生产单克隆抗体及其在间接竞争 ELISA 检测海产品中 okadaic 酸和麻痹性贝类毒素-1 中的应用。
Production of monoclonal antibody and application in indirect competitive ELISA for detecting okadaic acid and dinophytoxin-1 in seafood.
机构信息
Key Laboratory for Zoonosis Research, Ministry of Education, Institute of Zoonosis, Jilin University, Changchun, 130062, People's Republic of China.
出版信息
Environ Sci Pollut Res Int. 2011 Aug;19(7):2619-26. doi: 10.1007/s11356-012-0819-y. Epub 2012 Jul 21.
BACKGROUND, AIM, AND SCOPE: Okadaic acid (OA) and analogues of dinophysistoxin (DTX) are key diarrheic shellfish poisoning (DSP) toxins, which possibly arouse DSP symptoms by consuming the contaminated shellfish. Because of the stable toxicity in high temperature and the long-term carcinogenicity, the outbreaks of DSP related to consumption of bivalve mollusks contaminated by DSP toxins pose a hazard to public health. Therefore, it is worth developing a fast and reliable analytical method for the detection of OA and analogues in shellfish. In this paper, an indirect competitive enzyme-linked immunosorbent assay (ELISA) (icELISA) for detecting OA and DTX-1 in seafood was developed based on monoclonal antibody (McAb).
METHODS
The OA was conjugated to human immunoglobulin G (IgG) and bovine serum albumin (BSA) by the active ester method as the immune antigen and the detective antigen. The spleen cells from BALB/c mice immunized with OA-IgG were fused with SP2/0 myeloma cells. A hybridoma cell line, which secreted McAb against OA, was selected by "limiting dilution" cloning. An icELISA was developed based on immobilized conjugate (OA-BSA) competing the McAb with the free OA in seafood sample.
RESULTS
A hybridoma cell line, which secreted IgG1 subclass monoclonal antibody (McAb) against OA, was selected. The IC(50) of the McAb for OA and dinophytoxin-1 (DTX-1) were 4.40 and 3.89 ng/mL, respectively. Based on the McAb, an indirect competitive ELISA for detection of OA and DTX-1 in seafood was developed. The regression equation was y = 54.713x - 25.879 with a coefficient correlation of R (2) = 0.9729. The linear range and the limit of detection were 0.4-12.5 and 0.45 ng/mL, respectively. The average recovery of OA and DTX-1 spiked shellfish was 82.29% with the coefficient of variation of 7.67%.
CONCLUSION
The developed icELISA is a fast, sensitive, and convenient assay for detecting of total amount of OA and DTX-1 in seafood.
背景、目的和范围：冈田酸（OA）和麻痹性贝类毒素（PSP）类似物是关键的腹泻性贝类毒素（DSP）毒素，通过食用受污染的贝类可能会引起 DSP 症状。由于其在高温下稳定的毒性和长期的致癌性，与食用受 DSP 毒素污染的双壳贝类相关的 DSP 爆发对公众健康构成了危害。因此，开发一种快速可靠的贝类中 OA 和类似物检测分析方法是值得的。本文基于单克隆抗体（McAb）建立了一种间接竞争酶联免疫吸附试验（icELISA），用于检测贝类中的 OA 和 DTX-1。
方法
采用活泼酯法将 OA 与人免疫球蛋白 G（IgG）和牛血清白蛋白（BSA）偶联，作为免疫原和检测原。用 OA-IgG 免疫的 BALB/c 小鼠脾细胞与 SP2/0 骨髓瘤细胞融合，通过“有限稀释”克隆法筛选出分泌针对 OA 的 McAb 的杂交瘤细胞系。基于固定化结合物（OA-BSA）与贝类样品中游离 OA 竞争 McAb，建立了 icELISA。
结果
筛选出分泌针对 OA 的 IgG1 亚类 McAb 的杂交瘤细胞系。该 McAb 对 OA 和戴文毒素-1（DTX-1）的 IC50 分别为 4.40 和 3.89ng/mL。基于 McAb，建立了一种检测贝类中 OA 和 DTX-1 的间接竞争 ELISA。回归方程为 y=54.713x-25.879，相关系数 R2=0.9729。线性范围和检测限分别为 0.4-12.5ng/mL 和 0.45ng/mL。OA 和 DTX-1 加标贝类的平均回收率为 82.29%，变异系数为 7.67%。
结论
所建立的 icELISA 是一种快速、灵敏、方便的检测贝类中 OA 和 DTX-1 总量的方法。
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