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Enzyme-linked immunospot assay for detection of human respiratory syncytial virus f protein-specific gamma interferon-producing T cells.

ABSTRACT: Respiratory syncytial virus (RSV) causes significant disease in elderly adults, and we have previously reported that individuals 65 years of age and older have reduced RSV F protein-specific gamma interferon (IFN-?)-producing T cells compared to healthy younger adults. To measure RSV F-specific memory T cell responses in the elderly following infection or vaccination, we optimized and qualified an IFN-? enzyme-linked immunospot (ELISPOT) assay. Since peripheral blood mononuclear cells (PBMC) from the elderly could be more fragile, we established optimal cryopreservation techniques and minimal viability acceptance criteria. The number of cells per well, types and concentrations of stimulation antigens, and incubation times were evaluated to maximize assay sensitivity and precision. The optimized assay uses 300,000 cells/well, 2 ?g/ml of an RSV F peptide pool (RSV Fpp), and incubation for 22 ± 2 h in serum-free CTL-Test medium. The assay was qualified by 3 analysts using 3 RSV F-responding donor PBMC samples (high, medium, and low responders) tested on 5 different assay days. The assay sensitivity or limit of detection (LOD) was determined to be 21 spot-forming cells (SFC) per 10(6) PBMC, and the lower limit of quantitation (LLOQ) was estimated to be 63 SFC/10(6) PBMC. The intra- and interassay percent coefficients of variation (CV) were <10.5% and <31%, respectively. The results of the qualification study demonstrate that a robust, precise, and sensitive IFN-? ELISPOT assay has been developed that is fit for measuring RSV F-specific IFN-? T cell responses in subjects enrolled in a vaccine clinical trial or in epidemiology studies.

SUBMITTER: Patton K

PROVIDER: S-EPMC4018879 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Enzyme-linked immunospot assay for detection of human respiratory syncytial virus f protein-specific gamma interferon-producing T cells.

Patton Kathryn K Aslam Shahin S Lin Jim J Yu Li L Lambert Stacie S Dawes Glenn G Esser Mark T MT Woo Jennifer J Janetzki Sylvia S Cherukuri Anu A

Clinical and vaccine immunology : CVI 20140226 5

Respiratory syncytial virus (RSV) causes significant disease in elderly adults, and we have previously reported that individuals 65 years of age and older have reduced RSV F protein-specific gamma interferon (IFN-γ)-producing T cells compared to healthy younger adults. To measure RSV F-specific memory T cell responses in the elderly following infection or vaccination, we optimized and qualified an IFN-γ enzyme-linked immunospot (ELISPOT) assay. Since peripheral blood mononuclear cells (PBMC) fro ...[more]

PMID: 24574540

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Project description:Influenza continues to be the most important cause of viral respiratory disease, despite the availability of vaccines. Today's evaluation of influenza vaccines mainly focuses on the quantitative and functional analyses of antibodies to the surface proteins haemagglutinin (HA) and neuraminidase (NA). However, there is an increasing interest in measuring cellular immune responses targeting not only mutation-prone surface HA and NA but also conserved internal proteins as these are less explored yet potential correlates of protection. To date, laboratories that monitor cellular immune responses use a variety of in-house procedures. This generates diverging results, complicates interlaboratory comparisons, and hampers influenza vaccine evaluation. The European FLUCOP project aims to develop and standardize assays for the assessment of influenza vaccine correlates of protection. This report describes the harmonization and qualification of the influenza-specific interferon-gamma (IFN-γ) Enzyme-Linked ImmunoSpot (ELISpot) assay. Initially, two pilot studies were conducted to identify sources of variability during sample analysis and spot enumeration in order to develop a harmonized Standard Operating Procedure (SOP). Subsequently, an assay qualification study was performed to investigate the linearity, intermediate precision (reproducibility), repeatability, specificity, Lower and Upper Limits of Quantification (LLOQ-ULOQ), Limit of Detection (LOD) and the stability of signal over time. We were able to demonstrate that the FLUCOP harmonized IFN-γ ELISpot assay procedure can accurately enumerate IFN-γ secreting cells in the analytical range of 34.4 Spot Forming Units (SFU) per million cells up to the technical limit of the used reader and in the linear range from 120 000 to 360 000 cells per well, in plates stored up to 6 weeks after development. This IFN-γ ELISpot procedure will hopefully become a useful and reliable tool to investigate influenza-specific cellular immune responses induced by natural infection or vaccination and can be an additional instrument in the search for novel correlates of protection.

Dataset Information

Enzyme-linked immunospot assay for detection of human respiratory syncytial virus f protein-specific gamma interferon-producing T cells.

Publications

Enzyme-linked immunospot assay for detection of human respiratory syncytial virus f protein-specific gamma interferon-producing T cells.

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