title: Generation and characterization of a monoclonal antibody against canine tissue factor.
abstract: Tissue factor (TF, coagulation factor III) has recently identified roles in innate immunity and cancer. We generated a murine mAb against canine TF (cTF) cloned from Madin-Darby canine kidney cells and expressed in Chinese Hamster Ovarian (CHO) cells, with an equine IL-4 tag. One clone was selected for purification based on initial screening of CHO cell supernatants. The mAb was further characterized with flow cytometry, immunofluorescent microscopy, immunoblotting and immunohistochemical staining of normal and neoplastic canine tissue. The mAb labeled high, but not low, TF-expressing canine breast cancer (CMT25) and osteosarcoma (HMPOS) cells with flow cytometry and immunofluorescent microscopy. Immunoblotting revealed a 42kDa protein with homogenized canine brain and CMT25, but not HMPOS, lysates. The mAb labeled renal tubules and glomeruli, intestinal and dermal epithelium, and arteriolar adventitial cells in frozen tissues. Using immunofluorescent microscopy, increased numbers of labeled PBMCs were observed after LPS stimulation. Our results indicate that the anti-cTF mAb detects a protein with the expected tissue distribution and molecular weight of TF in normal, LPS-stimulated and neoplastic canine cells. This mAb may prove useful for exploring the role of TF in neoplastic and infectious disorders in dogs.
authors.affiliation: Departments of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, United States. Electronic address: ts23@cornell.edu.
authors.forename: Tracy
authors.initials: T
authors.lastname: Stokol
authors.affiliation: Departments of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, United States. Electronic address: janelle.daddona@gmail.com.
authors.forename: Janelle
authors.initials: J
authors.lastname: Daddona
authors.affiliation: Departments of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, United States. Electronic address: cd27@cornell.edu.
authors.forename: Christine
authors.initials: C
authors.lastname: DeLeonardis
authors.affiliation: Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, United States. Electronic address: ld295@cornell.edu.
authors.forename: Lynn
authors.initials: L
authors.lastname: Dong
authors.affiliation: Departments of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, United States. Electronic address: bw73@cornell.edu.
authors.forename: Bettina
authors.initials: B
authors.lastname: Wagner
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism; D016466:CHO Cells; D045744:Cell Line, Tumor; D003412:Cricetulus; D004285:Dogs / Q000235:genetics / Q000276:immunology*; D015151:Immunoblotting; D007150:Immunohistochemistry; D061985:Madin Darby Canine Kidney Cells; D008856:Microscopy, Fluorescence; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D013925:Thromboplastin / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D014018:Tissue Distribution
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D013925:Thromboplastin
keywords: Antibodies; Coagulation factor III; Flow cytometry; Hemostasis; Monoclonal