Development of an enzyme-linked immunosorbent assay for detection of antibody to the core protein (P24) of bovine leucosis virus

Abstract

Enzootic bovine leucosis (EBL) is a lymphoproliferative disease of cattle which is invariably associated with infection by an exogenous, C-type retrovirus, bovine leucosis virus (BLV) (Miller, 1969). The disease occurs throughout the world (Ferrer, 1980) and affects mainly dairy cattle. A 1983 serological survey suggested that 71 % of Queensland dairy herds were affected (Trueman et al., 1984 ). This project was undertaken to develop an assay for antibody to the major BL V core protein, p24. The absence of a sensitive and specific test for anti-p24 antibody has been seen as a major obstacle to the use of vaccines to control the spread of BLV (Burny et al., 1985a; Miller, 1986; De Boer et al., 1987). Vaccination with subunit preparations of the viral surface glycoprotein, gp51, has been shown to induce protective antibodies (Onuma et al., 1984). However, the agar delimmunodiffusion (AGID) test (Miller and Van der Maaten, 1976), which is the internationally recognized method for the diagnosis of BLV infection (Hoff-Jorgensen, 1987), detects anti-gp51 antibodies. If vaccination strategies for the control of EBL were implemented, a diagnostic test based on the detection of anti-p24 antibodies would be essential to differentiate infected from vaccinated cattle. An enzyme-linked immunosorbent assay (ELISA) was developed using a p24 antigen purified from BLY-infected cell culture supernate by lectinaffinity chromatography and gel filtration. The p24-ELISA detected BLV infection with a sensitivity and specificity, relative to the gp51-AGID test, of 98.1 % and 96.7%, respectively. Enzymatic cleavage experiments were conducted to identify antigenic regions on BLV-p24, with a view to producing a synthetic peptide for use as an ELISA antigen. These experiments, coupled with hydrophilicity predictions (Hopp and Woods, 1981; Kyte and Doolittle, 1982), suggested that p24 contained a major antigenic region near its amino-terminus. It was concluded that a synthetic peptide based on this portion of p24 may provide a useful alternative to ELISA antigen prepared from cell culture supernate.The possibility of producing ELISA antigen by bacterial expression of p24 was also explored. The portion of the BL V gag gene coding for p24 was successfully cloned, but all attempts to express the protein in E. coli were unsuccessful. Possible reasons for the inability to express p24 in a bacterial system are discussed