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Bovine leukemia virus (BLV) infection in cattle is seldom manifested clinically, and is routinely diagnosed by serologic tests such as enzyme-linked immunosorbent assay or Western blot (WB). Because of the difficulty in interpreting WB results, the aim of the present study was to determine which of the bands observed in WB were specifically produced by BLV and which corresponded to nonspecific proteins, either derived from medium components or of a cellular nature. Five different BLV antigen preparations from 2 cell lines (FLK-BLV and BLV-bat2) frequently used for the production of BLV antigen were compared. The protein profiles of these antigen preparations were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and WB. Fetal calf serum, required for cellular growth and important in induction of viral transcription in vitro, was identified as a source of irrelevant proteins. In this study, 15 nonspecific protein bands in the growth medium were observed. These bands interfered with the interpretation of results. A nonspecific protein (25 kD) that was highly reactive in cell lysate preparation from BLV-bat2 was also detected. The unequivocal identification of protein bands, both specific and nonspecific, seen in WB is important not for understanding the protein profile of antigen preparations but also for determining if an animal is BLV positive or negative.

A total of 59 monoclonal antibodies (mAbs) specific against the bovine leukaemia virus (BLV) using different antigen preparations was produced. The five antigen preparations for immunizing BALB/c mice were: live cells (CEL), sonicated and ultracentrifuged cells (SOC), cell lysates (LYS), semi-purified BLV (PV), and formalin-treated cells (FOR) from two cell lines permanently infected with BLV (FLK-BLV and BLV-bat2). These viral component presentations were selected to obtain mAbs against specific BLV proteins: located on the cell surface (FOR and CEL), in free virus particles (PV) and intracellular viral proteins (SOC and LYS). Two antigen preparations (SOC and LYS) were lethal to the mice following the intravenous and intrasplenic routes. Six fusions were performed in this study that rendered specific antibodies against BLV. The highest number of hybridomas was produced with SOC; however, the majority of the hybridomas produced (> 90%) were against cellular proteins. Even though immunization with PV gave the lowest number of hybridomas, the majority of them were specific against BLV. Based on the reactivity of the mAbs in Western blot (WB), we classified the mAbs into five groups, namely anti-gp51SU (39 mAbs), anti-gp30TM (six mAbs), anti-Pr72env (nine mAbs), anti-Pr66gag-pro (one mAb) and anti-Prgag (four mAbs). A very high percentage of the mAbs produced (48 of 59) reacted with gp51SU, suggesting that this is the most immunogenic and accessible BLV protein presented in the different antigen preparations. The majority of our mAbs recognized more than one band in WB, suggesting that, aside from reacting with mature proteins, the mAbs also recognized viral precursors.

Bovine leukemia virus (BLV) has a long latency period during which animals are inapparently infected, may spread the disease, and are only detected by serological techniques or by the most cumbersome molecular biology techniques. We have compared techniques for detecting either total antibodies (ELISA), anti-p24 and Gag-related proteins (Western blot), or anti-gp51 (agar gel immunodiffusion, AGID, and syncytia inhibition, SI) in rabbits inoculated experimentally with inocula of variable immunogenicity. The two tests to detect antibodies to gp51 correlated well in sera clearly positive or clearly negative by either one, but correlation was poor in the intermediate groups. All sera positive by AGID were also positive by ELISA, but results did not agree in sera negative by AGID, ELISA proving to be more sensitive. Western blot was a good technique for detecting antibodies against Gag-related proteins. However, no band was identified to clearly correspond to anti-Env-related proteins. As for other retroviruses, testing of animals for infection with BLV should include the detection of antibodies anti-Gag and anti-Env proteins.