Abstract
Interferon-gamma (IFN-γ) is a glycoprotein generated by lymphocytes that possesses anti-tumor, antiviral and immunomodulatory functions. IFN-γ assays are broadly employed in immunological research and clinical diagnostic tests. Intracellular IFN-γ staining, in particular, is an important immune assay that allows simultaneous determination of cellular phenotype and antigen-specific T cell response. Aptamers have great potential for molecule detection and can bind to target molecules with high affinity and specificity. In this study, a novel 59-mer DNA aptamer (B1–4) was developed for assay of intracellular IFN-γ. The selected aptamer bound to IFN-γ with a Kd of 74.5 nM, with minimal cross-reactivity to albumin. The aptamer was also found capable of binding with paraformaldehyde-fixed IFN-γ. Moreover, B1–4 could enter permeated and paraformaldehyde-fixed lymphocytes, and bound to intracellular IFN-γ produced by these cells. When FITC-labeled B1–4 was used to stain a group of lymphocytes, the average fluorescence of the cells was positively correlated with the number of PMA-stimulated lymphocytes within the group. A standard curve could thus be established for assessing the fraction of IFN-γ-producing cells in a cluster of lymphocytes. The selected aptamer hence provides a novel approach for assaying intracellular IFN-γ generated by a group of lymphocytes, and may have application potential in both scientific research and clinical laboratory test.
Highlights
Assays of IFN-c are commonly used in both immunological research and clinical diagnosis
Streptavidin-coated magnetic beads were purchased from Promega (USA). 1-Ethyl-3-(3-dimethyllaminopropyl)-carbodiimide hydrochloride (EDC), phorbol 12-muristate 13-acetate (PMA), and ionomycin were purchased from Sigma (US)
To monitor the enrichment of IFN-c-binding DNAs during each round of selection, the beads were incubated with FITC-labeled singlestranded DNA (ssDNA) pool and subjected to flow cytometry analysis
Summary
Assays of IFN-c are commonly used in both immunological research and clinical diagnosis. Current IFN-c detection methods are based on specific IFN-c antibodies, and include enzyme-linked immunosorbent assay (ELISA) [4,5,6], enzyme-linked immunospot assay (ELISPOT) [7,8], and intracellular cytokine staining (ICS) [9,10]. Among these methods, ICS is a commonly used important assay for assessing T cell reactivity [11,12,13], since it is the only immunological technique that can simultaneously determine both the cellular phenotype and the antigen-specific T cell response. The major reagents used in ICS are fluorescent dyes, which are safer than radioactive compounds such as 3H-thymidine or 51Cr
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