Enzyme-linked immunosorbent assay for monitoring toxic dioxin congeners in milk based on a newly generated monoclonal anti-dioxin antibody.
Enzyme-linked immunosorbent assay for monitoring toxic dioxin congeners in milk based on a newly generated monoclonal anti-dioxin antibody.
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DOI：
10.1021/ac0303620
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发表时间：
2004-02
影响因子：
7.4
通讯作者：
M. Okuyama;N. Kobayashi;Wakako Takeda;T. Anjo;Y. Matsuki;J. Goto;A. Kambegawa;S. Hori
中科院分区：
文献类型：
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作者：
M. Okuyama;N. Kobayashi;Wakako Takeda;T. Anjo;Y. Matsuki;J. Goto;A. Kambegawa;S. Hori
为了开发一种酶联免疫吸附试验（ELISA），用于监测由于多氯二苯并二恶英和二苯并呋喃污染的人类母乳中的毒性，我们已经产生了新的单克隆抗体使用一些半抗原衍生物连接到牛血清白蛋白通过C-1或C-2的位置上的二恶英骨架。用免疫原反复免疫BALB/c或A/J小鼠，取脾细胞与P3/NS 1/1-Ag 4 - 1骨髓瘤细胞融合。经过五次融合实验，建立了一个分泌抗体D9 - 36组的杂交瘤克隆，该抗体特异性识别主要有毒同系物2，3，7，8-四氯二苯并-对-二恶英（2，3，7，8-TCDD）、1，2，3，7，8-五氯二苯并-对-二恶英和2，3，4，7，8-五氯二苯并呋喃。基于竞争性和标记抗原形式开发了ELISA。通过一种新型的提取盒和过氧化物酶标记的二恶英类似物从黄油或牛奶样品中提取的有毒同系物依次与固定量的D9 - 36在Triton X-100的存在下反应。将结合的级分捕获在微量滴定板上，固定第二抗体，并比色测定酶活性。该ELISA法具有实用的灵敏度（可测量范围为1 - 100 pg/次;检测限为1.0 pg/次，相当于2，3，7，8-TCDD）。牛奶和黄油样品的含量测定值与通过高分辨率气相色谱/高分辨率质谱法测定的每种同系物的毒性当量之和合理一致。
To develop an enzyme-linked immunosorbent assay (ELISA) for monitoring the toxicity due to polychlorinated dibenzo-p-dioxins and dibenzofurans contaminated in human breast milk, we have generated novel monoclonal antibodies using some haptenic derivatives linked to bovine serum albumin via the C-1 or C-2 position on the dioxin skeleton. BALB/c or A/J mice were repeatedly immunized with the immunogen, and spleen cells were fused with P3/NS1/1-Ag4-1 myeloma cells. After five fusion experiments, a hybridoma clone was established that secretes an 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-antigen format. The toxic congeners extracted from butter or milk specimens by a novel extraction cartridge and a peroxidase-labeled dioxin analogue were sequentially reacted with a fixed amount of D9-36 in the presence of Triton X-100. The bound fraction was captured on a microtiter plate, immobilizing a second antibody, and the enzyme activity was colorimetrically determined. This ELISA afforded a practical sensitivity (measurable range, 1-100 pg/assay; detection limit, 1.0 pg/assay as 2,3,7,8-TCDD equivalent). The assay values for milk and butter samples were in reasonable accordance with the sum of the toxicity-equivalent quantity of each congener, which had been determined by a high-resolution gas chromatography/high-resolution mass spectrometry method.