Studies on mechanisms of auto-immune disease in dogs

抗犬T细胞表面抗原的单抗对犬自身免疫性疾病的研究具有重要意义。本研究针对犬胸腺细胞免疫的小鼠胸腺细胞与小鼠骨髓瘤细胞(P3X63-Ag.653)采用聚乙二醇法融合的方法，尝试制备犬T细胞的单抗。建立了11株抗犬淋巴细胞单抗的杂交瘤细胞株。对杂交瘤细胞表面抗原的免疫沉淀、抗体阳性细胞的功能分析以及阳性细胞在组织和血液中的分布进行了鉴定。单抗7F1、6H6和8C12分别为小鼠免疫球蛋白的IgG1(K)、IgG3(K)和IgG1(K)亚型。还原…下单抗7F1和6H6分别从外周血淋巴细胞表面沉淀出一种72 kDa、55 kDa的单体蛋白更多的条件。免疫组织化学显示，PBMC表面有33 kDa和38 kDa异二聚体蛋白沉淀，7F1阳性细胞分布于胸腺髓质，6H6和8C12阳性细胞分布于胸腺皮质。在功能分析中，7F1或6H6阳性细胞经细胞分选仪分选后，增殖反应增强。8C12阳性细胞的增殖反应低于8C12阴性细胞。CTLL-2细胞生物学检测显示，7F1和6H6阳性细胞IL-2活性较高，而8C12阳性细胞IL-2活性低于抗体阴性细胞。单抗7F1、6H6和8C12识别63.7(SY+-)。[)2.9，42.5(]SY.+-.[)3.6和14.5()sy.+-。根据这些结果，7F1、6H6和8C12分别识别犬PBMC上的CD5、CD4和CD8。其他抗体(来源于8个杂交瘤)的鉴定仍在继续。这三种抗体可用于细胞表面抗原分析、免疫组织化学和犬淋巴细胞的功能研究。我们正在使用这些抗体来研究诱导口服耐受、自身免疫性疾病犬的外周血液分析以及肠道和气管支气管粘膜的免疫组织化学。较少

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 … More 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