The principles, classification, and advantages of immunohistochemistry!
1. Basic principles of immunohistochemistry technology
Apply the principles of immunology and histochemistry to conduct in-situ qualitative, localization or quantitative research on certain chemical components in tissue sections or cell specimens. This technology is called immunohistochemistry or immunocytochemistry.
It is well known that the binding between antibodies and antigens is highly specific. Immunohistochemistry takes advantage of this characteristic, that is, it first extracts certain chemicals from tissues or cells, uses them as antigens or haptens to immunize mice and other experimental animals, prepares specific antibodies, and then uses these antibodies (First antibody) Use an antigen to immunize animals to prepare a second antibody, treat it with a certain enzyme (commonly used horseradish peroxidase) or biotin, and then combine it with the aforementioned antigen component to amplify the antigen. Since the antibody binds to the antigen The immune complex formed later is colorless, so the antigen-antibody reaction site must be displayed using histochemical methods (the commonly used chromogenic agent DAB appears as brown-yellow particles).
Through antigen-antibody reaction and color reaction, the chemical components in cells or tissues can be displayed. The antigen-antibody reaction products occurring in cells can be clearly seen under the microscope, so that the distribution and content of certain chemical components can be determined in situ in cells or tissues. . All substances in tissues or cells that can serve as antigens or haptens, such as proteins, polypeptides, amino acids, polysaccharides, phospholipids, receptors, enzymes, hormones, nucleic acids and pathogens, can be detected with corresponding specific antibodies.
2. Immunohistochemistry staining method
1. According to the type of labeling substances, such as fluorescent dyes, radioactive isotopes, enzymes (mainly horseradish peroxidase and alkaline phosphatase), ferritin, colloidal gold, etc., it can be divided into immunofluorescence method, radioimmunoassay, Enzyme labeling method and immunogold and silver method, etc.
2. According to the dyeing steps, it can be divided into direct method (also called one-step method) and indirect method (two-step, three-step or multi-step method); compared with the direct method, the sensitivity of the indirect method is much improved.
3. According to the binding method, it can be divided into antigen-antibody binding, such as peroxidase-anti-peroxidase (PAP) method and affinity connection, such as avidin-biotin-peroxidase complex (ABC) method, Streptoavidin-peroxidase conjugation (SP) method, etc., among which the SP method is the most commonly used method.
3. Principles of several commonly used immunohistochemistry methods
1. Immunofluorescence method
It is the earliest established immunohistochemistry technique. It uses the principle of specific binding of antigens and antibodies. First, known antibodies are labeled with fluorescein, which is used as a probe to check the corresponding antigens in cells or tissues and observed under a fluorescence microscope. When the fluorescein in the antigen-antibody complex is irradiated by excitation light, it will emit fluorescence of a certain wavelength, so that the location of a certain antigen in the tissue can be determined, and further quantitative analysis can be performed. Because immunofluorescence technology has strong specificity, high sensitivity, rapidity and simplicity, it is widely used in clinical pathological diagnosis and examination.
2. Immunoenzyme labeling method
The immunoenzyme labeling method is a technology developed in the 1960s after immunofluorescence. The basic principle is to first use enzyme-labeled antibodies to interact with tissues or cells, and then add enzyme substrates to generate colored insoluble products or particles with a certain electron density. Through light or electron microscopy, various types of substances on the cell surface and within the cells are detected. Conduct localization studies on antigenic components. Immunoenzyme labeling technology is currently the most commonly used technology.
The main advantages of this method compared with immunofluorescence technology are: accurate positioning, good contrast, stained specimens can be stored for a long time, and it is suitable for light and electron microscopy studies.
The development of immunoenzyme labeling methods is very rapid, and a variety of labeling methods have been derived. With the continuous improvement and innovation of methods, their specificity and sensitivity are constantly improving, and their use is becoming more and more convenient. Currently, the PAP method, ABC method, SP method, etc. are widely used in pathological diagnosis.
3. Immunocolloidal gold technology
Immuno-colloidal gold technology uses colloidal gold, a special metal particle, as a marker. Colloidal gold refers to a hydrosol of gold, which can quickly and stably adsorb proteins without significant impact on the biological activity of the protein. Therefore, using colloidal gold-labeled primary antibodies, secondary antibodies, or other molecules that can specifically bind immunoglobulins (such as Staphylococcus A protein) as probes can characterize, locate, and even quantify antigens in tissues or cells. Research. Since colloidal gold has particles of different sizes and the electron density of colloidal gold is high, immunocolloidal gold technology is particularly suitable for single-label or multi-label localization studies under immunoelectron microscopy. Since colloidal gold itself is light to deep red in color, it is also suitable for light microscopy observation. For example, the silver-enhanced immune gold-silver rule is more convenient for light microscopy observation.
4. Advantages of immunohistochemistry
1. Strong specificity The basic principle of immunology determines that the binding between antigens and antibodies is highly specific. Therefore, immunohistochemistry is theoretically a specific display of antigens in tissue cells, such as keratin (keratin) showing epithelium. Components, LCA shows lymphocyte component. Cross-reactivity occurs only when cross-antigens are present in tissue cells.
2. High sensitivity. In the initial stage of applying immunohistochemistry, due to technical limitations, there were only low-sensitivity techniques such as direct methods and indirect methods. At that time, the antibodies could only be diluted several times or dozens of times; now Due to the emergence of ABC method or SP method, the antibody can be diluted thousands, tens of thousands or even hundreds of millions of times and still combine with the antigen in tissue cells. Such highly sensitive antibody-antigen reaction makes immunohistochemistry methods more and more popular. Conveniently used in routine pathological diagnosis work.
3. Accurate positioning, combining form and function. This technology can accurately position antigens in tissues and cells through antigen-antibody reactions and color reactions, so it can simultaneously observe the positioning of different antigens in the same tissue or cells, so that It is possible to conduct research combining morphology and function, and it is very meaningful to conduct in-depth research on pathology.
