同亚型抗血清参考品在甲型H7N9禽流感病毒血凝素含量检测中的适用性研究

Quantification of HA in H7N9 influenza vaccine using heterogeneous antiserum of the same HA sub- type virus

目的：在无甲型H7 N9禽流感病毒特异性抗血清的情况下，研究现有的H7亚型抗血清用于单向免疫扩散试验（ SRID）的适用性，以建立应急情况下检测抗原含量的方法。方法获得英国国家生物制品检定所（NIBSC）已有的4种H7亚型抗血清参考品，对其制备用毒株与H7N9毒株的血凝素序列进行对比，之后通过双向免疫扩散试验比较其效价，根据同源性比对结果及双扩效价确定适用的抗血清，并以SRID方法验证检测H7N9抗原的可行性。结果 NIBSC 07/278号参考品制备使用的毒株（H7N3亚型）与H7N9血凝素氨基酸序列同源性最高，为97．14％；与H7N9抗原的双扩效价达到1∶8。以此抗血清进行SRID试验，H7N9抗原浓度10～40μg/ml时，能够形成清晰的沉淀环，回归曲线线性R2＞0．99。结论在无H7N9特异性抗血清的情况下，NIBSC 07/278编号抗血清能够用于H7 N9疫苗的血凝素含量的应急检测。

Objective To study the possibility of using heterogeneous antiserum in single radial immunodiffusion （SRID） for quantitative detection of HA in H7N9 influenza vaccine product when H7N9-specific antiserum is not available in order to establish a testing method for the detection of H 7N9 antigen in any urgent situation.Methods Antisera specific for H7N1, H7N2, H7N3 and H7N7 were obtained from NIBSC and used for SRID assay .Amino acid sequences of hemagglutinins were comparatively analyzed be-tween H7N9 virus and other viruses used to prepare heterogeneous antiserum .The titers of antisera against H7N9 and their homogenous antigens were detected by double immunodiffusion method .Based on the results of homology analysis and cross-reaction, a suitable antiserum was selected and its applicability was further validated by the SRID assay using H7N9 antigen.Results Influenza A virus subtype H7N3 that used for preparation of 07/278 antiserum showed the highest HA homology with H7N9 （97.14%）.The titer of 07/278 antiserum against H7N9 antigen was 1 ∶8 as detected by double immunodiffusion assay .The H7N9 anti-gen and the 07/278 antiserum could form a clear precipitation line in SRID assay .The detection of H7N9 antigen in the range of 10 to 40μg/ml showed a good linearity in the standard curve .Conclusion The 07/278 antiserum from NIBSC can be used as an alternative reagent for the quantitative detection of hemaggluti -nin in H7N9 influenza virus vaccine .