I. In vitro assays for delayed-type hypersensitivity reactions
Skin test and contact allergy elicitation are the two commonly used methods for detecting delayed-type hypersensitivity reactions (DTH). Skin testing induces a re-response to an antigen that has previously sensitized the patient, whereas contact sensitization tests the recipient's ability to become sensitized to a substance to which he or she has never been exposed.
1. Skin test Diagnosis of DTH by skin test, commonly used antigens are tuberculosis pure protein derivative (PPD), mumps virus, candidin, etc., in human test in the forearm intradermal injection of a small amount of soluble antigens, 24 ~ 48 small, measure the size of the redness and swelling of the hard nodules, hard nodules greater than 10mm in diameter that is regarded as positive. Indicates that the subject has a certain cellular immunity to the pathogen, if the skin test is not responsive, the test can be repeated with a higher concentration of antigen, if there is still no reaction that is negative, need to exclude the skin test technical error, may be the subject has never been exposed to this antigen, but also due to cellular immunity deficits, or due to cellular immunity deficits, or due to severe infections (measles, chronic disseminated tuberculosis) caused by the non-responsive .
2. Contact allergy is often induced by the use of low molecular weight compounds such as dinitrochlorobenzene (DNCB). The compound binds to skin proteins and causes a DTH reaction. In animal testing, the initial application of DNCB on the skin and then stimulate the stimulus 7-10 days later, the skin appears to be positive. This test is no longer used in humans. When the blood was centrifuged on top of the lymphocyte layering solution, the red blood cells (1.092), polymorphonuclear leukocytes (1.090), and lymphocytes (1.070) were separated from each other due to the difference in specific gravity. Lymphocytes and monocytes form a thin layer at the junction of plasma and stratified fluid. The cells in this thin layer are carefully separated, of which lymphocytes account for 80%, monocytes account for 20%, T cells account for 80% of the lymphocytes, B cells account for 4% to 10%, and its as non-DT, non-B cells.
1. T-cell count
(1) E wreath method: human T-cells have SRBC receptor (CD2) on the surface of the SRBCs can be combined with SRBCs to form a rosette-like structure, will be separated by the stratified liquid now RBM suspension and SRBCs in the serum containing balanced saline mixture, cultured at 37 degrees Celsius 5 to 10 minutes to put 4 degrees Celsius overnight, take the cells to count the suspension, the peripheral blood About 70%～80% of the lymphocytes in the lymphocyte wreaths are T cells. At present, this method has been used to isolate T cells without doing T cell counting.
(2) T-cell counting with monoclonal antibody: human PBM was divided into three equal parts, and mouse anti-human CD3, CD4 and CD8 monoclonal antibody was used as the primary antibody to bind to the cells, and then FITC-labeled rabbit anti-mouse IgG antibody was used as the secondary antibody for indirect immunofluorescence staining, and the results were detected by fluorescence microscope or flow cytometer, and the cells that were stained with fluorescence by the CD3 antibody in PBM were called CD3 cells. The cells in the PBM that were fluorescently stained by the CD3 antibody were called CD3+ cells, i.e. total T cells. Normal people have 70% to 80% of T cells in PBM. The sum of CD4+ cells and CD8+ cells should be the same as the number of CD3+ cells in a normal person. the ratio of CD4+ cells to CD8+ cells in a normal person is about 2/1, while in AIDS patients, the ratio is less than 1.7.
2. T-cell activation assay: T-cells can be activated by nonspecific substances called mitogens and transformed into lymphoblastoid cells. the process of T-cell transformation can be accompanied by the synthesis of DNA, RNA, and proteins. The process of T-cell transformation can be accompanied by an increase in the synthesis of DNA, RNA, and proteins, most often leading to cell division. The number of transformed lymphocytes can be counted under a light microscope, or the rate of lymphocyte transformation can be determined by doping dividing lymphocytes with tritium-labeled thymidine nucleoside (3HTdR), checking the doping of dividing lymphocytes with a liquid-flash detector, and checking the amount of 3H-TdR doped with a liquid-measuring device. Recently, there has been a method of examining the proliferative response of lymphocytes without using isotopes and with instrumental measurements, known as the MTT assay. MTT is a metronidazole salt, which is a substrate for cellular mitochondrial dehydrogenase, and enzymes within the cell can break down MTT to produce a blue-black colored methyl (fromazan) product. The amount of this product is positively correlated with the number of active cells. The result can be measured with an enzyme labeling detector (595mm) to measure the density of HSBC as a check for the MTT method. The results of this method parallel the 3H-TdR doping method and reflect the number of viable cells in the test (Table 20-3).
Table 20-3 Stimulants of the Lymphocyte Proliferative Response
Schizogen
T Cells
B Cells
Phytohaemagglutinin (PHA)
+
-
Cabbage Protein A (ConA)<
+
-
Peridotrichum americanum (PWM)
+
+
Staphylococcal Protein A (SPA)
±
+
Sparaspora paratyphi B (SPB)
±
+
Note: PWH is mainly a T-cell schizogen, but it can also induce B-cell proliferation and differentiation by stimulating T-cell secretion of soluble factors;
＊SPA does not require T-cell assistance to induce B-cell division, but T-cell assistance is required to induce B-cell activation of antibody-secreting cells
3. Cytotoxicity assays TC cells, NK cells, LAK cells, and TIL cells have a direct cytotoxicity to TC cells, NK cells, LAK cells, TIL cells have a direct cytotoxic (killing) effect on their target cells. The commonly used method to measure the cytotoxic effect is 51Cr-Na2Cro4 saline solution mixed with the target cells, incubated at 37 ℃ for about 1 hour, 51Cr can enter the target cells, and the cytoplasmic protein binding, after washing away the free 51Cr, you can get 51Cr-labeled target cells, will be examined for cytotoxicity of the cell mixed with 51Cr-labeled target cells (the ratio of 50:1 or 100:1) target cells are killed. (100:1) The more target cells are killed, the more liquid free 51Cr is released into the supernatant and cannot be absorbed by other cells. The cpm value in the supernatant is detected by γ-ray meter, and the killing activity of the cells to be examined can be calculated.
The cytotoxicity test to detect the soundness of Tc cell effector function, and the ADCC effect mediated by IgG, or the role of NK cells in anti-tumor immunity is meaningful.
4. Mixed lymphocyte response (MIR) A better way to study T cells in vitro, two-way MLR is often used to screen donors for bone marrow transplantation. Lymphocytes from different donors are mixed and cultured with the patient's lymphocytes for 4 to 5 days respectively, and the reactivity of T cells is measured in the last 8 hours by doping 51TdR doping method. Or use cytotoxic method to observe the stimulated T cells mixed with live target cells (target cells from the same individual as the stimulated cells) If the T cells are stimulated to produce cytotoxic T cells, which can kill the live cells, according to the amount of 51Cr released by the target cells to calculate the T cell movement inhibitory factor) and LIF (leukocyte movement inhibitory factor) to assess the cellular immune function. In recent years, immunoenzymatic techniques have been applied to measure IL-2, which are simple to perform and can be quantified to replace the measurement of MIF and LIF. Individual nucleated cells are incubated with schizonts for 24 hours, and then the IL-2 activity in the supernatant is measured. IL-2 secretion is markedly reduced in deficient cellular immunity, especially in AIDS patients. In some diseases, such as multiple sclerosis, rheumatoid arthritis, and transplant rejection, IL-2 levels in serum are elevated, indicating increased T-cell activity. Urinary IL-2 can also be elevated in patients with transplant rejection.
Enzyme-linked immunosorbent assay (ELISA) can also be used to measure the IL-2 receptor (CD25) shed by activated T cells in various body fluids. Generally, IL-2 levels and IL-2 receptor levels are parallel, and IL-2 and IL-2 receptor testing can be used to monitor certain diseases, such as graft rejection, autoimmune diseases, and patients undergoing immunosuppressive therapy.
A cytokine production capacity assay for cells cultured in vitro examines the biological activity or antigenicity of cytokines in the cell culture supernatant. Now available nucleic acid hybridization technology, that is, from the tissue or cell RNA extraction, and isotope or enzyme-labeled cDNA probe of the cytokine for molecular hybridization test, that is, blotting (dot blotting) or Northern blotting, if you find that there is a certain factor of the presence of mRNA, that is, that the cell in the culture conditions have the ability to produce a certain type of cytokine.