Differentiation of two bovine lentiviruses by a monoclonal antibody on the basis of epitope specificity

Abstract

Bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV) are bovine lentiviruses that are closely related genetically. A recombinant fusion protein containing the capsid protein of BIV expressed in Escherichia coli was used to immunize mice and produce monoclonal antibodies. Six hybridomas specific for BIV capsid protein were identified, and one antibody, designated 10H1, was characterized further. Competitive binding assays were performed to analyze the topography of antigenic determinants by enzyme-linked immunosorbent assay and demonstrated the existence of at least three distinct antigenic determinants on capsid protein. The monoclonal antibody reacted specifically with both BIV capsid and the recombinant fusion protein in Western immunoblot analyses. However, it did not react with the recombinant capsid fusion protein of JDV, indicating that BIV contains at least one unique epitope in the capsid protein that is absent in JDV. Further mapping of the epitope by chemical cleavage analysis identified that the epitope is located at the 6.4-kDa N terminus of the 29-kDa capsid protein. This monoclonal antibody assay will be valuable for distinguishing the two closely related lentiviruses by Western blotting.

FIG. 1

Differentiation of BIV and JDV gag protein by immunoblotting. The recombinant capsid proteins of BIV and JDV were reacted with polyclonal antibodies of BIV (A) and monoclonal antibody against BIV capsid (B). The capsid BIV and JDV were expressed as 29- and 58-kDa proteins, respectively (lanes 1 and 2).

FIG. 2

Chemical cleavage of the capsid BIV protein and identification of the positive reaction bands by monoclonal antibody. (A) Western blot of acid and CNBr-cleaved capsid BIV. Five micrograms of capsid BIV protein was incubated overnight with 75% formic acid at 37°C. Another 5 μg was incubated overnight at room temperature with CNBr (10 mg/ml) in 70% formic acid. All samples were solubilized in SDS-PAGE sample buffer and analyzed by Tricine gel electrophoresis. The molecular mass (in kilodaltons) of each of the peptides is indicated. CK, uncut control; uncut, uncleaved capsid protein; Partial cut, partially digested fragments. (B) Physical map of the 29-kDa BIV capsid protein. The location of the single Asp-Pro linkage cleavable by acid and the molecular masses (in kilodaltons) of the two fragments are indicated; so are the locations of three methionine residues and the molecular masses of the four fragments generated by the CNBr cleavage. The positions of the peptides that reacted positively are indicate by the filled box.

FIG. 3

Alignment of amino acid sequences of BIV and JDV capsid proteins. Peptide sequences were aligned by using the Genetics Computer Group GAP program. Vertical lines indicate amino acids that are conserved in the two sequence.