The Cytokine Enzyme-Linked ImmunoSpot (ELISPOT) Assay: Potential New Technology for Diagnosis and Studying Immune Function in Disease States
In certain disease processes, particularly those directly involving a cell-mediated immune response, the ability to study the function of T-cells and the cytokine products they secrete in response to specific antigen stimulation (for instance, a type 1 diabetes auto-antigen or a Mycobacterium tuberculosis antigen) can provide useful information about immune function and disease state. T-cells, based upon the cytokines they happen to be secreting, tell a tale of disease state (in the case of type 1 diabetes and other autoimmune disorders) or indicate presence of memory T-cells specific to exposure to antigen (in the case of Mycobacterium tuberculosis antigen), useful in diagnosing a particular condition.
The cytokine ELISPOT assay is relatively new technology currently being developed for studying T-cell and immune function that may, in the near future, be available for physicians as a diagnostic tool. It is based upon a modified version of the popular Enzyme-Linked ImmunoSorbent Assay (ELISA) and allows for the identification and enumeration of cytokine-producing cells at the single cell level. Simply put, at appropriate conditions, the ELISPOT assay allows visualization of the secreted product (cytokines) of individual activated T-cells to a specific peptide antigen. How do you “see” what cytokines individual T-cells are producing? You incubate T-cells with an antigen of interest, during which the stimulated T-cells will release cytokine in response to antigen. You then transfer the T-cells to another culture dish that has anti-cytokine antibody stuck to it (similar method to the ELISA) and the culture is allowed to sit. The T-cells are lysed, washed away, and individual spots appear after treatment with chemicals (similar to those used in photography to develop a photo) that specifically make the spots appear dark brown. Each spot that develops in the assay represents a single cell that has reacted to your chosen peptide antigen and has secreted cytokine in response to stimulation by peptide. Thus, the ELISPOT assay provides exquisitely sensitive qualitative (type of cytokine – many different anti-cytokine antibodies are available) and quantitative (number of responding cells) information (1).
Based upon relative secretion of different cytokines, the ELISPOT assay is an effective tool to enumerate T-cells with either a Th1 or Th2 phenotype and observe for more of a tendency toward a Th1 or Th2 immune response. For instance, you might stick anti-IL-10 antibody to a culture plate to capture a Th2 response, and you might stick anti-interferon gamma antibody to capture a Th1 response and compare the response of the same population of cells to the two different cytokines. This is useful in patients with an autoimmune disease, such as type 1 diabetes, and makes it possible to characterize disease state at a more detailed level. Studies have shown that those who have or who are genetically “at risk” for developing type 1 diabetes will often exhibit more of a Th1 response to diabetes auto-antigens relative to a Th2 response (2). While the assay is still under experimental development for use as a diagnostic aid, the results are promising for use as a clinical indicator of possible type 1 diabetes detectable much sooner than any clinical method we currently have available (5).
ELISPOT technology is also currently being developed to detect latent and active Tuberculosis infections. Diagnosis and preventative treatment of people with early tuberculosis infection before they develop disease (active tuberculosis) is a crucial element of tuberculosis control (4). Often the standard test for TB exposure (PPD test) gives false positives and false negatives (3), and ELISPOT technology would allow for more accurate determination of Mycobacterium tuberculosis exposure by detecting T cells responding to ESAT-6, a protein expressed in the tuberculosis bacterium, but absent from all strains of BCG vaccine (TB vaccine). These T-cells, if present, will secrete interferon gamma in response to antigen, and this interferon gamma can be detected.
ELISPOT technology has many, many more potentially clinically useful applications and may be uniquely useful for monitoring antigen-specific responses applicable to a wide range of areas in immunology research, including cancer, transplantation, infectious disease, and vaccine development.
References:
1.) http://en.wikipedia.org/wiki/ELISPOT
2.) Schloot, N. et al. 2003. Comparison of cytokine ELISPOT assay formats for the detection of islet antigen autoreactive T-cells. Journal of Autoimmunity: 21 (2203) 365 – 367.
3.) Booth, H. 2004. How tuberculosis can be diagnosed. The Pharmaceutical Journal: 273
4.http://www.brightsurf.com/news/headlines/17092/ACCURATE_IDENTIFICATION_OF_EARLY_TUBERCULOSIS_INFECTION_POSSIBLE_WITH_NEW_BLOOD_TESTp_2017.html
5.) Roep, B. et al. 1999. Autoreactive T cell Responses in Insulin-Dependent (Type 1) Diabetes Mellitus. Journal of Autoimmunity: 13, 267 – 282
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5 comments:
A very interesting post. Did you mean to say you might stick anti-IL-10 antibody to a culture plate to capture a Th2 response, and you might stick anti-interferon gamma antibody to capture a Th1 response?
Oh my! You are indeed correct! My mistake; I reversed the two. INF-G would indicate a Th1 response and IL-10 a Th2. Thanks for catching my mistake!
I have edited the blog to reflect the CORRECT cytokines for each class of T-helper cells. Shame on me for not catching the typo! Thanks again!
A plug for my own research:
Zeng C, MaWhinney S, Baron A, McFarland B. J Immuno Meth. Evaluating ELISPOT summary measures with criteria for obtaining reliable estimates. 2005; 297:97-108.
When using an ELISPOT assay, it is critical to determine an optimal effector cell concentration (perhaps using a pilot study). Choosing too few cells could lead to no or few spots, but a concentration that is too high may result in confluent spots. All ELISPOT assays should be conducted at the optimal concentration or, if feasible, across a range of concentrations. In practice, researchers often use a different (convenient) number of effector cells for each subject and then normalize to a summary concentration --this approach turns out to be invalid.
Indeed you are correct and this is an important step in assay development. When the assay is considered as a new diagnostic aid for a particular disease process or drug effect, it must be carefully optimized (I have personal experience with this! It is very tedious!!) for adequet response from the cell population and extensive statistical analysis of the data (spots produced per culture well). Depending upon what you are studying, the number of cells to use depends upon the type of response you see when using a positive control - a mitogen like Staphlococcus enterotoxin B - SEB - or ConA. When an appropiate and countable number of spots is obtained with use of a mitogen, you can assume that you have a responsive population of cells and test antigens can be studied. Typically, test antigens are compared with a "no antigen" culture well and the number of spots in test antigen cultures is compared to the "no antigen" value and statistical analysis is used to determine a "positive" or "negative" response. With my studies, I was using about 1.2 million PBMC's per culture well and, for my purposes, this seemed to produce appropriate visualization of cell response.
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