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 cell lines, mostly to produce antigen. Two of the most widely used cell lines are FLK-BLV and BLV-bat2.


OBJECTIVE
the dynamics of the excretion of BLV proteins and whole virus by the persistently BLV-infected cell lines mentioned above was studied using an indirect ELISA in combination with eight monoclonal antibodies (mAbs) and cow and rabbit serum.


METHODS
tissue culture flasks were seeded with different concentrations of cells (13000-67000 cells per cm2, corresponding to 1-5 million cells per 75 cm2 flask) and were studied for 20 days. Samples (1.5 ml) were removed every 24 h and 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-BLV produced a complete monolayer as early as 4 days after passage, 3 days earlier than BLV-bat2. Using mAbs, the amount of viral proteins in the supernatant 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, which causes depletion of essential nutrients and acidity. In comparison to polyclonal serum, mAbs gave more clear and defined values and are useful for determining the dynamics of viral production.


CONCLUSIONS
when aiming for high viral yield, BLV should be harvested between days 6 and 8 after passage, when viral shedding is at its maximum. These results are very useful for preparing antigen for monoclonal antibody production, or for techniques such as ELISA or Western blot.
Llames
L
L
Departmento Patología Animal I, Facultad de Veterinaria, Universidad Complutense, 28040, Madrid, Spain.
Goyache
J
J
Domenech
A
A
Arjona
A
A
Suarez
G
G
Gomez-Lucia
E
E
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000914:Antibodies, Viral / Q000276:immunology*; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D016583:Enzootic Bovine Leukosis / Q000276:immunology / Q000821:virology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D015686:Gene Products, env / Q000276:immunology*; D015683:Gene Products, gag / Q000276:immunology*; D001909:Leukemia Virus, Bovine / Q000276:immunology*; D011817:Rabbits; D014709:Vero Cells; D017735:Virus Latency
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D015686:Gene Products, env; D015683:Gene Products, gag