Immuno-chromatographic Assay
Immunochromatographic Assay (ICA), also known as lateral flow or rapid tests, is a technique that enables quick and simple detection of specific target molecules, such as antigens or antibodies, in biological samples. A common example of immunochromatographic assay includes the RSID tests for the detection of human blood, semen, saliva, urine, etc.
Figure: RSID tests for the detection of human blood, semen, saliva and urine
Principle of Immunochromatographic Assay (ICA)
Immunochromatographic assays are based on the principles of capillary action and specific antigen-antibody interactions to enable rapid and visual detection of target analytes. This rapid test utilizes a test strip containing distinct zones, including a sample application pad, a conjugate pad containing labelled primary antibodies, a reaction or test zone with immobilized primary antibodies, and a control zone with immobilized secondary antibodies.
When a sample is added to the application pad it begins to migrate along the membrane facilitated by capillary action. The target antigens, if present in the sample, form an antigen-antibody complex with labelled primary antibodies on the conjugate pad. These complexes are captured at the test zone (T) and the control zone (C), resulting in the formation of coloured lines at these zones. The appearance or absence of these lines indicates the test result.
Figure: Principle of Immunochromatographic assay (ICA)
Procedure for Immunochromatographic Assay (ICA)
Sample Application: Apply the biological sample (e.g., blood, urine, or other fluids) to the sample pad on one end of the test strip.
Lateral Flow and Formation of Antigen-Antibody Complex: Capillary action facilitates the movement of the sample along the strip through the conjugate pad, which contains labelled primary antibodies specific to the target antigen. If the target antigen is present in the suspected sample, it binds to the primary antibody and forms an antigen-antibody complex that continues to flow toward the reaction pad on the test strip.
Capture of Complex: The reaction pad contains immobilized primary antibodies specific to the target antigen at the test line (T) and immobilized secondary antibodies specific to the primary antibody at the control line (C). As the formed complex continues to flow along the strip, it is captured by the immobilized primary antibodies at the test line.
Control: Immobilized secondary antibodies at the control line (C) bind to the primary antibodies. A positive reaction at the control line occurs even in the absence of the antigen-antibody complex. Therefore, it ensures proper flow and functionality of the test.
Result Interpretation: Results are interpreted based on the presence or absence of coloured lines at the test (T) and control zones (C). A line appearing at the test zone (T) indicates a positive result for the target antigen.
Applications of Immunochromatographic Assay 👩🏻🔬
Rapid identification of biological samples: Identification of human-specific antigens in biological samples (such as blood, semen, saliva) to determine whether a given sample is of human origin.
Medical Diagnostics: Rapid detection of infectious diseases (such as HIV, malaria, and influenza), pregnancy testing, and hormone level monitoring.
Drug Testing: On-site screening for the presence of drugs or drug metabolites.
Advantages of Immunochromatographic Assay 👍🏼
Rapid Results: Provides quick results, usually within minutes.
Ease of Use: Simple to operate, requiring minimal training.
Portability: Suitable for on-site and point-of-care testing, eliminating the need for a laboratory setting.
Long Shelf Life: Stable reagents and components contribute to a longer shelf life.
Cost-Effectiveness: Generally more cost-effective compared to traditional laboratory methods.
Limitations of Immunochromatographic Assay 👎🏼
Sensitivity Variances: Sensitivity may vary for different assays, and some may lack sensitivity for low concentrations of analytes.
Quantitative Limitations: Typically provides qualitative results; quantification may be challenging.
Limited Multiplexing: Limited ability to detect multiple analytes simultaneously in a single test.
Subject to Hook Effect: High concentrations of analytes may cause a “hook effect”, also known as the “prozone effect” or “high-dose hook effect”. In the presence of a high concentration of the analyte, the antibodies can become saturated, and excess unbound analyte prevents the formation of a complete sandwich immunocomplex. As a result, the signal produced is lower than expected, leading to a false-negative result.
Interference: Non-specific interactions or interference may occur, leading to false-positive or false-negative results.