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.

Keywords: Antibodies; Coagulation factor III; Flow cytometry; Hemostasis; Monoclonal.