Establishment of Double Sandwich ELISA for Detection of Bordetella bronchiseptica

To establish a stable double sandwich ELISA method with good specificity and sensitivity for detection of Bordetella bronchiseptica(Bb),rabbit anti-Bb hyperimmune serum and monoclonal antibody against Bb(Bb McAb)were prepared and purified.Chessboard titration crossing test was used to determine the optimal working concentrations of these two antibodies.Specificity,sensitivity and repeatability of the constructed ELISA were determined,and compared with PCR method further.The results showed that the best working concentration of the purified serum(2.5 mg/L) and Bb McAb(5.0 mg/L) were 1∶6 400 and 1∶800,respectively.The ELISA method developed in this study possessed high specificity and repeatability,with no cross-reaction with E.coli,P.psicicida and C.perfringens.The coincidence of ELISA and PCR method was 100%.