Development of sensitive and specific indirect ELISA tests for the detection of antibodies against bovine leukemia virus in serum and milk samples.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL), a disease characterized by lymphoproliferation and B-cell tumors. There is currently no available treatment or vaccine, making it a global animal health challenge and resulting in severe economic losses. The World Organization for Animal Health (WOAH) advocates for serological diagnostic tools and disease control programs to eradicate BLV. One major diagnostic technique recognized by the WOAH is the enzyme-linked immunosorbent assay (ELISA). We developed and optimized three indirect ELISAs for detecting anti-BLV antibodies in serum and milk. These assays utilize viral Env ectodomain and p24 recombinant proteins expressed in Drosophila melanogaster S2 cells and Escherichia coli systems, respectively. We compared their performance with a widely used commercial ELISA kit for completeness, which detects antibodies against BLV gp51 protein. Our immunoassay using recombinant p24 protein (anti-p24-S ELISA) showed comparable strength to the reference method (kappa index: 84.4 %, in 952 samples evaluated). Results for the anti-ectoEnv-S ELISA in serum samples closely matched the reference kit, with higher concordance than the anti-p24-S ELISA (kappa index: 94.9 %, in 1781 samples evaluated). Similarly, our in-house anti-ectoEnv-M ELISA in milk samples exhibited high concordance with both the commercial reference kit and the anti-ectoEnv-S (kappa index between the three tests: 95.6 %, in 479 blood and milk paired samples). Thus, we have developed, optimized, and validated three indirect in-house ELISAs for detecting anti-BLV antibodies in cattle, demonstrating good sensitivity and specificity values, using two different recombinant viral proteins.