氯噻啉酶联免疫分析方法研究
Development of an enzyme-linked immunosorbent assay for the detection of imidaclothiz
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摘要: 建立了基于多克隆抗体的氯噻啉间接竞争酶联免疫分析(ic-ELISA)方法。设计合成了氯噻啉半抗原,将其分别与牛血清蛋白(BSA)和卵清蛋白(OVA)偶联制备免疫原和包被原。用免疫原免疫新西兰大白兔,制备多克隆抗体,抗体效价为2.56×106。通过对有机溶剂含量、离子强度和pH等影响因素的优化,确定了氯噻啉ic-ELISA的最佳检测条件(含体积分数10%甲醇的磷酸盐缓冲液、Na+浓度为0.14 mol/L、 pH 7.4),并建立了氯噻啉标准竞争曲线,该方法的抑制中浓度(IC50)为0.18 mg/L,最低检测限(IC10)为0.001 8 mg/L。所得多克隆抗体除与吡虫啉有较大的交叉反应外,与其他结构相似的化合物无明显交叉反应。在水、土壤和甘蓝中分别添加0.05~1 mg/kg的氯噻啉标准溶液,平均回收率为91.42% ~113.82%,相对标准偏差(RSD)为0.72% ~8.68%,符合农药残留检测要求。该ELISA方法可用于环境及农产品中氯噻啉残留检测。Abstract: A sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on polyclonal antibody for detection of imidaclothiz was developed.A hapten of imidaclothiz was synthesized and conjugated with bovine serum albumin (BSA) as the immunogen,conjugated with ovalbumin (OVA) as the coating antigen.Polyclonal antibody was obtained by immunizing New Zealand rabbits with immunogen,and the titer was 2.56×106.The inhibition standard curve of imidaclothiz was obtained under the optimised conditions .The half-maximal inhibition concentration (IC50) and the limit of detection(IC10) were 0.18 mg/L and 0.001 8 mg/L of imidaclothiz,respectively.Very low cross-reactivity was found for some structurally related compounds except imidacloprid.At different spiked levels (0.05-1 mg/kg in water,soil and cabbage) recoveries of imidaclothiz were from 91.42% -113.82%,with rlative sandard deviation(RSD) of 0.72% -8.68%.The ELISA could be a convenient tool for monitoring imidaclothiz residues in environmental and agricultural samples.
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