狂犬病病毒抗体竞争ELISA检测方法的建立及初步应用

Development and Preliminary Application of Competitive ELISA Method for Rabies Virus Antibody

目的建立狂犬病病毒抗体竞争ELISA检测方法,用于不同株狂犬病疫苗免疫后抗体水平的检测。方法用市售的不同狂犬病疫苗株抗原免疫NIH小鼠,制备免疫血清,用不同的疫苗株抗原包被酶标板,分别测定小鼠血清抗体效价,并通过不同株抗原抗体的交叉中和实验,分析抗原的差异性;在此基础上,将不同毒株抗原按不同比例混合包被酶标板,分别测定阳性血清样本,并与快速免疫荧光抑制试验(RFFIT)检测结果比较,确定包被抗原模式,建立狂犬病病毒抗体竞争ELISA检测方法,绘制标准曲线,确定最佳定量范围及灵敏度,确定Cut-off值;并对其特异性、精密性、准确性及稳定性进行验证;用建立的方法与其他市售狂犬病病毒抗体检测试剂分别检测血清样品,分析检测结果的一致性及相关性。结果狂犬病病毒抗原AG∶CTN-1=4∶1为最适包被抗原模式,试剂经优化后,检测抗体效价范围在535.00～33.44 mIU/ml之间,具有良好的线性关系(r>0.99),最低检出限为8.36 mIU/ml,Cut-off值为0.735 mIU。该方法检测人血清白蛋白、破伤风阳性血清、乙肝表面抗原阳性血清、白喉阳性质控血清均未发生反应;试验内变异系数在6.68%～7.84%之间;回收率在97.25%～104.50%之间;试剂置37℃3 d,与4℃保存试剂测定结果差异无统计学意义(P>0.05)。该方法检测血清样品与市售狂犬病病毒抗体检测试剂比较,均具有良好的一致性;与RFFIT测定结果比较,二者差异无统计学意义(P>0.05),回归方程为Y=-0.475+3.246 X,相关系数为0.801。结论已建立了狂犬病病毒抗体竞争ELISA检测方法,可用于大规模狂犬病病毒抗体的筛查。

Objective To develop a competitive ELISA method for rabies virus （RV） antibody and use for determination of antibody levels after immunization with of rabies vaccines prepared from various virus strains. Methods NIH mice were immunized with the antigens of commercial rabies vaccine prepared with various strains, and the obtained immune sera were determined for antibody titer by using the EIA plate coated with the antigens of various vaccine strains. The antigen diversity was analyzed by cross neutralization test of antigens and antibodies of various strains, based on which the EIA plate was coated with antigens of various strains mixed at different ratios to determine positive serum samples, and the results were compared with those by rapid fluorescent focus in- hibition test （RFF1T） to determine the coating antigen, and a competitive ELISA method for RV antibody was developed, of which the optimal quantitative detection range and sensitivity were determined by plotting a standard curve, the cut-off value was determined, and the specificity, precision, accuracy and stability were verified. Serum samples were determined by the developed method, and the results were compared with those by other commercial kits for RV antibody to analyze the consistence and correlation. Results The coating pattern was optimized as mixing the antigens from AG and CTN1 strains at a ratio of 4 ： 1. After optimization, the linear detection range of the developed kit for antibody titer was 535. O0 33. 44 mlU/ml （r 〉 O. 99）, while the minimum detection limit and cut-off value were 8. 36 mlU / ml and 0. 735 mlU respectively. All the detection results of human serum albumin, tetanus positive serum, HBsAg positive serum and diphtheria positive quality control serum by the developed method were negative. The intervariation coefficient of detection result by the developed method was 6. 68% ～ 7. 84%, while the recovery rate was 97. 25%～104. 50%. The detection results by the developed kit after storage at 37℃ for 3 d showed no significant difference with that after storage at 4℃（P 〉 0.05 ）. The detection results of serum samples by the developed kit were consistent with those by commercial kits, which showed no significant difference with those by RFFIT （P 〉 O. 05）, with a regression equation of Y = -0. 475 ＋ 3. 246X and a correlation coefficient of 0. 801. Conclusion A competitive ELISA kit for RV antibody was developed, which might be used for screening of RV antibody in a large quantity.