Chinese query network microbial strains cytokines (Cytokine) are collectively referred to as a small molecule secreted by a cell protein material having biological activity. In the process of immune response, cytokines play an important role in physiological and pathological processes such as immune regulation, inflammatory response, and tumor metastasis. The detection of cytokines is not only a means of basic immune research, but also has important value in clinical disease diagnosis, disease course observation, efficacy judgment and cytokine therapy monitoring.
However, due to the small content of cytokines in the body, the detection of cytokines has brought difficulties. The detection of cytokines has not been widely carried out in clinical diagnosis. It is known that the currently used cytokine detection methods are imperfect and different. The results obtained by the detection method are quite different, which brings certain difficulties to clinical diagnosis and treatment. Therefore, it is necessary to understand the characteristics and influencing factors of various detection methods.
At present, there are mainly the following types of cytokine biological activity detection and concentration determination methods:
1. Biological detection method Biological detection, also known as biological activity detection, is a detection method designed according to the specific biological activity of cytokines. Since various cytokines have different activities, such as IL-2 promotes lymphocyte proliferation, TNF kills tumor cells, CSFci stimulates blood cell colony formation, and IFN protects cells from viral attack, thus selecting a unique biological activity of a certain cytokine, It can be detected. Biological activity detection methods can be further divided into the following categories:
1. Cell proliferation method Many cytokines have cell zhang factor activity, especially interleukins, such as IL-2 ci stimulating t cell zhang, IL-3 ci stimulating large cell zhang, IL-6 ci granulocyte zhang, etc. . Using this property, cells that respond to specific cytokines have been screened, and cell lines that depend on only one factor, that is, cell-dependent strains (referred to as dependent strains) have been established. These dependent strains are not normally viable and can only proliferate when a specific factor is added. For example, the IL-2 dependent strain ctll-2 is rapidly dying in the medium without IL-2, and the IL-2 can be cultured in the right in vitro. In a certain concentration range, cell proliferation is directly proportional to the amount of IL-2, so the content of IL-2 is identified by measuring cell proliferation (for example, using 3h-tdr incorporation method, MTT method, etc.). In addition to dependent strains, there are also short-term cultured cells, such as thymocytes, bone marrow cells, and lymphoblasts that promote mitogen ci stimuli, which can be used as target cells to measure certain cytokine activity.
2. Target cell killing is a test designed to kill target cells in vitro based on certain cytokines such as TNF. Usually, the target cells are selected from tumor cell lines passaged in vitro, and the killing rate of the cells is determined by means of isotope release or dye staining.
3. Cytokine induced product analysis
Certain cytokines can stimulate specific cells to produce biologically active substances, such as IL-2, IL-3, which induces synthesis of amines in bone marrow cells, and IL-6 induces synthesis of α1-antichymotrypsin by hepatocytes. Cytokine activity can be reflected by measuring the corresponding product induced.
4. Cytopathic inhibition The virus can cause damage to target cells, and interferon can inhibit cytopathic effects caused by viruses. Therefore, such factors can be detected by cytopathic inhibition.
Second, the immunological detection method Cytokines are proteins or peptides, with strong antigenicity. With the advent of recombinant cytokines, specific anti-serum or monoclonal antibodies of cytokines can be obtained conveniently, so that the cytokines can be quantitatively detected by immunological techniques using the characteristics of antigen-antibody-specific reactions. Despite the wide variety of cytokines, similar techniques can be used to work with specific antibodies (including polyclonal or monoclonal antibodies) for a certain factor. Common methods include ELISA, RIA, and immunoblotting. Currently, almost all common cytokine detection kits are commercially available. In addition, the in vivo detection and distribution of factors in cells can be detected by using an enzyme-labeled or fluorescently-labeled anti-cytokine monoclonal antibody, such as the recently developed intracellular staining and enzyme-linked immunospot (ELISPOT) technology. . The immunological assay can directly determine the specific cytokine content in the sample (in ng/ml), which is convenient for large-scale detection of cytokine levels in serum of clinical patients. This method only measures the antigenicity of cytokines, and is not necessarily parallel to the activity of the factor. Therefore, in order to understand the biological effects of cytokines, it is necessary to combine biological detection methods.
Molecular Biology Methods This is a technique for detecting the expression of specific cytokine genes using gene probes of cytokines. At present, all the genes of the recognized cytokines have been cloned, so that it is easier to obtain a cDNA probe of a certain cytokine or artificially synthesize an oligonucleotide probe based on a known nucleotide sequence. There are various methods for detecting cytokine mRNA expression by gene probes, such as dot blot hybridization, Northern blot, reverse transcription PCR, and in situ hybridization of cells or tissues. The key to the experiment is to prepare high quality nucleic acid probes and obtain acceptable analytes (extracted mRNA samples or cell/tissue specimens). A nucleic acid probe refers to a DNA fragment or single-stranded DNA or RNA which is labeled with a radioisotope or other label (such as biotin, digoxin, etc.) and which is complementary to the gene of interest. According to their sources, they can be classified into cDNA probes, oligonucleotide probes, genomic gene probes, and DNA probes. Among them, cDNA probes and synthetic oligonucleotide probes are commonly used for dot blot hybridization and Northern blot, and RNA probes are more suitable for in situ hybridization because of their good penetrability. The application of nucleic acid probe technology has been programmed. The main examples of cDNA probes include: 1 extraction of plasmid DNA; 2 separation of target DNA fragments; 3 target DNA fragment labeling; 4 extraction of sample mrna; 5 labeling cDNA Hybridization for the sample to be tested; 6 autoradiography or color analysis. The method of detecting specific mRNA by RT-PCR which has appeared in recent years is also widely used in the field of cytokine research. The method has the advantages of sensitivity and rapidity, and even the specific mRNA can be detected from 1 to 10 cells.
The above three methods have their own advantages and disadvantages and can complement each other. In practical applications, they can be selected according to their respective experimental purposes and laboratory conditions. The biological detection method is sensitive and can directly measure biological functions. It is the most reliable method and is suitable for various experimental purposes. It is the most commonly used technology in scientific research departments, but it requires zhang period to culture dependent cell lines. The steps are complicated and there are many influencing factors, so it is not easy to master. The immunological assay is relatively simple, rapid, and reproducible, but the measured amount represents only the amount of the corresponding cytokine and does not represent activity, and the sensitivity is also lower than the biological activity assay (about 10 to 100 times lower). Molecular biology can only detect the expression of genes, and can not directly provide information on the concentration and activity of related factors, mainly used for mechanism discussion. In the detection of cytokines, it must be considered that the role of cytokines has a network characteristic. It is necessary to clarify the cytokine components determined by the detection method, and consider the levels of inhibitors and soluble receptors, and combine them in various combinations. It is possible to get more reliable results.
However, due to the small content of cytokines in the body, the detection of cytokines has brought difficulties. The detection of cytokines has not been widely carried out in clinical diagnosis. It is known that the currently used cytokine detection methods are imperfect and different. The results obtained by the detection method are quite different, which brings certain difficulties to clinical diagnosis and treatment. Therefore, it is necessary to understand the characteristics and influencing factors of various detection methods.
At present, there are mainly the following types of cytokine biological activity detection and concentration determination methods:
1. Biological detection method Biological detection, also known as biological activity detection, is a detection method designed according to the specific biological activity of cytokines. Since various cytokines have different activities, such as IL-2 promotes lymphocyte proliferation, TNF kills tumor cells, CSFci stimulates blood cell colony formation, and IFN protects cells from viral attack, thus selecting a unique biological activity of a certain cytokine, It can be detected. Biological activity detection methods can be further divided into the following categories:
1. Cell proliferation method Many cytokines have cell zhang factor activity, especially interleukins, such as IL-2 ci stimulating t cell zhang, IL-3 ci stimulating large cell zhang, IL-6 ci granulocyte zhang, etc. . Using this property, cells that respond to specific cytokines have been screened, and cell lines that depend on only one factor, that is, cell-dependent strains (referred to as dependent strains) have been established. These dependent strains are not normally viable and can only proliferate when a specific factor is added. For example, the IL-2 dependent strain ctll-2 is rapidly dying in the medium without IL-2, and the IL-2 can be cultured in the right in vitro. In a certain concentration range, cell proliferation is directly proportional to the amount of IL-2, so the content of IL-2 is identified by measuring cell proliferation (for example, using 3h-tdr incorporation method, MTT method, etc.). In addition to dependent strains, there are also short-term cultured cells, such as thymocytes, bone marrow cells, and lymphoblasts that promote mitogen ci stimuli, which can be used as target cells to measure certain cytokine activity.
2. Target cell killing is a test designed to kill target cells in vitro based on certain cytokines such as TNF. Usually, the target cells are selected from tumor cell lines passaged in vitro, and the killing rate of the cells is determined by means of isotope release or dye staining.
3. Cytokine induced product analysis
Certain cytokines can stimulate specific cells to produce biologically active substances, such as IL-2, IL-3, which induces synthesis of amines in bone marrow cells, and IL-6 induces synthesis of α1-antichymotrypsin by hepatocytes. Cytokine activity can be reflected by measuring the corresponding product induced.
4. Cytopathic inhibition The virus can cause damage to target cells, and interferon can inhibit cytopathic effects caused by viruses. Therefore, such factors can be detected by cytopathic inhibition.
Second, the immunological detection method Cytokines are proteins or peptides, with strong antigenicity. With the advent of recombinant cytokines, specific anti-serum or monoclonal antibodies of cytokines can be obtained conveniently, so that the cytokines can be quantitatively detected by immunological techniques using the characteristics of antigen-antibody-specific reactions. Despite the wide variety of cytokines, similar techniques can be used to work with specific antibodies (including polyclonal or monoclonal antibodies) for a certain factor. Common methods include ELISA, RIA, and immunoblotting. Currently, almost all common cytokine detection kits are commercially available. In addition, the in vivo detection and distribution of factors in cells can be detected by using an enzyme-labeled or fluorescently-labeled anti-cytokine monoclonal antibody, such as the recently developed intracellular staining and enzyme-linked immunospot (ELISPOT) technology. . The immunological assay can directly determine the specific cytokine content in the sample (in ng/ml), which is convenient for large-scale detection of cytokine levels in serum of clinical patients. This method only measures the antigenicity of cytokines, and is not necessarily parallel to the activity of the factor. Therefore, in order to understand the biological effects of cytokines, it is necessary to combine biological detection methods.
Molecular Biology Methods This is a technique for detecting the expression of specific cytokine genes using gene probes of cytokines. At present, all the genes of the recognized cytokines have been cloned, so that it is easier to obtain a cDNA probe of a certain cytokine or artificially synthesize an oligonucleotide probe based on a known nucleotide sequence. There are various methods for detecting cytokine mRNA expression by gene probes, such as dot blot hybridization, Northern blot, reverse transcription PCR, and in situ hybridization of cells or tissues. The key to the experiment is to prepare high quality nucleic acid probes and obtain acceptable analytes (extracted mRNA samples or cell/tissue specimens). A nucleic acid probe refers to a DNA fragment or single-stranded DNA or RNA which is labeled with a radioisotope or other label (such as biotin, digoxin, etc.) and which is complementary to the gene of interest. According to their sources, they can be classified into cDNA probes, oligonucleotide probes, genomic gene probes, and DNA probes. Among them, cDNA probes and synthetic oligonucleotide probes are commonly used for dot blot hybridization and Northern blot, and RNA probes are more suitable for in situ hybridization because of their good penetrability. The application of nucleic acid probe technology has been programmed. The main examples of cDNA probes include: 1 extraction of plasmid DNA; 2 separation of target DNA fragments; 3 target DNA fragment labeling; 4 extraction of sample mrna; 5 labeling cDNA Hybridization for the sample to be tested; 6 autoradiography or color analysis. The method of detecting specific mRNA by RT-PCR which has appeared in recent years is also widely used in the field of cytokine research. The method has the advantages of sensitivity and rapidity, and even the specific mRNA can be detected from 1 to 10 cells.
The above three methods have their own advantages and disadvantages and can complement each other. In practical applications, they can be selected according to their respective experimental purposes and laboratory conditions. The biological detection method is sensitive and can directly measure biological functions. It is the most reliable method and is suitable for various experimental purposes. It is the most commonly used technology in scientific research departments, but it requires zhang period to culture dependent cell lines. The steps are complicated and there are many influencing factors, so it is not easy to master. The immunological assay is relatively simple, rapid, and reproducible, but the measured amount represents only the amount of the corresponding cytokine and does not represent activity, and the sensitivity is also lower than the biological activity assay (about 10 to 100 times lower). Molecular biology can only detect the expression of genes, and can not directly provide information on the concentration and activity of related factors, mainly used for mechanism discussion. In the detection of cytokines, it must be considered that the role of cytokines has a network characteristic. It is necessary to clarify the cytokine components determined by the detection method, and consider the levels of inhibitors and soluble receptors, and combine them in various combinations. It is possible to get more reliable results.
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