Preparation Of Cypermethrin And Deltamethrin Monoclonal Antibodies And Development Of Immunoassays

Deltamethrin and cypermethrin are highly effective pyrethroid insecticides with wide spectrum.They are widely used in agriculture and health insects because of their high insecticidal activity and fast knockdown speed.However,due to its extensive use,residual problems and hazards are increasingly exposed.Therefore,it is of great significance to establish an efficient and rapid detection technology for pyrethroid pesticide residues to ensure the safety of food,ecological environment and human health.Immunoassay for pesticide residues has become a hotspot in the field of pesticide residue detection due to its characteristics of simple operation,high sensitivity,convenience and quickness.Antibody is the core reagent in immunoassay,and its quality directly determines the characteristics of the assay method.Monoclonal antibody（m Ab）is the preferred antibody type for immunoassay because of its high specificity,sensitivity and stability.Enzyme-linked immunosorbent assay（ELISA）and gold immunochromatography strip（GICS）are the two most commonly used analytical methods,which are widely used in food,chemical,medical and other detection fields.At present,no m Abs have been reported for cypermethrin,and a few m Abs have been reported for deltamethrin,but its quality is difficult to meet the actual testing needs and needs to be improved.In response to this problem,this study prepared m Abs against cypermethrin and deltamethrin,and evaluated its application effects in the enzyme-linked immunoassay and colloidal gold immunochromatographic,which provide reference for the development of rapid detection methods.The main findings are as follows:After antigen immunization of mice and cell fusion experiments,8 cell lines that stably secrete cypermethrin m Ab,and 9 cell lines that stably secrete deltamethrin m Ab were obtained.The anti-cypermethrin antibody secreted by the cell line 2G7H9 and the antibody anti-deltamethrin secreted by 4D4E11 showed the highest sensitivity by the determination of the cell culture supernatant.IC₅₀values of cypermethrin and deltamethrin were 19 ng/m L and 25 ng/m L,respectively.These two cell lines were selected to prepare ascites and purified to obtain m Abs.The subtypes of cypermethrin and deltamethrin m Abs were Ig G2b,and the affinity constants were 3.64×10⁸L/mol and 1.17×10⁸L/mol,respectively.Based on the m Abs of cypermethrin and deltamethrin,indirect competitive ELISAs（ic-ELISAs）were established to detect cypermethrin and deltamethrin,respectively.In order to make the established ic-ELISA with better sensitivity,8 kinds of coating agents were used as the homologous or heterologous coating agents（H1～H8-BSA）.The best combinations of coating antigen and antibody were selected on the basis of IC₅₀values.After the optimization of the working buffer conditions,a standard curve was established,and the sensitivity,specificity and accuracy of the method were evaluated.The optimized conditions of the ic-ELISA for cypermethrin were H5-BSA as the coating antigen,the coating concentration of 0.5μg/m L,and the antibody concentration of 0.25μg/m L.The sodium ion concentration,p H and methanol content（organic solvent）were 0.3 mol/L,7.4and 10%,respectively.Under the optimized conditions,the IC₅₀,limit of detection（LOD）and linear range(IC₁₀-IC₉₀)were 2.49 ng/m L,0.40 ng/m L and 0.40～7.87 ng/m L,respectively.Cross-reactivity（CR）experiment showed that the ic-ELISA for cypermethrin was no obvious CR with the analogues of cypermethrin except for cyfluthrin,cyhalothrin and fenvalerate.The average recoveries of the samples at the spiked concentrations of100～400 ng/g were 78.8%～87.6%,and the ic-ELISA had good correlation with high performance liquid chromatography（HPLC）in authentic sample detection.For deltamethrin ic-ELISA,the optimal conditions were H1-BSA as the coating antigen,the coating concentration of 0.25μg/m L,and the antibody concentration of 0.5μg/m L.The sodium ion concentration,p H and methanol content were 0.2 mol/L,7.4 and 20%,respectively.Under the optimized conditions,IC₅₀,LOD and linear range were 10.70ng/m L,5.9 ng/m L and 1.58～57.12 ng/m L,respectibely.CR showed that the specificity of the ic-ELISA for deltamethrin was strong because the ic-ELISA was no obvious CR with the analogues.The average recoveries of the samples at the spiked concentrations of150～600 ng/g were 79.8%～92.6%.In addition,the ic-ELISA had good correlation with HPLC in the detection of authentic samples.The ic-ELISAs established in this study have high sensitivity and high accuracy,which can be used for the rapid detection of cypermethrin and deltamethrin residues in tomato,lettuce,and cabbage samples.The GICSs for cypermethrin and deltamethrin were developed by using the anti-cypermethrin and anti-deltamethrin-based m Abs and the optimal coating agents through optimizing the main parameters of the GICSs and the conditions of the working buffer.Among them,the optimal working buffer conditions for cypermethrin GICS were p H 7,0.01 mol/L phosphate buffer containing Tween-20 and 10%DMSO.Under the optimal conditions,the LOD was 0.1μg/m L.The GICS for cypermethrin showed CRs with cyhalothrin and cyfluthrin.At the spiked concentrations of 0.2～2μg/g,the detection results of the GICS were consistent with the spiked concentrations in the samples of lettuce,cabbage and tomato.Compared with the maximum residue limits（MRLs）of cypermethrin in the foods,the result was indicated that the GICS could be used for the determination of cypermethrin in the samples of lettuce,cabbage and tomato.For deltamethrin GICS,the optimal working buffer conditions were p H 7.4,0.01 mol/L phosphate buffer containing Tween-20,1mol/L Na⁺and 10%methanol.Under the optimal conditions,the LOD was 0.5μg/m L.The GICS for deltamethrin showed no CR with the analogues.At the spiked concentrations of 1～5μg/g,the detection results of the GICS were consistent with the spiked concentrations in the samples of lettuce,cabbage and tomato.Compared with the MRLs of deltamethrin in the foods,the result was indicated that the GICS could be used for the determination of deltamethrin in the sample of lettuce.Although the developed GICSs show lower sensitivity than ic-ELISAs,the GICSs have the advantages of simple and fast operation,and they are very suitable for the on-site rapid detection of a large number of samples.