Evaluation of virus excretion by cells persistently infected with the bovine leukaemia virus (BLV) using monoclonal antibodies

Background: bovine leukaemia virus (BLV) is the causative agent of enzootic bovine leukaemia. Studies in vitro usually require the use of infected cel l lines, mostly to produce antigen. Two of the most widely used cell lines are FLK-BLV and BLV-bat(2). Objective: the dynamics of the excretion of BLV proteins and whole virus by the persistently BLV-infected cell lines menti oned above was studied using an indirect ELISA in combination with eight mo noclonal antibodies (mAbs) and cow and rabbit serum. Study design: tissue c ulture flasks were seeded with different concentrations of cells (13 000-67 000 cells per cm(2), corresponding to 1-5 million cells per 75 cm(2) flask ) and were studied for 20 days. Samples (1.5 ml) were removed every 24 h an d the presence of BLV proteins was determined using an indirect ELISA assay in which the antigen reaction with the monoclonal antibodies was evidenced by peroxidase labeled anti-mouse immunoglobulins. Results: cell line FLK-B LV produced a complete monolayer as early as 4 days after passage, 3 days e arlier than BLV-bat(2). Using mAbs, the amount of viral proteins in the sup ernatant showed a cyclic pattern, with two evident peaks at days ca. 8 and 16. These peaks occurred even in the absence of passage or medium change, w hich causes depletion of essential nutrients and acidity. In comparison to polyclonal serum, mAbs gave more dear and defined values and are useful for determining the dynamics of viral production. Conclusion : when aiming for high viral yield, BLV should be harvested between days 6 and 8 after passa ge, when viral shedding is at its maximum. These results are very useful fo r preparing antigen for monoclonal antibody production, or for techniques s uch as ELISA or Western blot. (C) 2001 Elsevier Science B.V. All rights res erved.