Herstellung und Charakterisierung monoklonaler Antikörper gegen equine Leukozyten Tierärztliche Fakultät - Digitale Hochschulschriften der LMU - Teil 01/07
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Production and characterization of monoclonal antibodies specific for equine leucocytes
This work was initiated to produce new monoclonal antibodies for the characterization of equine leucocyte-subpopulations. For immunisation, leucocytes isolated from the peripherial blood, lymph nodes and the intestinal epithelium of horses, have been used. Out of six cell-fusions 78 clons were isolated. To characterize the mAbs, the attention was focused on immunofluorescence analysis. Antibodies of the clones 4-13, 4-58, 1-19 and 6-39 have been purified by protein G-sepharoses chromatography and, except mAb 4-58, biotinylated.
MAb 4-13, mAb 4-58 and mAb 5-50 are binding specifically to equine T-cells. Flow cytometer analyses demonstrated an increased frequency following the stimulation with Concanavalin A. The frequency of mAb 4-13- and mAb 4-58-positive cells matched the total frequency of CD4- and CD8-positive cells. The number of cells, marked by mAb 5-50, is slightly lower. Most likely these three mAbs recognize three different, as yet unknown antigens. MAb 4-58 stimulated the in vitro proliferation of leucocytes. Moreover mRNA for interleukin-4 and interferon gamma was detected in these cells after the stimulation.
By contrast the antibodies 1-19, 2-52, 4-36, 4-55 and 6-39 are B-cell-specific, since they were present on cells that were Ig-positive.
The monoclonal antibodies 6-5 and 6-17 stain all equine leucocytes. Possibly they are binding to an equine CD45-molecule. The antibodies of clone 4-18 recognize an antigen on granulocyctes. Preliminary data suggests that mab 4-39 is binding to a lektin, that can be found mainly on B-Lymphocytes and endothelial cells. No group of leucocytes and no antigen could be assigned to the other antibodies.
The antibodies described here will be valuable tools to characterize different equine leucocyte-subpopulations by flow cytometry.
Production and characterization of monoclonal antibodies specific for equine leucocytes
This work was initiated to produce new monoclonal antibodies for the characterization of equine leucocyte-subpopulations. For immunisation, leucocytes isolated from the peripherial blood, lymph nodes and the intestinal epithelium of horses, have been used. Out of six cell-fusions 78 clons were isolated. To characterize the mAbs, the attention was focused on immunofluorescence analysis. Antibodies of the clones 4-13, 4-58, 1-19 and 6-39 have been purified by protein G-sepharoses chromatography and, except mAb 4-58, biotinylated.
MAb 4-13, mAb 4-58 and mAb 5-50 are binding specifically to equine T-cells. Flow cytometer analyses demonstrated an increased frequency following the stimulation with Concanavalin A. The frequency of mAb 4-13- and mAb 4-58-positive cells matched the total frequency of CD4- and CD8-positive cells. The number of cells, marked by mAb 5-50, is slightly lower. Most likely these three mAbs recognize three different, as yet unknown antigens. MAb 4-58 stimulated the in vitro proliferation of leucocytes. Moreover mRNA for interleukin-4 and interferon gamma was detected in these cells after the stimulation.
By contrast the antibodies 1-19, 2-52, 4-36, 4-55 and 6-39 are B-cell-specific, since they were present on cells that were Ig-positive.
The monoclonal antibodies 6-5 and 6-17 stain all equine leucocytes. Possibly they are binding to an equine CD45-molecule. The antibodies of clone 4-18 recognize an antigen on granulocyctes. Preliminary data suggests that mab 4-39 is binding to a lektin, that can be found mainly on B-Lymphocytes and endothelial cells. No group of leucocytes and no antigen could be assigned to the other antibodies.
The antibodies described here will be valuable tools to characterize different equine leucocyte-subpopulations by flow cytometry.
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