Establishment Of A NP Based Sandwich ELISA For Detection Of Avian Influenza Virus

Based on the viral surface antigen hemagglutinin(HA)and neuraminidase(NA),Avian Influenza virus(AIV)can be divided into 16 HA and 9 NA subtypes.AIV,can be further clustered into low pathogenic avian influenza(LPAI)and highly pathogenic avian influenza(HPAI)according to the pathogenicity.Although vaccination has effectively limited the spread of AIV and its pandemic,AIV infections still causes huge economic losses to the poultry industry annually It is should be noted that more and more AIVs could been recently isolated from the immunized chickens.Compared with the vaccine strains,the immune escape AIV strains have HA and NA proteins with altered antigenicity.Rapid and high-throughput detection for these antigenic variant AIVs is critical for the prevention and controlling of the AIV in China.Unlike the prone-mutated HA and NA proteins,NP protein of AIV is relatively conserved.Therefore,NP can be used as an effective target for detection of the immune escape AIV.In this study,mice were immunized with the H9N2 avian influenza virus,and DF1 cells transfected with the NP eukaryotic expression plasmid were used as screening antigen for monoclonal antibody(mAb)against NP protein.And then,a NP based sandwich ELISA for rapid and highly sensitive detection of AIV was generated.1.Development and characteristics of mAb against NP protein of AIVTo prepare mAb against NP protein of AIV,mice were firsiiy immunized with the H9N2 AIV,and DF1 cells transfected with the NP eukaryotic expression plasmid were used as screening antigen.Two positive hybridoma cell lines were screened by indirect immunofluorescence(IFA)and named as NP-2F10 and NP-4A7 respectively.In the IFA,both mAbs NP-2F10 and NP-4A7 showed efficient reaction with the MDCK cells infected with the H9N2 or with DF1 cells transfected with the NP eukaryotic expression plasmid with bright green fluorescence.The IFA titer of NP-2F10 and NP-4A7 was 1:6400 and 1:12800 respectively.Notably,mAbs NP-2F10 and NP-4A7 could not only recognize the denatured linear NP protein in the MDCK cells transfected with NP eukaryotic expression plasmid or H9N2 by Western blot,but also capture the non-denatured natural NP protein in the MDCK cells infected with H9N2 by co-immunoprecipitation.The generation of the two mAbs NP-2F10 and NP-4A7 against NP protein lays a foundation for developing a sandwich ELISA for the detection of AIV.2.Preliminary establishment of NP based sandwich ELISA for detection of AIVTo establish a rapid and highly sensitive diagnostic technique for the detection of AIV,the two mAbs NP-2F10 and NP-4A7 against NP protein were purified and labeled,and then a double-sandwich ELISA was generated.In the ELISA,mAb NP-2F10 was used as a capture antibody(concentration 2g/mL)and HPR-labeled NP-4A7 was used a detection antibody(concentration 1:16000).Specificity assay showed that the established sandwich ELISA only reacted with H1N1,H3N2 and H9N2 AIV,not reacted with other pathogens such as sera type 4 fowl adenovirus(FAdV-4),sera type 8 fowl adenovirus(FAdV-8),avian leukosis virus(ALV),avian reticuloendotheliosis virus(REV),Marek's disease virus(MDV)and avian infectious bronchitis virus(IBV)tested.Sensitivity analysis revealed that the limit of detection of the ELISA was 5x 102 TCID50/mL for AIV.This sensitivity of the ELISA was 1.25-50 times higher than that of the hemagglutination assay(HA).Moreover,the ELISA could be efficiently applied in detecting the clinical tissue samples for diagnostics of AIV infection.The coincidence rate between the ELISA and the viral isolation was 68.6%when 35 clinical tissue samples were tested.All these data demonstrated that the NP based sandwich ELISA has an applaud application in the specific and highly sensitive detection of AIV.