Abstract: An indirect ELISA coated with 121 ℃ high pressure Haemophilus parasuis serovar 4 and 5 hot-torlerance protein was established. The results of chessboard titration tests showed that coating antigen at a concentration of 10 μg/mL incubated optimally at 37 ℃ for 2 h and then coated for 30 min with 20 g/mL skim milk powder. The optimal dilution of serum was 1∶80. The reaction time of coating antigen with serum 30 min. The optimal dilution of ELISA secondary antibody was 1∶12000. The reaction time was 30 min. The time of color development reaction was 15 min. Reproducibility,specificity,sensitivity tests and detection for 200 sera by the established ELISA confirmed that the developed ELISA had good stability and specificity. Compared with indirect haemagglutination test,the indirect ELISA was more sensitive and the clinical test result was consistent to overseas ELISA reagent kits. So this assay can be used as a tool for diagnosis and quarantine of H.parasuis.

Key words: Haemophilus parasuis; antibody; indirect ELISA