【作者】 陈秀金；

【导师】 胥传来；

【作者基本信息】 江南大学 ， 食品营养与安全， 2013， 博士

【摘要】 拟除虫菊酯作为一类重要的杀虫剂，广泛地用于农业和卫生业。毒理学研究表明，拟除虫菊酯对环境和人类具有潜在危害，故迫切需要建立拟除虫菊酯的各种检测技术。免疫分析法用于检测拟除虫菊酯具有快速、高通量、多残留和现场检测的优势，因此，现已发展成为拟除虫菊酯残留检测的研究热点之一。本文设计了6种拟除虫菊酯的群选性半抗原，借助计算机软件Discovery Studio2.5和Gaussian04对半抗原和菊酯的结构进行空间模拟和原子电荷计算，选择与菊酯空间结构和电荷分布最匹配的半抗原1为免疫半抗原，选择二者差异较大的为包被半抗原。根据模拟和计算结果，选择并合成了半抗原1，3，4，5。此外，本文还设计并合成了针对氯氟氰菊酯的2种特异性半抗原。将合成的半抗原与载体蛋白（血蓝蛋白、牛血清白蛋白和卵清蛋白）偶联，采用紫外法鉴定后，免疫BALB/c小鼠，四免后，测定抗血清的效价和抑制，选择效价高并且抑制好的小鼠进行融合。通过细胞融合和筛选，获得拟除虫菊酯的群选性单克隆抗体和抗氯氟氰菊酯的特异性单克隆抗体。测得拟除虫菊酯群选性单克隆抗体的亲和常数为3.0108L/mol，抗体重链和轻链的亚型分别为IgG1和Kappa链。测得氯氟氰菊酯特异性单克隆抗体的亲和常数为2.8106L/mol，抗体重链和轻链的亚型分别为IgG2b和Kappa链。试验优化了包被原的种类和浓度、缓冲液的离子强度、pH、有机溶剂的种类和含量。利用酶联免疫分析法在最适条件下，测得抗体交叉反应较高六种菊酯的IC50分别为：氯氰菊酯1.660.76ng/mL、甲氰菊酯14.031.68ng/mL、顺氰戊菊酯45.764.07ng/mL、联苯菊酯191.811.2ng/mL、溴氰菊酯199.610.75ng/mL、氰戊菊酯298.515.08ng/mL。该法用于水样的添加回收，三种菊酯（氯氰菊酯、甲氰菊酯和顺氰戊菊酯）平均回收率在77.3%-111.3%之间，变异系数小于15%。对影响Au体系稳定性和试纸条灵敏度的因素（pH、抗体用量及包被原浓度）进行了优化，接着将菊酯群选性抗体标记到金纳米粒子上，通过组装，制备了检测拟除虫菊酯多残留的金标免疫层析试纸条。该试纸条借助便携式的读卡器，对氯氰菊酯的检测限达到了12.86ng/mL；该试纸条对氯氰菊酯、甲氰菊酯和顺氰戊菊酯的肉眼检测限分别为60ng/mL、200ng/mL和400ng/mL。在优化的色谱条件下，利用高效硅胶柱和Sino-Chiral OD手性柱经过二次拆分制备了8个氯氰菊酯单体、4个氰戊菊酯单体和2个甲氰菊酯单体。此外，利用Sino-Chiral OD手性柱并结合文献，确定了单体的构型和纯度。通过测定氯氰菊酯、氰戊菊酯和甲氰菊酯共14个单体的抑制曲线来研究菊酯群选性单克隆抗体的立体选择性。结果表明，抗体只能识别氯氰菊酯的单体6（CP6,1S3S S）和单体7（CP7,1S3S R）、甲氰的单体2（FP2, S型）以及氰戊菊酯的单体1（FV1, S S），而且CP7比CP6灵敏度要高很多。基于菊酯群选性单克隆抗体交叉反应的结果，构建了三种定量构效模型（QSAR）即二维定量构效模型（2D-QSAR）、全息定量构效模型(HQSAR)和虚拟定量构效模型（topomer QSAR）。由2D-QSAR模型得出，抗体活性和菊酯的疏水性有很大的相关性，而且疏水性越小，越容易被抗体识别。HQSAR模型从亚结构水平分析得出，在免疫反应中暴露比较少的片断，对抗体活性也有一定的影响。2D-QSAR模型和HQSAR模型的交叉验证系数q2=0.92，表明这两个模型具有很好的预测能力。构建topomer QSAR模型通过计算分子特定片段（R2）的空间场和静电场的等高势，来解释拟除虫菊酯群选性抗体的交叉反应。优化了包被原的种类和浓度、封闭剂的种类、缓冲液的离子强度、pH和甲醇含量。在最适条件下，采用酶联免疫法测得单克隆抗体对氯氟氰菊酯的IC50为13.261.23ng/mL，检测限为1.83ng/mL。测得抗体只对氟胺氰菊酯和苯醚氰菊酯有低于5%的交叉反应。三种水样（河水、自来水和饮用水）的添加回收率均高于75%，变异系数小于12%。更多还原

【Abstract】 As an important pesticide, synthetic pyrethroids are widely applied in agriculture andhygiene industry. Many toxicological studies indicate that synthetic pyrethroids are potentiallyharmful to environment and human health. Therefore, it is highly necessary to establishvarious detection techniques of synthetic pyrethroids. Immunoassays for pyrethroiddetermination have many advantages including rapid, high-throughput, multi-residues andon-site detection. Therefore, immunoassay has attracted increasing attention in the field ofsynthetic pyrethroid detection.In this paper, six general haptens for synthetic pyrethroids were designed. ThenDiscovery Studio2.5and Gaussian04software were empolyed to evaluate the theoreticalgeometries and electronic distributions of pyrethroids and haptens. Hapten1that had matchedgeometries and electronic properties with the analytes was chosen as an immunizing hapten.Other haptens were selected as coating haptens. In addition, two specific haptens forcyhalothrin were designed and synthesized in the present study.All haptens were conjugated with various carrier proteins containing keyhole limpethemocyanin, bovine serum albumin and ovalbumin to prepare antigens. Then these conjugateswere characterized with a UV-visible spectrometer and used to inject BALB/c mice. Ten daysafter the last immunization, the titre and inhibition of antisera were tested by indirectcompetitive enzyme-linked immunosorbent assay. Finally, the mouse producing antisera withthe highest titer and the lowest50%inhibition concentration was sacrificed for monoclonalantibody preparation. The process of cell fusion and screening referred to the classic measure.The chosen hybridomas which could steadily secrete anti-pyrethroids (or anti-cyhalothrin)antibody were obtained successfully and ascites were produced using in vivo induction. Theaffinity constant of broad-specificity monoclonal antibody3E9against pyrethroids wascalculated to be3.0108L/mol. The isotype of antibody3E9based on the commercial kitindicated that the heavy chain belongs to IgG1while the light chain is Kappa. The affinityconstant of monoclonal antibody2C8against cyhalothrin was calculated to be2.8106L/mol.The subtype of antibody2C8was characterized that the heavy chain is IgG2bwhile the lightchain is Kappa.Several important parameters that influenced assay performance were optimizedincluding coating antigens and their concentration, ionic strength, pH, organic solvents andtheir contents in assay buffer. Under the optimized conditions, six pyrethroids were highercross-reactivity with ascites3E9and their inhibition curves were plotted with IC50value of1.660.76ng/mL for cypermethrin,14.031.68ng/mL for fenpropathrin,45.764.07ng/mLfor esfenvalerate,191.811.2ng/mL for bifenthrin,199.610.75ng/mL for deltamethrin,298.515.08ng/mL for fenvalerate. In the spiked recoveries, the average recoveries were77.3%-111.3%for the fortified samples. The coefficient of variation values were less than15%.Several key factors that affected system stability and the sensitivity of test strip wereoptimised such as pH, antibody amount and the concentration of coating antigen. Based on the obtained anti-pyrethroids monoclonal antibody, the immunochromatographic test strip formulti-pyrethroids analysis was prepared using gold nanoparticals as a detector probe, and thetest strip for cypermethrin is with the detection limit of12.86ng/mL by aid of the portablereader, And the detection limits are of60ng/mL for cypermethrin,200ng/mL forfenpropathrin and400ng/mL for esfenvalerate with naked eyes.Under the optimized chromatographic conditions, the diastereomers of cypermethrin andfenvalerate have firstly been resolved on an HP silica column, and then injection of separateddiastereomers onto Sino-Chiral OD column were ultimately separated completely withreasonable retention times (60min). The order of elution was established and eachconfiguration was identified by comparison with the related literatures. Under the sameconditions, injection of fenpropathrin onto the Sino-Chiral OD chiral system achievedexcellent separation of the two enantiomers. The stereoselectivity of antibody3E9wasdetermined by comparing standard inhibition curves of fourteen isomers from cypermethrin,fenvalerate and fenpropathrin. Results demonstrated that only two of cypermethrin isomer6(CP6,1S3S S) and isomer7(CP7,1S3S R), one of fenpropathrin isomer2(FP2, S) andfenvalerate isomer1(FV1, S S) were detected, respectively and the general monoclonalantibody aganist CP7was higher sensitive than CP6.Based on cross-reactivity results of anti-pyrethroid monoclonal antibody, threequantitative structure-activity relationship (QSAR) models were constructed including2D-QSAR (two dimensional QSAR) model, HQSAR (hologram QSAR) model and topomerQSAR model. According to the2D-QSAR model, there was a significant correlation betweenantibody activity and pyrethroid hydrophobicity, specifically, synthetic pyrethroids could berecognized more easily when the hydrophobicity was weaker. From the substructure levelanalysis using HQSAR model, fragments of hapten linked with carrier protein could play animportant role on antibody recognition. Both of models showed highly predictive abilitieswith cross-validation coefficient q2values of0.92, respectively. According to topomer QSARmodel, the cross-reactivity of antibody3E9can be explained by calculating the steric andelectrostatic contours of fragments R2.To establish the enzyme-linked immunosorbent assay for cyhalothrin, coating antigensand their concentrations, blocking agent, ionic strength, pH value and the content of organicsolvent in assay buffer were optimized. Under the optimized conditions, an inhibitionstandard curve for cyhalothrin was plotted with the IC50value13.261.23ng/mL and with thedetection limit of1.83ng/mL. The monoclonal antibody2C8manifested good specificity tocyhalothrin with little cross-reactivity (<5%) only to tau-fluvalinate or cyphenothrin. Therecoveries were more than75%and the coefficient of variation value was less than12%.更多还原