Development Of Monoclonal Antibodies To Pymetrozine And Pretilachlor And Establishment Of Enzyme-Linked Immunosorbent Assay

Pymetrozine（PY）,a triazinone insecticide,has excellent control effects on aphids,planthoppers,whitefly and leafhoppers.Pretilachlor（PR）,a highly selective herbicide used exclusively in rice fields,is safe to rice and used to control grasses and broad-leaved weeds.These two drugs are widely used in agriculture（especially in rice）in China,and their residues are serious,causing water pollution,endangering aquatic organisms,causing serious losses to fisheries,and causing harm to human body through the enrichment of food chain,such as cancer.The detection of these two drugs is mostly by liquid chromatography,and the related reports are few.This method has the characteristics of complex operation,high detection cost and large amount of organic reagent waste liquid.The enzyme-linked immunosorbent assay（ELISA）,with its simple operation,low detection cost,less organic solvent waste,and high sensitivity and specificity can also reach or even surpass chromatographic methods,and suitable for residue detection monitoring in the screening analysis and field detection,and therefore become the mainstream technology and methods in the field of residue detection today.The ELISA for detection of these two drugs has rarely been investigated.Therefore,it is important to provide indirect competitive ELISA（ic-ELISA）methods for the detection of PY and PR in food in order to ensure food safety.In this study,through hapten design and antigen synthesis,we obtained two monoclonal antibodies（m Ab）that can recognize PY and PR,respectively.Based on the m Ab,ic-ELISA was established respectively,it can be used to detect the residues of broccoli,cabbage,wheat,corn,rice,chicken,fish,crayfish and lake water.The main findings are as follows:1.Pymetrozine hapten（PYC）with carboxyl group was synthesized from triazinone hydrochloride and 5-formylpyridine-3-carboxylic acid.A carboxyl-containing pretilachlor（PR-SC）hapten was synthesized from 3-mercaptopropionic acid and pretilachlor.PYC-BSA,PR-SC-BSA,PYC-OVA and PR-SC-OVA were prepared by coupling PYC and PR-SC with carrier protein by activated ester method.The coupling ratios were 10.8,15.4,7.9and 10.2,respectively.2.Female Balb/C mice were immunized with PYC-BSA and PR-SC-BSA respectively,and 3 mice were selected for cell fusion after serum detection.Two hybridoma cell lines,PY/2D2-D8 and PR/114 were obtained by cell screening.3.Established of ic-ELISA for pymetrozine:The isotype of m Ab obtained based on cell PY/2D2-D8 was Ig G1.The optimal concentration of PYC-OVA was 0.25μg/m L,the optimal PY/2D2-D8 m Ab concentration was 2μg/m L,and the sensitivity of phosphate buffer（PBS）as diluent of PY was the highest.The equation was Y=0.87209-0.37919X,R²=0.9961,IC₅₀ was 9.58±0.60μg/L with a linear range of 2.5-40μg/L.The m Ab showed no cross-react with metamitron,hexazinone,metribuzin,acetamiprid,nitenpyram and imidacloprid.The detection limits of PY in broccoli,cabbage,wheat,corn,rice,chicken,fish,crayfish and lake water were 1.56-2.72μg/L,the quantitative limit were 4.46-10.13μg/L,the recovery was 78.2%-103.1%and the coefficient of variation was less than 11.1%.4.Established of ic-ELISA for pretilachlor:The isotype of m Ab obtained based on cell PR/114 was Ig G1.The optimal concentration of PR-SC-OVA was 0.0625μg/m L,the optimal concentration of PR/114 m Ab was 1μg/m L,and the sensitivity of ic-Elisa was the highest when PBS containing 5%methanol was used as the diluent of competing drugs.The equation was Y=1.08288-0.387X,R²=0.9915,IC₅₀ was 31.47±2.35μg/L with a linear range of 6.25-100μg/L.The m Ab showed no cross-reactivity to alachlor,acetochlor,metalaxyl and diflufenican,the cross-reaction rate of butachlor and metolachlor was less than 2.0%.In order to detect PR in lake water,rice and soil,the samples should be diluted10 times,the actual samples linear range was 62.5-1000μg/l,the detection limit ware 48.3-52.3μg/l,and the quantitative limit ware 101.3-109.8μg/l,the recoveries ranged from 78.3%to 91.3%,and the coefficient of variation was less than 9.5%.5.The stability test showed that PY/2D2-D8 m Ab,PR/114 m Ab,PYC-OVA and PR-SC-OVA could be stored at 4℃for 12 months,and PY standard solution could be preserved for 8 months at 4℃,PR standard solution could be stored at 4℃for 2 months.This study designed and synthesized PYC as an original study,prepared two m Abs,can recognize PY and PR respectively.Based on this,PY and PR extraction methods and ic-ELISA assays in food were developed.The reliability of two ic-ELISA methods was verified by liquid chromatography,and the sensitivity of two methods was equal to that of liquid chromatography,which made up the gap of the PY immunoassay method,and provides a more sensitive detection method in the field of PR immune detection and a supervisory means for food safety,which has higher application value and prospect.