| Objective:To establish a method detecting infection of Mycobacterium tuberculosis by enzyme linked immunospot (ELISPOT) and manufacture a kit of ELISPOT. Evalue the clinical value of self-made kit for diagnosis of tuberculosis. Methods:To select 30 healthy volunteers who have not be infected Mycobacterium tuberculosis, 26 patients and 4 volunteers postive for PPD, 60 blood samples were taken for T SPOT-TB kit test.The special T lymphocyte of Mycobacterium tuberculosis will secrete IFN-γif it is stimulated by special antigen, detecting the IFN-γby ELISPOT will make sure wether there have be effective T lymphocyte or not. To pro-Coat 96 pores pyroxylin filter membranes ELISPOT plate with monoclonal capture antibody. Appropriate cells are pipetted into the wells. Recombinant ESAT-6 protein and CFP10-ESAT6 fusion protein will stimulate the T lymphocyte, then the immobilized antibody in the immediate vicinity of the secreting cells binds secreted IFN-γ. After washing away cells and unbound substances, a biotinylated polyclonal antibody specific for IFN-γis added to the wells. Follow to remove unbound biotinylated antibody by washing, and alkaline-phosphatase conjugated to streptavidin is added. Unbound enzyme is subsequently removed by washing and substrate solution (BClP/NBT) is added. Blue-black colored precipitate will form at the sites of cytokine localization and appear as spots, and each individual spot represents an individual IFN-γsecreting cell. The spots can be counted with an automated reader system of ELISPOT or manual stereomicroscope.To change the condition of experiment and find the best number of cell, the special antigen, the combinative antibody, the best stimulated time, the best incubated time and so on, and a diagnostic kit will be established .Selecting 32 negative control and 31 patients who were diagnosed active pulmonary tuberculosis, and their blood samples were taken for T SPOT-TB kit test and self-made kit test. The result of two different kits test is compared.Results:28 (93.3%) of 30 patient had a positive result by T SPOT-TB kit test, and 29 (96.6%) of 30 Healthy volunteers had a negetive result. To change the condition of experiment and find that the best numbers of cell is 2×105, the best incubated time is 20 hour and the best density of proteinum is 20μg/ml. 58(92.1%)of 63 cases have agreemented results between T SPOT-TB kit and the self-made kit. 5(7.9%)of 63 have different results. Appling the chisquare test(χ2), the result is P>0.05. So there are no differences in detection between two kits. 27(87.1%)of 31 patients had a positive result by T SPOT-TB kit test and 28(90.3%)had the same one by self-made kit test. 6(18.8%)of 32 Untuberculosis-infection cases had a positive result by T SPOT-TB kit test and 8(25.0%)had the same one by self-made kit test. The sensitivity of the self-made kits is higher than T SPOT-TB kit. The false positive of the self-made kits is higher than T SPOT-TB kit, but the patients included the tumour and elder who maybe have lower immunity than health, so they may be latent tuberculosis infection (LTBI). The result of the patients detected by the self-made kits implied that the spots of positive sputum was higher than negative sputum, P<0.05. The number of the special cell secreting IFN-γwas elevated.Conclusion:Oxford Immunotec T SPOT-TB kit have highly sensitivity and specificity; The self-made kit have highly sensitivity and specificity in tubercular diagnoses too and it can gain the same purpose as T SPOT-TB kit; ELISPOT has more sensitivity and specificity than PPD skin test for diagnosis of latent Mycobacterium tuberculosis infection. |
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