Development and application of blocking ELISA to detect antibodies against Salmonella Enteritidis

Salmonella is a foodborne zoonotic pathogen that poses a significant threat to public health. Contaminated poultry meat and eggs are major sources of Salmonella infection in humans. In recent years, Salmonella Enteritidis (S. Enteritidis) has become prevalent in large-scale poultry farms worldwide, highlighting the urgent need for a rapid detection method. However, no serological detection methods are currently available specifically for S. Enteritidis infection. In this study, we aimed to prepare monoclonal antibodies (mAbs) against the H:m flagellar antigen of Salmonella using classical B-cell hybridoma technology to develop a novel blocking enzyme-linked immunoassay (bELISA) for S. Enteritidis detection. After mAb preparation, epitopes were identified via dot-blot analysis, and a bELISA against S. Enteritidis was developed using S. Enteritidis flagellin as the coating antigen and biotin-2F12 as the blocking antibody. mAb 2F12 showed good specificity, and the antibody with the highest titer was subsequently purified and biotin-labeled. A percentage inhibition (PI) value above 17.05 % was considered positive. The assay demonstrated high specificity and sensitivity, detecting S. Enteritidis-positive sera at a 1:417 dilution, with a detection limit for mAb 2F12 of 399.15 ng/mL. The intra- and inter-batch CVs were below 15 %, confirming strong reproducibility. The bELISA detected S. Enteritidis antibodies earlier than the plate agglutination test, yielding positive results by day seven post-infection. The assay showed a 99.62 % coincidence rate with a commercial test kit, establishing it as a highly specific and sensitive tool for rapid S. Enteritidis detection. The test holds significant potential and value for Salmonella prevention and control in poultry farming.