Development of ELISA Milk-Based Test for Reference Sera Preparation and for Serological Detection of Bovine Brucellosis

The non-invasive nature and practical applicability of milk-based serological tests provide significant advantages over blood tests for the serological screening of brucellosis in dairy animals. Accordingly, several novel rapid chromatographic and filtration-based serological test formats have recently been applied and increasingly used in milk and milk serum (whey) for brucellosis detection. This study aimed to develop an ELISA test (wELISA) for the detection of anti-Brucella antibodies in milk and to establish whey collection for use in brucellosis diagnostics. For this purpose, wELISA was optimized and validated, and its performance characteristics were compared with those of the serum ELISA, which is recognized as an alternative in the reference test list for anti-Brucella antibody detection. Optimization was carried out based on the detection limit and serum dilution rates, using the percentage inhibition (PI) values in spiked samples and cut-off criteria. During the optimization process, the ELISA PI values of blood sera spiked into milk were compared with serum ELISA optical density (OD) values. Following optimization, all whey samples, including those from the control group were evaluated to determine the performance characteristics of wELISA. The whey samples used in the wELISA was prepared using the World Organization for Animal Health (WOAH, formerly OIE) reference sera and control sera. In conclusion, the lower detection limit of wELISA and its compatibility with the serum ELISA indicate that this test has potential for detecting anti-Brucella antibodies in milk and can be used for preparing reference collections for point-of-care tests in brucellosis diagnosis.