The Preparation Of Monoclonal Antibody And Development Of Immunoassays For Halosulfuronmethyl

Halosulfuron-methyl is a highly efficient and low toxicity sulfonylurea herbicide,mainly used for the removal of noxious weeds in corn,sugarcane,wheat,rice and tomato fields.The advantages of fast metabolism in target crops and soil,low toxicity and low application rate have made halosulfuron-methyl an excellent product in the field of herbicides.Halosulfuron-methyl has broad market and application prospect as the registered products in China,and the applicable crops are expanding.Although the metabolism rate of halosulfuron-methyl in the target crop field is fast,its residual in the soil and aquatic environment is long,and the damage to non-target crops is strong.A variety of metabolites may bring potential harm to subsequent crops and aquatic products in ecological farming.Therefore,it is necessary to develop rapid,sensitive and simple detection methods to monitor the residues of halosulfuron-methyl.Currently,instrumental confirmation methods(chromatography and mass spectrometry)with high sensitivity and accuracy are mainly used for the detection of halosulfuron-methyl,but the extraction and purification procedures in different matrices and the detection process are tedious,requiring professional operators and expensive instruments,which is not suitable for popularization in the grassroots testing department.Monoclonal antibody-based enzyme-linked immunosorbent assay and colloidal gold test strips exhibit many advantages,including high-throughput screening,high sensitivity,simplicity,rapid pre-treatment and convenience for non-professionals,which have gradually been a hot spot of the current research for the rapid detection of pesticide residues.In this research,halosulfuron-methyl hapten has been synthesized using halosulfuron-methyl metabolite-pyrazole sulfonamide-as the specific structure.The monoclonal antibody has been prepared by immunizing mice using hapten-protein conjugate as the immunogen,and fused by hybridoma technique.Based on the halosulfuron-methyl monoclonal antibody,indirect competitive enzyme-linked immunosorbent assay(ic ELISA),direct competitive enzyme-linked immunosorbent assay(dc ELISA)and colloidal gold test strip were established.The main studies are as follows.(1)Preparation and characterization of halosulfuron-methyl hapten and antigenConsidering the instability of the structure of halosulfuron-methyl,pyrazole sulfonamide was chosen as the specific structure of the happen.The amidation reaction between the amino group on pyrazole sulfonamide and succinic anhydride was used to synthesize the halosulfuron-methyl hapten.The properties of the hapten were characterized by HPLC?HRMS and NMR.The results showed that the purity of the hapten was 92.5%.The accurate molecular weight was 353.0084.The structure was authenticated by ¹H NMR and ¹³C NMR.The hapten was activated by DCC and NHS,and then conjugated to BSA and OVA to prepare the immunogen and coating antigen,respectively.The molar ratio between hapten and carrier were 3:1 and 2:1 for immunogen and coating antigen,respectively,determined by matrix-assisted laser-resolved mass spectrometry.In order to obtain the immune effect,EDC and sulfo-NHS was used as the activating reagents and the material ratio was improved.Finally,an immunogen with a molar ratio of 7:1 is obtained,which is used for mouse immunization and the preparation of corresponding antibodies.(2)Preparation and characterization of monoclonal antibodies,and development of ic ELISAMice were immunized with the prepared immunogen,and the titer of antiserum and inhibition rate were evaluated.Mouse spleen cells with serum titer>2.56×10⁵ and inhibition rate>85%were selected for cell fusion.The fusion of splenocytes and SP2/0 myeloma cells was performed in vitro.The positive hybridoma cells with inhibition were screened by ic ELISA.The screened hybridoma cells were subcloned by the limited gradient dilution method until a monoclonal cell line was cultured.After another positive and inhibitory screening of all monoclonal cell lines,the monoclonal cell line 1A91H11,which was able to stably secrete a highly sensitive antibody against halosulfuron-methyl,was finally obtained.The monoclonal antibody cell line was expanded and cultured to produce antibody by in vivo induction method,and the 1A91H11 monoclonal antibody protein was purified by salting method.Based on the monoclonal antibody 1A91H11,the ic ELISA method was established.The working concentration of antibody antigen,p H of working buffer,ion concentration,methanol concentration and reaction time of ic ELISA were optimized in turn.When p H was 8.5,Na⁺concentration was 0.1 M and the methanol concentration was 10%,the established ic ELISA has the lowest IC₅₀.The standard curve of the ELISA for halosulfuron-methyl was established under the optimal conditions.The working range of the standard curve was between 8.1 and 44.9 ng/m L,and the semi-inhibitory concentration(IC₅₀)and the minimum detection limit(IC₂₀)was 16.5 ng/m L,and 8.1 ng/m L,respectively.The monoclonal antibody 1A91H11 showed no cross-reactivity(CR?0.06%)with the structural analogues and the metabolite of halosulfuron-methyl.(3)Development of dc ELISAA direct competitive ELISA was established using the prepared monoclonal antibody 1A91H11.The enzyme-labeled hapten was prepared by activating the hapten with EDC and sulfo-NHS reagents and coupling with horseradish peroxidase(HRP),and the molar ratio of enzyme and hapten of HRP-hapten conjugate was identified as 1:1.The reaction p H,ion concentration and methanol content of the dc ELISA were optimized.When p H was 8.5,Na⁺concentration was 0.1 M without methanol,the established dc ELISA has the lowest IC₅₀.The standard curve of the ELISA for halosulfuron-methyl was established under optimal working conditions with a working concentration range(between IC₂₀-IC₈₀)of 0.7-10.7 ng/m L,and IC₅₀ of 2.7 ng/m L and a minimum limit of detection(LOD,IC₂₀)of 1.27 ng/m L.Compared with ic ELISA,the sensitivity of dc ELISA method is 6 times higher,and the detection time is shortened by 45 min.The spiking recovery experiment was performed in tomato matrices and maize matrices by adding 0.01 mg/kg?0.05mg/kg and 0.1 mg/kg halosulfuron-methyl.The recoveries were within 78.9%-87.9%in tomato and 103.0%-107.4%in maize,respectively.The coefficients of variation ranged from 1.1%-6.8%and 2.7%-6.4%,indicating that the developed method with good accuracy and precision was suitable for the detection of halosulfuron-methyl residue in tomato samples.(4)Preparation of colloidal gold test strip for halosulfuron-methylThe colloidal gold immunoassay strip was established using the monoclonal antibody1A91H11 to quantify the residue of halosulfuron-methyl.After optimizing the p H value,the concentration of the antigen and secondary antibody,and the concentration of the buffer,the test strip for halosulfuron-methyl was successfully prepared.The detection results could be obtained within 10 min,and the detecting range of the developed test strip was between 20 ng/m L-250ng/m L.The recovery experiments were carried out by extracting halosulfuron-methyl from spiked tomato samples with buffer salts,adjusting the p H of the extracts and then directly detected by the test strip.The tomato had little matrix effect on the test strip,and the recoveries in tomato matrix ranged from 91.6%to 102.6%with coefficients of variation between 8.9%and 17.5%,which could meet the rapid detection of halosulfuron-methyl in tomato samples.The established immunoassays in this research can be used for the rapid detection of halosulfuron-methyl in agricultural and environmental samples,providing theoretical and data support for the development of halosulfuron-methyl kits and test strips.