Biological assays of cytokines and their receptors

The principle is based on the specific biological activity of cytokines, using the corresponding indicator system, such as various dependent cell lines or target cells, and at the same time comparing with the standard to determine the activity level of cytokines in the sample. Unit (U / mL) said. The target cells used in the biological activity detection method can be directly isolated from the tissue, such as the colony formation test of bone marrow cells and the proliferation test of thymocytes (spleen cells), or cytokine-dependent strains grown in vitro that rely on cytokines to grow and Cells sensitive to cytokine killing are used as target cells. The measurement methods can be divided into methods for promoting cell proliferation and proliferation inhibition, antiviral activity measurement, colony formation, chemotaxis measurement, and cytokine induction product measurement.

(1) Promoting cell proliferation and proliferation inhibition method: The established methods that rely on cell lines to detect various cytokines have basically the same operation process. To detect cytokine growth-promoting activity or growth-inhibiting activity is to incubate the test sample or cytokine standard with different dilutions together with the cultured cell strain for a certain period of time, and then detect the number of proliferating cells. The number of proliferating cells in the former is directly proportional to the content of cytokines, while the number of proliferating cells in the latter is inversely proportional to the content of cytokines. Commonly used methods for detecting cell proliferation include 3H-TdR incorporation method, colorimetric method, staining method and direct counting method. Among them, colorimetric methods for measuring the activity of cellular metabolic enzymes reflecting the number of cell proliferation include MTY, XTF, MTS, and NAG, which can be automatically detected on a microplate reader without contacting nuclides, and are more commonly used. In addition, the method of measuring the fluorescence intensity of metabolites can also detect the number of proliferating cells, such as ATP method and cAMP method. Cytokines can also induce target cells to express certain surface molecules or secrete some proteins. You can use fluorescein-labeled antibodies to detect the number of cells expressing the corresponding molecules, or use specific methods to determine the proteins secreted by the cells to indirectly understand the activity of cytokines.

Various colony stimulating factors (CSF) act on different cells of the hematopoietic system to promote their proliferation and colony formation of bone marrow cells cloned and cultured in a semi-solid agar gel system, so colony formation experiments can be used to study the level of CSF.

(2) Cytotoxic activity assay: Many cytokines have cytolytic or cell growth inhibitory activity against transformed cells and virus-infected cells. To detect cytokine cytolytic Hatta cytotoxic activity or growth inhibitory activity is to incubate the test samples or cytokine standards at different dilutions with the cultured cell strain for a certain period of time, and then detect the number of surviving target cells and compare with the control Obtain the percentage of lysed or inhibited cell growth, or plot the OD value against the dilution of the sample, draw the dose response curve of the standard product, and find the corresponding sample content from the curve. The method of detecting the number of viable target cells is the same as the method of promoting cell proliferation.

(3) Antiviral activity determination method: The most commonly used method for detecting the content of interferon (1FN) in the sample is to detect its antiviral activity. The detection principle is to treat susceptible cells with cytokine samples to make the cells establish an antiviral state. Then the cells are attacked with an appropriate amount of virus, and the amount of virus replication or the degree of suppression of the cytopathy caused by the virus can be evaluated to determine the biological activity of the cytokines in the sample. Cell lines commonly used to detect IFN antiviral activity include WISH, Hep2 / c, L929, A549, Ratec and MDBK. Among them, WISH and Hep2 / c cell lines are used to detect human interferon, L929 cell line is used to detect mouse interferon, Ratec cell line is used to detect rat interferon, and MDBK cell line can be used to detect multiple genera. Viruses commonly used by IFN-a and IFN-Yo to attack cells include follicular stomatitis virus (VSV), rat brain myocarditis virus (EMCV), and Sindbisvirus. Specific methods for detecting antiviral activity include measuring cytokines to inhibit the cytopathic effect (CPE) of the virus, inhibiting the formation of viral plaques, or inhibiting the production of the virus, etc. The antiviral activity of IFN is usually expressed in units per milliliter (U / mL), and the purity of IFN is expressed in units per milligram of protein (U / mg) or the amount of IFN (ug / mg) ) Means. The definition of IFN antiviral activity unit refers to the reciprocal of the highest dilution of IFN that can inhibit 50% cytopathic or 50% viral plaque formation effect.

(4) Chemotaxis activity assay: multiple cytokines have chemotactic activity, which can induce directional migration of neutrophils, monocytes and macrophages, respectively. The ways in which chemokines induce cell movement include chemotaxis and chemical activation. Chemotaxis refers to inducing cells to move in the direction of high chemical concentration of chemokines. Agarose and microporous chamber chemotaxis assays can be used to determine the chemotactic activity of cytokines; chemical activation (chemokinesis) is Refers to enhancing the random movement of cells, which can be detected by agarose droplet chemical kinetics test.