How the Test Works

The principles of our T-SPOT assay system, are shown below, using blood as the bodily fluid in the example. The process starts with a blood sample, from which PBMCs (specifically Peripheral Blood Mononuclear Cells) or WBC components containing T cells are separated, washed and counted. A pre-determined number of PBMCs and antigens specific to the disease or condition of interest are then added to the wells of a microtiter plate to which antibodies to interferon-gamma, or IFN-γ, are bound. The test is based on the principle that the T cells of an individual who carries an active infection will respond to the antigens and secrete interferon-gamma. The secretion of interferon-gamma by the T cells of the subject is captured by the anti-interferon-gamma antibodies coated to the floor of each well. The numbers of individual reacting T cells are enumerated through visualizing the footprint of each T cell by this secretion of interferon-gamma.

How It Works

  1. A blood sample is collected using routine phlebotomy and a standard blood collection tube from which a subset of white blood cells, known as peripheral blood mononuclear cells, are isolated. The cells are washed, counted and normalized to create a standard cell suspension.
  2. A standard number of cells are added into specially designed plates and stimulated with antigens specific to the disease under study. Cells responding to these antigens release a chemical messenger known as a cytokine.
  3. Cytokine antibodies are used to directly capture the cytokine as it is released by the cells. A secondary labelled antibody is added and binds to the captured cytokine.
  4. A detection reagent is added and reacts with the secondary labelled antibody. This reaction produces spots, which are a footprint of where the cytokine was released. Spots are then enumerated.