基于N8亚型禽流感病毒神经氨酸酶蛋白单克隆抗体的阻断ELISA方法的建立OA

To establish an accurate and sensitive ELISA method for detecting antibodies against the N8 subtype of avian influenza virus(AIV),the antigenic epitopes of the N8 subtype AIV neuraminidase(NA)protein were predicted using the analysis and prediction tools on the IEDB website.A gene fragment with relatively concentrated epitopes was cloned into the pET expression vector.The target protein(designated NA8)was expressed using a prokaryotic expression system.The purified and refolded recombinant NA8 protein was immunized into BALB/c mice.Positive hybridoma cells were screened using both ELISA and immunofluorescence(IFA)techniques,yielding a monoclonal antibody(mAb)of the IgM class,N8/17,which stably secreted the antibody.This mAb exhibited good specificity and broad-spectrum activity,as demonstrated by ELISA,IFA,and Western-blot,with an IC50 of 4.96 mol/L.Using N8/17 as the blocking mAb,the reaction conditions for the ELISA were optimized,leading to the establishment of a blocking ELISA method for detecting N8 subtype AIV antibodies.The optimal blocking effect was achieved when the anti-gen coating concentration was 5 μg/mL,the blocking mAb N8/17 was diluted at a ratio of 1∶80,the test serum was diluted at a ratio of 1∶1,and the sample was blocked with 10％fetal bovine serum(FBS)for 120 min followed by substrate incubation for 10 min.The intra-and inter-assay coefficient of variation were below 10％.Detection results from clinical simulation samples showed a positive rate of 100％.This method exhibits good specificity and broad-spectrum activity,making it suitable for early diagnosis of N8 subtype AIV and demonstrating potential for clinical application.