【作者】 王姝婷；

【导师】 朱国念； 蔡磊明；

【作者基本信息】 浙江大学 ， 植物保护， 2008， 博士

【摘要】 本研究首先合成了四种农药的半抗原:三唑磷半抗原THHe、克百威半抗原BFNB、毒死蜱半抗原CHBu以及甲基对硫磷半抗原MP5。为制备出能够识别多种农药的“宽谱”特异性抗体,在人工抗原的制备上采用了一种全新的思路:采用碳二亚胺法,在同一载体蛋白上,依次偶联了4种不同的半抗原,在国内外首次制备得到了含有多种抗原决定簇的农药“多簇”人工抗原。通过将多簇人工抗原单独或混合免疫的方式获得了对应能够同时识别相应农药的高效价的宽谱特异性多克隆抗体,这为农药等小分子化合物多残留免疫快速测定技术研究开辟了一条新的途径。采用间接竞争ELISA法分别测试BSA宽谱多抗（即免疫原为BSA多簇人工抗原,下同）、HSA宽谱多抗、KLH宽谱多抗和BSA&KLH宽谱多抗对相应农药的亲合性,对克百威、三唑磷、毒死蜱、甲基对硫磷这4种农药的I₁₀范围分别为:0.0024-0.0451μg/mL、0.0001-0.0013μg/mL、0.0062-0.0516μg/mL、0.0019-0.0211μg/mL;I₅₀范围分别为0.0617-1.2208μg/mL、0.0242-0.1388μg/mL、0.4567-30.6194μg/mL、0.1156-7.5054μg/mL。将4种半抗原分别与载体蛋白偶联制备“单簇”抗原,再将单簇抗原混合免疫实验动物,同样获得了高效价的宽谱特异性多克隆抗体,但使用直接或间接竞争ELISA测试由混合单簇抗原免疫所得宽谱抗体对农药的亲合性,未检测出农药对抗原-抗体反应有明显的抑制作用。采用多簇抗原免疫实验动物制备单克隆抗体未获得成功,由单簇抗原分别免疫小鼠,成功制备出针对三唑磷、克百威、毒死蜱和甲基对硫磷的高特异性单抗,腹水效价均＞10⁵,其I₁₀分别为:0.0207、0.2325、0.7556和0.4004ng/mL;I₅₀分别为0.1356、3.4086、8.3890和3.2045 ng/mL。选择金免疫层析法作为实现农药多残留检测的技术手段。采用柠檬酸三钠还原法制备理论粒径为25nm的胶体金颗粒。使用所制备的宽谱多抗、高特异性单抗、双单抗（本所制备）,OVA-半抗原偶连物分别制备金探针。经过一系列的工作条件优化,采用多膜复合以及单膜多元受体固定的方式,在国内外农药残留检测领域首次开发出了能同时检测三唑磷和克百威的多残留金标试纸条。以单抗为金探针的试纸条对三唑磷和克百威的检测限分别为0.004μg/mL、0.031μg/mL,以双单抗为金探针的试纸条对三唑磷和克百威的检测限分别为0.008μg/mL和0.063μg/mL。更多还原

【Abstract】 Haptens of chlorpyrifos（CHBu）,Triazophos（THHe）,carbofrua（BFNB）and parathion-methyl（MP5）were synthesized in this study.Four kinds of multi-determinant artificial antigen was prepared by those haptens conjugating to the carrier protein（BSA,HSA,KLH）in turn.Male New Zealand white rabbits were immunized with those multi-determinant immunogen separately or mixed to produce the polyclonal antibodies（PAbs）,which can recognize the four pesticides.Four kinds of broad specificity PAbs which we obtained displayed high level for each relative hapten-OVA conjugate.Characterization studies of those PAbs showed that they have acceptable affinity and specificity to the four relative pesticides.An indirect competitive ELISA was developed for multi-residue determination.The I₅₀ range of four PAbs for the four pesticides was 0.0617-1.2208μg/mL,0.0242-0.1388μg/mL,0.4567-30.6194μg/mL and 0.1156-7.5054μg/mL,with the detection limit(I₁₀) range of 0.0024-0.0451μg/mL,0.0001-0.0013μg/mL,0.0062-0.0516μg/mL and 0.0019-0.0211μg/mL for carbofuran,triazophos,chlorpyrifos and parathion methyl, respectively.Results indicated that,this study provided a new strategy to develop immunoassays through artificial antigen design for pesticides multi-residue determination.The mixture immunogen（CHBu-BSA,THHe-BSA,BFNB-BSA and MP5-BSA, 1:1:1:1:1,m/m）were also immunized to rabbits as controls.Relevant broad specificity PAbs with high titer was obtained.Direct and indirect competitive ELISA method was used to study the characterization of this PAbs,obviously inhibitions of four pesticides was not found,and the ELISA curves therefore could not established either.Four kinds of monoclonal antibodies（McAbs）derived from BFNB-BSA,THHeBSA, CHBu-BSA and MP5-BSA respectively,were obtained.The I₅₀value for the four pesticides was 0.1356,3.4086,8.3890 and 3.2045 ng/mL,with the detection limit (I₁₀)of 0.0207,0.2325,0.7556 and 0.4004ng/mL for triazophos,carbofuran, chlorpyrifos and parathion methyl,respectively. Gold immuno-chromatography assay（GICA）based on nanocolloidal gold-labeled antibody or antigen for detection of multi-residue of pesticides was developed.The nanocolloidal gold,with an average particle diameter of 25 nm（G25）was labeled to broad specificity PAbs,McAbs,bispecific-McAb（bis- McAb）and OVA-hapten conjugate respectively.After comditions optimization,two kinds of strips were finally developed for the residue determination of triazophos and carbofuran.The limit of detection（LOD）for triazophos and carbofuran of the strip were 0.004μg/mL and 0.031μg/mL which based on gold-labeled McAbs,and were 0.008μg/mL and 0.063μg/mL which based on gold-labeled bis- McAb,respectively.更多还原