Method for detecting tuberculosis effector T lymphocytes by using enzyme-linked immunospot assay
An enzyme-linked immunospot and lymphocyte technology, applied in the field of medicine, can solve the problems of affecting the treatment of pulmonary tuberculosis patients, high misdiagnosis rate, poor patient compliance, etc., and achieve the effect of improving the efficiency and scope of clinical diagnosis and improving the survival rate.
Inactive Publication Date: 2013-10-23
FOSHAN DIAN MEDICAL LAB
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[0002] Existing routine tuberculosis detection methods require a large amount of blood from patients and poor patient compliance; in addition, the traditional method has a high misdiagnosis rate for tuberculosis-effect T lymphocytes and immunocompromised patients, which seriously affects the treatment of tuberculosis patients
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[0024] A. Collect 4-8ml of heparin lithium anticoagulant peripheral blood, centrifuge at 1600-1800r / min for 28-30min; pour 4ml of heparin lithium anticoagulant peripheral blood into a 15ml centrifuge tube containing 4ml of RPMI1640 equilibrated to room temperature, and mix well; Carefully add the above blood sample to the upper layer of 4ml of Ficoll lymphocyte separation medium at a ratio of 2-3:1 to ensure that the above blood sample does not mix with the Ficoll lymphocyte separation medium; then centrifuge at 2280r / min at 18°C for 22min;
[0025] B. After centrifugation, draw the cloudy mononuclear cell layer into a 15ml centrifuge tube, add RPMI1640 to 10ml, mix well, and centrifuge at 1780r / min for 7min at 18°C;
[0026] Firstly, the white blood cell layer between the red blood cells and plasma is absorbed by centrifugation to increase the number of lymphocytes, so that the collection of mononuclear cells in anticoagulant whole blood can achieve the maximum effect of...
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The invention relates to a method for detecting tuberculosis effector T lymphocytes by using enzyme-linked immunospot assay. The method comprises the steps of: collecting lithium heparin peripheral anticoagulation blood; carrying out centrifugation, and then, absorbing a middle cloudy mononuclear cell layer to a centrifuge tube; carrying out centrifugation, then, discarding supernatant, adding RPMI1640, and carrying out centrifugation; discarding supernatant, and adding AIM-V culture fluid; preparing diluted cell working liquid; adding the AIM-V culture fluid, an antigen A, an antigen B and positive control into a culture plate; and washing for four times by using sterile PBS, flushing with distilled water so as to terminate reaction, drying the culture plate at room temperature, observing results by using a magnifier, and counting spots. According to the method, the number of the lymphocytes is increased through centrifugally absorbing a leucocyte layer between erythrocytes and blood plasma, so that mononuclear cells in whole anticoagulation blood are collected to the maximum, the clinical diagnosis application efficiency in tuberculosis screening is increased remarkably, and the clinical diagnosis application range in tuberculosis screening is widened remarkably.
Description
technical field [0001] The invention relates to the technical field of medicine, in particular to a method for detecting tuberculosis-effect T lymphocytes using enzyme-linked immunospot method. Background technique [0002] Existing routine tuberculosis detection methods require a large amount of blood from patients and poor patient compliance; in addition, the traditional method has a high misdiagnosis rate for tuberculosis-effect T lymphocytes and immunocompromised patients, which seriously affects the treatment of tuberculosis patients. [0003] T-SPOT.TB uses tuberculosis-specific antigens (ESTA-6, CFP-10) to detect whether there are tuberculosis-effect T lymphocytes in the subject by enzyme-linked immunospot method (ELISPOT), so as to judge whether the subject is currently A new method of infection with Mycobacterium tuberculosis (current infection). The existing conventional detection method is mainly to extract lymphocytes from heparin lithium and heparin sodium anti...
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IPC IPC(8): G01N33/543
Inventor 戴宁榆叶爱玲李思李贵州
Owner FOSHAN DIAN MEDICAL LAB