HIT: a versatile proteomics platform for multianalyte phenotyping of cytokines, intracellular proteins and surface molecules.
We have developed a multianalyte fluid-phase protein array technology termed high-throughput immunophenotyping using transcription (HIT). This method employs a panel of monoclonal antibodies, each tagged with a unique oligonucleotide sequence that serves as a molecular bar code. After staining a sample, T7 polymerase amplifies the tags, which are then hybridized to a DNA microarray for indirect measurement of each analyte. Although there are many potential applications for this technology, here we report its suitability for profiling cytokines, intracellular molecules and cell surface markers. Using HIT, we profiled 90 surface markers on human naive T helper cells activated in vitro. The markers identified in this screen are consistent with previously described activation markers and were validated by flow cytometry. Additionally, a HIT screen of surface markers expressed on T helper cells activated in the presence of transforming growth factor-beta identified downregulation of CD26 in these cells. HIT arrays are an ideal platform for rapidly identifying markers for further characterization and therapeutic intervention.
Department of Medicine, Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, California 94305, USA.
Michael G
MG
Kattah
John
J
Coller
Regina K
RK
Cheung
Neekaan
N
Oshidary
Paul J
PJ
Utz
D015415:Biomarkers; D002462:Cell Membrane / Q000378:metabolism*; D016207:Cytokines / Q000032:analysis / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D042541:Intracellular Space / Q000378:metabolism*; D009841:Oligonucleotides / Q000235:genetics; D010641:Phenotype; D011506:Proteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D014158:Transcription, Genetic / Q000235:genetics*
D015415:Biomarkers; D016207:Cytokines; D009841:Oligonucleotides; D011506:Proteins