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A Competitive Immunoassay for the Determination of the Herbicide Fluazifop in Drinking Water and Groundwater A competitive solid-phase enzyme immunoassay using rabbit polyclonal antibodies was developed for the detection of the herbicide fluazifop [(RS)-2-[4-(5-trifluoromethyl-2-pyridyloxy)phenoxy]propionic acid] in drinking water and groundwater. Present regulatory limits for drinking water in Germany were taken as the critical level. The carrier protein was bovine serum albumin; horseradish peroxidase was used as marker enzyme with 3,3′,5,5′-tetramethylbenzidine as substrate. A high concentration of high-affinity antibodies in the serum, optimization of test conditions (antibody and enzyme tracer concentration, incubation time etc.), and very low cross reactivities to substances of similar structures led to a highly sensitive and specific ELISA with a detection limit below 0.1 μg/L for fluazifop as free acid. On testing the suitability of the assay's use as a screening test with one hundred drinking-water samples, the three samples which had been spiked in the laboratory were recognized as positive with respect to their fluazifop content. Confirmation by gas chromatography-mass spectrometry showed the test results of two other samples to be false positive. False negative results did not appear. The concentration was in the detection limit region of 0.1 μg/L. 相似文献
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从cDNA文库中获得了梅氏新贝尼登虫卵黄铁蛋白(NmYF)的编码序列。序列全长739个核苷酸,3′-末端具有polyA尾,单一大开放阅读框编码一个由226个氨基酸组成的分子量为26.1kDa的蛋白。序列比较表明,NmYF与卫氏并殖吸虫卵黄铁蛋白最相似,氨基酸序列同源性为23.7%。系统进化树分析表明,NmYF、卫氏并殖吸虫卵黄铁蛋白和头槽绦虫卵黄铁蛋白形成了一个小的进化簇,而体铁蛋白序列形成了一个大的进化簇,揭示了卵黄铁蛋白和体铁蛋白间的差异以及进化上的相关性。这是梅氏新贝尼登虫卵黄铁蛋白基因序列的首次报道。随后,构建了插入有全长NmYF基因开放阅读框序列的原核表达质粒pET-22b-NmYF,转化大肠杆菌BL21pLysE菌株,经IPTG诱导表达后,SDS-PAGE检测表明预期大小的目的蛋白大量表达。随后制备了目的蛋白的多克隆抗血清,它能与目的蛋白起强的特异性反应,但与细菌自身蛋白不起反应,可用于后续研究。 相似文献
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大黄鱼(Pseudosciaena crocea)热激蛋白的基因克隆、原核表达及抗血清的制备 总被引：1，自引：1，他引：1
应用trizol裂解法提取象山港网箱养殖大黄鱼肝脏总RNA,采用RTPCR获得大黄鱼cDNA;通过设计特异引物进行PCR扩增,将PCR产物转化到质粒并直接测序,得到1.7kb的目的基因;同时将此目的基因连接到表达质粒pSBET中,在大肠杆菌BL21中进行诱导表达以及表达产物的分析。克隆基因的表达产物经SDSPAGE分析表明,HSP基因的蛋白分子量为60kDa左右,与阅读框的编码大小一致;表达蛋白经纯化后免疫小鼠制备抗血清。Westernblotting检测结果表明,制备的抗血清与HSP蛋白起较强的特异性反应。HSP基因的系统进化分析表明,大黄鱼与非洲爪蟾最为接近,从侧面印证了脊椎动物中两栖纲动物与鱼纲动物的亲源关系。通过大黄鱼HSP基因的提取和分析,可为以后制备出核酸探针、筛选和克隆出一批具有优良性状的基因、构建基因文库等研究工作奠定基础。 相似文献
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