Direct blotting with viable cells of protein mixtures separated by two-dimensional gel electrophoresis.
A procedure is described which combines the high resolution power of two-dimensional (2D) gel electrophoresis with the advantage of direct probing with viable cells. This device permits the transfer by electroelution of 480 distinct fractions from a 2D gel into soluble phase. Transferred fractions are virtually nontoxic, thus allowing direct probing with viable cells. Using this procedure it was shown that T cells from normal healthy individuals recognized a multitude of Mycobacterium tuberculosis antigens and that the fine antigen recognition pattern of T cells changed after short-term culture in vitro. The application of this procedure to the verification of antigen purity at the T cell level and to the identification of antigens within crude bacterial lysates which are recognized by cloned T cells is described. This approach should be applicable to the rapid identification and characterization of any interesting T cell antigen, for example from important pathogens against which a subunit vaccine is desirable. Moreover, it could be helpful for the analysis of interactions between soluble ligands and their target cells.
Gulle
H
H
Department of Medical Microbiology and Immunology, University of Ulm, F.R.G.
Schoel
B
B
Kaufmann
S H
SH
D000942:Antigens, Bacterial / Q000276:immunology; D000945:Antigens, Differentiation, T-Lymphocyte / Q000032:analysis* / Q000276:immunology; D002478:Cells, Cultured; D002999:Clone Cells; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006360:Heat-Shock Proteins / Q000276:immunology; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D008089:Listeria monocytogenes / Q000276:immunology; D009169:Mycobacterium tuberculosis / Q000276:immunology; D011994:Recombinant Proteins; D013601:T-Lymphocytes / Q000276:immunology*
D000942:Antigens, Bacterial; D000945:Antigens, Differentiation, T-Lymphocyte; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins