Immunochromatographic analysis

The principle of operation is that when a test strip is immersed in a biological fluid (or other liquid sample), it begins to migrate along the strip according to the principle of thin-layer chromatography. Labeled specific antibodies coated on the bottom of the test strip move with it, which affinity bind to the analyte.

There are 2 IHA formats: direct and competitive method.

1. In the direct (sandwich) ICA scheme , an antibody- labeled conjugate is applied onto the conjugate membrane. Antibodies specific for this analyte are immobilized on the test line, and anti-species antibodies specific for primary antibodies are immobilized on the control line. When applying a sample containing the analyte, when the sample enters the membrane with a conjugate, the analyte binds to the At-label conjugate. Then the immune complex enters the test zone, where it binds to specific antibodies, forming an At-Ag-At-tag “sandwich”. Excess unbound conjugate binds to anti-species antibodies on the control line. Thus, the detection of 2 lines on the test strip is a positive test result. In the absence of analyte in the sample, the conjugate binds to anti-species antibodies only on the control line, forming one line on the test strip.

   The direct IHA method is used to detect macromolecular compounds - viruses, incl. HIV various hormones (for example, in pregnancy tests), pathogens of infectious diseases.

2. The competitive ICA method used to determine low molecular weight compounds is based on competition between the analyte and the immobilized analyte: carrier protein conjugate for a limited number of specific antibody binding sites contained in the At-label conjugate. When a sample containing the analyte is applied, it binds to the At-label conjugate on the membrane with the conjugate. Further, the immunocomplex passes through the test zone, where the analyte: carrier protein conjugate is immobilized. The immunocomplex cannot contact this conjugate due to steric difficulties: low molecular weight compounds usually have one antigenic determinant and, accordingly, antibodies have one binding site to the antigen, which is already occupied by the analyte. Next, the immune complex binds antiviral antibodies located on the control line. As a result, the absence of a colored band in the test zone and the presence of color in the control zone indicates that the concentration of the analyte in the test sample exceeds its threshold value for this test.

In the absence of the analyte in the sample, the At-label conjugate binds to the conjugate Ar: carrier protein immobilized in the test line area. The unbound At-label conjugate enters the control line area and binds to anti-species antibodies there. Thus, the presence of two colored lines (test and control) is a negative result of the analysis.

The competitive IHA format is used to detect low molecular weight compounds, including metabolites of narcotic compounds in urine, oral fluid, and tissue extracts.

The advantage of the method is its quickness and ease of use, the ability to use non-instrumented ICA formats with a visual assessment of the analysis result. In this case, the use of any equipment is not required and the analysis can be carried out by a layman in any conditions, including "field".

There are also instrumental semi-quantitative and quantitative ICA formats in which special readers are used to record the label intensity in the test zone of the test strip.

Various particles with the following properties are used as labels in the ICA:

1. Dyes (nano-particles of colloidal gold or carbon, or particles of colored latex). In this case, visual detection of the result is used, or instrumental colorimetric determination (or scanning). The use of various color markers attached to the latex particles allows a multianalysis in which lines of different colors correspond to different analytes. The most commonly used label is colloidal gold nano particles.
2. Fluorescent, phosphorescent and bioluminescent labels covalently bonded to latex particles. These tags are used only in instrument versions of the IHA, when the result is recorded by a special reader. Among the above, the most common fluorescent labels.
3. Paramagnetic tags (also attached to latex particles). This type of labels is used in the ICA with the use of devices that record the strength of the magnetic field.
4. Enzyme labels are used on the same principle as in ELISA. The enzymatic reaction is recorded by staining the substrates, and the result of the analysis is visual, or read using a reader.
5. A new direction in the development of various types of ICA is the use of liposomes as carriers of various labels (staining, fluorescent, enzymatic, electroactive, etc.).

• Raphael C. Wong l Harley Y. Tse (Editors) Lateral Flow Immunoassay. Springer, USA, 2009.

• Immunochromatographic analysis (rus.) (Doc). Date of treatment January 1, 2010. Archived on April 14, 2012.