus subject areas

  • Insect Science
  • Business, Management and Accounting(all)
  • Industrial and Manufacturing Engineering
  • Agricultural and Biological Sciences(all)
  • Polymers and Plastics


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