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目的建立呼吸道合胞病毒(respiratory syncytial virus,RSV)抗体效价的3种检测方法,并进行比较。方法棋盘滴定法优化ELISA法的包被抗原及酶标二抗的浓度,并检测该方法的线性范围及重复性;同时建立微量细胞中和试验法和蚀斑减少中和试验法,检测两种方法的重复性。用建立的3种方法及市售RSV抗体ELISA检测试剂盒同时检测55份健康人血清样本,并将两种ELISA法与两种中和抗体效价检测方法间的相关性进行两两比较。结果 ELISA法中包被抗原为10μg/ml及HRP标记的山羊抗人Ig G稀释度为1∶30 000时是最佳组合;在38.00~2.375 U/ml范围内,抗体浓度与A_(450/630)值呈良好线性关系,R~2=0.981 4;高、中、低3个浓度样本的变异系数(CV)均<10%。微量细胞中和试验7 d后,细胞病变明显,细胞成片脱落;连续6次检测抗RSV多克隆抗体中和效价的CV值为4.2%。RSV感染Vero细胞7 d后出现明显的蚀斑形态,结晶紫染色后蚀斑边缘清晰;连续6次检测兔抗RSV抗血清效价的CV值为6.81%。本实验建立的ELISA法与市售ELISA试剂盒的相关系数(R)为0.787,微量细胞中和试验与蚀斑减少中和试验的R为0.937。结论成功建立了3种RSV抗体效价检测方法,本实验建立的ELISA法与市售ELISA试剂盒间及两种中和抗体检测方法间具有良好的相关性。
OBJECTIVE To establish three detection methods for the antibody titer of respiratory syncytial virus (RSV), and to compare them. Methods The substrate titration method was used to optimize the concentration of coated antigen and enzyme-labeled secondary antibody in ELISA. The linear range and repeatability of the method were also tested. At the same time, the method of neutralization of microtubules and neutralization of plaque was established, Method repeatability. Three healthy human serum samples were tested simultaneously by the three established methods and the commercially available RSV antibody ELISA kit, and the correlations between the two ELISA methods and the two neutralizing antibody titers were compared. Results The best combination of 10μg / ml antigen and HRP-labeled goat anti-human Ig G was 1:30 000. The antibody concentration was in the range of 38.00-2.375 U / ml with A 450 / 630), R ~ 2 = 0.981 4. The coefficient of variation (CV) of high, medium and low concentration samples were all less than 10%. After 7 days of micro-cell neutralization test, the cytopathic effect was obvious and the cells became exfoliated. The CV value of neutralizing titers of anti-RSV polyclonal antibody for 6 times in a row was 4.2%. RSV infected Vero cells 7 days after obvious plaque morphology, crystal violet staining plaque edge clear; six consecutive detection of rabbit anti-RSV antiserum titers CV value of 6.81%. The correlation coefficient (R) between ELISA and commercially available ELISA kit established in our experiment was 0.787, and the R value of neutralization test and plaque reduction neutralization test was 0.937. Conclusion Three RSV antibody titer tests were successfully established. The ELISA assay established in this study has good correlation with the ELISA kit and the detection of two neutralizing antibodies.