Researcher reviewing data on his computer screen

How the T-SPOT.TB test works

1 small tube. 1 million ways to find the truth.2

The immune response to infection with Mycobacterium tuberculosis (MTB) is mediated predominantly through T cell activation. As part of this response, T cells are sensitised to MTB antigens and the activated effector T cells, both CD4 and CD8, produce the cytokine interferon-gamma when stimulated by these antigens. The T-SPOT.TB test uses T-SPOT technology, a modified version of the enzyme-linked immunospot (ELISPOT) methodology to enumerate M. tuberculosis-sensitised T cells by capturing interferon-gamma in the vicinity of CD4 and CD8 T cells from which it was secreted.2

When your test looks for signs of TB infection using T cells, it matters how many T cells you include. The T-SPOT.TB test, unlike other tests, uses the same number each time. And it’s a big number: 250,000 cells per well, four wells per test.2 That’s a million witnesses to infection – and a moment of truth for TB.


The principles of the T-SPOT.TB test:

T-SPOT.TB test technology

Step 1

A blood specimen 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 (PBMC), are isolated. The cells are washed, counted and normalised to create a standard cell suspension.

T-SPOT.TB test technology

Step 2

A standard number of cells are added into specially designed plates and stimulated with TB-specific antigens, ESAT-6 and CFP10. Cells responding to these antigens release interferon-gamma.

Step 3

Interferon-gamma antibodies are used to directly capture interferon-gamma as it is released by the cells. A secondary enzyme labeled antibody is added and binds to the captured interferon-gamma.

Step 4

A detection reagent is added and reacts with the enzyme labeled antibody. This reaction produces spots, which are a footprint of where the interferon-gamma was released. Spots are then enumerated.


All of the above can now be automated using the T-Cell Select reagent kit, see below the different workflows available to perform the T-SPOT.TB test:

Comparison of the different workflows available to perform the T-SPOT.TB test, showing total hands on time, total time per sample and hands on time per sample for each of the 3 processes (note full automation timings are calculated for 48 samples).


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T-SPOT.TB test

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