Preparation Of Monoclonal Antibody Immunochromatographic Strip For The Carbendazim Detection

Carbendazim（CBZ）,a benzimidazole-type compound,is commonly used as broad spectrum pesticides in agricultural production.CBZ has shown obvious effect on various fungal diseases,thus can effectively promote the yield and quality of crops.CBZ is hard to be decomposed and can be found in the environment and agricultural products for a long time due to its high chemical stability,and thereby posing a serious threat to food safety and human health.To date,various instrument-dependent methods have been established for the detection of CBZ in various food samples because of their unique merits of good accuracy and high precision.However,these methods are commonly suffered from the expensive instrument,skilled operators,high cost,time consuming and also require professional laboratory conditions,and,thus limiting its applications in large-scale screening and on-site real-time detection.In contrast,immunoassays based on antigen-antibody interation are widely used for rapid and on-site detection of pesticide residues.Among them,immunochromatographic assay（ICA）has been regarded as the most effective point of care method owing to its several merits of simplicity,rapidity,cost-effectiveness and naked-eye interpretention.Therefore,the preparation of anti-CBZ monoclonal antibody and sensitive ICA method for CBZ screening determination are of great significance.Conventional ICA method based on gold nanoparticles（Au NPs）as signal report shows relatively low sensitivity due to its low signal intensity of Au NPs,and thus limiting for the detection of the analytes with trace concentration.Fluorescent materials such as fluorescent microspheres and quantum dot microspheres have been widely used for signal amplification labels for improving the detection performance of ICA.However,the signal intensities of these labels are restricted to the aggregation-caused quenching（ACQ）properties.In contrast,aggregation-induced emission（AIE）dyes doped fluorescent microbeads（AIEFMs）can achieve higher signal intensity via loading more AIE dyes,which is conducive to enhancing the sensitivity of ICA.In addition,the AIEFMs)also has some other optical advantages of large Stokes shift,high photobleaching resistance,and low background interference.In this study,the CBZ immunogen was synthesized,and was used to immunize mices for producing anti-CBZ monoclonal antibodies（m Abs）.First,the diaminobenzimidazole,p-nitrophenol carbonate and aminobutyric acid were used as raw materials to synthesize the carboxyl modified CBZ.Then,the CBZ-hemocyanin（KLH）,CBZ-bovine serum albumin（BSA）and CBZ-ovalbumin（OVA）conjugations were prepared by coupling the carboxyl group of CBZ derivant to the amino groups of KLH,BSA and OVA via a carbodiimine method.The CBZ-KLH conjugations were used to inject the Balb/c mice,and the titers of anti-serums were evaluated by a competing ELISA,and the spleen cells of the mouse with highest titer was selected to fuse with myeloma cells.After subcloning and cell line screening,an anti-CBZ cell strain named 6C5,was successfully obtianed.The m Abs-6C5 was identified as Ig G1type by isotype identification kit.The titer of m Abs-6C5 was achieved at 1:243000and the binding affinity of m Abs-6C5 toward CBZ-BSA was 1.03×10⁸ L mol^-1.Under the optimal conditions,a competing ELISA was developed for the CBZ quantitative determination with a dynamic linearity from 3.13 ng m L^-1to 50 ng m L^-1,The IC₅₀and limit of detection（LOD）values of the proposed ELISA were 13.56 ng m L^-1and 1.92 ng m L^-1,respectively,and the as-prepared m Abs-6C5 exhibited a 5.4%cross-reaction to its analogue of benomyl and a negligible cross-reaction to other common benzimidazoles.Thereafter,the nanoprobes were fabricated by coupling the m Abs-6C5 with the highly luminescent AIEFMs,and the AIEFMs-ICA was developed for the CBZ determination,and the Au NPs-ICA was established for a comparison.Under the optimal conditions,the proposed AIEFMs-ICA can quantify the CBZ in a linearity from 1.56 ng m L^-1to 25 ng m L^-1 with a LOD of 0.75 ng m L^-1,while the dynamic linearity of Au NPs-ICA is in a range of 6.25 ng m L^-1to 50 ng m L^-1 with a LOD of4.78 ng m L^-1,and this data is 6.3-fold higher than that of AIEFMs-ICA.The inter-and intra assays were assassed by analyzing the CBZ spiked lettuce,and the average recoveries of AIEFMs-ICA were in a range of 91%to 112.3%with the coefficient of variation（CV）in a range of 2.5%to 13.2%,indicating an accepted accuracy and precision of the developed AIEFMs-ICA.In addition,the proposed AIEFMs-ICA also showed a good specificity for CBZ determination.Furthermore,,the practicability of proposed AIEFMs-ICA was evaluated by analyzing twenty-one of real lettuce samples fortified by CBZ.The data obtained from AIEFMs-ICA were further compared with ELISA kit,and the results comfirmed that the proposed AIEFMs-ICA can be used for CBZ quantitative determination.