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.
Subject(s)
Antibodies, Monoclonal/immunology , Antibody Specificity , Cattle Diseases/diagnosis , Immunodeficiency Virus, Bovine/immunology , Lentivirus Infections/diagnosis , Animals , Antigens, Neoplasm , Capsid/immunology , Cattle , Cattle Diseases/immunology , Diagnosis, Differential , Enzyme-Linked Immunosorbent Assay , Epitopes/immunology , Hybridomas , Immunodeficiency Virus, Bovine/isolation & purification , Immunoglobulin kappa-Chains , Lentivirus Infections/immunology , Lentivirus Infections/veterinary , Mice , Mice, Inbred BALB C , Tumor Cells, CulturedABSTRACT
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.
Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Gene Products, gag/genetics , Gene Products, gag/immunology , Immunodeficiency Virus, Bovine/genetics , Animals , Cattle , Cloning, Molecular , Gene Products, gag/analysis , Recombinant Proteins/analysis , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Sensitivity and SpecificityABSTRACT
A rapid, inexpensive enzyme-linked immunosorbent assay (ELISA) to quantitate antibodies to porcine respiratory and reproductive syndrome virus (PRRSV) in serum was developed using a recombinant PRRSV nucleoprotein (rN). The sensitivity (85.3%) and specificity (81.7%) of the Kansas State University ELISA were good, correlating well (82.4%) with the IDEXX HerdChek ELISA.
