|Budget Amount *help
¥4,300,000 (Direct Cost : ¥4,300,000)
Fiscal Year 1995 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1994 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1993 : ¥2,500,000 (Direct Cost : ¥2,500,000)
Monoclonal antibodies for canine T cell surface antigen are important for research of canine auto-immune disease. In this study, we tried making monoclonal antibody for canine T cell, because of a few monoclonal antibodies were available for the research.
Spleen cells from immunized mouse with canine thymo-cytes were fused with murine myeloma cells (P3X63-Ag.653) by polyethelenglycol method. Eleven strains of hybridomas that producing monoclonal antibody against canine lymphocytes were established. Three antibodies produced by these hybridomas were characterized by immuno-precipitation of cell surface antigen, functional analysis of each antibody positive cells and distribution of positive cells in tissues and blood. Monoclonal antibody (MAb) 7F1,6H6 and 8C12 were the IgG1 (k), IgG3 (k) and IgG1 (k) isotype of mouse immunoglobulin respectively. MAb 7F1 and 6h6 precipitated a 72 kDa, 55 kDa monomeric protein respectively from the surface of peripheral blood lymphocytes (PBMC) under reduc
ing conditions. MAb 8C12 precipitated 33 kDa and 38kDa hetero dimer protein from the surface of PBMC.7F1 positive cells were rich in medulla of thymus, on the other hand 6H6 or 8C12 positive cells were rich in cortex of thymus respectively by immunohistochemistry. In functional assay, proliferative responses was enhanced in enrichment of 7F1 or 6H6 positive cells by sorting with cell-sorter. 8C12 positive cells were lower proliferative response than 8C12 negative cells. IL-2 activities by biological assay using CTLL-2 cells were higher in 7F1 or 6H6 positive cells, however the activities were lower in 8C12 positive cells compared to each antibody negative cells. MAb 7F1,6H6 and 8C12 recognized 63.7(]SY.+-。[)2.9,42.5(]SY.+-。[)3.6 and 14.5(]SY.+-。[)1.0% of PBMC respectively by cell analyzer.
From these results, we concluded that 7F1,6H6 and 8C12 recognized CD5, CD4 and CD8 on canine PBMC respectively. Characterization of the other antibodies (derived from 8 hybridomas) are continuing now.
These three antibodies are useful for analysis of cell surface antigen, imunohistochemistry and functional studies of canine lymphocytes. We are using these antibodies for studies of inducing oral tolerance, peripheral blood analysis of autoimmune-disease dogs and immunohistochemistry of intestinal and tracheobronchial mucosa. Less