Development And Potentially Applicaton Of Monoclonal Antibodies Against Haemophilus Paragallinarum

The 8-week-old female BALB/c mice were immunized with Hpg14 (serotype A) and Hpg668 (serotype C) strains, which were inactivated by 0.01% Merthiolate. Spleen cells collected from immunized mice were fused with SP2/0-Ag-14 myeloma cells after the fourth immunization, the test of indirect ELISA and slide agglutination test were used to screen hybridoma cells, limited dilution method was performed to subclone the positive clones. Nine strains of hybridoma secreted moloclonal antibodies (McAbs) against the Haemophilus paragallinarum were obtained after several subcloning. Eight of which against all serotype of Haemophilus paragallinarum, and were designated as 3D3, 1D6, 5C9, 4C3, 4G11, 6G2, 5D2 and 1E8 respectively, and one of them was against serotype C, and was designated 1F5. The titers of obtained McAbs from the mouse ascitic fluid were 6×10~3～1×10~5 in indirect ELISA test, and 23～26 in direct agglutination test. By using the immunoglobulin subtypes kit, 4C3, 4G11 and6G2 were identified to be IgG1, 1E8 to be IgG3, 1D6 to be IgG2a and 3D3, 5C9 and 1F5 to be IgM.The specificity test suggested that nine McAbs were specific to Haemophilus paragallinarum, but not reacted with other strains of bacterias, Such as Pasteurella multocida, Haemophilus gallinarum and Actinobacillus. .The indirect test of nine McAbs with 4 strains isolated and 3 strain separated Haemophilus paragallinarum showed that all the eight McAbs reacted with all the senven strains of Hpg, and 1F5 reacted with 3 of the 7 strains Haemophilus paragallinarum, and the 3 strains were serotype C.A Blocking enzyme-linked Immunosorbent assay (B-ELISA) based on one monoclonal antibodies was developed in this study to detect antibodies to Haemophilus paragallinarum with B-ELISA positive serums to 8 usual pathogens and many serum samples derived from vaccinated and artificially infected chickens and sick clinical chickens were tested. The results showed that this B-ELISA, with good specificity, sensitivity and simplicity, would be a useful assay for diagnosis, moni-toring and epidemiologic analysis of infectiou scoryza.