Evidence of tissue structures, antibodies and pathogens through immune labeling is popular, modern and accurate. Immunofluorescence refers to immune labeling with prepared, fluorescent antibodies that are made to glow under UV light.
At the Direct immunofluorescence detection the substrate is examined directly with luminous antibodies, without primary antibodies or artificial antigens.
What is direct immunofluorescence detection?
Direct immunofluorescence detection is a diagnostic method based on knowledge of immunology, immunostaining and immunohistochemistry. The focus here is on the ability of antibodies to bind to specific points in tissue or to antigens in a serum. These places are epitopes.
In biochemical diagnostics, there are artificial antibodies or mimetics (singular: mimetic) that enable these antibody-antigen bonds to be marked by fluorescence or radioactivity. Artificial antibody conjugates bind on the one hand to the epitopes and on the other hand have a fluorescent marker in the case of immunofluorescence. This is an alternative to using radioactive markers. The special thing about immunofluorescence direct detection compared to indirect detection is that the antibody that binds to the epitope of the antigen in the examined material also represents the antibody conjugate with the fluorescent marker. Additional antibodies are not necessary for direct detection.
Fluorescein, which shines under UV light, and fluorescein isothiocyanate (FITC) are used as dyes for making the artificial antibody conjugates in immunofluorescence. So far it reads a bit complicated, but direct immunofluorescence detection is standard medical laboratory diagnostic method for a wide variety of medical questions. The antibodies with the fluorescent dye are ready for sale.
Function, effect & goals
Immunofluorescence direct detection is available for tissue examinations for staining certain structures in tissue. But they are also available for single cells. Flow cytometry plays a major role here. Finally, there are the solid and liquid phase immunassays. Immunofluorescence examinations of tissues are especially important in oncology, i.e. the medical treatment of cancer diseases.
Tumor-specific antigens in tumor tissue can be detected directly with immunofluorescence. These examinations on tissue samples from tumors are often important in order to find out where the existing metastases came from in the body, or in order to make a judgment as to whether a tumor is benign or malignant. The examination of individual cells with direct immunofluorescence detection is used to find virus antigens, bacterial antigens and other epitopes.
For example, you can find out whether the cells are infected by a virus and what stage of the infection cycle the cells are in. The name FACS (= Fluorescence-Activated Cell Sorting) is a highly efficient flow cytometry method in which the cells marked with fluorescence are distributed to different test tubes depending on the type of color. This procedure is important in immunology, hematology and infectious diseases.
Immunofluorescence immunassays allow the direct detection of environmental toxins, genetically modified organisms and certain additives in food. In this experimental setup there is always a solid and a liquid phase. Numerous pathogens, including the HIV virus that causes AIDS, could also be detected directly. When detecting infectious and autoimmune diseases, however, it is often a matter of detecting antibodies rather than antigens. These are the defense molecules formed by the body's immune system. According to the definition presented here, such proofs are then not direct proofs, since the fluorescent antibodies do not couple directly to the body's own antigens, but to the antigens of the test arrangement.
These antigens in the experimental set-up are in turn linked to the body's own antibodies. Immunofluorescence direct detection for common infections, for example HIV viruses and chlamydia, is only used in special detection and confirmation tests. There are tests for many more diseases. In most situations, indirect detection of antibodies is more suitable for infectious diseases, as the body's own immune system has an ingenious ability to remember previous infections. In other situations, direct detection of antigens and indirect detection of antibodies complement each other. The latter show that an infection has occurred before, while the former provide more precise information about the current status of the activity of the pathogen.
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With direct immunofluorescence detection, as with all medical evidence, there are two risks: the risk of a false, positive result and the risk of a false, negative result. False, positive results cause psychological discomfort and great stress for the patient.
Therefore, additional test procedures are added to positive results, especially if the diagnosis leads to drastic life changes. The risk of a false, negative result is that the patient does not learn about the threat to his own health and perhaps even to public health in good time. Therefore, it is good to do a lot of research and to make and offer a large number of different immunofluorescence direct detections ready for sale. Together with other direct and indirect detection methods for diseases and pathologies, this increases the accuracy of the diagnosis.
Direct detection is based on the antibody conjugate, which on the one hand binds to the epitopes of the antigens and on the other hand also causes the fluorescence. Such a product is therefore only used in one type of test procedure and is not used for other types of test procedure.This is an important procedural difference to the indirect evidence, in which the fluorescent antibodies are preceded by primary antibodies for epitope binding. The antibody conjugate is therefore suitable for different tests. This procedural difference is different from the medical difference between the indirect detection of antibodies and the direct detection of antigens.
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