Heterologous expression of the membrane protein OmpA from bovine Pasteurella multocida and establishment of an indirect ELISA for antibody detection

Reliable serological tools are needed for surveillance of Pasteurella multocida antibodies. Four P. multocida membrane proteins were expressed in a prokaryotic system and screened by Western blotting and indirect ELISA. Among them, a dilution-refolded OmpA preparation retained serum immunoreactivity and met the antigen-selection criterion under the tested conditions. The optimized assay used a nominal total-protein coating concentration of 1 µg/mL, 1% skim milk blocking at 37 °C for 1 h, serum diluted 1:400, secondary antibody diluted 1:25,000, and 15 min colour development. In the preliminary analytical-specificity panel, the available PmB-positive serum and positive sera against eight other bovine respiratory pathogens remained below the lower decision threshold. PmA-positive serum remained test-positive at a dilution of 1:1280, and the intra-assay and inter-assay coefficients of variation were below 5%. Within the vaccination-defined 60-serum development panel, ROC analysis yielded an AUC of 0.969 and apparent sensitivity and specificity of 95.00% each relative to the predefined serum groups. These estimates are preliminary because the panel was derived from a single ranch, the same 20 predefined negative-reference sera were also used to establish the decision thresholds, and no independent reference method was applied.