Enzyme-Linked Immunosorbent Assay for Monitoring Toxic Dioxin Congeners in Milk Based on a Newly Generated Monoclonal Anti-Dioxin Antibody

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• 作者：Mitsunobu Okuyama ; Norihiro Kobayashi ; Wakako Takeda ; Takako Anjo ; Yasuhiko Matsuki ; Junichi Goto ; Akira Kambegawa ; and Shinjiro Hori
• 刊名：Analytical Chemistry
• 出版年：2004
• 出版时间：April 1, 2004
• 年：2004
• 卷：76
• 期：7
• 页码：1948 - 1956
• 全文大小：224K
• 年卷期：v.76,no.7(April 1, 2004)
• ISSN：1520-6882

文摘

To develop an enzyme-linked immunosorbent assay(ELISA) for monitoring the toxicity due to polychlorinateddibenzo-p-dioxins and dibenzofurans contaminated inhuman breast milk, we have generated novel monoclonalantibodies using some haptenic derivatives linked to bovineserum albumin via the C-1 or C-2 position on the dioxinskeleton. BALB/c or A/J mice were repeatedly immunizedwith the immunogen, and spleen cells were fused withP3/NS1/1-Ag4-1 myeloma cells. After five fusion experiments, a hybridoma clone was established that secretesan antibody D9-36 group specifically recognizing the major toxic congeners, 2,3,7,8-tetrachlorodibenzo-p-dioxin(2,3,7,8-TCDD), 1,2,3,7,8-pentachlorodibenzo-p-dioxin,and 2,3,4,7,8-pentachlorodibenzofran. An ELISA is developed on the basis of the competitive and labeled-antigenformat. The toxic congeners extracted from butter or milkspecimens by a novel extraction cartridge and a peroxidase-labeled dioxin analogue were sequentially reactedwith a fixed amount of D9-36 in the presence of TritonX-100. The bound fraction was captured on a microtiterplate, immobilizing a second antibody, and the enzymeactivity was colorimetrically determined. This ELISAafforded a practical sensitivity (measurable range, 1-100pg/assay; detection limit, 1.0 pg/assay as 2,3,7,8-TCDDequivalent). The assay values for milk and butter sampleswere in reasonable accordance with the sum of thetoxicity-equivalent quantity of each congener, which hadbeen determined by a high-resolution gas chromatography/high-resolution mass spectrometry method.