B型流感病毒核衣壳蛋白ELISA捕获法的建立和临床应用

摘要

Objective To prepare and characterize monoclonal antibodies (mAbs) against nucleocapsid (N) protein of influenza B virus and to establish mAbs - based sandwich antigen capture ELISA for detecting N protein of influenza B virus. Methods BALB/c mice were immunized with purified recombinant N protein in combination with virus antigen of influenza B virus for producing mAbs. The identification of mAbs was performed using indirect enzyme - linked immunosorbent assay (ELISA) , indirect fluorescent -antibody assay (IFA) , and Western immunoblotting (WB). The binding epitopes of mAbs were analyzed by competitive inhibition assay, and all combinations of mAbs were evaluated to determine a best matched pair of capture antibody and detecting antibody, to establish a sandwich ELISA for detecting N antigen of influenza B virus. Results A total of 12 mAbs specifically against N of influenza B virus were obtained, which bound to at least eight different epitopes on the N antigens of influenza B virus. On the basis of the eight groups of epitopes among the 12 mAbs, N antigen capture ELISA was established, in which combination of mAbs BN4 and BN8 were selected as capture antibodies and combination of horseradish peroxidase (HRP) - conjugated mAbs BN1 and BN5 as detecting antibodies. The sensitivity of influenza B virus N antigen detection in fresh specimen and freeze thawing specimen from respiratory tracts were 100% and 83.9% , with reference to results of virus isolation and IFA, respectively, and the specificity of the assay was 100% with reference to 130 normal respiratory specimens. No cross - reaction with other respiratory viruses such as influenza A virus and parainfluenze virus were observed when tested with the clinical specimens or virus culture. Conclusion A sensitive and specific antigen capture ELISA was established for detecting N antigen of influenza B virus by using well - characterized mAbs specific to N protein of influenza B virus, which can be used for early diagnosis of influenza B virus infection and epidemiological research.%目的 制备和鉴定B型流感病毒核衣壳(N)蛋白单克隆抗体(mAb),建立双抗体夹心捕获抗原EUSA,用于B型流感病毒感染的早期诊断.方法 用重组B型流感病毒N蛋白和B型流感病毒抗原免疫BALB/c小鼠制备mAb,采用ELISA间接法、免疫荧光(IFA)和免疫印迹(WB)进行筛选和鉴定,通过竞争抑制试验分析抗体识别表位,并采用抗体配对试验建立双抗体夹心捕获抗原ELISA检测B型流感病毒N抗原.结果 获得12株特异性针对B型流感病毒N蛋白的mAb,识别8个不同的抗原位点,根据mAb识别抗原表位,对mAb进行多种组合的配对试验,筛选出2株单抗BN4和BN8混合作为捕获抗体,辣根过氧化物酶标记的单抗BN1和BN5混合作为测定抗体,建立了双抗体夹心ELISA,测定新鲜的漱口液标本的灵敏度达100%,而对冻融的漱口液标本检测灵敏度为83.9%,与其他呼吸道病毒如A型流感病毒、副流感病毒等无交叉反应,特异度达100%.结论 获得特异性好的mAb,经过抗体的配对和优化,建立了一种灵敏度高、特异性强的B型流感病毒抗原的ELISA捕捉法,可用于B型流感病毒感染的早期实验室诊断及流行病学研究.