呼吸道合胞病毒抗体效价3种检测方法的建立及比较

Development and comparison of three methods for determination of antibody titer against respiratory syncytial virus

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 develop and compare three methods for determination of antibody titer against respiratory syncytial virus （RSV）. Methods The concentration of coating antigen and enzyme-labeled the secondary antibody in ELISA were optimized by chessboard titration, and the method was determined for linear range and reproducibility. Meanwhile, micro-neutralization test and plaque reduction neutralization test were developed and determined for reproducibility. Fifty- five healthy human serum samples were determined by the developed three methods and commercial ELISA kit for RSV antibody separately, and the correlations between the test results were compared. Results The optimal concentration of coating antigen and optimal dilution of HRP-labeled goat anti-human IgG were 10 p,g/ml and 1 ： 30 000 respectively. The antibody concentration at a range of 38. 00 - 4. 375 U/ml showed good linear relationship to A 45o/63o value, with a R2 value of 0. 981 4. All the coefficients of variation （CVs） of determination results of samples at high, moderate and low concentrations were less than 10%. Obvious CPE appeared 7 d after micro-neutralization test, while the confluent cells fell off. The CV of neutralizing titer of polyclonal antibody against RSV in six consecutive tests was 4. 2%. Obvious plaques were observed in Vero cells 7 d after infection with RSV, of which the borders were clear after crystal violet staining. The CV of antiserum titer against RSV in six consecutive tests was 6. 81%. The correlation coefficient （R） of determined results by the developed ELISA and the commercial ELISA kit was 0. 787, while that by micro-neutralization test and plaque reduction neutralization test was 0. 937. Conclusion Three methods for determination of antibody titer against RSV were developed successfully, and good correlations were observed between the determination results by the developed ELISA method and commercial ELISA kit and between those by micro-neutralization test and plaque reduction neutralization test.