Development and application of a competitive ELISA for the detection of antibodies against Salmonella Abortusequi in equids

Abstract

ABSTRACT The high abortion rate associated with Salmonella Abortusequi ( S . Abortusequi) infection in equids has re-emerged over the past 10 years and has caused serious economic losses to China. Our previous studies showed that the flagellin FljB gene could distinguish S . Abortusequi from most Salmonella serotypes. In this study, the flagellin antigen was used to develop a competitive enzyme-linked immunosorbent assay (cELISA) that could be used to detect both horse and donkey serum samples using a monoclonal antibody (MAb) that was found to bind to FljB. A cELISA was established using the purified MAb coating of the plate and incubation of the mixture of horseradish peroxidase (HRP)-conjugated FljB antigen with the undiluted serum sample. The performance of the cELISA and the tube agglutination test (TAT) assay was compared with respect to sensitivity and specificity, by testing a panel containing 660 S . Abortusequi-positive and 515 S . Abortusequi-negative serum samples, all of which had been characterized by Western blotting. Receiver operator characteristic (ROC) analyses were performed to determine the cutoff value and estimate the detection specificity (Sp) and sensitivity (Se). ROC analysis showed that the area under the ROC curve (AUC) values of cELISA [AUC = 0.9941; 95% confidence interval (CI), 0.9898–0.9984] were higher than those of TAT (AUC = 0.7705; 95% Cl, 0.7437–0.7972). A cutoff value of 39.5% was selected with Sp and Se values of 100 (95% Cl, 99.26–100.00) and 97.58 (95% Cl, 96.10–98.50), respectively. The cELISA has excellent futures compared with TAT, such as shortened detection time, no need for pre-treatment of sera, and easy interpretation of the results, and is more suitable for disease surveillance.