Indirect ELISA developed to detect antibodies against Mycoplasma synoviae P50 protein via immunoproteomic screening

Mycoplasma synoviae infection is a chronic disease of poultry with significant economic impacts. An efficient diagnostic tool for M. synoviae infection is in great demand. This study aimed to develop a novel indirect enzyme-linked immunosorbent assay (iELISA) method based on antigens identified by pull-down assay combined with mass spectrometry. Using these methods and anti-M. synoviae serum, we identified an uncharacterized protein with a molecular weight of 53 kDa (named P50 protein) and then established a recombinant P50 protein-based ELISA (rP50-ELISA) to detect antibodies against P50 protein. A receiver operating characteristic (ROC) analysis was performed to estimate the optical density (OD) cut-off value that maximized the sensitivity (Se) and specificity (Sp) of the rP50-ELISA, which had a mean Se of 93% (95% confidence interval (CI) = 86.25-96.57%) and a mean Sp of 100% (95% CI = 91.80-100%), with an area under the curve (AUC) of 0.9979 (95% CI = 99.41-100%). The rP50-ELISA showed no cross-reactivity with antibodies against other avian pathogens. Serum samples from 164 clinical chickens were tested with the rP50-ELISA, and the results revealed a high concordance rate of 93.29% with commercial diagnostic kits. KEY POINTS: • Screening for the major antigen of M. synoviae for ELISA development. • The P50 protein was selected as a coating antigen for ELISA. • rP50-ELISA was successfully developed for detecting anti-M. synoviae antibodies with high sensitivity and specificity.