| Chemical soil barrier as a kind of effective termite control method,is widely used in termite prevention for new buildings.Bifenthrin,a pyrethroids insecticide and acaricide,which possesses the characteristics of broad-spectrum,high efficiency and strong poison on termites,is one of the most commonly used termite control chemicals in China even all over the world.Currently,instrument-based methods such as gas chromatography(GC)and high performance liquid chromatography(HPLC)to detect bifenthrin have been reported,but the cost is expensive,and the methods are difficult to apply outside the laboratory.To achieve the rapid and on-site detection of bifenthrin,monoclonal antibody against bifenthrin was prepared and a sensitive ic-ELISA and a gold immunochromatographic strip were developed in this study,which provided reliable tools for the rapid detection of bifenthrin in chemical soil barrier.Lambda cyhalthrin acid,2-methyl-3-biphenylmethyanol and bifenthrin were used as materials to synthesize hapten GFd,LBc and LBs,respectively.All the haptens were conjugated to bovine serum albumin(BSA)by active ester method to produce immunogens.The haptens and hapten LBy were conjugated to ovalbumin by mixed anhydride method to produce coating antigens.The formations of the conjugates were confirmed by UV-vis spectroscopy and the molar ratios were estimated.Used the immunogens to immunize BALB/c mice by intraperitoneal injection and chose mice for cell fusion according to their titers.After several times of screening and subcloning,one hybridoma cell line that could stably produce monoclonal antibody(MAb)against bifenthrin was selected,and named 3D4.The MAb produced by 3D4 was IgG1 and kappa type.A sensitive monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay(ic-ELISA)was developed to detect bifenthrin.Experimental parameters such as concentrations of coating antigens and antibodies,coating antigen,blocking solution and PBS buffer were studied to improve the sensitivity of the ic-ELISA.Under the optimum conditions,the calibration curve was constructed.The linear equation and correlation coefficient were B/Bo(%)=-0.3510LogC+0.0454 and R2=0.9903,respectively.The detection range was 0.004-0.70 mg/L.The IC50 and IC10 were 0.05 mg/L and 0.004 mg/L.The specificity was evaluated by measuring the cross reactivities(CRs)with some analogues of bifenthrin,and all the CRs were lower than 0.7%,which meant the mAb had a high specificity for bifenthrin.Loess,red soil and black soil were spiked with bifenthrin standards at 2-10 mg/L.The recoveries were in the range of 83.5-104.7%and the relative standard deviations(RSDs)were 4.8-13.6%.In order to evaluate the correlation between the ELISA and GC-μECD,two methods were used to detect the concentrations of bifenthrin in loess samples.The line equation and correlation coefficient obtained from linear regression of the combined ELISA and GC data for bifenthrin were y=1.0275x-0.2038 and R2=0.9704,respectively.The high degree of linearity indicates that the developed ic-ELISA is reliable and accurate.A gold immunochromatographic strip for the rapid detection of bifenthrin was developed in this study.Experimental parameters such as coating antigens,aperture of nitrocellulose filter membrane,grain size of colloidal gold particle and working buffer(ionic strength,pH value,concentration of PEG 20000 and methanol)were studied to improve the sensitivity of the strip.Under the optimum conditions,the complete inhibition concentration of bifenthrin was 60 mg/L.Although its sensitivity was lower than ic-ELISA,the strip is superior in detecting time and operation steps.A large number of studies have shown that,under the condition of same antigen and antibody,the sensitivity of ELSIA is usually higher than that of strip.The reason is the enzyme marker in ELISA makes the signal amplification.It can also through the silver enhancement method to improve the sensitivity of the gold immunochromatographic strip. |
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