What is immunochromatographic analysis (IHA)? We will examine in more detail in this matter.
IHA is an immunochemical method of analysis, which is based on the method of thin-layer chromatography. It includes the reaction between the corresponding antibody and antigen in biological materials. It is carried out by means of special test strips, test cartridges or panels. IHA is an express test.
The essence of this research method
Its action is based on the fact that when immersed in a biological fluid (or other liquid sample) of the test, it begins to move along the strip according to the method of thin-layer chromatography. Specific labeled antibodies applied to it move with it, which bind affinity to the component being analyzed.
Two analysis formats are distinguished: direct and competitive.
Direct method
In the format of a sandwich (direct) immunochromatographic blood test, an antibody-labeled conjugate is used, which is applied to the membrane for conjugate. The immobilized antibodies on the strip, with a specific analyte, on the control line are anti-species antibodies specific for primary antibodies.
When a sample containing the conjugate (the desired substance) is introduced onto the membrane, the desired component binds to the At-label conjugate. After that, the formed immune complex penetrates into the test zone, where it binds to specific antibodies, and an At-Ag-At tag is formed. Then, an excessive amount of unbound conjugate is coupled on the control line with anti-species antibodies. If two lines appear on the test strip, the test result is positive. If the analyte is absent in the sample, then the conjugate binds to antivirus antibodies only on the control line, and one line is formed.
Direct IHA is used to determine the causative agents of infectious pathologies, viruses - macromolecular compounds, including various hormones, HIV.
Competitive research method
The method of competitive immunochromatographic analysis is used to establish low molecular weight compounds. It is based on the competition of the immobilized analyte conjugate and analyte: carrier protein for a limited number of peripheral antibody binding sites contained in the At-label conjugate. When a sample containing an analyte is applied, the latter binds to the conjugate with the At-label conjugate on the membrane. The immunocomplex then passes through the test zone in which the analyte conjugate, i.e., the carrier protein, is immobilized.
The immunocomplex cannot contact this conjugate due to steric difficulties: low molecular weight compounds most often have one antigenic determinant, so immunoglobulins have one center of contact with the antigen that has already been occupied with the analyte. The immune complex is subsequently bound by anti-species immunoglobulins, which are on the control line. If there is no colored strip in the test area and there is color in the control zone, this indicates that the content of the determined component in the test sample is higher than the threshold value for such a test. If there is no analyte in the sample in the analyte, then the At-label conjugate is coupled with the Ag conjugate: carrier protein immobilized in the region of the test line. The at-label free conjugate enters the control line, where it binds to anti-species antibodies. If there are two colored lines (control and test), then we can talk about a negative test result.
The competitive ICA method is used to determine low molecular weight compounds, including tissue extracts.
The advantage of this method is the ease and speed of use, the ability to use non-instrumented ICA methods with a visual assessment of the result. No additional equipment is required, and the analysis can be carried out by a person without a medical education at any time.
What labels are used in IHA?
In immunochromatographic analysis, different particles and substances are used as labels.
- Dyes (colored latex particles, or carbon nanoparticles, or colloidal gold). This option allows you to visually track the result or determine the colorimetric device. The use of ink marks that attach to the medium allows for multi-analysis, where multi-colored lines correspond to different analytes. The most commonly used label is colloidal gold nanoparticles.
- Bio-luminescent, phosphorescent and fluorescent labels covalently bound to latex particles. They are used only in instrumental methods of ICA, in which the result is recorded by a special reader. The most common are fluorescent labels.
- Paramagnetic labels attached to latex particles. The method is used in the ICA using instruments that record the strength of the magnetic field.
- Liposomes used as carriers of different labels (electroactive, enzymatic, fluorescent, staining, etc.) This is a new direction among the types of analysis.
- Enzyme labels used on the basis of ELISA. Registration of the reaction is carried out by staining the substrates. As a result, a visual result is obtained, or is read by a reader.
The advantages of this research method
The main advantages of using the method of immunochromatographic analysis are:
- Convenience and simplicity - allows you to get a result (initial presentation and analysis of the cause of the disease) without special skills and equipment.
- Reliability - the reliability of the tests reaches 99.8%, each test at the same time has internal built-in control.
- Anonymity - this is especially important when determining sexually transmitted diseases, other infectious diseases, as well as establishing the use of narcotic substances.
- Profitability - everyone can get a test, time is saved for analysis.
- Independence - does not require prior consultation and prescription.
Drug test how it is carried out: features and disadvantages of the IHA method
The most popular service is the analysis of urine for drugs through the method of immunochromatographic analysis. This allows you to examine urine for 14 types of drugs.
Immunochromatographic analysis for drugs is also considered an express diagnostic method. The test result can be found after 15 minutes. For this study, special test strips are used that are saturated with indicator substances. If there are traces of a narcotic substance in the urine, the color of the strip changes. The main drawback of such an analysis is the inability to determine the concentration of the substance and the degree of drug intoxication, thus, the results of immunochromatographic analysis cannot be used as evidence in court.
What is the result of such an analysis?
If two pink stripes appear on the indicators, then it is concluded that the drug is not contained in the urine, or its concentration is insufficient for determination by the immunochromatographic method. When one pink strip occurs, one can speak of the presence of a narcotic substance: marijuana, derivatives of ephedron, opiates, derivatives of barbituric acid, amphetamines, cocaine, phencyclidine, benzodiazepines, ecstasy.
What conclusions can be drawn?
Immunochromatographic analysis (rapid test) is an effective diagnostic tool, it allows you to visually identify and evaluate the concentration of antigens, hormones, antibodies and other diagnostically important components in the human body for several minutes. Rapid tests have a high degree of accuracy and sensitivity, more than a hundred types of diseases are found, as well as the entire spectrum of narcotic substances, and the reliability of the analysis is very high.