Development of a Highly Sensitive Enzyme-Linked Immunosorbent Assay Based on Polyclonal Antibodies for the Detection of Polychlorinated Dibenzo-p-dioxins

Yukio Sugawara, Shirley J. Gee, James R. Sanborn, S. Douglass Gilman, Bruce D. Hammock

Research output: Contribution to journalArticle

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Abstract

The development of an enzyme-linked immunosorbent assay (ELISA) based on polyclonal antibodies for the polychlorinated dibenzo-p-dioxins is described. We previously reported the synthesis of haptens and generation of antibodies for detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Antisera were screened with seven different coating antigens (hapten-protein conjugates), including trans-3-(7,8-dichlorodibenzo-p-dioxin-2-yl)-cis-2-methylpropenoic acid (VII) and 5-(3,7,8-trichlorodibenzo-p-dioxin-2-yl)penta-trans,trans-2,4-dienoic acid (X). All inhibition screening and optimization studies were conducted using a less toxic surrogate standard for TCDD [2,3,7-trichloro-8-methyl-dibenzo-p-dioxin (TMDD; XVII)] which responded similarly to 2,3,7,8-TCDD in the EUSA. The most sensitive assay from the screening studies [coating antigen VII-BSA, 0.1 μg/mL, and antiserum 7598 (anti-X-LPH), 1:10000] was further optimized and characterized. It exhibited an IC50 value of 12 pg/well (240 pg/mL), with working range from 2 to 240 pg/well (40 to 4800 pg/mL). The influence of various physical and chemical factors (time, solvent, detergent) was investigated. The optimized assay was then used to assess cross-reactivity by congeners of halogenated dioxins and related structures. DMSO up to concentrations of 37.5% decreased the IC50 value in the assay, whereas methanol to concentrations of 30% did not lead to improved IC50 values.

Original languageEnglish (US)
Pages (from-to)1092-1099
Number of pages8
JournalAnalytical Chemistry
Volume70
Issue number6
StatePublished - Mar 15 1998

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Immunosorbents
Assays
Antibodies
Haptens
Enzymes
Immune Sera
Screening
Antigens
Coatings
Acids
Dioxins
Poisons
Dimethyl Sulfoxide
Detergents
Methanol
1,4-dioxin
dibenzo(1,4)dioxin
Proteins

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Development of a Highly Sensitive Enzyme-Linked Immunosorbent Assay Based on Polyclonal Antibodies for the Detection of Polychlorinated Dibenzo-p-dioxins. / Sugawara, Yukio; Gee, Shirley J.; Sanborn, James R.; Gilman, S. Douglass; Hammock, Bruce D.

In: Analytical Chemistry, Vol. 70, No. 6, 15.03.1998, p. 1092-1099.

Research output: Contribution to journalArticle

Sugawara, Yukio ; Gee, Shirley J. ; Sanborn, James R. ; Gilman, S. Douglass ; Hammock, Bruce D. / Development of a Highly Sensitive Enzyme-Linked Immunosorbent Assay Based on Polyclonal Antibodies for the Detection of Polychlorinated Dibenzo-p-dioxins. In: Analytical Chemistry. 1998 ; Vol. 70, No. 6. pp. 1092-1099.
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