Multiple cellular antigen detection by ICP-MS.
There is a great need in cell biology for the simultaneous detection of many intracellular and extracellular proteins within single cells. Current optical methods based on fluorescence activated flow cytometry are difficult to multiplex. We have developed a novel application of ICP-MS-linked metal-tagged immunophenotyping which has great potential for highly multiplexed proteomic analysis. Expression of intracellular oncogenic kinase BCR/Abl, myeloid cell surface antigen CD33, human stem cell factor receptor c-Kit and integrin receptor VLA-4 were investigated using model human leukemia cell lines. Antigens to which specific antibodies are available and are distinguishably tagged can be determined simultaneously, or multiplexed. Four commercially available tags (Au, Sm, Eu, and Tb) conjugated to secondary antibodies enable a 4-plex assay assuming that the primary antibodies are not cross-reactive. Results obtained by ICP-MS were compared with data from FACS. ICP-MS as an analytical detector possesses several advantages that enhance the performance of immunoassays, which are discussed in detail. Although multiplexing using metal-conjugated reagents is in a very early stage of research and feasibility studies, it is already apparent that more than four antigens could be accurately detected simultaneously using the ICP-MS instrument.
Ornatsky
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Institute for Biomaterials and Biomedical Engineering, University of Toronto, 4 Taddlecreek Rd., Rm. 407, Toronto, ON, Canada, M5S 3G9. olga.ornatsky@utoronto.ca
Baranov
V I
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Bandura
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Tanner
S D
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Dick
J
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D000818:Animals; D000911:Antibodies, Monoclonal; D000941:Antigens / Q000032:analysis*; D015703:Antigens, CD / Q000032:analysis; D015214:Antigens, Differentiation, Myelomonocytic / Q000032:analysis; D002460:Cell Line; D002469:Cell Separation; D005434:Flow Cytometry; D016044:Fusion Proteins, bcr-abl / Q000032:analysis; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D039041:Integrin alpha4beta1 / Q000032:analysis; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D019009:Proto-Oncogene Proteins c-kit / Q000032:analysis; D063268:Sialic Acid Binding Ig-like Lectin 3
D000911:Antibodies, Monoclonal; D000941:Antigens; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; C571163:CD33 protein, human; C571165:Cd33 protein, mouse; D039041:Integrin alpha4beta1; D063268:Sialic Acid Binding Ig-like Lectin 3; D019009:Proto-Oncogene Proteins c-kit; D016044:Fusion Proteins, bcr-abl