Preparation And Identification Of Monoclonal Antibodies Against NP Protein Of Avian Influenza Virus

The 8-week-old female BALB/c mice were immunized intraperitoneally with 200 μg formaldehyde-inactivated H9N2 subtype avian influenza virus emulsified in Freund' s complete adjuvant Two weeks later, the same antigen emulsified in Freunds incomplete adjuvant were immunized intraperitoneally. Four weeks later, the same antigen was administered intravenously without adjuvant, and three days after final immunization, spleen cells from immunized mice were fused with Sp2/0-Ag-14 myeloma cells. By using indirect ELISA and HI to screen hybridoma cells, and by using limiting dilution method to subclone positive clones three times, we acquired three positive clones, denominated C2C5, D3B10 and C2H8 respectively. The liters of their aschic fluids were 5 ×214, 210 and 5×212 respectively in indirect ELISA. They were of IgG2, subtype. In Dot-ELISA, C2H8 and D3B10 could react with AIV H9N2 and H4N1, but neither could react with NDV, IBV, IBDV, EDSV-76, or with SPF chicken embryo allantoic fluid. The results suggested that these two McAbs were probably specific forthe NP protein of AIV. Another McAb could react with ATV H9N2, but not react with AJV H5N1, NDV, IBV, BDV, EDSV-76, or with SPF chicken embryo allantoic fluid C2C5 was probably specific to AIV H9.In SDS-PAGE and western-blotting analysis, C2H8 and D3B10 reacted with NP protein of AIV. The results in this study indicated that the two McAbs would be useful as a tool for the detection of AIV strains, serological investigation and other epidemiological study. And C2C5 could not react with polypeptide bands of AIV in SDS-PAGE and western-blot.