Analysis of bovine herpesvirus 4 (DN 599) major antigens with monoclonal antibodies and polyclonal immune serum

Abstract

Monoclonal antibodies (MAbs) and polyclonal immune sera were produced and used to identify the major antigens of bovine herpesvirus type 4 (BHV-4). SDS-polyacrylamide gel electrophoresis of immunoprecipitates of radiolabeled lysates from infected cells resolved 24 peptide bands varying from 12 kDa to over 300 kDa. Six peptides were identified as major viral antigens by immunoprecipitation. Based on the pattern of radioimmunoprecipitation, MAbs were assigned into four groups. Group 1 precipitated a tunicamycin-sensitive glycoprotein complex which contained six components (245, 190, 152, 123, and 48/46 kDa). Deglycosylation with endoglycosidase F revealed two peptides with Mr of 93 and 38 kDa as the basic peptides of the glycoprotein complex. In addition, a 115 kDa glycopeptide containing glycan-peptide bonds of mixed type was identified. Group 2 precipitated a non-glycosylated protein complex consisting of three monomers (33/31/30 kDa). Groups 3 and 4 reacted with single monomeric non-glycosylated peptides with Mr of 48 and 14 kDa, respectively. Although none of the MAbs exhibited significant neutralizing activity, some reacted strongly in immunosorbent and/or immunohistochemical assays, suggesting they may be good candidates for use in diagnostic assays.

Investigation of sheep-associated malignant catarrhal fever virus infection in ruminants by PCR and competitive inhibition enzyme-linked immunosorbent assay

“…Fetal mouflon sheep kidney (FMSK) cells and the Minnesota isolate of MCFV (MN-MCFV), originally derived from an outbreak of MCF in cattle in Minnesota (6), were kindly provided by W. Heuschele, Center for Reproduction of Endangered Species, Zoological Society of San Diego, San Diego, Calif. A putative SA-MCFV isolate from Austria (29) (designated here as Au-732) was kindly provided by J. Pearson, National Veterinary Service Laboratory, Animal and Plant Inspection Service, U.S. Department of Agriculture, Ames, Iowa. The viruses were propagated on FMSK cells in high-glucose Dul-becco's modified Eagle medium with 10% fetal bovine serum and were purified by sucrose gradient centrifugation (14).…”

Competitive inhibition enzyme-linked immunosorbent assay for antibody in sheep and other ruminants to a conserved epitope of malignant catarrhal fever virus

“…Radioimmunoprecipitation. Radioimmunoprecipitation of labeled viral proteins was performed as previously described (20). Briefly, monolayers of FMSK cells were infected with MN-MCFV at a multiplicity of infection of 3.0.…”

Identification and characterization of the major proteins of malignant catarrhal fever virus

“…Adult BALB/c mice were each injected subcutaneously with 20 gg/mouse of MN-MCFV antigens, emulsified in 0.1 ml of Ribi MPL+TDM Emulsion (Ribi Immunochem Research). Hybridomas were produced by previously described methods (Davis, 1988;Li et al, 1991). Hybridomas were screened for reactivity to MCFV antigens in an enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence assay, and further characterized by immunoprecipitation and immunoblot.…”