| Benzothiostrobin, a fungicide developed by the Central China Normal University, is a novel strobilurin fungicide which has highly efficient controls of most fungal diseases in crops. In order to further study the safe use of benzothiostrobin, residual control and determination of the maximum residue limits (MRL), this paper has developed a HPLC method for the quantitative determination of benzothiostrobin and two immunoassays based on anti-benzothiostrobin MAb.The content of benzothiostrobin was determined quantitatively by HPLC with SB-C18 column at UV 230 nm, and acetonitrile-trifluoroacetic acid (65:35, V/V) as the mobile phase at a flow rate of 0.8 mL/min. The assay exhibited a good linearity in benzothiostrobin concentration range from 0.1 to 10 mg/L with a correlation coefficient of 0.9999. The standard deviation was 0.0172, the variation coefficient was 0.332% and the average recovery was 102.7%.The hapten of benzothiostrobin was synthesized and conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) to generate immunogen (hapten-BSA) and coating antigen (hapten-OVA). The conjugation confirmed by UV-Vis spectroscopy showed that the carrier protein and hapten had been coupled successfully. The molar ratios were estimated as 17:1 and 8:1 for immunogen and coating antigen, respectively. The immunogen was used to immunized BALB/c mice and obtained the anti-benzothiostrobin MAb. When the concentration of coating antigen was 4 mg/L, the titer of benzothiostrobin antibodies was 6.3×106 by using the indirect noncompetitive enzyme-linked immunosorbent assay (ELISA). An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed to detect benzothiostrobin pesticides using a specific MAb antibody. A variety of conditions including the working concentration, organic solvent, ionic intension and pH value on the affinity of benzothiostrobin to antibody were determined to optimum assay conditions, subsequently. Under optimal conditions (5% methanol,0.1 M Na+, pH 5.5), the half-maximal inhibition concentration (IC50) and the limit of detection (IC10) of the developed ic-ELISA were 7.55 and 0.428 μg/L, respectively. The cross-reactivities (CR) were less than 0.05% with tested analog compounds and regarded as negligible except pyraclostrobin with 0.34%. The recoveries of benzothiostrobin ranged from 80.43 to 113.83% in environmental and agricultural samples, conformed to the requirements of residue detection. The correlation of ELISA and high-performance liquid chromatography (HPLC) was satisfactory in authentic samples detection. The present study indicates that the ic-ELISA established is a potentially useful tool for detecting benzothiostrobin in environmental and agricultural samples.Based on the gold-conjugated MAb and an optimal antigen, a simple and rapid (5 min) immunochromatography assay (ICA) for detection of benzothiostrobin in different agricultural products was developed. On the optimized condition,100% inhibitory concentration of ICA established is 0.1 mg/L and the LOD is 0.05 mg/L. The selectivity of the ICA was evaluated by measuring its cross-reactivity (CR) with 7 strobilurin fungicides, and the results showed the ICA had specificity for the benzothiostrobin. Comparing the result of spike recoveries of five kinds of spiked samples (Paddy water, rice, soil pears, tomatoes) and the real samples (pear and soil) with HPLC, the results show that the real concentration of samples and the real phenomenon of the test strip are consistent and it can be used to detect benzothiostrobin in agricultural products. Thus, the method for detecting benzothiostrobin is simple, rapid, high sensitivity, less special equipment and Solutions, low cost, less demanding quality of personnel and better promotion prospects. |
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