五氯硝基苯残留酶联免疫吸附分析研究
摘要
本文通过合成具有五氯硝基苯分子结构特征的半抗原，采用活性酯法将半抗原与牛血清白蛋白(BSA)或卵清蛋白(OVA)进行偶联，分别制备了免疫原和包被抗原。免疫原免疫新西兰大耳白兔，获得五氯硝基苯的抗血清，ELISA法测的其效价为1∶1.2×10~6，用多克隆抗体建立了五氯硝基苯的间接ELISA法。间接ELISA法五氯硝基苯的抑制中浓度IC_(50)为76.7 ng／mL，五氯硝基苯的检测限(IC_(10))为4.7ng／mL。多克隆抗体与其结构类似物的交叉反应为五氯酚：3.23％，六氯苯：12.22％，2，4-D：＜2％，标准曲线的线形范围为10-2000 ng／mL。研究了间接ELISA法的影响因素(pH值、离子强度、有机溶剂)：pH值为中性的情况对ELISA的影响很小，5％的乙腈对ELISA基本上没有影响。初步建立了水样(井水、河水)和土壤样品中五氯硝基苯间接ELISA法的提取方法，回收率分别是86.7％、81.3％和78.9％。对实际样品进行了检测，并与气相色谱进行比较，二者检测结果无显著性差异。如果进一步研究开发试剂盒，可适用于现场大量样品的快速检测。
The specifc polyclonal antibodies(PcAbs) for pentachloronitrobenzene were produced in this study. The hapten keeping the molecule structure of pentachloronitrobenzene was synthesized and the structure was identified.Then the NHS esterification was employed to conjugate hapten to large carrier protein-BSA or OVA.immunogen and coating conjugate have been synthesized.The immunogen was injected into New Zealand white rabbits,anti-serum had been obtained.The titer of antisera was 1:1.2×106 determined by indirect ELISA.For indirect ELISA.the IC50 of pentachloronitrobenzene is 76.7 ng/mL and the detection limit(IC10) is 4.7ng/mL.The cross reactivities of PcAbs with pentachlorophenol .hexachlorobenzene and 2,4-D are 3.23%,12.22% and <2% respectively.The influence of ionic strength,pH,organic solvent on indirect ELISA were tested in this paper.The extraction of water samples(well,lake),soil samples on indirect ELISA for pentachloronitrobenzene were established, the recoveries were 86.7%,81.3% and 78.9%,respectively.The result from the analysis of soil samples by ELISA was similar with by GC.If the ELISA test kit for pentachloronitrobenzene is developed,it will be used to screen large number of samples on the spot.
The specifc polyclonal antibodies(PcAbs) for pentachloronitrobenzene were produced in this study. The hapten keeping the molecule structure of pentachloronitrobenzene was synthesized and the structure was identified.Then the NHS esterification was employed to conjugate hapten to large carrier protein-BSA or OVA.immunogen and coating conjugate have been synthesized.The immunogen was injected into New Zealand white rabbits,anti-serum had been obtained.The titer of antisera was 1:1.2×106 determined by indirect ELISA.For indirect ELISA.the IC50 of pentachloronitrobenzene is 76.7 ng/mL and the detection limit(IC10) is 4.7ng/mL.The cross reactivities of PcAbs with pentachlorophenol .hexachlorobenzene and 2,4-D are 3.23%,12.22% and <2% respectively.The influence of ionic strength,pH,organic solvent on indirect ELISA were tested in this paper.The extraction of water samples(well,lake),soil samples on indirect ELISA for pentachloronitrobenzene were established, the recoveries were 86.7%,81.3% and 78.9%,respectively.The result from the analysis of soil samples by ELISA was similar with by GC.If the ELISA test kit for pentachloronitrobenzene is developed,it will be used to screen large number of samples on the spot.
引文
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Diflubenzuron and BAY SIR8514.J Agric Food Chen,1982,30:949-957
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