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目的建立甲型副伤寒沙门菌抗体间接ELISA检测方法,并进行验证及初步应用。方法以甲型副伤寒沙门菌体脂多糖(lipopolysaccharide,LPS)作为包被抗原,HRP标记的羊抗小鼠IgG作为二抗,TMB作为显色剂,建立检测甲型副伤寒沙门菌抗体的间接ELISA法,优化间接ELISA法的试验条件,并进行验证。取2批甲型副伤寒结合物原液,分别经NIH小鼠大腿内侧皮下各免疫3次,间隔14 d,第2、3次免疫后7 d采血,按建立的间接ELISA法检测血清中甲型副伤寒沙门菌抗体水平,计算抗体阳转率。结果抗原的最适包被浓度为2μg/ml,血清最适稀释倍数为1∶80,酶标二抗的最适稀释比例为1∶6 000;包被抗原与血清的最适反应时间为2 h,血清与酶标二抗的最适反应时间为1 h,封闭液室温下最适封闭时间为2 h。用建立的方法检测20只小鼠甲型副伤寒沙门菌全菌体超免血清,血清稀释500倍阳性检出率仍不低于80%;检测小鼠甲型副伤寒沙门菌全菌体超免血清的试验内和试验间变异系数分别为4.6%和7.5%,检测阴性血清样品的试验内和试验间变异系数分别为9.4%和13.9%,均低于15%;检测小鼠甲型副伤寒沙门菌全菌体超免血清和甲型副伤寒结合物原液免疫阳性血清的结果为阳性,检测乙型副伤寒结合物小鼠免疫阳性血清、伤寒Vi多糖结合物小鼠免疫阳性血清及阴性血清的结果为阴性。两批甲型副伤寒结合物原液免疫小鼠在第2针免疫后7 d的血清阳转率均为10%,第3针免疫后7 d的血清阳转率均为100%。结论建立的检测甲型副伤寒沙门菌抗体的间接ELISA法灵敏度较高,精密性良好,特异性较强,可用于评价甲型副伤寒结合疫苗的免疫原性。
Objective To establish an indirect ELISA method for the detection of Salmonella paratyphi A antibody, and to verify and preliminary application. Methods The detection of Salmonella paratyphi A antibody was established by using lipopolysaccharide (LPS) as coating antigen, HRP-labeled goat anti-mouse IgG as secondary antibody and TMB as developing reagent ELISA method to optimize the indirect ELISA test conditions, and verify. Take two batches of Paratyphus Paratyphides Conjugate stock solution, were subcutaneously immunized subcutaneously NIH mice three times each subcutaneous immunization at an interval of 14 d, the second and third immunization 7 d after the blood collection, according to the establishment of indirect ELISA method in serum A Paratyphoid Salmonella antibody levels calculated antibody positive conversion rate. Results The optimum concentration of antigens was 2μg / ml, the optimum dilution of serum was 1:80, the optimum dilution ratio of enzyme secondary antibody was 1: 6000, and the optimal reaction time of coating antigen and serum was 2 h, the optimal reaction time of serum and ELISA secondary antibody was 1 h, and the optimal blocking time was 2 h at room temperature. Twenty mice with Salmonella paratyphi A were detected by established method. The positive detection rate of serum-diluted 500 times was still not lower than 80%. Detection of Salmonella paratyphi A The coefficient of variation (CV) in serum-free and intra-assay were 4.6% and 7.5%, respectively. The coefficient of variation (CV) between the two assays were 9.4% and 13.9% Salmonella typhimurium whole cell free serum and Parap
Paratyphoid conjugate liquid positive serum immunization results were positive for Paratyphoid B conjugate immune positive sera, typhoid Vi polysaccharide conjugate immune-positive sera and negative Serum results are negative. The seroconversion rates of the two groups of vaccines against Paratyphoid Conjugate were 7% 7 days after the second immunization. The seroconversion rates of the 3rd immunization were all 100% after 7 days. Conclusion The indirect ELISA method established for the detection of Salmonella paratyphi A antibody has high sensitivity, good precision and strong specificity and can be used to evaluate the immunogenicity of the Paratyphorin A conjugate vaccine.
Paratyphoid conjugate liquid positive serum immunization results were positive for Paratyphoid B conjugate immune positive sera, typhoid Vi polysaccharide conjugate immune-positive sera and negative Serum results are negative. The seroconversion rates of the two groups of vaccines against Paratyphoid Conjugate were 7% 7 days after the second immunization. The seroconversion rates of the 3rd immunization were all 100% after 7 days. Conclusion The indirect ELISA method established for the detection of Salmonella paratyphi A antibody has high sensitivity, good precision and strong specificity and can be used to evaluate the immunogenicity of the Paratyphorin A conjugate vaccine.