1. Key Laboratory of Western Characteristic Biological Resources Protection and Utilization, Ministry of Education, Ningxia University, Yinchuan 750021, China; 2.Institute of Special Wild Economic Animals and Plants, Chinese Academy of Agricultural Sciencea, Changchun 130112, China
Abstract：In order to prepare monoclonal antibodies against Lipopolysaccharide, we extracted and purified LPS by phenol water method and cold methanol secondary precipitation method. The concentration of LPS was measured by the lipopolysaccharide quantitative detection kit, and the purity of LPS was detected by SDS-PAGE and silver staining. We used a dose-purified LPS subcutaneous injection to immunize Balb/c mice, and detected the serum titer of the immunized mice by indirect ELISA. Then, the mice with the highest titer were selected for intraperitoneal injection. We obtained cell fusion of spleen cells from immunized mice and myeloma cells SP2/0 under the action of PEG1500, and screened positive hybridoma cell lines by indirect ELISA. A 10-week-old female Balb/c mouse was intraperitoneally injected with a positive hybridoma cell line, and the ascites was collected after the abdomen of the mouse was significantly inflated. The monoclonal antibodies obtained from the collected ascites were subjected to titer analysis, subclass determination, purity detection and specificity test. The results showed that the concentration of LPS was 1 mg/ml with high purity. The affinity constants of the two monoclonal antibodies were 1.12×109 and 1.11×109, with the titers of 1∶6400 and 1∶12800 respectively. The subclasses for the two monoclonal antibodies were G3 and G1, with the same κ type light chain and greater purity than 90%. And both monoclonal antibodies can be captured by Brucella A, M antigen sites. The results of immunofluorescence assay showed that the selected monoclonal antibodies had good reactogenicity, and immunofluorescence was observed after incubation with Brucella 16M, Brucella 2308 and Brucella S2 for 1 hour. In the conclusion, two monoclonal antibodies against Brucella 16M LPS were successfully obtained, with high potency, good purity, high sensitivity and strong reactivity for the subsequent characteristics study. It will be also helpful for the development of Brucella rapid pathogens test kits and antibody detection methods.
2019, Vol. 35 
