TSPOT.TB Test Kit

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T-SPOT.TB Test Kit

• Flexible, 96-well format
• Twelve, 8-well strips
• 4 wells used per patient 24 patients per kit
• Positive and Negative control for each patient
  test
• Utilizes standard blood collection tubes
• No special lab equipment required

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The Science behind T-SPOT
Simplified, validated ELISpot method
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Performing the T-SPOT.TB Test

Plasma

• Peripheral blood mononuclear cells (PBMCs) are
  separated from whole blood and washed
• Removes any source of background interference
• Washed PBMCs are counted to ensure a
  standardized number of cells are added to the
  assay

Lymphocyte and monocyte band
Density gradient fluid
Gel barrier
Erythrocytes and neutrophils
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Performing the T-SPOT.TB Test

• T cells are stimulated using TB antigens (ESAT-6
  and CFP 10) not present in BCG and most
  non-tuberculosis mycobacteria
• 4 wells per sample
• Negative Control
• Panel A (ESAT-6)
• Panel B (CFP 10)
• Positive Control

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Performing the T-SPOT.TB Test

• Wells coated with high affinity antibodies to
  IFN-?
• Incubate inoculated plates in
  humidified incubator at 37oC
  with 5 CO2 for 16-20 hours

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Performing the T-SPOT.TB Test

• Plate is washed
• Conjugated second antibody is added
• Binds to INF-? secreted by T cells
• Incubate at 2 - 8oC for 1 hour
• Plate is washed
• Unbound conjugate removed

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Performing the T-SPOT.TB Test

• Add Substrate
• Incubate at Room Temp for 7 minutes
• Substrate cleaved by bound enzyme to form a spot
  of insoluble precipitate at the site of the
  reaction
• Each spot represents where IFN-? was secreted in
  the vicinity of T cells
• Plate is washed thoroughly to stop the detection
  reaction and allowed to dry

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Interpretation of Results
Negative Result
Positive Result