Cytokines can be quantitatively or qualitatively detected by immunoassay technology by utilizing the specific binding properties of antigen and antibody. Although there are many types of cytokines, immunoassays can be used for detection as long as specific antibodies (including polyclonal or monoclonal antibodies) against a cytokine are obtained. Commonly used methods include ELISA, RIA, Western blotting, immunofluorescence, and flow cytometry analysis based on immunofluorescence techniques.
1. ELISA method
ELISA is the most widely used heterogeneous enzyme-labeled immunoassay technology. One or more steps of antigen-antibody reaction and one-step enzymatic reaction constitute the basic steps of ELISA, which can be used for qualitative or quantitative analysis. The double-antibody sandwich method is the most commonly used method for cytokine determination. The antibodies used in this method can be polyclonal antibodies directed against the same antigen or monoclonal antibodies directed against different epitopes of the same antigen.
Specimens for cytokine determination mainly include two categories, one is serum (plasma), synovial fluid, pleural fluid, cerebrospinal fluid or peritoneal fluid and other body fluids, which can be used for the detection of cytokines and soluble adhesion molecules; the other is cell culture after in vitro culture. The supernatant is only used for the detection of cytokines.
ELISA method has the advantages of specificity, simplicity, easy promotion and standardization. In addition, biologically inactive cytokine precursors, decomposed fragments and conjugates with corresponding receptors can also be detected by this method. The disadvantage is that the sensitivity is low and the biological activity of cytokines cannot be judged.
2. Flow cytometry
Flow cytometry is a method based on fluorescent antibody staining technology and the sensitive resolution of flow cytometry. This method is mainly used for the detection of intracellular cytokines and cell surface adhesion molecules. Through specific fluorescent antibody staining, the detection of cytokines or adhesion factors at the single cell level can be performed simply and quickly, and the detection of different cell subsets can be accurately determined. Expression of cytokines and membrane molecules. According to the properties of fluorescent antibodies, fluorescent antibody staining can be divided into direct and indirect methods. The former uses fluorescently labeled cytokines or specific antibodies for adhesion molecules, while the latter uses fluorescently labeled secondary antibodies. The direct method is more commonly used, although its sensitivity is not as good as the indirect method, but its specificity is strong.
When this method detects intracellular cytokines, it mainly includes the following basic steps:
1. Isolation and culture of cells to be tested
2. Cell fixation The commonly used cell fixative is 4% paraformaldehyde.
3. Block non-specific binding sites Resuspend the fixed cells with PBS solution containing 5% nonfat milk powder and calcium and magnesium ions.
4. Staining and analysis Fluorescein-labeled cytokine-specific monoclonal antibody was used for fluorescent antibody staining, and the percentage of fluorescent positive cells and fluorescence intensity were analyzed by flow cytometry. In addition, if using different fluorescein-labeled monoclonal antibodies of two or more cytokines, it is possible to simultaneously detect two or more different cytokines in the same cell.
This method can also be used to distinguish cell subsets with different secretory properties, such as Th1 and Th2 cells.
3. ELISPOT
Enzyme-linked immunospot test (ELISPOT), derived from ELISA, but breaking through traditional ELISA, is an extension and development of quantitative determination of antibody-forming cells, and has been increasingly used in rheumatoid factor, IFN-γ and other cytokines Quantitative assay of secretory cells.
ELISPOT is superior to traditional antibody, cytokine or other soluble molecule-secreting cell detection methods, and can detect 1 cell that secretes corresponding molecules from 200,000 to 300,000 cells. If biotin and avidin systems are introduced, it is sensitive. Sex will also be greatly improved. When doing ELISPOT, the selected specific antibody should have the characteristics of high affinity, high specificity and low endotoxin. The chosen cell activator must not affect the secretory function of the cell.
4. Methodological evaluation of immunological assays
Immunoassays can be used for the detection of almost all cytokines. Compared with biological activity assays, the main advantages and disadvantages are:
Advantages: ①High specificity, the use of specific monoclonal antibodies can be used for the detection of single cytokines; ②The operation is simple and fast, and does not need to rely on cell lines, so it does not need to maintain culture, which greatly increases the operability of the method and is easy to popularize and facilitate the census; ③ the influencing factors are relatively few and easy to control, the repeatability is good, and the method is easy to standardize.
Disadvantages: ① What is measured is only the protein content of cytokines, which is not necessarily proportional to its biological activity; ② The measurement results have a great relationship with the source and affinity of the antibody used. When using mAbs with different affinities, the same sample was detected. The results may be different; ③ the sensitivity is relatively low, about 10 to 100 times lower than that of the biological activity method, and the lower limit of determination is generally 100 pg; ④ If there are soluble receptors for cytokines in the sample, it may affect the specific antibodies to cytokines combination.
In addition to the methods described above, molecular biology techniques, which are also biological assay methods, are also widely used in the detection of cytokines or adhesion molecules, such as PCR/RT-PCR, dot blot, Northern-blot, and FISH. cell or tissue in situ hybridization, etc.
Other immunoassays: polarized fluorescence detection techniques; use of confocal laser microscopy; luminescence techniques; immunohistochemical staining. All these methods play an active role in the detection of adhesion molecules, cytokines and their secreting cells.