Characterization of a Canine CD44 Specific Monoclonal Antibody

Abstract

Monoclonal antibodies (mAbs) were generated against a CD44 mRNA expressing (RT‐PCR) macrophage/monocyte cell line (DH82) from a dog with malignant histiocytosis. The mAbs, that reacted with DH82 cells by FACS analysis were tested on formalin‐fixed, paraffin‐embedded tissues. Exclusively the incubation of DH82 cell pellets with mAbs from clone 2D10 resulted in a cell membrane associated immunoreaction. Immunoelectron microscopy specified, that the antibody bound exclusively to the cell membrane and processes of DH82 cells. The mAb was tested on a variety of normal canine tissues, including lymphoid, urinary, alimentary, respiratory, and endocrine organs, nervous tissues, liver, pancreas, skin, and muscles. Furthermore, tumour and inflamed tissues were tested for immunoreaction with the mAb. Immunohistologically, the 2D10 mAb reacted with macrophages/monocytes, subsets of lymphocytes, epithelial cells, and central nervous system white matter. FACS analysis of canine peripheral blood leukocytes showed, that a high proportion of lymphocytes and granulocytes were positive with this mAb. Western blot analysis revealed, that the 2D10 mAb bound to a protein with a molecular weight of about 85 kDa. The results of FACS and Western blot analyses, RT‐PCR, immunohistology and immunoelectron microscopy strongly suggest that the antigen detected by the 2D10 mAb is most likely the canine equivalent of human CD44, a cell bound hyaluronan binding proteoglycan.

V3 versican isoform expression alters the phenotype of melanoma cells and their tumorigenic potential

“…Anti‐human CD44 used for Western blot and immunocytochemistry was provided by Dr. R. Vilella (Hospital Clinic de Barcelona, Spain). The rat monoclonal anti‐canine CD44 antibody was provided by Dr. S. Alldinger (Justus‐Liebig‐Universitat Giessen, Germany) 22. The antibodies against p21 and Cyclin D1 were provided by Dr. J. Gil (Facultat de Medicina, Universitat de Barcelona).…”

Up-regulation of the hyaluronate receptor CD44 in canine distemper demyelinated plaques

“…The primary antibodies used in this study were: polyclonal antibody (pAb) against glial fibrillary acidic protein (GFAP; Dako, Hamburg, Germany, Z0334, 1 : 500); anti-myelin basic protein (MBP) pAb (Dianova, Hamburg, Germany; 1 : 1000); monoclonal antibody (mAb) against CDV nucleoprotein (CDV-NP; clone 3991; 1 : 6000; [1]); and anti-CD44 mAb [3]. Prior to incubation with the primary antibodies paraffin sections were rehydrated through graded alcohols.…”

“…A monoclonal CD44 specific antibody directed against a CD44 mRNA-expressing [3] macrophage/monocyte cell line from a dog with malignant histiocytosis [27] bound to macrophages/monocytes, subsets of lymphocytes and epithelial cells. Additionally, the antigen was expressed on normal CNS white matter [3].…”

DH82 Canine and RAW264.7 Murine Macrophage Cell Lines Display Distinct Activation Profiles Upon Interaction With Leishmania infantum and Leishmania amazonensis

“…TLR4, was already detected in these cells (Wellman et al, 1988;Alldinger et al, 1999;Fujimoto et al, 2012); other receptors need to be characterized. Although the analysis of parasites per cell in 24 and 48 h post-infection showed that the number of parasites in L. amazonensis-infected DH82 cells was higher compared with that in RAW264.7 ones, there is no significant difference in the number of parasites per cell in 24 and 48 h in L. amazonensisinfected DH82 cells, which suggests that the parasite is not multiplying.…”