Immunochromatographic Test (ICT): Introduction, Principle, Test Requirements, Procedure, Result -Interpretation, Uses and ICT Footages

Introduction of Immunochromatographic Test (ICT)

The full form of ICT is the immunochromatographic test and it is the most common test in the diagnostic laboratory all over the world for the rapid finding in the field of diagnosis and treatment of patients. Lateral flow assays also known as lateral flow immunochromatographic tests, are simple test devices intended to detect the presence (or absence) of a target analyte (antigen or antibody) in the sample (matrix) without the need for specialized and costly equipment.

Immunochromatographic Test (ICT): Introduction, Principle,Test Requirements, Procedure, Result -Interpretation and Uses
Fig. Urine Pregnancy Immunochromatographic Test (ICT): Left test device-Negative and Right test device-Positive

Principle of immunochromatographic Test

The immunochromatographic test (ICT) principle is the same as the sandwich ELISA (enzyme-linked immunoassay) method. The only difference is that immunological reaction is carried out on the chromatographic paper by capillary action. For this system, two kinds of specific antibodies against the antigen are applied. One of the antibodies is immobilized on the chromatographic paper while another is labeled with colloidal gold and infiltrated into a sample pad. An immunochromatographic unit is completed by binding the sample pad at the end of the membrane.

The liquid sample is dropped on the sample pad. The antigen in the sample forms an immunocomplex with the antibody labeled with colloidal gold. Its complex moves along with the liquid sample and makes a contact with the antibody immobilized on the membrane. It is followed by forming an immuno- complex with the immobilized antibody. It results in generating a colored red-purple line.  The appearance of a red-purple line on the membrane indicates the presence of an antigen of interest in the sample. The liquid of the sample migrates through the membrane very fast, which makes it possible to detect the presence or absence of antigen within 15 minutes as shown above image.

Components of Immunochromatographic Test Device

Sample pad: sample pad acts as a sponge and holds an excess of sample fluid. Once soaked, the fluid migrates to the second element. Conjugate pad: A dried format of bio-active particles in a salt-sugar matrix that contains everything to guarantee an optimized chemical reaction between the target molecule (e.g. an antigen) and its chemical partner (e.g., antibody) that has been immobilized on the particle surface. Control: It contains an antibody that picks up free latex/gold in order to confirm the test has operated correctly. Test: It contains a specific capture molecule and only captures those particles onto which an analyte (antigen or antibody ) molecule has been immobilized.

Test Requirements

  • Test device kit
  • Specimen-for skin puncture: Lancet, alcohol pad while for serum/plasma-needle, syringe, collection tube, tourniquet, alcohol swab, iodine solution, centrifuge
  • Timer
  • Waste discarding bin

The procedure of the Immunochromatographic test

Note: For those immunochromatographic tests in which whole blood is applicable for example Malaria rapid diagnostic test in the diagnosis of Malaria for detection of Plasmodium species (vivax or falciparum) antigens. Take an alcohol swab and disinfect the site of the prick. Take a pricker and puncture the area below the fingertip. Ooze the blood. In a case where there is a need for serum or plasma, follow this procedure of blood collection. Clean the venipuncture site with chlorhexidine or povidone-iodine using a back-and-forth friction scrub for 30 seconds. Allow it to dry for 30 seconds. Do not retouch the site; if you must repalpate for vein, the area must be recleaned using the above procedure.

Perform venipuncture. When the required amount of blood is received, remove the needle from the skin. Hold pressure on the site with gauze and apply an adhesive bandage when the bleeding stops. Inject blood specimens into a red-protected vial for serum or EDTA purple vial for plasma. After clotting the blood, centrifuge the vial in case of serum but not waiting for clotting in case of plasma separation. Discard the needle and syringe into a sharp container. Take two drops (the volume depends on the nature of the test and manufacturer guidelines) with a sucker and dispensed them in the sample hole of the strip. Add buffer to the sample. Wait for 10-15 minutes. Observe results and interpret accordingly.

Result interpretation of Immunochromatographic test

Negative test: Only on the band at the control region

Test positive: Both bands at test and control regions

Invalid: No band at all or band only at the test region

Note: The test is negative and positive as shown above image because of having a single band at the control region in the left test device while both bands at the control and test regions or lines in the right side test device respectively.

Advantages and disadvantages of  Immunochromatographic test

Immunochromatographic tests are a promising tool for a reliable level of diagnostic performance.
Simple. Rapid assays can be completed in 15-20 minutes. They reduce the need for trained examiners and costly equipment. It can be used under harsh field conditions. ICT assays are not as sensitive as other immunoassays like ELISA or RIA (radioimmunoassay) or molecular tests like PCR. The results of this test should be confirmed by other reference tests.

Uses of Immunochromatographic test

An immunochromatographic test is widely adopted as the quick or spot or screening test for laboratory diagnosis of various diseases etiological agents as well as the conditions and they are-

  • HBsAg test
  • HCV Ab test
  • HIV Ab test
  • Urine pregnancy test
  • Helicobacter pylori test
  • Scrub typhus test
  • Dengue test
  • K39 test for visceral leishmaniasis (Kala-azar)
  • Malarial parasite test
  • Leptospira test
  • HAV test
  • HEV test
  • SARS-CoV-2 Antigen
  • SARS-CoV-2 Antibodies
  • TORCH test, etc.

ICT Footages

Hepatitis B surface antigen (HBsAg ) – Reactive or Positive

Hepatitis B surface antigen (HBsAg ) - Reactive or positive
Fig. Hepatitis B surface antigen (HBsAg ) – Reactive or positive

Hepatitis C Virus (HCV)-Antibody Test-Reactive/Positive

 Hepatitis C Virus (HCV)-Antibody test-Positive
Fig. Hepatitis C Virus (HCV)-Antibody test-Positive

Hepatitis C Virus (HCV) Tridot-Antibody test-Non-Reactive or Negative

 Hepatitis C Virus (HCV) Tridot-Antibody test-Non-Reactive or Negative
Fig. Hepatitis C Virus (HCV) Tridot-Antibody test-Non-Reactive or Negative

Hepatitis C Virus (HCV) Tridot-Antibody test-Reactive or Positive

 Hepatitis C Virus (HCV) Tridot-Antibody test-Reactive or Positive
Fig. Hepatitis C Virus (HCV) Tridot-Antibody test-Reactive or Positive

Human Immunodeficiency Virus (HIV) Tridot- Antibody Test: Reactive/ non-Reactive or Positive or Negative

Human Immunodeficiency Virus (HIV) Tridot- Antibody Test: Reactive or Positive
Fig. Human Immunodeficiency Virus (HIV) Tridot- Antibody Test: Reactive or Positive (5) while non-reactive or negative (23)

HBsAg, HCV, HAE, and HEV tests-Non-reactive or negative

HBsAg, HCV, HAE and HEV tests-Non-reactive or negative
Fig. HBsAg, HCV, HAE, and HEV tests-Non-reactive or negative

HIV, HBsAg, and HCV Tests: Non-reactive or negative

HIV, HBsAg and HCV Tests: Non-reactive or negative
Fig. HIV, HBsAg, and HCV Tests: Non-reactive or negative

 TORCH Panel Rapid Test

The TORCH Panel Rapid Test is a lateral flow chromatographic immunoassay for the qualitative detection and differentiation of antibodies (IgM) to Toxoplasma gondii, other, rubella (German measles), cytomegalovirus, and herpes simplex viruses.

 TORCH Panel Rapid Test
Fig. TORCH Panel Rapid Test

HAV IgM Test Procedure

HAV IgM Test Procedure
Fig. HAV IgM Test Procedure

HEV IgM Test Procedure

HEV IgM Test Procedure
Fig. HEV IgM Test Procedure

rK39 Test for Visceral Leishmaniasis-Negative

rK39 Test for Visceral Leishmaniasis-Negative
Fig. rK39 Test for Visceral Leishmaniasis-Negative

Rapid rK39 immunochromatographic test showing positivity for Leishmania species

Rapid rK39 immunochromatographic test showing positivity for Leishmania species
Fig. Rapid rK39 immunochromatographic test showing positivity for Leishmania species

Rapid Immunochromatographic Test for Malaria

Rapid Immunochromatographic Test for Malaria
Fig. Rapid Immunochromatographic Test for Malaria

Helicobacter pylori Antibody Test

Helicobacter pylori Antibody Test-
Fig. Helicobacter pylori Antibody Test-Negative

Scrub Typhus Antibody Test-Positive

Scrub Typhus Antibody Test-Positive
Fig. Scrub Typhus Antibody Test-Positive

Dengue NS1antigen and IgM and IgG Antibodies Test

Dengue NS1antigen and IgM and IgG antibodies Test
Fig. Dengue NS1antigen and IgM and IgG antibodies Test showing NS1 antigen Positive

 SARS-CoV-2 antigen test-Left (weak) and right (strong) positive

Abbott sars-cov-2 antigen test
 Fig. SARS-CoV-2 antigen test-Left (weak) and right (strong) positive

SARS-CoV-2 IgM and IgG Antibodies Test

SARS-CoV-2 IgM and IgG Antibodies Test
Fig. SARS-CoV-2 IgM and IgG Antibodies Test: Test device 1-Negative, 2-IgG: Positive, 3-IgM (weak) and IG (strong): Positive, and 4-IgG: Positive

Leptospira IgM and IgG Rapid Diagnostic Test

Leptospira IgM and IgG Rapid Diagnostic Test
Fig. Leptospira IgM and IgG Rapid Diagnostic Test

Further Reading 

  1. https://www.sciencedirect.com/topics/chemistry/immunochromatography
  2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4488388/
  3. https://www.creative-diagnostics.com/Immunochromatography-guide.htm
  4. https://www.scielo.br/scielo.php?script=sci_arttext&pid=S0102-311X2020000602001
  5. https://pubmed.ncbi.nlm.nih.gov/16711337/
  6. https://www.rapidmicrobiology.com/test-method/lateral-flow-immunoassays

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