A molecular assay for sensitive detection of pathogen-specific T-cells
Kasprowicz VO., Mitchell JE., Chetty S., Govender P., Huang KHG., Fletcher HA., Webster DP., Brown S., Kasmar A., Millington K., Day CL., Mkhwanazi N., McClurg C., Chonco F., Lalvani A., Walker BD., Ndung'u T., Klenerman P.
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. © 2011 Kasprowicz et al.