Development and Preliminary Application of Apd-ELISA Kit for Antibody Detection of Haemophilus parasuis

The present study aimed to develop an ELISA for detecting antibodies of Haemophilus parasuis by optimization of reaction conditions, determination of its cutoff value and evaluation of its application with autotransporter passenger domain (Apd) as the coating antigen. Firstly, the porcine sera from immunization and challenge experiments were used as HPS-positive and -negative control sera to optimize the Apd-ELISA reaction parameters and conditions. Next, the cutoff value was determined based on testing of the serum samples with confirmed background, and then the blocking solutions and the packing methods of plates were selected. Finally, Apd-ELISA was used to detect clinical serum samples and then compared with the Biocheck OppA-ELISA kit from Netherland. With the coating antigen at 0.5 μg·mL^-1, the tested sera being diluted at 1:200 and incubation for 45 min and HRP-labeled goat anti-pig second antibody being diluted at 1:15 000, Apd-ELISA displayed the improved discriminative capability and the reduced background signal. The cutoff value was determined to be 0.33 by calculation formula (S-N)/(P-N) and the OD_{630 nm} value of the 27 positive porcine sera and 40 negative porcine sera with the confirmed background. The blocking solution K and the vaccum package can preserve ELISA plates for 4 days at 50 ℃ (comparable to 22 months at 4 ℃). Detection of 1 179 clinical sera with Apd-ELISA indicated that the positive rates were 83.76%, 61.09% and 27.48% in sows, fattening pigs and piglets, respectively. Compared with the OppA-ELISA kit from Biocheck (OppA-ELISA), Apd-ELISA showed 100% identity to the results of OppA-ELISA kit in clinically negative sera and experimentally positive sera from the pigs immunized with the inactivated vaccine, but 4.76%(6/126)identity to the results in the sera from clinically positive sera. However, Apd-ELISA displayed 73.02%(92/126)identity to the results of OppA-Western blot in the sera from clinically and experimentally infected pigs. The present study obtained the Apd-ELISA kit that can effectively distinguish HPS-positive and -negative sera and can be stably preserved, which would be applied for antibody monitoring among pigs immunized with HPS-inactivated vaccine and subjected to infection of H. parasuis.