Adaptation of a sandwich enzyme-linked immunosorbent assay to determine the concentration of bovine leukemia virus p24 and optimal conditions for p24 expression in short-term cultures of peripheral blood mononuclear cells

Abstract

Bovine leukemia virus (BLV) is a common retroviral infection of cattle. Infection is accompanied by integration of BLV into the host cell genome and is persistent for the life of the individual as is the presence of anti-BLV antibodies. Lymphosarcoma occurs in a small fraction of infected adult individuals but otherwise there is little or no associated disease. Viremia is undetectable, however, BLV is expressed readily once infected cells are cultured in vitro. A sandwich enzyme-linked immunosorbent assay (sELISA) was optimized, using murine monoclonal antibodies, to quantify the major internal structural protein (p24) produced in short-term cultures of peripheral blood mononuclear cells (PBMCs). Optimal production of BLV p24 was achieved utilizing RPMI supplemented with 10% fetal bovine serum (FBS), pH 7, and 5×10 6 cells per ml. Cultures were terminated at 24 h. The sELISA was linear between 30 and 900 ng/ml and the limit of detection was 1.2 ng/ml. At three concentrations of p24, intra- and inter-assay coefficients of variation (CV) varied between 9.2 and 13.3 and 5.1 and 12.9%, respectively.