联合酶联免疫的微量病毒中和法检测大流行流感疫苗血清中和抗体的可行性研究

目的: 建立联合酶联免疫的微量病毒中和法(ELISA-MVN法),用于大流行流感疫苗流感病毒中和抗体效价的检测,并进行方法学验证。方法: 采用4种羊抗流感病毒血清参考品,与人感染禽流感H5N1病毒疫苗株(A/Vietnam/1194/2004-NIBRG-14)病毒液,分别于37 ℃孵育18 h后,固定细胞,ELISA检测细胞内流感病毒核蛋白表达,确定其中和抗体效价,考察该方法的特异性;通过对高中低不同抗体滴度的血清样本的多次平行检测来评价该方法的重复性;通过ELISA-MVN法和血凝抑制试验(HI试验)分别检测大流行流感疫苗接种者血清样本,比较2种方法的灵敏性和相关性。结果: ELISA-MVN结果显示羊抗H5N1的血清中和抗体检测滴度为5 120,其他3种血清的中和抗体滴度小于10,该方法特异性良好;采用ELISA-MVN法对3种不同滴度的血清进行重复检测,组内重复性试验的6次平行试验结果的RSD为3.5%。组间重复性5次结果,RSD为4.8%~8.6%。2种方法测定疫苗接种者血清样本抗体效价的结果具有显著相关性(P<0.001),相关系数r=0.932。ELISA-MVN法检测结果的几何平均滴度高于HI试验检测结果,该方法较HI试验在检测大流行流感疫苗血清样本灵敏度高。结论: ELISA-MVN可满足大流行流感疫苗血清学检测的要求,并可用于评价大流行流感疫苗的免疫效果。

Objective: To develop and validate the enzyme linked immunosorbent assay-based micro-virus neutralization (ELISA-MVN) tests for investigating the immune responses to vaccination against influenza A (H5N1) virus. Methods: Specificity of MVN assay was tested for the purified hemagglutinin of the viruses incubated with influenza H5N1 at 37 ℃ for 18 h with sheep antisera as reference substances.After the cells were fixed, the expression of influenza nucleoprotein was tested by ELISA for determination of neutralizing antibody titer in sera.Reproducibility was evaluated by parallel testing of serum samples at high, medium and low antibody titers.ELISA-MVN assay and hemagglutination inhibition (HI) assay were separately used to test the antibody potencies in human sera immunized with influenza H5N1 (A/Vietnam/1194/2004-NIBRG-14) vaccine to evaluate and compare the precisions and correlation of the two assay methods. Results: ELISA-MVN method is specific for sheep antisera to influenza H5N1 virus with a titer of 5 120 and showed negative reaction for all other three antisera (the titers were less than 10), which manifested good specificity.The relative standard deviation of inter-reproducibility was 3.5% for 6 parallel tests of sera at different titers by ELISA-MVN, the RSD for intra-reproducibility assessed by 5 tests was 4.8%-8.6%.There was a significantly positive correlation between the results by these two methods for testing antibody titers of sera from vaccine inoculators (P<0.001), with the correlation coefficient of 0.93.The antibody titers measured by ELISA-MVN were higher than that by HI assay, reflecting higher sensitivity of ELISA-MVN. Conclusion: The established ELISA-MVN method can meet the serum detection requirements of pandemic influenza vaccine, which can be applied for assessing the immune effects of pandemic influezna vaccine.