H7N9亚型流感疫苗诱导的神经氨酸酶抑制抗体检测方法的建立

Measurement of anti-influenza neuraminidase antibody induced by H7N9 influenza vaccine

目的建立酶联凝集素测定法(enzyme-linked lectin assay,ELLA)检测接种流感疫苗志愿者血清中神经氨酸酶抑制(neuraminidase inhibition,NI)抗体。方法对神经氨酸酶切底物、酶标抗体孵育浓度和时间、最适病毒量以及病毒稀释液pH值等反应条件进行优化,确定合适的反应体系,采用建立的方法检测对接种H7N9大流行流感疫苗34名志愿者血清样本进行检测。结果胎球蛋白作为酶切底物最佳包被浓度为7.5μg/ml;酶标抗体最佳稀释度为1∶500,H7N9疫苗株作为抗原使用pH6.5的稀释液稀释至浓度4.5lgCCID50/ml。疫苗接种后血清样本抗N9型NI抗体滴度较接种前均有显著增高(P<0.001),在34名志愿者中,47%的人NI抗体接种后呈阳性反应(NI抗体滴度≥40),低于HI抗体反应阳转率(P<0.05)。结论建立的基于天然底物检测NI抗体的ELLA法简单实用,为流感疫苗和血清流行病学NI抗体免疫评价体系的建立提供实验资料。

Objective To develop an enzyme-linked lectin assay(ELLA)for measuring neuraminidase inhibition(NI)antibody titers in subjects vaccinated with H7N9 influenza vaccine.Methods Neuraminidase substrate,the dilution and incubation time of enzyme-labeled antibody,the concentration of influenza antigen for coating and pH value of the dilution buffer were optimized.Based on that,ELLA was established and used to detect anti-influenza neuraminidase antibody titers in serum samples of 34 subjects before and after vaccination with H7N9 influenza vaccine.Results The optimal neuraminidase substrate was fetuin at a coating concentration of 7.5μg/ml.The optimal dilution of enzyme-labeled antibody was 1∶500.The virus strain of influenza H7N9 vaccine was used as antigen at a concentrations of 4.5lgCCID50/ml in solution with a pH of 6.5.Influenza-specific NI titers detected after immunization with vaccine were significantly higher than those before vaccination(P<0.001).In the 34 subjects receiving H7N9 vaccine,the seroconversion rate of NI antibody was 47%(≥40 in NI titer),which was lower than that of HI antibody(P<0.05).Conclusions An ELLA with natural substrate for measurement of anti-influenza NI antibody was developed.It is simple and practical and might be used in the establishment of immune evaluation system for influenza vaccines and NI antibody.