A: According to different kinds of markers, immunolabeling techniques can be divided into radioimmunoassay, enzyme-labeled immunoassay, fluorescence-labeled immunoassay, chemiluminescence-labeled immunoassay and colloidal gold-labeled immunoassay. There are similarities and differences between them, which are summarized as follows.
First, the similarities mainly include the following aspects:
1, all of which have high specificity. The immune techniques of the five immunolabeling techniques all adopt antigen-antibody reaction, and the combination of antigen and antibody is one-to-one correspondence and specific combination, so they all have very high specificity.
2. Both have high sensitivity. Because the labeling techniques of the five immunolabeling techniques all use tracer labels, such as enzymes, radionuclides, fluorescein, colloidal gold, dense substances, etc., after reacting with the corresponding antibodies or antigens in the specimen, it is not necessary to measure the antigen-antibody complex itself, but only the markers in the complex, so that the invisible reaction can be amplified by chemical or physical means and converted into visible and measurable signals such as light, color, electricity and pulse, and it can be used.
3. The detection objects are the same. Except for radioimmunoassay, which can only detect antigen, the other four immunolabeling techniques can detect antigen or antibody, that is, through the specific binding of known antigen (or antibody) with the antibody (or antigen) to be detected, the antibody or antigen can be detected qualitatively or quantitatively.
4. The procedure of immunolabeling is basically the same. Includes a series of procedures, such as purification of antigen or antibody, determination of labeled substance (i.e. determination of final detection mode), labeling, separation and purification of labeled products, analysis and identification, etc.
Second, the differences mainly include the following aspects:
1, the detection principle is different. First of all, enzyme immunoassay is an immune method with enzyme-labeled antibody (or antigen) as the main reagent, combining the specificity of antigen-antibody reaction and the efficiency and specificity of enzyme-catalyzed substrate reaction. It has specific requirements for labeled enzymes, such as high activity and high purity. Radioimmunoassay is a labeled immunoassay method with radioisotopes as tracers. Immunofluorescence technology uses fluorescein as a marker to bind with known antibodies or antigens, and then uses fluorescein-labeled antibodies as standard reagents to detect and immobilize unknown antigens. There are also specific requirements for fluorescein used for labeling, such as high fluorescence efficiency, stable combination with protein, easy storage and so on. Luminescence immunoassay is a new type of ultra-micro analysis technology, which combines luminescence analysis with immune reaction. It is an immunoassay method in which an antibody (or antigen) is labeled with a luminous body and the antigen (or antibody) reaction is detected by luminescence. Immunocolloidal gold labeling technique is a new immunoassay method, which uses colloidal gold as tracer, which is negatively charged in alkaline environment, and is applied to antigen-antibody reaction because of the firm combination of static electricity and positive charge groups of antibody protein molecules.
2. Use different tags. Because the principles of immunolabeling are different, the markers used are necessarily different. The marker of fluorescence immunoassay is fluorescein, which is usually tetraethyl rhodamine, fluorescein isothiocyanate and phycoerythrin. The marker of enzyme immunoassay is enzyme, and generally horseradish peroxidase (HRP) or alkaline phosphatase (AP) is used. Radioimmunoassay uses radionuclide labeling, generally 13 1I, 125I, 14C and 3h; Luminescence immunoassay uses chemiluminescent substances for labeling, and commonly used chemiluminescent reagents include luminol, isoluminol and derivatives of luminol; The symbol of immune colloidal gold technology is colloidal gold, which is a suspension of gold particles formed by the reduction of gold salts into original gold. The size of colloidal gold particles is mostly 1- 100 nm.
3. Detection requires different instruments or methods. Enzyme immunoassay is to add enzyme substrate to develop color, and then compare color with enzyme label detector; Radioimmunoassay uses liquid scintillation counter, crystal scintillation counter or X-ray film for immunoassay. Immunofluorescence technology uses fluorescence colorimeter or fluorescence microscope for analysis; Luminescence analyzer is an instrument needed for luminescence immunoassay. Immunocolloidal gold technology is detected by immuno-electronic competition or dot immunogold staining.