Establishment Of An Universal Antigen Capture ELISA For Detection Influenza A Virus

Influenza A virus?IAV?is a highly infectious respiratory pathogen.Its seasonal epidemics and pandemics cause morbidity and mortality of people and animals worldwide,posing a great threat to global public health security,economic development and social stability.Influenza viruses can be transmitted through the saliva,nasal secretions,feces and blood of infected animals.The common symptoms of influenza virus infection are fever,headache,cough and so on.In recent years,influenza frequently occurs and poses a serious threat to public health safety.Therefore,the establishment of a convenient,rapid and accurate detection method for universal influenza A virus is of great significance for cross-species transmission,clinical diagnosis and epidemiological research of Influenza A.Nucleoprotein?NP?of IAV is encoded by the fifth RNA in the genome of influenza virus,with a molecular weight of 56 KD,rich in arginine,glycine and serine residues,and is a highly conserved structural protein.The maximum amino acid difference between virus strains isolated from different hosts is less than 11%.There are also significant similarities between the NP genes of influenza viruses A,B and C.NP protein contains antigen epitopes common to all subtypes of influenza viruses,which can induce the generation of cross-reactive antibodies and T-cell reactions.It is the main internal viral antigen recognized by CD8⁺T cells,and is often used in the immunodiagnosis of influenza virus and the research and development of new influenza virus vaccine.In this study,the highly conserved NP proteins in H3N8_XJ07 of equine influenza virus and H1N1_SC09 of human influenza A virus were selected to construct the eukaryotic expression plasmid of NP protein.The NP protein was purified by the eukaryotic expression system,and the monoclonal antibody?MAb?of IAV NP protein was prepared by immunizing the recombinant plasmid containing NP gene and then immunizing the NP protein expressed by the eukaryotic expression system.After three times subclone of H3N8_XJ07 and H1N1_SC09,a total of 21 positive monoclonal cells with stable antibody secretion were obtained by indirect ELISA method.H1N1_SC09NP segmented mutant was constructed,and three antibodies that could recognize different amino acid fragments of NP protein were screened by western blot test.Three monoclonal cells?XJ07-4,SC09-6 and SC09-12?were selected for extended culture.By optimizing 3 strains of MAb and the reaction conditions,using SC09-6 as the capture antibody and XJ07-4 as the detecting antibody,an antigen capturing ELISA method for detecting NP protein of influenza A virus was established.In this method,the concentration of captured antibody was 2 mg/mL and 0.1 g/well and the concentration of detected antibody was 1mg/mL and 0.05 g/well.The concentration of NP protein was from 6.07 ng/mL to 101.57 ng/mL,showing a good linear relationship with OD_450nm and the correlation coefficient was R2?0.99.The antigen capturing ELISA method was evaluated by specificity,broad-spectrum,sensitivity and repeatability.The results showed that antigen capturing ELISA method could specifically detect IAV but had no cross-reaction with influenza B virus,newcastle disease virus,equine infectious anemia virus,equine herpesvirus and equine arteritis virus.Multiple subtypes of influenza A virus can be detected broadly;Compared with the hemagglutination test,the antigen capturing ELISA method for IAV is highly sensitive and can detect 20 ng/mL of viral NP protein.The coefficient of variation of repeatability in and between batches was less than 8%.Compared with RT-PCR,the coincidence rate was 71.43%.In summary,the antigen capturing ELISA method established in this study has strong universality,specificity,sensitivity and repeatability,which can be used for the rapid diagnosis of influenza a disease in a variety of hosts and can also provide technical support for the functional research and quantitative detection of NP protein.