Preparation Of Antibodies To Metalachlor Enantiomer With Enantioselective Recognition Characteristics

Metolachlor occupies one of the top ten pesticide varieties in world sales,and also one of the amide herbicides with the largest production and use in China.It consists of two chiral elements,thus resulting in four stereoisomers.Metolachlor shows enantioselectivity in biological activity and environmental fate;therefor it is of great significance from the perspective of enantiomers to investigate the interaction between the single enantiomer and the corresponding receptor to explain the difference.Moreover,inactive enantiomers may cause potential damage to crops,environmental and even do great harm to human beings through the food chain.In order to reduce toxicity and side effects,it is necessary to establish an efficient method for separation and analysis of chiral substances,and then investigate the mechanism of chiral pesticide enantiomers exhibiting different effects.It has always been great challenges to separate and analyze chiral compounds with similar physical and chemical properties.Immunoassay based on antigen-antibody interactions has shown good application prospects in the separation and analysis of chiral substances,due to its advantages of high specificity,rapidity and easy operation.However,hampered by lack of antigen-antibody recognition mechanism,the practical application of enantioselective antibody recognizing chiral compound enantiomers is still very limited.Antibody can be a specific receptor.If the chiral recognition mechanism between antibody and metolachlor enantiomer can be well revealed,it will provides guidance for the study of chiral molecule enantiomers in vivo,and also be conducive to the application of enantioselective antibodies in food safety,pharmacology and other fields.In this paper,an indirect competitive ELISA analysis method was established,relying on polyclonal and monoclonal antibodies obtained by using the derivatives of metolachlor and its stereoisomers as hapten.Based on the binding activities of 5 polyclonal antibodies against 44 structurally similar compounds,a three-dimensional quantitative structure-activity relationship（3D QSAR）was constructed.The research work in this paper is as follows:（1）Synthesis of metolachlor haptenMetolachlor hapten was prepared by metolachlor via the spacer of 3-mercaptopropionic acid.AD-H column was used for the separation of metolachlor,and the absolute configuration order of hapten was determined asɑRS,ɑRR,ɑSS andɑSR by comparing the experimental ECD with the calculated ECD spectra and combining with chromatogram of metolachlor.（2）Preparation of metolachlor antibody with enantioselectivityMetolachlor and four enantiomers haptens were coupled with the carrier protein（BSA or OVA）by the direct EDC method to prepare for immunogens and coating antigens respectively.Immunogens were used to immunize rabbits and mice to obtain corresponding polyclonal antibodies.Mice with good effect were selected for the generation of monoclonal antibodies by cell hybridization techniques.Finally,five types of p Abs and m Abs（mice）againstɑSS andɑSR-MET were successfully produced,respectively.（3）Indirect competitive enzyme-linked immunoassay for metolachlor and its stereoisomersThe established ic-ELISA calibration curve for metolachlor and four stereoisomers（ɑRS,ɑRR,ɑSS,ɑSR）were conducted by antibody exhibited IC₅₀ value of 14.64ng/m L,28.89ng/m L,13.34 ng/m L,27.07 ng/m L,5.68 ng/m L and LOD of 0.8ng/m L,0.86 ng/m L,0.85ng/m L,0.91 ng/m L,0.22 ng/m L.The enantioselectivity and specificity of 5 polyclonal antibodies towards 44 metolachlor structural analogues were evaluated by cross reactivity.It is demonstrated that these five antibodies can recognize 30,30,31,27 and 32metolachlor structural analogues,respectively.At the same time,the ic-ELISA standard curves for six monoclonal antibodies（ɑSS-MET-1、ɑSS-MET-3、ɑSS-MET-8、ɑSR-MET-2、ɑSR-MET-3 andɑSR-MET-6）were established with IC₅₀ value of 52.72 ng/m L,9.85ng/m L,29.04 ng/m L,81.15 ng/m L,42.82 ng/m L and 92.98 ng/m L,LOD value of 1.62ng/m L,1.26 ng/m L,1.05 ng/m L,0.90 ng/m L,1.01 ng/m L and 0.93 ng/m L,respectively.The cross-reactions between four metolachlor antibodies and five stereoisomers suggested that the antibodies could enantioselectively recognize these stereoisomers.（4）The recognition mechanism between five metolachlor antibodies and structural analoguesBased on the binding activities of antibodies against similar compounds,a three-dimensional quantitative structure-activity relationship（3D QSAR）was constructed,including comparative molecular field analysis（Co MFA）and a comparative molecular similarity indices analysis（Co MSIA）.The 3D QSAR model for racemic antibody showed that there was not only one specific binding site between antibody and small molecules..The affinity of antibody against small molecules will be improved by increasing the negative electricity,reducing the hydrophobicity around the chloroacetyl side chain,decreasing the radical volume around the alkoxy side chain and the hydrophobicity around C-6 site on benzene ring,which will be conducive to antibody recognition.The structure of the arm is crucial to antibody recognition.Antibodies with broad-specificity can be obtained by derivation at chloroacetyl and alkoxy groups,while specific antibodies may be gained through derivation on benzene rings.Co MSIA model was applied to investigate the cross-reaction between four stereoisomers of metolachlor and antibodies.It can be revealed that the four antibodies possess enantioselectivity.Four stereoisomer antibodies recognized carbon chirality stronger than axis chirality.Among these stereoselective antibodies,ɑRR-MET polyclonal antibodies had the strongest stereoselectivity and better specificity.