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目的建立间接竞争酶联免疫吸附分析(ELISA)方法测定海水样品和海洋贝类中赤潮毒素记忆缺失性贝毒主要成分软骨藻酸(DA)。方法采用碳二亚胺法,将半抗原DA分别与载体蛋白卵清蛋白(OVA)和牛血清白蛋白(BSA)偶联,得到包被抗原和免疫抗原。以DA-BSA做为免疫抗原,免疫BALB/c小鼠,制备多克隆抗体,建立间接竞争ELISA方法分析检测海水样品和海洋贝类中的软骨藻酸。结果最低检出限为10.0ng/ml(对于贝相当于4μg/g);海水样品回收率为83.2%～124.7%,批内变异系数在4.7%～5.9%之间;贝类样品回收率为85.9%～99.9%,批内变异系数2.4%～7.1%之间。结论建立的ELISA方法检测海洋贝类中DA可以满足国际规定的安全限值。
Objective To establish an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the determination of domoic acid (DA), the main component of the memory-deficient shellfish poisoning in red tide toxin in marine samples and marine shellfish. Methods The carbodiimide method was used to couple the hapten DA with carrier protein ovalbumin (OVA) and bovine serum albumin (BSA), respectively, to obtain coated antigen and immune antigen. BALB / c mice were immunized with DA-BSA as an immunogen and polyclonal antibodies were prepared. An indirect competitive ELISA was developed to detect domoic acid in seawater samples and marine shellfish. Results The detection limit was 10.0 ng / ml (equivalent to 4 μg / g for shellfish), the recoveries of seawater samples ranged from 83.2% to 124.7%, and the intra-assay CVs ranged from 4.7% to 5.9%. The recovery of shellfish samples was 85.9% ~ 99.9%, within the variation coefficient of 2.4% ~ 7.1%. Conclusion The established ELISA method for the detection of DA in marine shellfish can meet the international safety limits.