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J Proteome Res. 2010 Nov 5;9(11):5629-37. doi: 10.1021/pr100447b. Epub 2010 Oct 4.

Novel cellular microarray assay for profiling T-cell peptide antigen specificities.

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  • 1Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH 43210, USA.

Abstract

We present a novel cellular microarray assay using soluble peptide-loaded HLA A2-Ig dimer complexes that optimizes the avidity of peptide-HLA binding by preserving the molecular flexibility of the dimer complex while attaining much higher concentrations of the complex relative to cognate T-cell receptors. A seminal advance in assay development is made by separating the molecular T-cell receptor recognition event from the binding interactions that lead to antigen-specific cell capture on the microarray. This advance enables the quantitative determination of antigen-specific frequencies in heterogeneous T-cell populations without enumerating the number of cells captured on the microarray. The specificity of cell capture, sensitivity to low antigen-specific frequencies, and quantitation of antigenic T-cell specificities are established using CD8 T-cell populations with prepared antigen-specific CTL frequencies and heterogeneous T cells isolated from peripheral blood. The results demonstrate several advantages for high-throughput broad-based, quantitative assessments of low-frequency antigen specificities. The assay enables the use of cellular microarrays to determine the stability and flux of antigen-specific T-cell responses within and across populations.

PMID:
20836567
[PubMed - indexed for MEDLINE]
PMCID:
PMC3018781
Free PMC Article

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