Helicobacter pylori stool antigen test: Procedure


Helicobacter pylori stool antigen test: Procedure

  • Ensure tests, specimens, buffer, and/or controls are at room temperature (15-30°C) prior to testing.

Specimen Collection and Pre-treatment:

  • Using Specimen Dilution Tubes:
    • Utilize the specimen dilution tubes provided in the test kit for collection.
    • Aim to perform the assay within 6 hours after collection for best results.
  • Specimen Handling:
    1. Remove the dilution tube applicator carefully to avoid spilling or splattering.
    2. Collect specimens by inserting the applicator stick into at least three different sites of the feces to gather approximately 50 mg (equivalent to 1/4 of a pea).
    3. Reinsert the applicator into the tube and securely tighten the cap, ensuring not to break the tip of the tube.
    4. Vigorously shake the specimen dilution tube to mix the specimen with the extraction buffer.

Testing Procedure:

  • Test Setup:
    1. Remove the H. pylori Ag test cassette from its sealed pouch and place it on a clean, level surface.
    2. Label the test cassette with patient or control identification.
    3. Perform the assay within one hour for optimal results.
  • Specimen Application:
    1. Use a tissue paper to break the tip of the dilution tube.
    2. Hold the tube vertically and dispense 2 drops of the solution into the specimen well (S) of the test cassette, avoiding air bubbles and not allowing the solution to enter the result window.
  • Observation and Results:
    1. Observe as the test operates and color moves along the membrane.
    2. Read the result at 10 minutes; do not interpret results after 20 minutes.


  • If the specimen does not migrate due to the presence of particles, centrifuge the extracted specimens. Collect 80 µL of the supernatant, dispense it into the specimen well (S) of a new test cassette, and repeat the testing instructions.

Helicobacter pylori stool antigen test: Result interpretation

  • Positive:

    • Two colored lines appear on the membrane.
      • One line appears in the control line region (C).
      • Another line appears in the test line region (T).
  • Negative:

    • Only one colored line appears.
      • This line is in the control line region (C).
      • No colored line appears in the test line region (T).
  • Invalid:

    • No control line appears.
      • Tests without a control line at the specified reading time are considered invalid and must be discarded.
      • Review the procedure and repeat the test with a new test cassette.
      • If the issue persists, stop using the kit and contact your distributor.


  • The intensity of the color in the test line region (T) can vary depending on the concentration of analytes present in the specimen.
    • Any shade of color in the test line region should be considered a positive result.
    • This test is qualitative only and does not measure analyte concentration.
  • Potential causes for control line failure include insufficient specimen volume, incorrect operating procedures, or expired tests.

Last update: 16 April 2024, 09:24


Gastroenterologist - Hepatologist, Thessaloniki

PhD at Medical School, Aristotle University of Thessaloniki, Greece

PGDip at Universitair Medisch Centrum Utrecht, The Netherlands

Ex President, Hellenic H. pylori & Microbiota Study Group