Cloning of the Bovine Immunodeficiency Virus gag Gene and Development of a Recombinant-Protein-Based Enzyme-Linked Immunosorbent Assay

Abstract

An enzyme-linked immunosorbent assay (ELISA) was established for the rapid detection of specific bovine immunodeficiency virus (BIV) antibodies in cattle, using recombinant Gag protein as an antigen. The gag coding region from BIV was cloned into an expression vector, pQE32, which expressed high levels of recombinant protein from Escherichia coli. The ELISA was standardized by a checkerboard titration against known BIV-positive and -negative sera from cattle and a monoclonal antibody to the Gag protein. A total of 139 cattle serum samples, from the diagnostic laboratory at Kansas State University, Manhattan, and from the Dairy Station, Louisiana State University, Baton Rouge, were compared by ELISA and immunoblot assays for the detection of BIV-specific antibodies. Of 26 cattle sera samples which tested positive using the immunoblot assay, 23 were positive by ELISA, thus establishing a strong correlation between the two tests. The sensitivity and specificity of ELISA relative to immunoblotting were 0.88 and 0.93, respectively. ELISA proved to be as specific as immunoblotting but was much less time-consuming and easier to perform.

Serological survey for bovine immunodeficiency virus in dairy cattle from Poland

“…Since these sera were also positive by TM-ELISA derived from the FL112 isolate (Scobie et al 1999), the conclusion can therefore be made that our ELISA is ideal for BIV serodiagnosis. In this study, the ELISA achieved 94% of specificity and this was slightly higher than obtained by Zheng et al (2000). This enhanced specificity is probably due to the modification, in which all sera tested were preincubated with E. coli lysate.…”

“…Zheng et al (2000) first reported the use of a recombinant BIV Gag protein-based ELISA that can be used for such an analysis. Our study has adopted and modified such an ELISA in order to investigate the prevalence of BIV infection in dairy cattle in Poland and the possible association between BIV and BLV infection.…”

“…ELISA was performed using the protocol described by Zheng et al (2000). Briefly, ELISA plates (Immulon 2HB, Dynex Technologies) were coated with 400 ng of purified recombinant Gag protein, diluted in sodium carbonate buffer (pH 9.6), and incubated overnight at 4 o C. The plates were washed with PBS containing 0.05% Tween 20 (PBST) buffer, blocked with 0.05% glycine in PBS, and incubated at 37 o C for 30 min.…”

Differentiation of Two Bovine Lentiviruses by a Monoclonal Antibody on the Basis of Epitope Specificity

“…The capsid protein was expressed as a 67-kDa fusion protein to the TrpE protein. Another capsid expression vector, pQE32, was constructed recently in our laboratory (31) and contains the same 0.8-kb capsid insert as the pATH vector. This construct expressed a 29-kDa capsid protein with a small fusion of 13 amino acid residues at the N terminus.…”

Unique Epitope of Bovine Immunodeficiency Virus Gag Protein Spans the Cleavage Site between p16 MA and p2L

“…The BIV gag gene has been cloned, and monoclonal antibodies (MAbs) to recombinant Gag protein have been developed (22,25,26). One of our MAbs, designated 10H1, reacts specifically with the BIV Gag protein but not with the JDV Gag protein, indicating that BIV has at least one epitope different from JDV epitopes.…”