title: Generation by self re-fusion of bovine³ × murine² heterohybridomas secreting virus-neutralizing bovine monoclonal antibodies to bovine herpesvirus 1 glycoproteins gB, gC, and gD.
abstract: Seventy-eight heterohybridomas (HH) stably secreting bovine monoclonal antibodies (BomAb) to Bovine herpesvirus 1 (BHV1) were produced by fusing lymph node cells from a BHV1 hyperimmunized calf with 3 types of non-secreting fusion partners. Seven were generated through fusion with the murine × murine (murine(2)) hybridoma SP2/0, 3 through fusion with bovine-murine(2) HH previously generated using cells from the same calf, and 68 through fusion with bovine(2)-murine(2) HH previously generated by sequential fusions using cells from the same calf. The chromosome number of example HH increased with increasing numbers of input fusions. A variety of indirect fluorescent antibody assay patterns was observed using the BomAb, suggesting diverse antigen specificity. Three bovine(3)-murine(2) HH secreted IgG1 BomAb neutralizing BHV1 without complement, and were chosen for further characterization. SDS-PAGE of detergent-solubilized BHV1 proteins bound to the 3 neutralizing BomAb demonstrated their individual specificities for BHV1 envelope glycoproteins gB, gC, and gD, the major neutralization targets for BHV1. The 3 HH stably secreted the BomAb in culture for over one year, and pilot-scale production of the BomAb was accomplished by in vivo and in vitro methods. A cocktail of the 3 BomAb was administered intravenously (i.v.) to a 6-month-old calf and its serum neutralization activity decreased with a half-life consistent with non-immune clearance, suggesting that BomAb may be useful for passive immune treatment of disease in cattle. Rabbits were passively protected by i.v. injection with each of the anti-gB and anti-gD BomAb when challenged i.v. with BHV1 24h later. Self re-fusion was shown to be advantageous for efficiently producing HH stably secreting host monoclonal antibodies. The BomAb described should prove useful in studies of the host immune response to BHV1, as reagents, and as sources of bovine immunoglobulin sequences.
authors.affiliation: Science, Technology and Analysis Services, Veterinary Services (VS), Animal and Plant Health Inspection Service (APHIS), United States Department of Agriculture (USDA), 1920 Dayton Ave., Ames, IA 50010, United States. Electronic address: Randall.L.Levings@aphis.usda.gov.
authors.forename: Randall L
authors.initials: RL
authors.lastname: Levings
authors.affiliation: Center for Veterinary Biologics, VS, APHIS, USDA, 1920 Dayton Ave., Ames, IA 50010, United States.
authors.forename: Ione R
authors.initials: IR
authors.lastname: Stoll
authors.affiliation: National Veterinary Services Laboratories, VS, APHIS, USDA, 1920 Dayton Ave., Ames, IA 50010, United States.
authors.forename: Janet V
authors.initials: JV
authors.lastname: Warg
authors.affiliation: Center for Veterinary Biologics, VS, APHIS, USDA, 1920 Dayton Ave., Ames, IA 50010, United States.
authors.forename: Peggy A
authors.initials: PA
authors.lastname: Patterson
authors.affiliation: National Animal Disease Center, Agricultural Research Service, USDA, 1920 Dayton Ave., Ames, IA 50010, United States.
authors.forename: Lea Ann
authors.initials: LA
authors.lastname: Hobbs
authors.affiliation: Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, United States.
authors.forename: Merlin L
authors.initials: ML
authors.lastname: Kaeberle
authors.affiliation: Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, United States.
authors.forename: James A
authors.initials: JA
authors.lastname: Roth
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods; D000939:Epitopes / Q000276:immunology*; D005260:Female; D006566:Herpesviridae Infections / Q000276:immunology / Q000662:veterinary* / Q000821:virology; D007242:Herpesvirus 1, Bovine / Q000276:immunology*; D006825:Hybridomas / Q000276:immunology*; D007114:Immunization / Q000662:veterinary; D007621:Karyotyping / Q000662:veterinary; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009500:Neutralization Tests / Q000662:veterinary; D011817:Rabbits; D014764:Viral Proteins / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D014764:Viral Proteins; C046998:bovine herpesvirus type-1 glycoproteins
keywords: Bovine herpesvirus 1; Bovine monoclonal antibody; Heterohybridoma; Passive immunity; Virus glycoprotein; Virus neutralization