Evaluation of biotinylated cells as a source of antigens for characterization of their molecular profile.
Biotinylated lymphoid cells have been suggested as a useful source of antigen for the immunochemical characterization of their molecular profile. Labelling with biotin eliminates the problems associated with the use of radioactivity. However, this method has not been widely used. This reflects: (1) difficulties in optimizing the signal/background ratio because of the lack of a simple method to quantify biotinylated proteins in a cell lysate, (2) the loss of reactivity with monoclonal antibody of antigen following biotinylation, because of steric hindrance, and (3) the lack of information about the utility of other biotinylated cells as an antigen source. To overcome these limitations, we developed an ELISA to quantify biotinylated proteins in cell lysates and optimized the signal/background ratio. The validity of this approach was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of a number of cell surface antigens immunoprecipitated from lymphoid cells by an optimal amount of monoclonal antibody. Furthermore, we showed that biotinylated melanoma cells are a useful source of antigen for immunoprecipitation experiments and that ligation of biotin to antigen does not affect reactivity with monoclonal antibody. Lastly, biotinylated antigens in cell lysates stored at -80 degrees C for 6 months maintained their reactivity with monoclonal antibodies. Biotinylated cells thus represent a useful source of antigen for characterizing the immunochemical profile and analyzing the specificity of antibodies with immunochemical methods.
Perosa
F
F
Department of Biomedical Sciences and Human Oncology (DIMO), University of Bari Medical School, Italy.
Luccarelli
G
G
Neri
M
M
De Pinto
V
V
Ferrone
S
S
Dammacco
F
F
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000032:analysis / Q000276:immunology; D000954:Antigens, Surface / Q000032:analysis / Q000276:immunology; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology*; D001710:Biotin; D015153:Blotting, Western; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes; D006650:Histocompatibility Testing / Q000379:methods; D006801:Humans; D019169:Jurkat Cells; D008545:Melanoma / Q000175:diagnosis / Q000276:immunology; D011233:Precipitin Tests
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D000939:Epitopes; D001710:Biotin