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 journalArticle

9 Citations (Scopus)

Abstract

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
Volume74
Issue number1
StatePublished - Feb 2005

Fingerprint

Ecdysteroids
Immunosorbents
ecdysteroids
Assays
Enzymes
Enzyme-Linked Immunosorbent Assay
enzyme-linked immunosorbent assay
tracer techniques
Convolvulus
Agrius
rabbits
Ecdysterone
Rabbits
Ecdysone
Manduca
Molting
Casein
ecdysone
ecdysterone
Hemolymph

Keywords

  • 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

Cite this

Shiotsuki, T., Hua, Y. J., Tsugane, T., Gee, S., & Hammock, B. D. (2005). Optimization of an enzyme-linked immunosorbent assay for ecdysteroids. Journal of Insect Biotechnology and Sericology, 74(1), 1-4.

Optimization of an enzyme-linked immunosorbent assay for ecdysteroids. / Shiotsuki, Takahiro; Hua, Yue Jin; Tsugane, Taneaki; Gee, Shirley; Hammock, Bruce D.

In: Journal of Insect Biotechnology and Sericology, Vol. 74, No. 1, 02.2005, p. 1-4.

Research output: Contribution to journalArticle

Shiotsuki, T, Hua, YJ, Tsugane, T, Gee, S & Hammock, BD 2005, 'Optimization of an enzyme-linked immunosorbent assay for ecdysteroids', Journal of Insect Biotechnology and Sericology, vol. 74, no. 1, pp. 1-4.
Shiotsuki, Takahiro ; Hua, Yue Jin ; Tsugane, Taneaki ; Gee, Shirley ; Hammock, Bruce D. / Optimization of an enzyme-linked immunosorbent assay for ecdysteroids. In: Journal of Insect Biotechnology and Sericology. 2005 ; Vol. 74, No. 1. pp. 1-4.
@article{94b433ac6be64915ae3285ea1a460ba1,
title = "Optimization of an enzyme-linked immunosorbent assay for ecdysteroids",
abstract = "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.",
keywords = "Agrius convolvuli, Bombyx mori, Ecdysteroid titer, Enzyme-linked immunosorbent assay (ELISA)",
author = "Takahiro Shiotsuki and Hua, {Yue Jin} and Taneaki Tsugane and Shirley Gee and Hammock, {Bruce D.}",
year = "2005",
month = "2",
language = "English (US)",
volume = "74",
pages = "1--4",
journal = "Journal of Insect Biotechnology and Sericology",
issn = "1346-8073",
publisher = "Japanese Society of Sericultural Sciences",
number = "1",

}

TY - JOUR

T1 - Optimization of an enzyme-linked immunosorbent assay for ecdysteroids

AU - Shiotsuki, Takahiro

AU - Hua, Yue Jin

AU - Tsugane, Taneaki

AU - Gee, Shirley

AU - Hammock, Bruce D.

PY - 2005/2

Y1 - 2005/2

N2 - 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.

AB - 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.

KW - Agrius convolvuli

KW - Bombyx mori

KW - Ecdysteroid titer

KW - Enzyme-linked immunosorbent assay (ELISA)

UR - http://www.scopus.com/inward/record.url?scp=18844432174&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=18844432174&partnerID=8YFLogxK

M3 - Article

AN - SCOPUS:18844432174

VL - 74

SP - 1

EP - 4

JO - Journal of Insect Biotechnology and Sericology

JF - Journal of Insect Biotechnology and Sericology

SN - 1346-8073

IS - 1

ER -