Establishment Of An Indirect ELISA For Detection Of Antibodies Against Swine Influenza A (H3N2) Virus And Development Of Monoclonal Antibodies To Nuclear Protein

Using a pair of specific primers designed according to the relevant nucleotide sequence of A/Swine/Heilongjiang/1/05(H3N2) from GenBank, the HA1 gene of H3N2 subtype swine influenza virus (SIV) was amplified with RT-PCR method. The PCR product was cloned into pET-32a(+) and a recombinant plasmid pET-HA1 was generated. The target gene was expressed in the host cell BL21 successfully when induced with IPTG.The expression was optimized with proper inducing condition of 0.8mmol/L IPTG and 4 hours . SDS-PAGE result showed that the HA1 gene was expressed in prokaryotic expression system, and the target protein take the proportion of 40%(42KD) in the whole protein by scanning assay. Western blot analysis proved the recombinant protein has good reactivity against H3N2 subtype SIV positive serum.The indirect ELISA was established to detect antibody of H3 subtype SIV using purified protein. The optional working circumstances for the indirect HA-ELISA assay (antigen concentration: 3.75μg/ml; serum dilution:1:100) was confirmed.The working concentration of HRP-labeled rabbit anti-swine IgG was 1:1000.The positive criterion of this ELISA assay is ODthe tested serum >0.254. The tests showed no cross-reactivity with antibodies to H1N1 SIV; H5N1 SIV; H9N2 SIV; SFV;PRRSV and PRV.Compared with HI,the diagnostic sensitivity, specificity, and accuracy of HA1 ELISA were more optimization after detecting 309 serum samples collected from different coughing herds in China.The coincidence rate was 89% between indirect and HI test ,indicating the recombinant HA1 protein could be used for primary diagnoses of pig infection with H3 subtype SIV.In this experiment, several hybridoma cell strains were developed by fusion of SP2/0 mouse myeloma cells with spleen cells isolated from a BALB/C mouse which was immunized by the purified SIV(SGD/16406).Four McAb cell strains were determined by indirect ELISA coated with purified virus: D4E11,B8G6,B8H5,C8G8. The subtypes of NP McAbs were determined by Western blot analysis, using purified SIV(SGD/16406)as antigen.The result of Western blot show that there is only one strip in 56KD. the specificial McAbs of NP(ELISA titers) were: 1:32000, 1:32000, 1:8000, 1:16000. the number of chromosome were about 98 to 106 and is the same as total chromosome number of SP2/0 mouse myeloma cells and spleen cells.The McAbs ,B8G6 and B8H5 were proved to belong to immunoglobulin subclass IgG2.D4E11 and C8G8 were proved to belong to immunoglobulin subclass .The tests by indirect ELISA showed no cross-reactivity with antibodies to SFV;PRRSV and PRV. The result proved that 4 McAbs had good specificity of type for SIV.