| Along with the increasing level of technology, the use of pesticides has become an indispensable and important link of agriculture. Through pest control and improve agricultural production, pesticides improve the quality of people's lives. At present, China has become the world's second largest pesticide user states. The annual consumption is estimated at 30 million tons. In recent years, with the increasing of pest resistance, the frequency and concentration of pesticides use is Continue to improve. This leads to a large number of residues of harmful substances remain in the environment. What's more, the pollution was expanded through the food chain, and affects the ecological balance of the environment. The accumulation and enrichment of pesticides in the human body can cause various diseases, such as cancer, which direct impact on human health. The problems caused by the pesticide residue have been attracting people's attention. Therefore, it is imperative to establish a method which is simple, rapid and sensitive to detect pesticide residues.Currently, Bensulfuron-Methyl (BSM) is a king of herbicide which is most widely used in rice filed. However, long-term, large-scale use is bound to produce residues in the environment, and endanger the security of the entire ecosystem. Therefore, the real-time monitoring of this herbicide is necessary. At present, the detection method of BSM residues is chromatography method. While chromatography is sensitive and accurate, but it has some drawbacks, such as time-consuming, procedure-complicated and high cost and so on. In contrast, as an immunological method, the ELISA techniques make up for the shortcomings of chromatography. It is not only simple, rapid, sensitive and can detect large quantities of samples at the same time. Therefore, it has been more and more used to detect pesticide residues in the environment.In this study, the glutaraldehyde was used as coupling agent to prepare artificial cross-linking antigen (BSM-OVA) for BSM. Different way was used to identify the antigens were prepared successfully. Then, the BALB/c mice were immunized with immunogen.7 days after the five times immunization, the titers of antiserum was 1:1.28×105 which detected by indirect ELISA, and the value of IC50 was 0.032μg·mL-1 determined by Indirect competitive ELISA. PEG-mediated method was used to fuse the immunized spleen cells and SP2/0 myeloma cells. After screening, one hybridoma cell line which could uniformly secrete monoclonal antibody of BSM was got. We numbered this cell line as 2H1, and the type of antibodies is IgG2b which detected by capture ELISA.Induced in vivo ascites method and caprylic acid-ammonium sulfate methods were used to prepared and purify monoclonal antibodies of BSM. The indirect competitive ELISA was used to determine the best working conditions of the monoclonal antibodies of BSM. Under the optimal conditions, the indirect competitive ELISA method which used monoclonal antibodies to detect the residues of BSM was established. The standard curve of indirect competitive ELISA to detect the herbicide residues was got. The regression equation is y=13.816x+34.753, the detection range is 1～1883ng·mL-1, and the minimum detection limit is 0.2ng·mL-1. Added concentration of 10-1～103ng·mL-1 of BSM to the East Lake water, the recovery deyected was 96.53%～107.2%, the coefficient of variation value was 3.711%～8.637%. These dates show that the method we have established has a high accuracy and sensitivity for detection of BSM residues. We found BSM residues in some samples when the method was used to detect the residue value of BSM in some grains crop samples.This study explores a new way using immunological method to detect the residues of BSM. Compared with the traditional method, the best feature of this method is rapid, sensitive, and can detect samples in large quantities at the same time. |
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