[Expression of Mycobacterium tuberculosis early secretory antigenic target-6 protein and its application in detection of Mycobacterium tuberculosis antigen-specific interferon-gamma response].
OBJECTIVE
To obtain the purified early secretory antigenic target-6 (ESAT-6) protein and to evaluate its application in detection of Mycobacterium tuberculosis antigen-specific interferon-gamma (IFN-gamma) response.


METHODS
ESAT-6 protein was expressed by genetic engineering. The antigen specificity and reactivity of ESAT-6 were evaluated by Western blot. Using ESAT-6 as the antigen, the antigen-specific IFN-gamma response in patients with tuberculosis, healthy medical workers, and village residents was detected by the Elispot method. The results were also compared with those obtained by a commercial kit (QuantiFERON-TB-GOLD, QFT-G).


RESULTS
ESAT-6 protein was successfully expressed and purified, and the antigen specificity of ESAT-6 was confirmed by its recognition by the antigen-specific antibody (anti-ESAT-6). The specificity and sensitivity of the Elispot assay using ESAT-6 as the antigen in detecting the IFN-gamma response was comparable with those of the commercial kit (QFT-G). The positive rates of the Elispot assay for patients with tuberculosis, healthy medical workers and villagers were 36/49 (73.5%), 11/62 (17.7%), and 17/194 (8.8%), respectively, while the rates of the OFT-G method for patients with tuberculosis and healthy medical workers were 38/49 (77.6%) and 14/58 (24.1%), respectively. The sensitivity (73.5%, 77.6%; chi2 = 0.381, P > 0.05) and specificity (82.3%, 75.9%; chi2 = 0.406, P > 0.05) of these two methods did not differ significantly.


CONCLUSIONS
Recombinant ESAT-6 protein was expressed and purified. Elispot using recombinant ESAT-6 protein as antigen showed high sensitivity and specificity for detection of Mycobacterium tuberculosis antigen-specific IFN-gamma response. The purified ESAT-6 can be used for diagnosis of Mycobacterium tuberculosis infection.
Zhang
Jing
J
Respiratory Department, Shenzhen Donghu Hospital, Shenzhen 518020, China.
Liu
Wei-Long
WL
Zhou
Bo-Ping
BP
Zhang
Ming-Xia
MX
Yang
Qian-Ting
QT
Zhu
Xiu-Yun
XY
Lu
Jian
J
Deng
Qun-Yi
QY
Chen
Xin-Chun
XC
D000293:Adolescent; D000328:Adult; D000368:Aged; D000942:Antigens, Bacterial / Q000302:isolation & purification* / Q000378:metabolism; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D016022:Case-Control Studies; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D006801:Humans; D007371:Interferon-gamma / Q000097:blood / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D009169:Mycobacterium tuberculosis / Q000276:immunology* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D014376:Tuberculosis / Q000175:diagnosis* / Q000276:immunology; D055815:Young Adult
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D011994:Recombinant Proteins; D007371:Interferon-gamma