HIT: a new versatile protein array platform for multi-analyte phenotyping of surface molecules
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE10665 | GEO | 2008/03/03
SECONDARY ACCESSION(S): PRJNA107635
REPOSITORIES: GEO
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Project description:We have developed a multi-analyte 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 barcode. After staining a sample, T7 polymerase amplifies the tags which are then hybridized to a DNA microarray for indirect measurement of each analyte. Here we screened 90 antibodies directed against a panel of cell surface markers as well as 4 isotype controls to compare resting human naive CD4+ T cells versus CD4+ T cells activated for 48 h with anti-CD3/anti-CD28 coated beads. Keywords: protein profiling, response to stimulus Two-condition experiment, activated versus resting cells. Biological replicates: 3 normal human donors, each donor served as an internal control. One replicate per array. One of the biological replicates was performed as a dye-swap to monitor dye-bias. We also performed a self-self comparison between activated cells from two different donors.
2010-05-26 | E-GEOD-10665 | biostudies-arrayexpress
Project description:We have developed a multi-analyte 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 barcode. After staining a sample, T7 polymerase amplifies the tags which are then hybridized to a DNA microarray for indirect measurement of each analyte. Here we screened 90 antibodies directed against a panel of cell surface markers as well as 4 isotype controls to compare human naive CD4+ T cells activated in the presence or absence of TGF-β. Keywords: response to stimulus
2008-03-08 | GSE10762 | GEO
Project description:We have developed a multi-analyte 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 barcode. After staining a sample, T7 polymerase amplifies the tags which are then hybridized to a DNA microarray for indirect measurement of each analyte. Here we screened 90 antibodies directed against a panel of cell surface markers as well as 4 isotype controls to compare human naive CD4+ T cells activated in the presence or absence of TGF-β. Keywords: response to stimulus Two-condition experiment, with or without TGF-β. Biological replicates: 3 normal human donors, each donor served as an internal control. One replicate per array. One of the biological replicates was performed as a dye-swap to monitor dye-bias.
2010-05-26 | E-GEOD-10762 | biostudies-arrayexpress
Project description:We have developed a multi-analyte 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 barcode. After staining a sample, T7 polymerase amplifies the tags which are then hybridized to a DNA microarray for indirect measurement of each analyte. Here we coupled 44 of the tags to aliquots of an IgG1 isotype negative control antibody and the 4 remaining tags we coupled to anti-CD3, anti-CD4, anti-CD19, and anti-CD20 to create a 48-plex HIT cocktail. We then used this cocktail to stain 1 x 10^6 T or B cells and during amplification incorporated either cyanine 3-UTP (Cy3-UTP) or Cy5-UTP. Additionally, we snap froze and thawed an aliquot of the cocktail (FT). Keywords: protein profiling, cell type comparison Two-condition experiment, T cells versus B cells, including dye-swaps and self-self experiments. We also tested an array that had been frozen and thawed.
2010-05-26 | E-GEOD-10761 | biostudies-arrayexpress
Project description:We have developed a multi-analyte 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 barcode. After staining a sample, T7 polymerase amplifies the tags which are then hybridized to a DNA microarray for indirect measurement of each analyte. Here we coupled 44 of the tags to aliquots of an IgG1 isotype negative control antibody and the 4 remaining tags we coupled to anti-CD3, anti-CD4, anti-CD19, and anti-CD20 to create a 48-plex HIT cocktail. We then used this cocktail to stain 1 x 10^6 T or B cells and during amplification incorporated either cyanine 3-UTP (Cy3-UTP) or Cy5-UTP. Additionally, we snap froze and thawed an aliquot of the cocktail (FT). Keywords: protein profiling, cell type comparison
2008-03-08 | GSE10761 | GEO
Project description: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.
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