竞争抑制酶联免疫吸附法快速检测金黄色葡萄球菌肠毒素P

Rapid determination for establishment of competitive inhibition enzyme linked immunosorbent assay for staphylococcal enterotoxin P

中文关键词:  单克隆抗体  酶联免疫吸附法  金黄色葡萄球菌  金黄色葡萄球菌肠毒素P

英文关键词:monoclonal antibody  enzyme linked immunosorbent assay  Staphylococcus aureus  staphylococcal enterotoxin P

中文摘要:

      目的 建立竞争抑制酶联免疫吸附法(enzyme linked immunosorbent assay, ELISA)快速检测金黄色葡萄球菌肠毒素P (staphylococcal enterotoxin P, SEP)的分析方法。方法 根据ELISA的检测程序, 利用交叉方阵滴定法确定SEP抗原的最佳包被浓度及单克隆抗体的最佳倍比稀释浓度, 再对辣根过氧化物酶标记的羊抗鼠IgG (HRP-IgG)最佳稀释浓度及最佳反应时间进行筛选, 然后通过测定450 nm处不同SEP抗原包被条件(包被液类型、包被环境)、封闭液类型及浓度、封闭时间、竞争反应温度、反应方式(预混反应、直接反应)下的OD值对检测条件进行优化, 最后用灵敏度、批内变异、批间变异和加标样品回收率对方法进行评价。结果 SEP抗原最佳包被浓度为2.500 μg/mL, 鼠单抗血清稀释度1:6000 (V:V), 酶标抗稀释度1:3000 (V:V); 反应1 h, 最佳包被条件为磷酸盐缓冲液4 ℃过夜, 10%脱脂乳封闭2 h, 37 ℃直接竞争反应。此方法的线性回归方程为: Y=0.4166X-0.7415 (r2=0.9908), 灵敏度为0.954 μg/mL, 批内及批间变异系数均低于3%, 对人工污染的脱脂牛奶、LB (Luria-Bertani)液体培养基中肠毒素蛋白SEP的回收率高于98%。结论 该方法具有较好的回收率和稳定性, 可应用于肠毒素蛋白SEP的快速检测。

英文摘要:

      Objective To establish a method for the rapid determination of staphylococcal enterotoxin P (SEP) by competitive inhibition enzyme linked immunosorbent assay (ELISA). Method According to the detection procedure of ELISA, the optimal coating concentration of SEP antigen and the optimal dilution concentration of monoclonal antibody were determined by cross-square titration method. The optimal dilution concentration and optimal reaction time of goat anti-mouse IgG (HRP-IgG) labeled with horseradish peroxidase were screened. Further, the detection conditions by measuring the absorbance under different SEP antigen coating conditions (coating solution type, coating environment), blocking solution type and concentration, blocking time, competitive reaction temperature, and reaction mode (premix reaction, direct reaction) at 450 nm were optimized. Finally, the method was evaluated by sensitivity, intra-assay variation, inter-assay variation, and recovery of spiked samples. Results The best coating concentration of SEP antigen was 2.500 μg/mL, and anti-SEP monoclonal antibody was 1:6000 (V:V). The best dilution of enzyme-labeled antibody was 1:3000 (V:V), and the reaction time was 1 h. The best coating conditions were phosphate buffer solution 4 ℃ overnight, and the blocking conditions were 10% degreasing 2 h, 37 ℃ direct competition reaction. The linear regression equation of this method was Y=0.4166X-0.7415 (r2=0.9908), which sensitivity was 0.954 μg/mL. The intra-assay and inter-assay coefficients of variation were less than 3%. The recoveries of enterotoxin protein SEP in artificially contaminated skimmed milk and LB (Luria-Bertani) liquid medium were higher than 98%. Conclusion The method has good recovery and stability, and can be applied to the rapid detection of enterotoxin protein SEP.