A molecular assay for sensitive detection of pathogen-specific T-cells |
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| Authors: | Kasprowicz Victoria O Mitchell Jessica E Chetty Shivan Govender Pamla Huang Kuan-Hsiang Gary Fletcher Helen A Webster Daniel P Brown Sebastian Kasmar Anne Millington Kerry Day Cheryl L Mkhwanazi Nompumelelo McClurg Cheryl Chonco Fundisiwe Lalvani Ajit Walker Bruce D Ndung'u Thumbi Klenerman Paul |
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| Institution: | Ragon Institute of MGH, MIT and Harvard, Harvard Medical School, Boston, Massachusetts, United States of America. vkasprowicz@partners.org |
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| Abstract: | Here we describe the development and validation of a highly sensitive assay of antigen-specific IFN-γ production using real time quantitative PCR (qPCR) for two reporters--monokine-induced by IFN-γ (MIG) and the IFN-γ inducible protein-10 (IP10). We developed and validated the assay and applied it to the detection of CMV, HIV and Mycobacterium tuberculosis (MTB) specific responses, in a cohort of HIV co-infected patients. We compared the sensitivity of this assay to that of the ex vivo RD1 (ESAT-6 and CFP-10)-specific IFN-γ Elispot assay. We observed a clear quantitative correlation between the two assays (P<0.001). Our assay proved to be a sensitive assay for the detection of MTB-specific T cells, could be performed on whole blood samples of fingerprick (50 uL) volumes, and was not affected by HIV-mediated immunosuppression. This assay platform is potentially of utility in diagnosis of infection in this and other clinical settings. |
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