Development And Application Of Immunoassays Based On Specific Monoclonal Antibody Against Fenpyroximate

Fenpyroximate is mainly used to prevent and control plant-feeding mites on apple,citrus,peach,pear and other fruit trees and crops,and it is a phenoxypyrazole acaricide.The fenpyroximate applied in agricultural production can directly or indirectly enter the soil and even groundwater causing environmental contamination.The toxicity of fenpyroximate to most mammals is weak,but it is more toxic to aquatic organisms.As an effective inhibitor of mitochondrial activity,fenpyroximate may lead to neurotoxicity,ATP loss,oxidative damage and even cell death of human nerve cells,and be closely related to Parkinson’s disease.Consequently,it is necessary to develop a fast,sensitive and simple monitoring method of fenpyroximate residue.At present,techniques such as liquid chromatography（LC）and liquid chromatography-mass spectrometry（LC-MS）are widely used for the detection of fenpyroximate in environmental and food samples,but instrumental methods suffer from expensive equipment,specialized operational requirements and complex pre-treatment processes.In contrast,the immunoassay method is effortless,swift,sensitive,inexpensive,and can be used for large-scale sample screening,making it a very viable assay.Consequently,it is of great importance to investigate the immunological technique of fenpyroximate.In this paper,four fenpyroximate haptens were designed and synthesized,and two icELISA methods and one CLEIA method were developed based on the monoclonal antibody against fenpyroximate prepared by immunizing mice.（1）Four kinds of fenpyroximate haptens were designed,and active groups（-COOH）were introduced into the haptens that could be coupled with carrier proteins.All the haptens were confirmed by nuclear magnetic resonance（NMR）and liquid mass spectrometry（MS）.Coated antigens and immunogens were obtained by coupling haptens with ovagmin（OVA）and bovine serum albumin（BSA）respectively by active ester method.After immunizing mice with immunogen hapten A-BSA,serum titer was determined to confirme that the artificial antigens were immunogenic and could effectively stimulate the production of antibodies against fenpyroximate.Mice with high serum titer and strong specificity screened was used for the preparation of monoclonal antibody after enhanced immunization.（2）A monoclonal cell strain 2G4C7 which can specifically recognize fenpyroximate was screened by hybridoma cell technology.The cell strain was expanded and propagated by in vivo induction in mice,and the icELISA was established after ascites was collected.The mAb 2G4C7 has high specificity and no cross reaction with common acaricides.Based on this antibody,an icELISA method was established.The optimal working range(IC₂₀～IC₈₀)was 4.77～187.28 ng/m L,and the median inhibitory concentration(IC₅₀)value of the method was 30.92 ng/m L.The recoveries were 88.9%～119.1%and 73.3%～115.4%with RSD of 1.1%～7.7%and 3.8%～6.8%in citrus and apples,respectively.Both icELISA and UPLC-MS/MS were used to detect citrus samples,and the results of the two methods were accordant.It turns out that the icELISA method based on mAb 2G4C7 was accurate and efficient,and could be applied to the detection of fenpyroximate residues in citrus and apples,meeting the requirements of high throughput rapid detection in the actual market.（3）An indirect competitive CLEIA for the detection of fenpyroximate was developed based on mAb 2G4C7.In the establishment of CLEIA method,the influences of the chemical luminous plate,the mass concentration of artificial antigen,the dilution ratio of enzyme-labeled secondary antibody,the organic solvent in buffer solution and the p H value of buffer solution on CLEIA method were explored.Under each optimization condition,CLEIA method was established based on mAb 2G4C7,and a standard curve was drawn.The IC₅₀ value of this method was 3.9 ng/m L,which was more than 7 times more sensitive than icELISA method for detecting fenpyroximate.The optimal working range(IC₂₀～IC₈₀)is 0.4～29.8 ng/m L.The recoveries were 78.4%～94.7%and 80.8%～103.9%with RSD of 0.2%～5.1%and 0.4%～4.6%in citrus and apple,respectively.CLEIA and UPLC-MS/MS were used to detect citrus samples at the same time,and the results of the two methods were consistent.The above showed that this CLEIA method for the detection of fenpyroximate residues in citrus and apples was successfully developed,which was sensitive and had important reference value for achieving rapid screening for the detection of fenpyroximate in food.（4）Due to the high sensitivity of mAb 2G4C7 to the hydrolysis product of fenpyroximate（Hapten A）,and the ability of fenpyroximate to be completely hydrolyzed to Hapten A under dichloromethane and trifluoroacetic acid conditions of3:1.An icELISA method for hapten A,a hydrolyzed product of fenpyroximate,was established based on mAb 2G4C7 to achieve indirect detection of fenpyroximate,which significantly improved the sensitivity of icELISA for the detection of fenpyroximate.The IC₅₀ value of this method is 0.34 ng/m L,which is more than 90 times more sensitive than the previous methods for detecting fenpyroximate itself,and the best working range(IC₂₀-IC₈₀)is 0.07～1.49 ng/m L.The recoveries were 77.0%～105.4%in citrus and 72.3%～106.4%in apple,with RSD of 1.4%～5.0%and 0.5%～4.7%, respectively.The correlation R² of icELISA and UPLC-MS/MS for citrus samples was0.98,which showed the consistency of the two methods.The above showed that the icELISA method for the indirect detection of fenpyroximate based on the fenpyroximate hydrolysis product Hapten A was accurate,efficient and sensitive and could be applied to the determination of fenpyroximate residues in citrus and apple,and also provided a new idea to improve the sensitivity of the immunoassay.