Enzyme-linked Immunospot Assay (ELISPOT): Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Summary

Identification of microbial targets of adaptive immunity in idiopathic diseases can be accomplished by the use of the enzyme-linked immunospot assay.

Abstract

Adaptive immunity is an important component to clearance of intracellular pathogens. The ability to detect and quantify these responses in humans is an important diagnostic tool. The enzyme-linked immunospot assay (ELISPOT) is gaining popularity for its ability to identify cellular immune responses against microbial antigens, including immunosuppressed populations such as those with HIV infection, transplantation, and steroid use. This assay has the capacity to quantify the immune responses against specific microbial antigens, as well as distinguish if these responses are Th1 or Th2 in character. ELISPOT is not limited to the site of inflammation. It is versatile in its ability to assess for immune responses within peripheral blood, as well as sites of active involvement such as bronchoalveolar lavage, cerebral spinal fluid, and ascites. Detection of immune responses against a single or multiple antigens is possible, as well as specific epitopes within microbial proteins. This assay facilitates detection of immune responses over time, as well as distinctions in antigens recognized by host T cells. Dual color ELISPOT assays are available for detection of simultaneous expression of two cytokines. Recent applications for this technique include diagnosis of extrapulmonary tuberculosis, as well as investigation of the contribution of infectious antigens to autoimmune diseases.

Protocol

For the following protocol, we use peripheral blood mononuclear cells (PBMC) as the cells of interest. However, this protocol may be used with other cell types. Perform the assay in a tissue culture hood using sterile technique.

Day 1: Preparation of Plate and Cells

1. Take out an ELISPOT plate and open it in the hood.
2. Wash the plate 3 times with 150 μL of 1X PBS using a multichannel pipettor, if available, otherwise single well pipetting is acceptable.
3. Take out a package of reservoirs and open it in the hood.
4. Add 2 μg/mL of the capture antibody, anti-human I....

Discussion

The ELISPOT assay has emerged as one of the most important and widely used assays to monitor immune responses in humans and a variety of other species. With the ELISPOT assay, immune cell frequencies can be measured at the single cell level without expansion or manipulation of cell populations. ELISPOT assay has been widely applied to investigate antigen specific immune responses in various diseases including infections, cancers, allergies and autoimmune diseases. The application of this assay has been largely applied t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
A. Preparation of Media
B. Preparation of Tris Buffer
Add 800 ml of ddH2O to 1 L beaker. Measure 12 grams of Tris and add to the beaker. Add 0.12 grams of magnesium chloride hexahydrate (MgCl2:6H2O) into the beaker. Stir with magnetic stirring rod on automatic stirring plate. Once dissolved, pH the solution to 9.5. Add 200 mL of ddH2O to the beaker, bringing it up to 1000mL. Filter the solution with a 0.22 μm filter.
C. Materials necessary for ELISPOT analysis: