ABSTRACT
Objective To sensitize the T-cell in the peripheral blood of the active tuberculosis pa-tients by rCFP-10/ESAT-6 fusion protein, phytohaemaggiutinin(PHA) and physiologic saline, and to detect the IFN-γ to approach the significance of the tuberculosis infection. Methods One hundred and eleven pa-tients were diagnosed by clinical definite, 292 undergraduate students were chosen by X-ray and PPD-selec-fion as volunteers. 3.0 ml of blood was taken from each volunteer, rCFP-IO/ESAT-6, PHA and physiologic saline were added into each 1.0 ml, respectively. The A valule and antibody of IFN-γ were assayed by ELISA. Results Treated with rCFP-10/ESAT-6 group: the A value average of patients group was 1. 3885±0.6236, students group was 0.2944±0.0917. Intergroup t'=16.4259, P<0.05, set>0.42 as cut-off, the positive rate of patients group was 93.58%, students group was 13.07%. Treated with PHA group: the A value average of patients group was 1.2463±0.5541, of which the other was 0.5613±0.064, t'=19.1797,P<0.05. Treated with physiologic saline group:the A value average of patients group was 0.0772±0.0444,of which the other was 0.0290±0.0235,t'=13.9487,P<0.05. All had significant deviation. The antibody positive rate of the patients group was 66.36%, the students group was 7.19%. Conclusion rCFP-10/ESAT-6 as specific antigen, the sensitivity of IFN-γ release assay by ELISA is above 90%. No matter specific or non-specific disposal, the active tuberculosis patients have higher IFN-γ, release level and antibody than the control group.
ABSTRACT
This study aimed to establish human IFN-gamma (hIFN-gamma) in vitro release assay and to apply it in diagnosis of human tuberculosis. Human IFN-gamma gene was cloned and expressed in Escherichia coli. The recombinant hIFN-gamma was purified and used as immunogen to immunize mice and rabbits respectively. Monoclonal and polyclonal antibodies were respectively developed and a sandwich ELISA was established. The heparized whole blood from 111 active tuberculosis patients and 292 clinical healthy controls were collected. The blood was stimulated with tuberculosis specific fused antigen ESAT-6/CFP-10 and the plasma was collected for IFN-gamma detection. The sensitivity for tuberculosis diagnosis was 95.5%, whereas the positive detection rate for the healthy controls was 16.7%. There was a significant difference between the patients and healthy controls (P<0.01) indicating that this assay had a high sensitivity and specificity, and thus could be promising in tuberculosis diagnosis.