Preparation Of The Monoclonal Antibodies Against GST And Establishment Of Double Sandwich ELISA For Detecing The Antibody Of H5 Subtype Avian Influenza Virus

The GST protein was expressed by using a pGEX-4T expression vector. Then female Balb/c mice were immunized with the purified GST protein. The purified GST protein was used to detect the titer of secreting antibody against GST by ELISA test.The hybridomas were obtained by fusing mouse myeloma cells SP2/0 with splenocytes from the mice immunized with GST. Four strains of hybridoma cells were obtained with secreting anti-GST antibody, named 1C1, 3H11, 4F5 and 4H11. The ascite of McAbs were prepared by injecting the hybridoma cells into mice abdomen. The indirect ELISA results showed that the McAbs revealed high affinity with GST at the titer of 1 : 819200. The characteristics of McAb was performed by ELISA test and SDS—PAGE electrophoresis. The results indicated that the subclass of the McAb was either IgG1 or IgG2a, the molecular weight was 150KD. Western-blot analysis proved that the McAb had good immunoreactivity against either GST or GST—HA1The recombinant fusion protein was highly expressed in E.coli BL21 four hours laterminduced by lmmol/L IPTG in the form of inclusion bodies. The molecular weight of GST—HA1 is 62KD. Western-blot analysis proved that the recombinant protein had good immunoreactivity against H5 ATV positive serum.Based on the GST—HA1 and McAb, the double sandwich enzyme-linked immun-osorbent assay (ELISA) for detection of H5 subtype ATV was developed. The best coating concentration of the GST—HA1 wasl:5000. The best working concentration of McAb was 1;160. The double sandwich ELISA did not react the IBDV positive serum,IBV positive serum, ICV positive serum, E.coli positive serum, NDV positive serum, H9 AIV positive serum and CAV positive serum. The variant coefficients of OD₄₉₀ value within assay were 3% ⁴%.The results showed that the assay was specific, sensitive, reliable, convenient, and had a high coincidence rate with HI⁹5% ( 20/21) . And the assay did not require specific equipment and technique so that it can be extended easily.The double sandwich ELISA of establishment replaced ATV with the GST—HA1, therefore the safety was better.The double sandwich ELISA had established the foundation for the related search .