Serodiagnosis of Enzootic Bovine Leucosis using recombinant p24.

Introduction: Bovine leukemia virus (BLV) is a retrovirus that causes enzootic bovine leukemia (EBL), a life-long infectious disease highly prevalent in dairy cattle. Serological testing and removing infected animals is central to disease control. In Brazil, agar gel immunodiffusion (AGID) using BLV is the only commercially available assay for diagnosis. Thus, we developed an indirect ELISA based on the recombinant capsid protein of BLV to detect antibodies in cattle. Methods: The nucleotide sequence coding for the BLV capsid protein was amplified by PCR, cloned an expressed in Escherichia coli. The protein was purified by the AKTA pure chromatography system, analyzed by SDS-PAGE and used to immunize rats. The antigenicity of the protein was evaluated by Western blot immunized using rat serum and serum from AGID-BLV positive cattle. The microplate type, antigen concentration and ideal serum dilution were determined by checkboard titration. The Receiver Operating Characteristic (ROC) curve analysis and the Area Under de Curve (AUC) analysis were used to evaluate the iELISA performance. Results: The expressed protein had a molecular weight of 24 KDa (p24). The serum from immunized rats and serum from cattle positive to BLV recognized the p24 protein by Western blot. The best performance of the p24 iELISA was found using PolySorp® microplates, 50 ng/well of antigen, and bovine sera diluted at 1:100. Our indirect p24 ELISA to BLV had a sensitivity of 100% and specificity of 98.7%. The ROC had an AUC value of 0.9989 (95% confidence interval of 0.9961 at 1.002; p<0.0001) which indicated a high level of accuracy. With the p24 iELISA we tested 790 bovine sera and found 237 positive and 553 negative samples. Conclusions: Our p24 iELISA is suitable to detect anti-BLV antibodies in bovine sera and might be used to develop a commercial kit aiming the control of EBL in cattle.