Production Of Monoclonal Antibodies Specific For Avian Influenza Virus H7N9 Subtype And Its N9 Subtype Immunochromatographic Strip Development

Influenza is an acute upper respiratory tract infection caused by influenza virus,which is a public health issue of great concern in China.Influenza viruses belong to orthomyxoviridae,and can be divided into four subtypes,A,B,C and D,according to the antigenicity of nuclear protein(NP)and matrix protein(M).Among them,the avian influenza virus(AIV)in the type A influenza virus is commonly found in birds and humanity,and can cause avian influenza,which belongs to a zoonotic disease.Hemagglutinin(HA)and neuraminidase(NA)are two important antigen proteins present on the surface of avian influenza virus.According to the differences between the two surface proteins,avian influenza virus is divided into 18 HA subtypes and 11 NA subtypes.The H7N9 subtype of AIV is one of the most harmful subtypes to poultry,not only endangering the health of poultry,leading to production and economic losses,but also being able to infect humanity through vectors,thereby posing a significant threat to human life and health.Therefore,it is particularly important to develop a rapid pathogenic detection method for H7N9 virus.In this study,BALB/c mice were immunized with purified H7N9 subtype AIV.Through indirect ELISA and immunoperoxidase monolayer cell assay(IPMA),14 hybridoma cells stably secreting monoclonal antibodies(m Abs)against H7N9 subtype AIV were obtained,including five strains targeting HA protein(3F3,4D7,4F6,10G9,16D6)and nine strains targeting NA protein(1D4,6H10,6G7,12A2,2E6,2E2,6C7,12F12,12A2).The indirect ELISA results showed that the ascites titers of five HA m Abs were all 1:1 000 000,and the ascites titers of nine NA m Abs were between 1:4 000 and 1:256 000;The results of IPMA showed that five HA m Abs reacted with H7 subtype AIV,nine NA m Abs reacted with N9 subtype AIV,and did not react with other subtype influenza viruses;The HI results showed that the HI titers of the five HA m Abs against the H7-Re3 antigen strain in ascites were 6 log2 to12 log2;The results of neuraminidase inhibition test showed that the 50% inhibition concentration of enzyme activity of nine NA m Abs ranged from 9.77 ng/m L to 10000ng/m L;The results of MDCK cell microneutralization test showed that five HA m Abs had neutralizing activity,while nine NA m Abs did not.Immunoglobulin subtype identification showed that the heavy chain of four HA m Abs was Ig G1,the heavy chain of one HA m Abs was Ig G2 b,and the light chain was κ;The heavy chain of five NA m Abs was Ig M,the heavy chain of four NA m Abs was Ig G1,and the light chain was κ.Nine NA m Abs were labeled with colloidal gold,and then paired with Dot-blot for screening.The m Ab 12F12 was selected as the gold labeled antibody and the m Ab12A2 as the blocking antibody.By optimizing the test paper parameters and using0.25 mg/m L m Ab 12A2 and 0.5 mg/m L SPA on the nitrocellulose membrane spray point detection line(T)and quality control line(C),a colloidal gold antigen detection test strip for N9 subtype AIV was successfully prepared,and a rapid detection method for N9 subtype AIV antigen was established.The specificity identification results showed that the test strip could specifically identify the N9 subtype AIV and did not intersect with H1N1,H9N2,H6N6,H10N7,NDV,IBDV,IBV,and MDV,indicating that the test strip had good specificity;The sensitivity test results showed that the detection limit of the test strip for H7N9 subtype AIV allantoic fluid was 1000 TCID50,which was 2-4 hemagglutination units higher than the HA test,indicating that the test strip had good sensitivity;The results of broad-spectrum identification showed that the test strip can not only identify AIV isolated of H7N9 subtype in 2020 and 2021,but also identify H7-Re1,H7-Re2,H7-Re3,and H7-Re4 strains used for inactivated vaccine of H7N9 subtype AIV,indicating that the test strip had good broad-spectrum characteristics;The stability test results showed that the sensitivity and specificity of the dried and packaged test strip did not change after being stored at room temperature for six months,indicating that the test strip had good stability.To sum up,this study successfully prepared a reliable,convenient,and rapid N9 subtype AIV antigen detection test strip,providing technical means for monitoring and diagnosing the N9 subtype avian influenza epidemic.