Development and Performance Characterization of Double Antigen Sandwich ELISA for Mycoplasma bovis Antibody Detection

Abstract
Background: Mycoplasma (M.) bovis is an important pathogen of cattle, which is associated with the occurrence of many. Currently there is no effective vaccine to prevent M. bovis infections, and no
commercial vaccines currently available, so the detection approach shows the importance for the control the disease and surveille the prevalence of M. bovis.
Objective: To develop and optimize an ELISA, and evaluate the analytical, diagnostic and epidemiological performance.
Animals: Field serum samples (n=368) from 9 cattle farms in different counties in Ningxia Province, China.
Methods: Different principles of antibody detection ELISA were compared, and the parameters of double antigen sandwich (DAS) ELISA with P48 recombinant antigen were optimized. All serum
samples were tested with DAS ELISA and reference kit. Accuracy, sensitivity and specificity were evaluated using ROC curve, and Box plot was used for the prevalence and epidemiological performance
analysis.
Results: ROC curve analysis for DAS ELISA showed the area under curve was 0.807 (p<0.001). With cutoff of OD 1.5, the diagnostic sensitivity was 69.16% (95% Confidence interval [CI], 59.5% to 77.7%) with a diagnostic specificity of 72.8% (95% CI, 67.0% to 78.1%). 6 of 9 farms infected with M. bovis were detected, and the relevance rate were 81.43%, 75.00%, 65.79%, 33.33%, 38.64% and 72.84% with DAS ELISA respectively, higher than 50.00%, 50.00%, 28.95%, 3.33%, 29.55, 40.74% with reference kit.

Conclusions: The optimized DAS ELISA provided unambiguous improved serodiagnostic performance and sufficient sensitivity, specificity, and it will be an improved tool for M. bovis surveillance
and prevention.