Development And Potentially Applicaton Of Monoclonal Antibodies Against Serum-type A Of Haemophilus Paragallinarum

The 8-week-old female BALB/c mice were immunized with 083 (serotype A) 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. Six strains of hybridoma secreted moloclonal antibodies (McAbs) against the serum-type A of Haemophilus paragallinarum were obtained after several subcloning. Five of which against serotype A of Haemophilus paragallinarum were got antigen by Page's method, and were designated as 1D5, 3F5, 4B3, 4F5, 4G2, respectively, and one of them was got antigen by Kume's method, and was designated 2-7B. The titers of obtained McAbs from the mouse ascitic fluid were 105～4×106in indirect ELISA test, and 23～25 in direct agglutination test. By using the immunoglobulin subtypes kit, 4F5 and 2-7B were identified to be IgG1, 3F5,4B3 and 4G2 to be IgG3, 1D5 to be IgM.The specificity test suggested that six McAbs were specific to serum-type A of Haemophilus paragallinarum, but not reacted with other strains of bacterias, Such as Pasteurella multocida, Haemophilus gallinarum, Actinobacillus and other types Haemophilus paragallinarum.A sandwich enzyme-linked Immunosorbent assay (sandwich ELISA) based on one monoclonal antibodies was developed in this study to detect antibodies to serum-type A of Haemophilus paragallinarum with sandwich ELISA positive serums to 4 usual pathogens and many serum samples derived from vaccinated and artificially infected chickens and sick clinical chickens were tested. The results showed that this sandwich ELISA, with good specificity, sensitivity and simplicity, would be a useful assay for diagnosis, moni-toring and epidemiologic analysis of infectiou scoryza.