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Principle and Analysis Method of Fluorescence Immunoassay (FIA)

Fluorescence immunoassay (FIA) has the advantages of high specificity of immune response and high sensitivity of fluorescence technology. (TRF) et al. Qualitative or quantitative measurement by fluorescence microscope, microscopic fluorescence spectrophotometer, flow cytometer and time-resolved fluorometer is widely used in the diagnosis of endocrine and metabolic diseases.

Basic principle: a new immunoassay technology using fluorescein-labeled antibodies or antigens as tracers, the principle is similar to ELISA. This method can not only quantify antigens and antibodies in liquids, but also qualitatively and quantitatively identify antigens and antibodies in tissue sections. Generally, due to the autofluorescence of samples and reagents and the scattering of excitation light, the background fluorescence is high, which affects the sensitivity of the assay. Lanthanides are generally used as fluorescent markers (tracers). After the tracer is combined with the corresponding antigen or antibody, the fluorescence phenomenon is observed or the fluorescence intensity is measured with the help of a fluorescence detector, so as to judge the existence, localization and distribution of the antigen or antibody or detect the content of the antigen or antibody in the tested sample.

The principle of FIA is the same as that of RIA, except that the label is changed from nuclide to fluorescein. Fluorescein is a substance that can produce strong fluorescence under the irradiation of laser light. After it absorbs light energy, it generates excited molecules within a very short time (10-8 to 10-9 seconds) and releases wavelengths higher than the excitation light. With longer visible light, the substance with this characteristic is chemically bound to the antibody (or antigen) molecule, and after the latter is combined with the matching antigen (or antibody), the fluorescence is observed or the fluorescence intensity is measured by a fluorescence detector, Determine the presence and distribution of antigens (or antibodies), or detect the content of antigens (or antibodies) in samples.

Fluorescence immunoassay is a method that combines the specificity of immunological reactions with the sensitivity of fluorescent techniques. Fluorescence immunoassay plays an important role in basic medical research and clinical diagnosis, and the development of labeled probes with excellent performance is a decisive factor in the development of this technology. In recent years, semiconductor fluorescent nanocrystals have attracted extensive attention due to their special physical and chemical properties, and have been widely used as a new generation of fluorescent markers in the fields of optics, biology, and medicine.

Fluorescence immunoassay is divided into four types: direct method/indirect method/complement method/double labeling method

1. Direct method: The specific fluorescent antibody is directly added to the fixed specimen (containing the antigen) to form an antigen-antibody complex to identify the unknown antigen. And according to the distribution and morphology of fluorescence, its antigenicity can be determined. The advantages of this method are that it is simple, rapid, and specific; the disadvantage is that the known antigen cannot be used to detect unknown antibodies, and the detection of each antigen requires the preparation of corresponding specific fluorescent antibodies, which is less sensitive. Commonly used for rapid inspection of bacteria and viruses.

2. Indirect method: also known as double antibody method. Using fluorescently labeled antibodies to identify unknown antigens or antibodies, fluorescently labeled antiglobulin antibodies can detect various unknown antigens or antibodies, and its sensitivity is 5 to 10 times higher than the direct method. The disadvantage is that there are many components participating in the reaction. It is complicated and requires many comparisons. Commonly used in the detection of various autoantibodies.

3. Complement method: use fluorescein to label anti-complement antibodies to identify unknown antigens or antibodies. This method only needs one labeled anti-complement antibody. Since complement binds to the antigen-antibody complex without species specificity, it is not limited by the known antibody or the animal species of the serum to be tested, so various antigen-antibody systems can be detected. . The disadvantage is that it is easy to produce non-specific interference, requires more controls, and is not easy to store for a long time due to the instability of complement.

4. Double labeling method: use fluorescent yellow isothiocyanate (FITC) and tetraethyl rhodamine (RB200) to label different antibodies to detect the same matrix sample, if there are two corresponding antigens, then Displays different colors of fluorescence.

Fluorescein-labeled antibody

An ideal fluorescein for labeling antibodies should have the following characteristics:

1. It has chemical groups that can form covalent bonds with protein molecules, which are not easy to dissociate after binding, and the unbound fluorescein and its degradation products are easy to remove;

2. It can maintain the inherent biochemical properties, immune activity and specificity of the antibody after binding with the antibody; the fluorescence efficiency (the number of photons of fluorescence emitted/the number of photons of absorbed light) is high, and there is no significant decrease after binding to the protein;

3. The fluorescent color contrasts with the color of the observation background, which is easy to judge;

4. The method of combining is simple, rapid, safe and non-toxic;

5. Reagents are readily available: Axispharm Fluorescent Dye.