| As an important pesticide, synthetic pyrethroids are widely applied in agriculture andhygiene industry. Many toxicological studies indicate that synthetic pyrethroids are potentiallyharmful to environment and human health. Therefore, it is highly necessary to establishvarious detection techniques of synthetic pyrethroids. Immunoassays for pyrethroiddetermination have many advantages including rapid, high-throughput, multi-residues andon-site detection. Therefore, immunoassay has attracted increasing attention in the field ofsynthetic pyrethroid detection.In this paper, six general haptens for synthetic pyrethroids were designed. ThenDiscovery Studio2.5and Gaussian04software were empolyed to evaluate the theoreticalgeometries and electronic distributions of pyrethroids and haptens. Hapten1that had matchedgeometries and electronic properties with the analytes was chosen as an immunizing hapten.Other haptens were selected as coating haptens. In addition, two specific haptens forcyhalothrin were designed and synthesized in the present study.All haptens were conjugated with various carrier proteins containing keyhole limpethemocyanin, bovine serum albumin and ovalbumin to prepare antigens. Then these conjugateswere characterized with a UV-visible spectrometer and used to inject BALB/c mice. Ten daysafter the last immunization, the titre and inhibition of antisera were tested by indirectcompetitive enzyme-linked immunosorbent assay. Finally, the mouse producing antisera withthe highest titer and the lowest50%inhibition concentration was sacrificed for monoclonalantibody preparation. The process of cell fusion and screening referred to the classic measure.The chosen hybridomas which could steadily secrete anti-pyrethroids (or anti-cyhalothrin)antibody were obtained successfully and ascites were produced using in vivo induction. Theaffinity constant of broad-specificity monoclonal antibody3E9against pyrethroids wascalculated to be3.0108L/mol. The isotype of antibody3E9based on the commercial kitindicated that the heavy chain belongs to IgG1while the light chain is Kappa. The affinityconstant of monoclonal antibody2C8against cyhalothrin was calculated to be2.8106L/mol.The subtype of antibody2C8was characterized that the heavy chain is IgG2bwhile the lightchain is Kappa.Several important parameters that influenced assay performance were optimizedincluding coating antigens and their concentration, ionic strength, pH, organic solvents andtheir contents in assay buffer. Under the optimized conditions, six pyrethroids were highercross-reactivity with ascites3E9and their inhibition curves were plotted with IC50value of1.660.76ng/mL for cypermethrin,14.031.68ng/mL for fenpropathrin,45.764.07ng/mLfor esfenvalerate,191.811.2ng/mL for bifenthrin,199.610.75ng/mL for deltamethrin,298.515.08ng/mL for fenvalerate. In the spiked recoveries, the average recoveries were77.3%-111.3%for the fortified samples. The coefficient of variation values were less than15%.Several key factors that affected system stability and the sensitivity of test strip wereoptimised such as pH, antibody amount and the concentration of coating antigen. Based on the obtained anti-pyrethroids monoclonal antibody, the immunochromatographic test strip formulti-pyrethroids analysis was prepared using gold nanoparticals as a detector probe, and thetest strip for cypermethrin is with the detection limit of12.86ng/mL by aid of the portablereader, And the detection limits are of60ng/mL for cypermethrin,200ng/mL forfenpropathrin and400ng/mL for esfenvalerate with naked eyes.Under the optimized chromatographic conditions, the diastereomers of cypermethrin andfenvalerate have firstly been resolved on an HP silica column, and then injection of separateddiastereomers onto Sino-Chiral OD column were ultimately separated completely withreasonable retention times (60min). The order of elution was established and eachconfiguration was identified by comparison with the related literatures. Under the sameconditions, injection of fenpropathrin onto the Sino-Chiral OD chiral system achievedexcellent separation of the two enantiomers. The stereoselectivity of antibody3E9wasdetermined by comparing standard inhibition curves of fourteen isomers from cypermethrin,fenvalerate and fenpropathrin. Results demonstrated that only two of cypermethrin isomer6(CP6,1S3S S) and isomer7(CP7,1S3S R), one of fenpropathrin isomer2(FP2, S) andfenvalerate isomer1(FV1, S S) were detected, respectively and the general monoclonalantibody aganist CP7was higher sensitive than CP6.Based on cross-reactivity results of anti-pyrethroid monoclonal antibody, threequantitative structure-activity relationship (QSAR) models were constructed including2D-QSAR (two dimensional QSAR) model, HQSAR (hologram QSAR) model and topomerQSAR model. According to the2D-QSAR model, there was a significant correlation betweenantibody activity and pyrethroid hydrophobicity, specifically, synthetic pyrethroids could berecognized more easily when the hydrophobicity was weaker. From the substructure levelanalysis using HQSAR model, fragments of hapten linked with carrier protein could play animportant role on antibody recognition. Both of models showed highly predictive abilitieswith cross-validation coefficient q2values of0.92, respectively. According to topomer QSARmodel, the cross-reactivity of antibody3E9can be explained by calculating the steric andelectrostatic contours of fragments R2.To establish the enzyme-linked immunosorbent assay for cyhalothrin, coating antigensand their concentrations, blocking agent, ionic strength, pH value and the content of organicsolvent in assay buffer were optimized. Under the optimized conditions, an inhibitionstandard curve for cyhalothrin was plotted with the IC50value13.261.23ng/mL and with thedetection limit of1.83ng/mL. The monoclonal antibody2C8manifested good specificity tocyhalothrin with little cross-reactivity (<5%) only to tau-fluvalinate or cyphenothrin. Therecoveries were more than75%and the coefficient of variation value was less than12%. |
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