| 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 (I50=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, I50 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 (I50=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, I50 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.
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