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目的制备肠炎沙门菌(Salmonella enteritidis,SE)脂多糖(Lipopolysaccharide,LPS)抗原,并鉴定其生物学特性。方法分别用热酚水法和水饱和冷酚法提取SE LPS抗原,免疫BALB/c小鼠,制备LPS单克隆抗体。采用蒽酮-硫酸法测定LPS多糖含量,考马斯亮蓝染色法测定其蛋白含量,SDS-PAGE分析其分子结构,Western blot分析其反应原性,间接ELISA法分析其特异性。结果热酚水法水相LPS粗提物中的多糖和蛋白含量分别约为3.413 mg/ml和46μg/ml,水饱和冷酚法水相LPS粗提物中的多糖和蛋白含量分别约为2.248 mg/ml和31μg/ml;提取的LPS呈现明显的梯度型条带;制备的2株抗LPS单抗(1D3和2E5)均能与提取的LPS发生特异性反应;制备的LPS除了与伤寒沙门菌A～F多价血清发生交叉反应外,与其他各种肠道菌单价或多价血清均未发生反应。结论热酚水法和水饱和冷酚法均可制备出活性、纯度均较好的LPS,但水饱和冷酚法操作简单,且提取的样品纯度高,特异性和反应原性良好,可用于肠炎沙门菌特异性抗原位点单抗的制备及免疫学检测。
Objective To prepare lipopolysaccharide (LPS) antigen of Salmonella enteritidis (SE) and identify its biological characteristics. Methods SE LPS antigen was extracted by hot phenolic water method and water-saturated cold phenol method, respectively. BALB / c mice were immunized to prepare LPS monoclonal antibody. The polysaccharide content of LPS was determined by anthrone-sulfuric acid method, the protein content was determined by Coomassie brilliant blue staining, the molecular structure was analyzed by SDS-PAGE, and the specificity was analyzed by indirect ELISA. Results The contents of polysaccharides and proteins in LPS crude extracts of hot phenol aqueous solution were about 3.413 mg / ml and 46 μg / ml, respectively. The content of polysaccharide and protein in LPS crude extract was about 2.248 mg / ml and 31μg / ml, respectively. The extracted LPS showed obvious gradient-type bands. The two anti-LPS mAbs (1D3 and 2E5) were able to react specifically with the extracted LPS. Fungus A ~ F polyvalent serum cross-reaction, with a variety of other intestinal bacteria monovalent or multivalent serum did not react. Conclusion Both hot phenol and water-saturated chilled phenol can produce LPS with good activity and purity. However, the water-saturated chilled phenol method has simple operation and high purity, specificity and reactogenicity and can be used in Preparation and immunological detection of Salmonella enteritidis specific antigenic monoclonal antibody.