title: A molecular assay for sensitive detection of pathogen-specific T-cells.
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.
authors.affiliation: Ragon Institute of MGH, MIT and Harvard, Harvard Medical School, Boston, Massachusetts, United States of America. vkasprowicz@partners.org
authors.forename: Victoria O
authors.initials: VO
authors.lastname: Kasprowicz
authors.forename: Jessica E
authors.initials: JE
authors.lastname: Mitchell
authors.forename: Shivan
authors.initials: S
authors.lastname: Chetty
authors.forename: Pamla
authors.initials: P
authors.lastname: Govender
authors.forename: Kuan-Hsiang Gary
authors.initials: KH
authors.lastname: Huang
authors.forename: Helen A
authors.initials: HA
authors.lastname: Fletcher
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Webster
authors.forename: Sebastian
authors.initials: S
authors.lastname: Brown
authors.forename: Anne
authors.initials: A
authors.lastname: Kasmar
authors.forename: Kerry
authors.initials: K
authors.lastname: Millington
authors.forename: Cheryl L
authors.initials: CL
authors.lastname: Day
authors.forename: Nompumelelo
authors.initials: N
authors.lastname: Mkhwanazi
authors.forename: Cheryl
authors.initials: C
authors.lastname: McClurg
authors.forename: Fundisiwe
authors.initials: F
authors.lastname: Chonco
authors.forename: Ajit
authors.initials: A
authors.lastname: Lalvani
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Walker
authors.forename: Thumbi
authors.initials: T
authors.lastname: Ndung'u
authors.forename: Paul
authors.initials: P
authors.lastname: Klenerman
mesh_terms: D054370:Chemokine CXCL9 / Q000235:genetics / Q000378:metabolism; D058501:Enzyme-Linked Immunospot Assay; D000939:Epitopes / Q000276:immunology; D005786:Gene Expression Regulation; D006678:HIV / Q000276:immunology; D015658:HIV Infections / Q000175:diagnosis / Q000276:immunology / Q000382:microbiology / Q000821:virology; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007165:Immunosuppression Therapy; D007371:Interferon-gamma / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000276:immunology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D018121:Receptors, Cytokine / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013601:T-Lymphocytes / Q000276:immunology*; D014376:Tuberculosis / Q000097:blood / Q000175:diagnosis / Q000276:immunology / Q000382:microbiology
chemical_list: C080810:CXCL9 protein, human; D054370:Chemokine CXCL9; D000939:Epitopes; C104577:IP10-Mig receptor; D018121:Receptors, Cytokine; D007371:Interferon-gamma