Cost-efficient quantification of enzyme-linked immunospot.

Enzyme-linked immunospot (ELISPOT) analysis is a sensitive technique for the detection and quantification of antigen-specific T cells (5,7) and also for the detection of various cytokines released by immune cells (2). It is used extensively to monitor peptide-specific T cell responses in vaccination protocols (3), melanoma patients (6), and adoptive cellular immunotherapy (9) because it can detect and quantify low numbers of antigen-specific T cells in freshly isolated blood lymphocytes with no need for prior in vitro expansion (9). The assay is based on the visualization of cytokine secretion by individual T lymphocytes after in vitro stimulation by an antigen. Briefly, the ELISPOT plates are coated with antibody that is specific for the cytokine being assayed. The antibody binds to the nitrocellulose base of the ELISPOT plate. The activated cells are then transferred to the plate, and cytokines are released during the incubation period. Cytokines released locally around each cell are captured by the specific antibody, and any excess cytokine is washed off. A second antibody that is also specific for the cytokine of interest is then added; this antibody is coupled to an enzyme that is capable of converting a substrate into an insoluble colored product. The plate is then washed again, and the enzyme substrate is added. The substrate gets converted into the insoluble product, forming colored spots representing the areas of captured cytokines secreted by activated cells. These spots are then counted for the quantification of cytokines or activated cells. Even though it is a widely used technique, most laboratories still rely on the manual quantification of the colored spots using a stereomicroscope, which limits the great potential of this otherwise powerful technique. A commercially available digital quantification system utilizing the AlphaImager System (Alpha Innotech, San Leandro, CA, USA) incorporating a charge-coupled device (CCD) camera and the software to quantify the spots has been recently reported (8). However, it is impractical for most laboratories to invest in such expensive systems (approximately AUS $80000 from Carl Zeiss, Thornwood, NY, USA) with a limited area of application. An alternative approach currently being used involves removal of the nitrocellulose membrane from each well of the microplate for enumeration. This is, however, very tedious and also restricts the possibility of storing the plates for future reference. We have found that the test plates can be stored wrapped in aluminium foil at 4°C for years without any effect on the quality of spots. We suggest an accurate and more cost-efficient approach for the enumeration of these spots using equipment that can be assembled in most laboratories. The ELISPOT plates are stained after culture, dried at room temperature, and illuminated from the top. The image of each well is captured by a CCD video camera (DAGE; AUS $1500; Maryland Telecommunications Inc., Michigan, IN, USA) directly attached to a microscope lens (25 mm, 1:1.3; AUS $200) and held by a clamp on a support stand approximately 10 cm from the plate. The lens magnifies the image of each well of the microplate to the full screen size. Optimum resolution can then be achieved by adjusting the contrast from the lens. Thereafter, the distance of the camera and the aperture size of the lens are kept constant for each experiment. The parameters set for the enumeration of the spots of interest are the intensity threshold and the diameter of spots. The intensity threshold is thus set optimally so as to eliminate any small artifacts. The image is then captured and the spots are counted automatically (Figure 1) using the Image-Pro Plus imaging software (AUS $5500; Media Cybernetics, Silver Spring, MD, USA). The data (number of spots and the area of each spot) is transferred to a Microsoft Excel spreadsheet, and the means and standard deviations are calBenchmarks