倍硫磷和速灭威半抗原分子设计及其免疫效果研究
摘要
有机磷农药倍硫磷由硫代磷酸酯结构(X)和芳香环结构(Y)两部分构成，从倍硫磷分子上5个不同位置各自引出连接手臂结构(Arm)，设计了5种形式的半抗原：X-Arm-Y(手臂结构位于近似整个分子中间的位置)、Arm-X-Y、X-Y-Arm、X-Arm和Arm-Y；合成了5种相应的倍硫磷半抗原，即4-[2-(二甲氧基硫代磷酰氧基)-4-甲基-5(甲硫基)苯胺基]-4-羰基丁酸(Hapten A)、6-{甲氧基[4-(甲硫基)苯氧基]硫代磷酰胺基}己酸(Hapten B)、4-[4-(二甲氧基硫代磷酰氧基)-2-甲基苯基胺基]-4-羰基丁酸(Hapten C)、4-[3-甲级-4-(甲硫基)苯氧基]丁酸(Hapten D)和4-(二甲氧基硫代磷酰胺基)丁酸(Hapten E)；质谱和核磁共振谱鉴定了这5种半抗原结构。
合成的倍硫磷5个半抗原中，Hapten A试图在人工抗原上平等地暴露倍硫磷的两个结构特征，即硫代磷酸酯基团X和苯环基团Y，Hapten B偏向于暴露X，Hapten C偏向于暴露Y，Hapten D只暴露Y，Hapten E只暴露X。利用活泼酯方法将Hapten A-C与牛血清白蛋白(BSA)偶联，将Hapten A-E与卵清蛋白(OVA)偶联。利用3个BSA偶联物共制备了6个鼠多克隆抗体，共测定了30个抗体／包被抗原组合对倍硫磷分析的灵敏度和特异性，筛选出一个最佳组合，该组合具有高度的灵敏度(I_(50)为0.08ng／mL)和特异性。结果表明，当目标分析物与免疫半抗原的结构差异性小于包被半抗原与免疫半抗原的结构差异性的时候，具有高灵敏度的异源ELISA方法就很有可能被建立。免疫结果表明，采用不同空间手臂连接位点的免疫半抗原有可能显著地提高抗体的质量，即灵敏度和／或特异性。结果还表明，半抗原连接位点附近取代基的变化对半抗原与抗体结合的削弱作用比远离半抗原结合位点基团的变化的削弱作用更大。
根据以上倍硫磷免疫与包被半抗原组合分析结果，选择利用活泼酯法将人工合成的2种倍硫磷半抗原4-[4-(二甲氧基硫代磷酰氧基)-2-甲基苯基胺基]-4-羰基丁酸(Hapten C)和6-{甲氧基[4-(甲硫基)苯氧基]硫代磷酰胺基}己酸(Hapten B)分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)共价偶联，以Hapten C-BSA为免疫原制备倍硫磷兔多克隆抗体，以Hapten B-OVA为包被抗原测定所得抗血清效价(抗血清呈现阳性的最大稀释倍数)都在64000倍以上，而且抗体仅与杀螟硫磷有4.5％的交叉反应，而与其他农药的交叉反应率都在0.1％以下；通过分析条件优化，确定了倍硫磷酶联免疫吸附分析(ELISA)的最佳工作条件，建立了定量测定倍硫磷的间接竞争ELISA方法，I_(50)为0.0107 ng／mL，检测限为0.0002 ng／mL。葡萄和桃的果实及水中的平均添加回收率在81.9-104.0％之间。本研究为倍硫磷农药在农产品和环境中残留量快速检测新增一种有效的方法。
针对氨基甲酸酯农药速灭威设计了五种结构半抗原。以3-甲基苯酚、双光气和3-氨基丙酸为主要原料合成了Hapten 1，即3-(3-甲苯基氧基羰基氨基)丙酸；以苯酚、双光气和6-氨基己酸为主要原料合成了Hapten 2，即6-(苯氧基羰基氨基)己酸；以2-异丙氧基苯酚、双光气和4-氨基丁酸为主要原料合成了Hapten 3，即4-((2-异丙氧基苯氧基)羰基氨基)丁酸；以3-甲基苯酚和4-溴丁酸乙酯为主要原料合成了Hapten 4，即4-(3-甲苯基氧基)丁酸；并把3-(3-羟基)苯丙酸设计为Hapten 5；质谱和(或)核磁共振谱鉴定了这些半抗原结构。
利用活泼酯法将速灭威的以上5种半抗原(Hapten 1-5)与载体牛血清白蛋白(BSA)或卵清蛋白(OVA)共价偶联制备成速灭威抗原，其中BSA-Hapten 1用来制备速灭威兔多克隆抗体MpAb01和MpAb02，而OVA-Hapten 1-5用作包被抗原，共测定并比较了10个抗原／抗体组合的相对亲和性、6个组合的灵敏度和3个组合的特异性。数据显示，MpAb01／OVA-Hapten 4组合的灵敏度最高(I_(50)=0.11μg／mL)且特异性最强(与混灭威等7种农药的CR值均为0)，但亲和性相对偏弱；Hapten 2和Hapten 1作为包被半抗原的亲和性及灵敏度相差不大，但Hapten 2的异源组合对混灭威、残杀威等6种农药都有不同程度交叉反应，而Hapten 1的同源ELISA组合只对混灭威有交叉反应(30％)。结合半抗原和分析物化学结构分析，结果表明：(1)在保证一定滴度值前提下，包被半抗原与免疫半抗原结构相差越远(尤其是半抗原空间手臂连接位点差异)可能越有利于建立较高灵敏度的ELISA；(2)免疫半抗原连接位点处及附近的基团在抗体-半抗原互作中可能起非常重要的作用。
根据以上速灭威免疫与包被半抗原组合分析结果，选择抗原抗体组合MpAb01／OVA-Hapten 1进行进一步研究、测定了离子强度、有机溶剂、pH值、封闭物等影响因子对免疫分析的影响，确定了速灭威酶联免疫吸附分析(ELISA)的最佳工作参数，并建立了定量测定速灭威的间接竞争ELISA方法，检测线性范围为1～10000ng／mL，I_(50)为40.738ng／mL，检测限为0.014～0.018ng／mL，批内变异系数4.0％，批间变异系数11.4％，土壤、稻谷和水中的添加回收率依次为80％、93％和107％。所建立的ELISA分析方法灵敏实用，为速灭威在环境和农产品中残留量快速检测新增一种有效的方法。
提出了半抗原-抗体互作模型，内容包括：(1)免疫半抗原结构(尤其指空间手臂连接位点不同的半抗原结构)与抗体反应特性之间是一一对应关系，特定空间手臂连接位点的半抗原与其相应抗体结合后的复合物就有一个特定的最稳定空间结合形式；(2)在半抗原-抗体的最稳定空间结合形式中，免疫半抗原空间手臂连接位点左右的基团靠近相应抗体的“活性中心”，并与之相互吻合；(3)与免疫半抗原结构相比，差异基团远离免疫半抗原空间手臂连接位点的类似物与抗体的结合能力较强，而差异基团靠近免疫半抗原空间手臂连接位点的类似物与抗体的结合能力较弱或根本就没有结合能力。利用倍硫磷抗体和速灭威抗体与反应半抗原的互作表现对以上模型进行实例证明。最后讨论了半抗原-抗体互作模型的局限性及前景。
综上所述，本文一方面为化学农药倍硫磷和速灭威的残留检测新增加了有效的免疫分析方法，另一方面为农药等小分子化学品免疫分析研究中的半抗原设计提供了理论基础。
Hapten spacer arm structures (Arm) were derived from five different attachment siteson fenthion molecule and five forms of hapten, such as X-Arm-Y, Arm-X-Y, X-Y-Arm,X-Ann and Arm-Y, were designed according to characteristic of organophosphoruspesticide fenthion composed of phosphorothionate (X) and aromatic circle (Y). The haptenswere synthesized correspondingly, which were 4-(2-(dimethoxyphosphorothioyloxy)-4-methyl-5-(methylthio) phenylamino)-4-oxobutanoic acid (Hapten A), 6-(methoxy(4-(methylthio) phenoxy) phosphorothioylamino) hexanoic acid (Hapten B), 4-(4-(dimethoxyphosphorothioyloxy)-2-methylphenylamino)-4-oxobutanoic acid (Hapten C),4-(3-methyl-4-(methylthio) phenoxy) butanoic acid (Hapten D), and 4-(dimethoxy-phosphorothioylamino) butanoic acid (Hapten E). These hapten's structures were identifiedby mass chromatography and nuclear magnetic resonance spectnma.
Among the above haptens, Hapten A was attempted to expose equally two features ofthe fenthion structure, the phosphorothioate group X and the aromatic group Y, in theresulting conjugates, Hapten B was designed to partially expose X, Hapten C was designedto partially expose Y, Hapten D to expose Y, and Hapten E to expose X. Haptens A-C wereconjugated with bovine serum albumin (BSA) and haptens A-E were conjugated withovalbumin (OVA). Six polyclonal antisera were raised against the three BSA conjugates,and thirty antibody/coating conjugate combinations were selected for studies of assaysensitivity and specificity for fenthion. The study revealed the best combination with highsensitivity (I_(50)=0.08 ng/mL) and high assay specificity, which indicated that whenstructural difference between the analyte and an immunizing hapten is more than thatbetween a coating hapten and the immunizing hapten, a high sensitive ELISA in theheterologous system may stand a good chance to be developed. The immunity resultsshowed that heterology in the hapten spacer-arm attachment site of the immunogen couldachieve a remarkable improvement in the quantity, sensitivity, and/or specificity of antibody,and that the moiety of an analyte, which is the same as the moiety near/on the immunizingspacer-arm hapten attachment site, contributes greatly to the interaction of antibody and hapten.
According to the analysis of combinations of immunizing and coating hapten forfenthion, two fenthion haptens synthesized, 4-(4-(dimethoxyphosphorothioyloxy)-2-methyl-phenylamino)-4-oxobutanoic acid (Hapten C) and 6-(methoxy (4-(methylthio)phenoxy)-phosphorothioylamino) hexanoic acid (Hapten B) were selected. Hapten C was conjugatedwith BSA and Hapten B with OVA by active ester method. Then Hapten B-OVA was usedas coating antigen, while Hapten C-BSA was used as immunogen. Titer values of theantisera were both above 64000, and there was only cross reactivity (CR) with fenitrothion(28.6％), and CRs with other pestisides were all below 0.1％. After optimization, aneffective competitive indirect Enzyme-Linked Immunosorbent Assay (ELISA) procedurefor determination of fenthion was established, I_(50) of which was 0.0107ng/mL, limit ofdetection (LOD) of which was 0.0002ng/mL. The recoveries obtained by standard fenthionaddition to the different samples such as grape, peach and river water were all from 81.9％to 104.0％. The development of ELISA procedure may become a new convenient andsatisfied analytical tool for monitoring fenthion residues in environment and agriculturalsamples.
Five haptens of metolcarb were designed and synthesized here: Hapten 1,3-(m-tolyloxycarbonylamino) propanoic acid, was synthesized with main materials ofm-cresol, trichloromethyl carbonochloridate, and 3-aminopropanoic acid; Hapten 2,6-(phenoxycarbonylamino) hexanoie acid, was synthesized with main materials of phenol,trichloromethyl carbonochloridate, and 6-aminohexanoic acid; Hapten 3,4-((2-isopropoxyphenoxy) carbonylamino) butanoic acid, was synthesized with mainmaterials of 2-isopropoxyphenol, trichloromethyl carbonochloridate, and 4-aminobutanoicacid; Hapten 4, 4-(m-tolyloxy) butanoic acid, was synthesized with main materials ofm-cresol and ethyl 4-bromobutanoate; the compound, 3-(3-hydroxyphenyl) propanoic acid,was directly used as Hapten 5. Structures of here synthesized haptens were verified by massspectra and (or) nuclear magnetic resonance.
These five haptens (Hapten 1-5) were conjugated with carrier protein BSA or OVA withactive ether method, and antigens of metolcarb were prepared. Polyclonal antibodies,named MpAb01 and MpAb02 were prepared against BSA-Hapten 1, while OVA-Hapten1-5 were used as coating antigens. It was determined and compared that ten combinationsof antibody/antigen for affinity, six combinations for sensitivity, and three combinations forspecificity. The data showed a combination of MpAb01/ OVA-Hapten 4 with high sensitivity (I_(50)=0.11；μg/mL) and strong specificity (CRs with seven tested pesticides, suchas dimethacarb and so on, were all 0), but low affinity, were revealed. There were littledifference for sensitivity and specificity of the combinations of Hapten 2 and Hapten 1 usedas coating hapten. The heterologous ELISA system of Hapten 2 showed certain extent ofCRs with six tested pesticides such as dimethacarb, propoxur, and so on, while thehomologous ELISA system of Hapten 1 showed only CR with dimethacarb (30％).Considering structure of haptens and analytes, the results here implyed that:1) Whenstructural difference between the analyte and an immunizing hapten is less than thatbetween a coating hapten and the immunizing hapten, a high sensitive ELISA in theheterologous system may stand a good chance to be developed; 2) The moiety of an analyte,which is the same as the moiety near/on the immunizing spacer-arm hapten attachment site,contributes greatly to the interaction of antibody and hapten.
According to the analysis of combinations of immunizing and coating haptens formetolcarb, the combination of MpAb01/OVA-Hapten 1 was selected for further study. Afteroptimization of the ELISA conditions such as ionic strengths, organic solvents, pH values,blocking agents and so on, the proper parameters of ELISA procedure were determined,and an assay protoleal for the N-methylcarbamate insecticide metolcarb was established.Based on statistical analysis, the dynamic range of determination was from 1 ng/mL to10000ng/mL, I_(50) was 40.738 ng/mL, LOD was 0.014～0.018ng/mL, intra-assay coefficientof variation (CV) reached 4.0％, and inter-assay CV reached 11.4％. The recoveriesobtained by standard metolcarb addition to the different samples as rice seed, water and soilwere 80％, 93％and 107％respectively. These results indicate that the ELISA here could bea convenient and satisfied analytical tool for monitoring metolcarb residues inenvironmental and agricultural samples.
An interaction model of hapten-antibody was expressed, containing: 1) The relationshipbetween structure of immunizing hapten and reactive characteristic of antibody wascorresponding one by one. And there is the most stable spacer form for the complexityformed from a hapten with certain spacer-arm attachment site and its correspondingantibody. 2) In the most stable spacer form of hapten-antibody, the moiety which is on ornear the spacer-arm attachment site of immunizing hapten is close to and matched with the"active center" of the corresponding antibody. 3) Compared with the structure ofimmunizing hapten, the moiety of an analyte, which is the same as the moiety near/on theimmunizing spacer-ann hapten attachment site, contributes greatly to the interaction of antibody and hapten. And then, some exemplifications about interaction of the antibodies offenthion with some reactive haptens and the antibody of metolcarb with some other reactivehaptens were described. Finally, limitations and prospects about the interaction model ofhapten-antibody were discussed.
According to the above, these studies provided not only the new immunoassayprotolcols for chemical pesticides fenthion and metolcarb, but also some theory of haptendesign for immunoassay researches of small molecular chemicals.
合成的倍硫磷5个半抗原中，Hapten A试图在人工抗原上平等地暴露倍硫磷的两个结构特征，即硫代磷酸酯基团X和苯环基团Y，Hapten B偏向于暴露X，Hapten C偏向于暴露Y，Hapten D只暴露Y，Hapten E只暴露X。利用活泼酯方法将Hapten A-C与牛血清白蛋白(BSA)偶联，将Hapten A-E与卵清蛋白(OVA)偶联。利用3个BSA偶联物共制备了6个鼠多克隆抗体，共测定了30个抗体／包被抗原组合对倍硫磷分析的灵敏度和特异性，筛选出一个最佳组合，该组合具有高度的灵敏度(I_(50)为0.08ng／mL)和特异性。结果表明，当目标分析物与免疫半抗原的结构差异性小于包被半抗原与免疫半抗原的结构差异性的时候，具有高灵敏度的异源ELISA方法就很有可能被建立。免疫结果表明，采用不同空间手臂连接位点的免疫半抗原有可能显著地提高抗体的质量，即灵敏度和／或特异性。结果还表明，半抗原连接位点附近取代基的变化对半抗原与抗体结合的削弱作用比远离半抗原结合位点基团的变化的削弱作用更大。
根据以上倍硫磷免疫与包被半抗原组合分析结果，选择利用活泼酯法将人工合成的2种倍硫磷半抗原4-[4-(二甲氧基硫代磷酰氧基)-2-甲基苯基胺基]-4-羰基丁酸(Hapten C)和6-{甲氧基[4-(甲硫基)苯氧基]硫代磷酰胺基}己酸(Hapten B)分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)共价偶联，以Hapten C-BSA为免疫原制备倍硫磷兔多克隆抗体，以Hapten B-OVA为包被抗原测定所得抗血清效价(抗血清呈现阳性的最大稀释倍数)都在64000倍以上，而且抗体仅与杀螟硫磷有4.5％的交叉反应，而与其他农药的交叉反应率都在0.1％以下；通过分析条件优化，确定了倍硫磷酶联免疫吸附分析(ELISA)的最佳工作条件，建立了定量测定倍硫磷的间接竞争ELISA方法，I_(50)为0.0107 ng／mL，检测限为0.0002 ng／mL。葡萄和桃的果实及水中的平均添加回收率在81.9-104.0％之间。本研究为倍硫磷农药在农产品和环境中残留量快速检测新增一种有效的方法。
针对氨基甲酸酯农药速灭威设计了五种结构半抗原。以3-甲基苯酚、双光气和3-氨基丙酸为主要原料合成了Hapten 1，即3-(3-甲苯基氧基羰基氨基)丙酸；以苯酚、双光气和6-氨基己酸为主要原料合成了Hapten 2，即6-(苯氧基羰基氨基)己酸；以2-异丙氧基苯酚、双光气和4-氨基丁酸为主要原料合成了Hapten 3，即4-((2-异丙氧基苯氧基)羰基氨基)丁酸；以3-甲基苯酚和4-溴丁酸乙酯为主要原料合成了Hapten 4，即4-(3-甲苯基氧基)丁酸；并把3-(3-羟基)苯丙酸设计为Hapten 5；质谱和(或)核磁共振谱鉴定了这些半抗原结构。
利用活泼酯法将速灭威的以上5种半抗原(Hapten 1-5)与载体牛血清白蛋白(BSA)或卵清蛋白(OVA)共价偶联制备成速灭威抗原，其中BSA-Hapten 1用来制备速灭威兔多克隆抗体MpAb01和MpAb02，而OVA-Hapten 1-5用作包被抗原，共测定并比较了10个抗原／抗体组合的相对亲和性、6个组合的灵敏度和3个组合的特异性。数据显示，MpAb01／OVA-Hapten 4组合的灵敏度最高(I_(50)=0.11μg／mL)且特异性最强(与混灭威等7种农药的CR值均为0)，但亲和性相对偏弱；Hapten 2和Hapten 1作为包被半抗原的亲和性及灵敏度相差不大，但Hapten 2的异源组合对混灭威、残杀威等6种农药都有不同程度交叉反应，而Hapten 1的同源ELISA组合只对混灭威有交叉反应(30％)。结合半抗原和分析物化学结构分析，结果表明：(1)在保证一定滴度值前提下，包被半抗原与免疫半抗原结构相差越远(尤其是半抗原空间手臂连接位点差异)可能越有利于建立较高灵敏度的ELISA；(2)免疫半抗原连接位点处及附近的基团在抗体-半抗原互作中可能起非常重要的作用。
根据以上速灭威免疫与包被半抗原组合分析结果，选择抗原抗体组合MpAb01／OVA-Hapten 1进行进一步研究、测定了离子强度、有机溶剂、pH值、封闭物等影响因子对免疫分析的影响，确定了速灭威酶联免疫吸附分析(ELISA)的最佳工作参数，并建立了定量测定速灭威的间接竞争ELISA方法，检测线性范围为1～10000ng／mL，I_(50)为40.738ng／mL，检测限为0.014～0.018ng／mL，批内变异系数4.0％，批间变异系数11.4％，土壤、稻谷和水中的添加回收率依次为80％、93％和107％。所建立的ELISA分析方法灵敏实用，为速灭威在环境和农产品中残留量快速检测新增一种有效的方法。
提出了半抗原-抗体互作模型，内容包括：(1)免疫半抗原结构(尤其指空间手臂连接位点不同的半抗原结构)与抗体反应特性之间是一一对应关系，特定空间手臂连接位点的半抗原与其相应抗体结合后的复合物就有一个特定的最稳定空间结合形式；(2)在半抗原-抗体的最稳定空间结合形式中，免疫半抗原空间手臂连接位点左右的基团靠近相应抗体的“活性中心”，并与之相互吻合；(3)与免疫半抗原结构相比，差异基团远离免疫半抗原空间手臂连接位点的类似物与抗体的结合能力较强，而差异基团靠近免疫半抗原空间手臂连接位点的类似物与抗体的结合能力较弱或根本就没有结合能力。利用倍硫磷抗体和速灭威抗体与反应半抗原的互作表现对以上模型进行实例证明。最后讨论了半抗原-抗体互作模型的局限性及前景。
综上所述，本文一方面为化学农药倍硫磷和速灭威的残留检测新增加了有效的免疫分析方法，另一方面为农药等小分子化学品免疫分析研究中的半抗原设计提供了理论基础。
Hapten spacer arm structures (Arm) were derived from five different attachment siteson fenthion molecule and five forms of hapten, such as X-Arm-Y, Arm-X-Y, X-Y-Arm,X-Ann and Arm-Y, were designed according to characteristic of organophosphoruspesticide fenthion composed of phosphorothionate (X) and aromatic circle (Y). The haptenswere synthesized correspondingly, which were 4-(2-(dimethoxyphosphorothioyloxy)-4-methyl-5-(methylthio) phenylamino)-4-oxobutanoic acid (Hapten A), 6-(methoxy(4-(methylthio) phenoxy) phosphorothioylamino) hexanoic acid (Hapten B), 4-(4-(dimethoxyphosphorothioyloxy)-2-methylphenylamino)-4-oxobutanoic acid (Hapten C),4-(3-methyl-4-(methylthio) phenoxy) butanoic acid (Hapten D), and 4-(dimethoxy-phosphorothioylamino) butanoic acid (Hapten E). These hapten's structures were identifiedby mass chromatography and nuclear magnetic resonance spectnma.
Among the above haptens, Hapten A was attempted to expose equally two features ofthe fenthion structure, the phosphorothioate group X and the aromatic group Y, in theresulting conjugates, Hapten B was designed to partially expose X, Hapten C was designedto partially expose Y, Hapten D to expose Y, and Hapten E to expose X. Haptens A-C wereconjugated with bovine serum albumin (BSA) and haptens A-E were conjugated withovalbumin (OVA). Six polyclonal antisera were raised against the three BSA conjugates,and thirty antibody/coating conjugate combinations were selected for studies of assaysensitivity and specificity for fenthion. The study revealed the best combination with highsensitivity (I_(50)=0.08 ng/mL) and high assay specificity, which indicated that whenstructural difference between the analyte and an immunizing hapten is more than thatbetween a coating hapten and the immunizing hapten, a high sensitive ELISA in theheterologous system may stand a good chance to be developed. The immunity resultsshowed that heterology in the hapten spacer-arm attachment site of the immunogen couldachieve a remarkable improvement in the quantity, sensitivity, and/or specificity of antibody,and that the moiety of an analyte, which is the same as the moiety near/on the immunizingspacer-arm hapten attachment site, contributes greatly to the interaction of antibody and hapten.
According to the analysis of combinations of immunizing and coating hapten forfenthion, two fenthion haptens synthesized, 4-(4-(dimethoxyphosphorothioyloxy)-2-methyl-phenylamino)-4-oxobutanoic acid (Hapten C) and 6-(methoxy (4-(methylthio)phenoxy)-phosphorothioylamino) hexanoic acid (Hapten B) were selected. Hapten C was conjugatedwith BSA and Hapten B with OVA by active ester method. Then Hapten B-OVA was usedas coating antigen, while Hapten C-BSA was used as immunogen. Titer values of theantisera were both above 64000, and there was only cross reactivity (CR) with fenitrothion(28.6％), and CRs with other pestisides were all below 0.1％. After optimization, aneffective competitive indirect Enzyme-Linked Immunosorbent Assay (ELISA) procedurefor determination of fenthion was established, I_(50) of which was 0.0107ng/mL, limit ofdetection (LOD) of which was 0.0002ng/mL. The recoveries obtained by standard fenthionaddition to the different samples such as grape, peach and river water were all from 81.9％to 104.0％. The development of ELISA procedure may become a new convenient andsatisfied analytical tool for monitoring fenthion residues in environment and agriculturalsamples.
Five haptens of metolcarb were designed and synthesized here: Hapten 1,3-(m-tolyloxycarbonylamino) propanoic acid, was synthesized with main materials ofm-cresol, trichloromethyl carbonochloridate, and 3-aminopropanoic acid; Hapten 2,6-(phenoxycarbonylamino) hexanoie acid, was synthesized with main materials of phenol,trichloromethyl carbonochloridate, and 6-aminohexanoic acid; Hapten 3,4-((2-isopropoxyphenoxy) carbonylamino) butanoic acid, was synthesized with mainmaterials of 2-isopropoxyphenol, trichloromethyl carbonochloridate, and 4-aminobutanoicacid; Hapten 4, 4-(m-tolyloxy) butanoic acid, was synthesized with main materials ofm-cresol and ethyl 4-bromobutanoate; the compound, 3-(3-hydroxyphenyl) propanoic acid,was directly used as Hapten 5. Structures of here synthesized haptens were verified by massspectra and (or) nuclear magnetic resonance.
These five haptens (Hapten 1-5) were conjugated with carrier protein BSA or OVA withactive ether method, and antigens of metolcarb were prepared. Polyclonal antibodies,named MpAb01 and MpAb02 were prepared against BSA-Hapten 1, while OVA-Hapten1-5 were used as coating antigens. It was determined and compared that ten combinationsof antibody/antigen for affinity, six combinations for sensitivity, and three combinations forspecificity. The data showed a combination of MpAb01/ OVA-Hapten 4 with high sensitivity (I_(50)=0.11；μg/mL) and strong specificity (CRs with seven tested pesticides, suchas dimethacarb and so on, were all 0), but low affinity, were revealed. There were littledifference for sensitivity and specificity of the combinations of Hapten 2 and Hapten 1 usedas coating hapten. The heterologous ELISA system of Hapten 2 showed certain extent ofCRs with six tested pesticides such as dimethacarb, propoxur, and so on, while thehomologous ELISA system of Hapten 1 showed only CR with dimethacarb (30％).Considering structure of haptens and analytes, the results here implyed that:1) Whenstructural difference between the analyte and an immunizing hapten is less than thatbetween a coating hapten and the immunizing hapten, a high sensitive ELISA in theheterologous system may stand a good chance to be developed; 2) The moiety of an analyte,which is the same as the moiety near/on the immunizing spacer-arm hapten attachment site,contributes greatly to the interaction of antibody and hapten.
According to the analysis of combinations of immunizing and coating haptens formetolcarb, the combination of MpAb01/OVA-Hapten 1 was selected for further study. Afteroptimization of the ELISA conditions such as ionic strengths, organic solvents, pH values,blocking agents and so on, the proper parameters of ELISA procedure were determined,and an assay protoleal for the N-methylcarbamate insecticide metolcarb was established.Based on statistical analysis, the dynamic range of determination was from 1 ng/mL to10000ng/mL, I_(50) was 40.738 ng/mL, LOD was 0.014～0.018ng/mL, intra-assay coefficientof variation (CV) reached 4.0％, and inter-assay CV reached 11.4％. The recoveriesobtained by standard metolcarb addition to the different samples as rice seed, water and soilwere 80％, 93％and 107％respectively. These results indicate that the ELISA here could bea convenient and satisfied analytical tool for monitoring metolcarb residues inenvironmental and agricultural samples.
An interaction model of hapten-antibody was expressed, containing: 1) The relationshipbetween structure of immunizing hapten and reactive characteristic of antibody wascorresponding one by one. And there is the most stable spacer form for the complexityformed from a hapten with certain spacer-arm attachment site and its correspondingantibody. 2) In the most stable spacer form of hapten-antibody, the moiety which is on ornear the spacer-arm attachment site of immunizing hapten is close to and matched with the"active center" of the corresponding antibody. 3) Compared with the structure ofimmunizing hapten, the moiety of an analyte, which is the same as the moiety near/on theimmunizing spacer-ann hapten attachment site, contributes greatly to the interaction of antibody and hapten. And then, some exemplifications about interaction of the antibodies offenthion with some reactive haptens and the antibody of metolcarb with some other reactivehaptens were described. Finally, limitations and prospects about the interaction model ofhapten-antibody were discussed.
According to the above, these studies provided not only the new immunoassayprotolcols for chemical pesticides fenthion and metolcarb, but also some theory of haptendesign for immunoassay researches of small molecular chemicals.
引文
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1. Abad A., Montoya A. (1994). Production of Monoclonal Antibodies for Carbaryl from a Hapten Preserving the Carbamate Group. J. Agric. Food Chem. 42:1818-1823
2. Abad A., Moreno M.J., Montoya A. (1998). Hapten Synthesis and Production of Monoclonal Antibodies to the N-Methylcarbamate Pesticide Methiocarb. J. Agric. Food Chem. 46:2417-2426
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7. Zhang Q., Li T.J., Zhu X.X., Xu L.N., Liu F.Q., Hu B.S., Jiang Y.H.; Cao B. (2006). The Determination of N-methylcarbamatelnsecticide Metolcarb by Enzyme-linked Immunosorbent Assay. Chinese J. of Anal. Chem. 34, 178-182
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10．刘曙照，冯大和，钱传范．(1999)．甲萘威酶联免疫吸附分析技术研究．农药学学报．1(1)：62-68
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2. Brun E.M., Marta Garces-Garcia, Rosa Puchades, Angel Maquieira (2004). Enzyme-linked immunosorbent assay for the organophosphorus insecticide fenthion. Influence of hapten structure. Journal of Immunological Methods, 295: 21-35
3. Kim Y. J., Cho Y. A., Lee H. S., Lee Y.T., Gee S.J., Hammock B.D. (2003a). Synthesis of haptens for immunoassay of organophosphorus pesticides and effect of heterology in hapten spacer arm length on immunoassay sensitivity. Anal. Chim. Acta., 475: 85-96
4. Kim Y. J., Cho Y. A., Lee H-S., Lee Y. T. (2003b). Investigation of the effect of hapten heterology on immunoassay sensitivity and development of an enzyme-linked immunosorbent assay for the organophosphorus insecticide fenthion. Anal. Chim. Acta., 494: 29-40
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3. Brady J.F., Fleeker J.R., Wilson R.A., Mumma R.O. (1989). Enzyme-immunoassay for Aldicarb, in Biological Monitoring of pesticide Exposure, Measurement, Estimation and Risk Reduction, eds. Wang R.G.M., Franklin C.A., Honeyeut R.C., Reinhert J,C. ACS Symposium Series382, American Chemical Society, Washtionton, DC, 262-284
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12．刘曙照，冯大和，钱传范．(1999)．甲萘威酶联免疫吸附分析技术研究．农药学学报．1(1)：62-68
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1. Brun E.M., Marta Garces-Garcia, Rosa Puchades, Angel Maquieira (2004). Enzyme-linked immunosorbent assay for the organophosphorus insecticide fenthion. Influence of hapten structure. Journal of Immunological Methods, 295:21-35
2. Ercegovich, C.D., Vallejo, R.P., Getting, R.R., Woods, L., Bogus, E.R., and Mumma, R.O., (1981). Development of a Radioimmunoassay for Parathion. J. Agric. Food Chem. 29, 559
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5. Kim Y.J., Cho Y.A., Lee H-S., Lee Y.T. (2003b). Investigation of the effect of hapten heterology on immunoassay sensitivity and development of an enzyme-linked immunosorbent assay for the organophosphorus insecticide fenthion. Anal. Chim. Acta., 494:29-40
6. Lee, N., McAdam, D.P., and Skerritt, J.H., (1998). Development of Immunoassays for Type Ⅱ Synthetic Pyrethroids. 1. Hapten Design and Application to Heterologous and Homologous Assays. J. Agric. Food Chem. 46, 520
7. Lee, W.Y., Lee, E.K., Kim, Y.J., Park, W.C., Chung, T., Lee, Y.T., (2006). Monoelonal antibody-based enzyme-linked immunosorbent assays for the detection of the organophosphorus insecticide isofenphos. Anal. Chim. Acta. 557:159
8. McAdam, D.P., Hill, A.S., Beasley, H.L., Skerritt, J.H., (1992). Mono-and polyclonal antibodies to the organophosphate fenitrothion: 1. Approaches to hapten-protein conjugation. J. Agric. Food Chem. 40, 1466
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11．刘凤权，许志刚，王金生．(1998)．定量测定甲胺磷残留的间接竞争ELISA的建立和初步应用．农业生物技术学报，6(2)：140-146
1. Brun E.M., Marta Garces-Garcia, Rosa Puchades, Angel Maquieira (2004). Enzyme-linked immunosorbent assay for the organophosphorus insecticide fenthion. Influence of hapten structure. Journal of Immunological Methods. 295: 21-35.
2. Kim Y.J., Cho Y.A., Lee H-S., Lee Y.T. (2003). Investigation of the effect ofhapten heterology on immunoassay sensitivity and development of an enzyme-linked immunosorbent assay for the organophosphorus insecticide fenthion. Anal. Chim. Acta., 494: 29-40.
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5. Wing, K.D. and B.D. Hammock. (1979). Stereoselectivity of a radioimmunoassay for the insecticide S-bioallethrin. Experientia. 35:1619-1620.
6. Zhang Q., Li T.J., Zhu X.X., Xu L.N., Liu F.Q., Hu B.S., Jiang Y.H.; Cao B. (2006). The Determination of N-methylcarbamateInsectieide Metolearb by Enzyme-linked Immunosorbent Assay. Chinese J. of Anal. Chem. 34, 178-182
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9．毕刚．(2001)．倍硫磷的气相色谱/红外光谱检测．分析科学学报．17(1)：88
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11．孙涛，周晓龙，王建梅．(2006)．气相色谱法测定牛初乳中甲胺磷、马拉硫磷、倍硫磷的残留．现代科学仪器．(1)：74-75
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7. Zhang Q., Li T.J., Zhu X.X., Xu L.N., Liu F.Q., Hu B.S., Jiang Y.H.; Cao B. (2006). The Determination of N-methylcarbamatelnsecticide Metolcarb by Enzyme-linked Immunosorbent Assay. Chinese J. of Anal. Chem. 34, 178-182
8. Zhang Q., Li T.J., Zhu X.X., Xu L.N., Liu F.Q., Hu B.S., Jiang Y.H.; Cao B. (2006). The Determination of N-methylcarbarnateInsecticide Metolcarb by Enzyme-linked Immunosorbent Assay. Agri. Sci. in China. 6(2): 175-179
9．刘曙照，冯大和，陈美娟，钱传范．(2000)．对克百威具高度特异性的免疫分析技术研究．分析科学学报．16(5)：373-378
10．刘曙照，冯大和，钱传范．(1999)．甲萘威酶联免疫吸附分析技术研究．农药学学报．1(1)：62-68
1. Abad A., Moreno M.J., Montoya A. (1998). Hapten Synthesis and Production of Monoclonal Antibodies to the N-Methylcarbamate Pesticide Methiocarb. J. Agric. Food Chem. 46:2417-2426
2. Brun E.M., Marta Garces-Garcia, Rosa Puchades, Angel Maquieira (2004). Enzyme-linked immunosorbent assay for the organophosphorus insecticide fenthion. Influence of hapten structure. Journal of Immunological Methods, 295: 21-35
3. Kim Y. J., Cho Y. A., Lee H. S., Lee Y.T., Gee S.J., Hammock B.D. (2003a). Synthesis of haptens for immunoassay of organophosphorus pesticides and effect of heterology in hapten spacer arm length on immunoassay sensitivity. Anal. Chim. Acta., 475: 85-96
4. Kim Y. J., Cho Y. A., Lee H-S., Lee Y. T. (2003b). Investigation of the effect of hapten heterology on immunoassay sensitivity and development of an enzyme-linked immunosorbent assay for the organophosphorus insecticide fenthion. Anal. Chim. Acta., 494: 29-40
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6. Moreno M. J., Abad A., Montoya A. (2001). Production of Monoclonal Antibodies to the N-Methylcarbarnate Pesticide Propoxur. J. Agric. Food Chem. 49: 72-78
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1. Abad A., Montoya A. (1994). Production of Monoclonal Antibodies for Carbaryl from a Hapten Preserving the Carbamate Group. J. Agric. Food Chem. 42: 1818-1823
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12．刘曙照，冯大和，钱传范．(1999)．甲萘威酶联免疫吸附分析技术研究．农药学学报．1(1)：62-68
13．刘凤权，许志刚，王金生．(1998)．定量测定甲胺磷残留的间接竞争ELISA的建立和初步应用．农业生物技术学报，6(2)：140-146
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18．王镜岩，朱圣庚，徐长法．(2002)．生物化学．高等教育出版社，北京
19．李春华，马晓慧，陈慰祖，王存新．(2006)．蛋白质-蛋白质分子对接方法研究进展．生物化学与生物物理进展．33(7)：616-621