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目的:流感病毒Vero细胞适应株A/Yunnan/1/2005va(H3N2)是一株以Vero细胞为培养基质能高产的病毒株,可用于制备以Vero细胞为基质的流感病毒裂解灭活疫苗;通过在低温下连续传代培养,可选育出流感病毒Vero细胞冷适应株,用于制备Vero细胞流感病毒减毒活疫苗。为了便于对Vero细胞冷适应株的进一步研究,建立一个检测该病毒株的ELISA方法。方法:以流感病毒Vero细胞适应株A/Yunnan/1/2005Va(H3N2)的纯化抗原为免疫原制备羊抗A/Yunnan/1/2005Va(H3N2)和鸡抗A/Yunnan/1/2005Va(H3N2)的抗血清。将抗血清先后经硫酸铵沉淀法和Protein G亲和层析柱纯化后,以纯化的羊抗A/Yunnan/1/2005Va(H3N2)IgG为包被抗体,鸡抗A/Yunnan/1/2005Va(H3N2)IgG为第二抗体,测定包被抗体、第二抗体和酶标抗体最佳工作浓度,建立双抗体夹心ELISA检测方法。结果:羊抗A/Yunnan/1/2005Va(H3N2)IgG的最佳工作浓度为5μg/mL,鸡抗A/Yunnan/1/2005Va(H3N2)IgG的最适浓度为10μg/mL,酶标抗体最佳稀释倍数为1:4000。对已知的阳性样品,经双抗体夹心ELISA法测定的病毒滴度比血凝方法测定病毒滴度灵敏度高。结论:通过测定包被抗体、第二抗体和酶标抗体最佳工作浓度,建立了双抗体夹心ELISA检测流感病毒Vero细胞适应株病毒含量的方法。该方法操作简单、方便快速、敏感性高,可应用于Vero细胞冷适应株选育时对流感病毒Vero细胞适应株A/Yunnan/1/2005va(H3N2)的检测,对于研制Vero细胞流感减毒活疫苗有重要意义。
OBJECTIVE: The influenza virus Vero cell adapted strain A / Yunnan / 1 / 2005va (H3N2) is a strain capable of high yielding with Vero cell culture medium, which can be used for preparing Vero cell-based influenza virus inactivated inactivated vaccine; Continuous subculture at low temperature, optional breeding of influenza virus Vero cells cold adapted strain for the preparation of live attenuated Vero cell influenza virus vaccine. In order to facilitate further research on Vero cell cold-adapted strains, an ELISA method for detecting the strain was established. Methods: The sheep anti-A / Yunnan / 1 / 2005Va (H3N2) and chicken anti-A / Yunnan / 1 / 2005Va (H3N2) were immunized with the purified antigen of influenza virus Vero cell adapted strain A / Yunnan / ) Antisera. After the antiserum was purified by ammonium sulfate precipitation and Protein G affinity chromatography, the purified goat anti-A / Yunnan / 1 / 2005Va (H3N2) IgG was coated with anti-A / Yunnan / 1 / 2005Va (H3N2) IgG as the second antibody to determine the optimal working concentration of coated antibody, secondary antibody and ELISA antibody. Results: The optimum working concentration of goat anti-A / Yunnan / 1 / 2005Va (H3N2) IgG was 5μg / mL, and the optimum concentration of anti-A / Yunnan / The best dilution factor is 1: 4000. For known positive samples, the titer of the virus as measured by the double antibody sandwich ELISA is higher than the titer of the virus determined by the hemagglutination assay. Conclusion: The method of double-antibody sandwich ELISA to detect the virus content of influenza virus Vero cell adapted strains was established by measuring the optimal working concentration of coated antibody, secondary antibody and ELISA antibody. The method has the advantages of simple operation, rapid and high sensitivity, and can be applied to the detection of the influenza virus Vero cell adapted strain A / Yunnan / 1 / 2005va (H3N2) during the breeding of the cold adapted strain of Vero cell, Live vaccines have important implications.