Development Of McAbs Against H3N2 Subtype Swine Influenza Virus And Establishment Of Antigen Capture ELISA For Detecting H3 Subtype Swine Influenza Virus

Swine influenza(SI) is an acute respiratory,diseases caused by swine influenza virus(SIV), which is manifested with high morbidity and low mortality.The severity of this disease depends on secondary infection with other bacteria and viruses.Moreover,pigs can be infected not only by human influenza virus but also by avian influenza virus,suggesting the pigs as an intermediate host of interspecies transmission or as mixing vessels,play significant role in the public health implications.In this study,seven monoclonal antibodies(McAbs)against H3N2 subtype SIV were prepared by fusing SP2/0 cell with cells of BALB/c mice that had been immunized with the purified antigen strain(A/Swine/Guangdong/9/2005).Positive hybridoma cell strains including 1C9,2C5,2F10, 3D3,4E8,5C7 and 5D12 were determined by HI and ELISA.These ascites ELISA titers of McAbs were 1:105,1:106,1:104,1:105,1:106,1:105 and 1:106 for 1C9,2C5,2F10,3D3,4E8,5C7 and 5D12,respectively.Among these McAbs,1C9,2C5,2F10,3D3 belonged to IgG1,the others belonged to IgG2a.All McAbs hadκchain.The results of Western-blot analysis showed that all the McAbs could reacted with HA protein of SIV.Furthermore,we also tested the specificities of these McAbs by detecting the expression of pCAGGS-H3HA in 293T cells using indirect immunofluorescence assay(IFA).Taking the multiclonal serum against H3N2 SIV as coating antibody,an antigen capture ELISA was developed for detecting H3 subtype SIV antigen using one of the prepared McAbs(4E8),in which 4E8 was purified with caprylic acid and sulfuric acid.The optimal concentration of the reagents were listed as below serum was 1:800.McAb was 0.5μg each well and HRP-labeled goat anti-mouse IgG was 1:5000.The optimal assay conditions were listed as below:McAb was 1.0h at 37(?)C,HRP-labeled goat anti-mouse IgG was 1.0h at 37(?)C and OPD was 10min at room temperature.5%skimmed milk-PBS solution was the best choice for blocking buffer.The test was highly specific and had no cross-reaction with PRRSV,PCV,CSFV,PPV,H1N1 SIV.H5N1 SIV and H9N2 SIV.The variation coefficients were less than 10%in the intro-bath and in the inter-bath tests, indicating good repeatability of the assay.The minimum detectable concentration of the purified antigen by this assay was 3.11ng.A total of 72 samples including swabs and lungs collected from SPF chickens infected with H3 subtype SIV were evaluated by the optimized ELISA,simultaneously with virus isolation.There was 90.28%coincidence rate between these two methods in the detection of different samples.Above these resuts demonstrate that the antigen capture ELISA is highly specific,sensitive and easy to perform for detecting H3 subtype SIV.