流感病毒神经氨酸酶活性及型别鉴定酶联凝集素法的建立  

Development of enzyme-linked lectin assay for activity and subtyping of influenza virus neuraminidase

作　　者：曹海丹 刘旭光 王平 赵丹红 孙宇 孙志伟 杨红育 李帅 张雪梅 吴业红 CAO Hai-dan;LIU Xu-guang;WANG Ping;ZHAO Dan-hong;SUN Yu;SUN Zhi-wei;YANG Hong-yu;LI Shuai;ZHANG Xue-mei;WU Ye-hong(Changchun Institute of Biological Products Co.,Ltd.y Changchun 130012,Jilin Province,China)

机构地区：[1]长春生物制品研究所有限责任公司,吉林长春130012

出　　处：《中国生物制品学杂志》2021年第5期585-589,共5页Chinese Journal of Biologicals

基　　金：国家科技重大专项(2018ZX09737008-003)。

摘　　要：目的建立酶联凝集素法(Enzyme-linked lectin assay,ELLA)用于流感病毒神经氨酸酶(neuraminidase,NA)活性及型别鉴定。方法以胎球蛋白包被酶标板作为NA底物,以辣根过氧化物酶标记的花生凝集素(HRP-PNA)作为显示酶结合物建立ELLA法,并验证该方法的耐用性、专属性和适用性。结果流感病毒裂解疫苗单价病毒原液NA活性均为阳性,且各型别批间一致性较好;H1N1型别毒株与3种NA抗血清(N1、N2和B)的NI50分别为1∶2560、1∶80、1∶10,H3N2型别毒株与3种NA抗血清(N1、N2和B)的NI50分别为1∶160、1∶2560、1∶10,B型毒株与3种NA抗血清(N1、N2和B)的NI50分别为1∶40、1∶40、1∶320,相应的NA血清能够明显抑制相应型别的流感病毒NA活性。HRP-PNA不同孵育时间对检测结果无明显影响;疫苗生产过程中的添加物对检测结果无影响;同一型别不同疫苗毒株型别鉴定结果基本一致。结论成功建立了ELLA法,可用于流感病毒NA活性及型别鉴定。该方法的建立为国内流感病毒裂解疫苗质量评估提供了新的评价方法。Objective To develop an enzyme-linked lectin assay(ELLA) for activity and subtyping of neuraminidase(NA)of influenza virus. Methods ELLA was developed using fetal globulin-coated microtiter plate as NA substrate and horseradish peroxidase-labeled peanut agglutinin(HRP-PNA)as enzyme conjugate,and verified for durability,specificity and suitability. Results The bulk of influenza virus split vaccine showed NA activity which was in agreement in various subtypes and batches. The NI50 of subtype H1N1 against NA antisera N1,N2 and B were 1∶2 560,1∶80 and 1∶10,while those of subtype H3N2 were 1∶160,1∶2 560 and 1∶10,and those of subtype B were 1∶40,1∶40 and 1∶320,respectively. The NA antisera significantly inhibited the NA activity of influenza virus of the corresponding subtype.The incubation time of HRP-PNA and the additives during vaccine production showed no significant influence on the test result. The subtyping results of various vaccine virus strains of the same subtype were basically in agreement. Conclusion An ELLA was successfully developed,which might be used for determination of NA activity and subtyping of influenza virus. It provided a novel method for quality evaluation of domestic influenza virus split vaccine.

关 键 词：神经氨酸酶 流感病毒 酶联凝集素法 型别鉴定 

分 类 号：R373.13[医药卫生—病原生物学] R392-33[医药卫生—基础医学]