Next Article in Journal / Special Issue
Simultaneous Detection of Antigen-Specific IgG- and IgM-Secreting Cells with a B Cell Fluorospot Assay
Previous Article in Journal
ELISPOT Cell Analysis Assay: Searching for Extracellular Footprints
Cells 2012, 1(1), 5-14; doi:10.3390/cells1010005
Article

ELISPOT Refinement Using Spot Morphology for Assessing Host Responses to Tuberculosis

,
,
,
,
,
 and *
Received: 9 February 2012; in revised form: 28 February 2012 / Accepted: 6 March 2012 / Published: 13 March 2012
(This article belongs to the Special Issue ELISPOT Research)
View Full-Text   |   Download PDF [236 KB, uploaded 13 March 2012]   |   Browse Figures
Abstract: Tuberculosis is a global health problem. The Mycobacterium bovis Bacille Calmette Guerin (BCG) vaccine has variable efficacy (0–80%) so there is a drive to develop novel vaccines. The cytokine, interferon gamma (IFNγ), is an essential component of the protective response to M. tuberculosis (M. tb) infection and is also produced in response to BCG vaccination. Induction of an IFNγ response is used as a biomarker of successful vaccination in the assessment of new tuberculosis (TB) vaccines. The IFNγ ELISPOT assay provides an important tool for TB research. It is used for both the diagnosis of infection (T.Spot assay), and for the evaluation of the immunogenicity of new TB vaccine candidates in human clinical trials, in the non-human primate (NHP) model of TB infection studies. The ELISPOT assay captures IFNγ produced by peripheral blood mononuclear cells (PBMCs) following specific stimulation, onto a membrane so individual cells can be enumerated and the frequency of responding cells determined. Hence spot forming units (SFU) per 106 cells provide the traditional measure for ELISPOT assays. The discriminatory power of SFU is limited. In some situations, the number of SFU in BCG vaccinated, and unvaccinated, subjects was found to be similar, although the spots were observed to be larger in vaccinated subjects. Spot size potentially provides a measure of the quantity of cytokine produced by individual cells. The AID ELISPOT plate reader software used to determine frequency of spots also has the capability to determine the size of each spot. Consideration of spot size in combination with spot forming units was investigated in our studies of BCG immunogenicity. This additional readout was found to enhance the discriminatory power of the ELISPOT assay, and provide more information on the immune response to BCG vaccination and infection with M.tb.
Keywords: ELISPOT; IFNγ; tuberculosis; BCG ELISPOT; IFNγ; tuberculosis; BCG
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Export to BibTeX |
EndNote


MDPI and ACS Style

Sibley, L.S.; White, A.D.; Marriott, A.; Dennis, M.J.; Williams, A.; Marsh, P.D.; Sharpe, S.A. ELISPOT Refinement Using Spot Morphology for Assessing Host Responses to Tuberculosis. Cells 2012, 1, 5-14.

AMA Style

Sibley LS, White AD, Marriott A, Dennis MJ, Williams A, Marsh PD, Sharpe SA. ELISPOT Refinement Using Spot Morphology for Assessing Host Responses to Tuberculosis. Cells. 2012; 1(1):5-14.

Chicago/Turabian Style

Sibley, Laura S.; White, Andrew D.; Marriott, Alice; Dennis, Michael J.; Williams, Ann; Marsh, Philip D.; Sharpe, Sally A. 2012. "ELISPOT Refinement Using Spot Morphology for Assessing Host Responses to Tuberculosis." Cells 1, no. 1: 5-14.


Cells EISSN 2073-4409 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert