Rapid and simple detection of a mycobacterium circulating antigen in serum of pulmonary tuberculosis patients by using a monoclonal antibody and Fast-Dot-ELISA.
OBJECTIVE
Several immunoassays have been established for detection of Mycobacterium tuberculosis (MTB) antigens in serum, sputum and cerebrospinal fluid of tuberculous patients using polyclonal or monoclonal antibodies raised against different mycobacterium antigens. Some of these assays display both high sensitivity and specificity for the detection of these antigens. However, these assays require special and highly expensive equipment and the procedures require long periods for their completion. Thus, the rationale of this study was to establish and evaluate Fast-Dot-Enzyme-Linked Immunosorbent Assay (FD-ELISA) as a fast, cheap and field applicable assay for detection of mycobacterium antigen in serum of patients with pulmonary TB.


METHODS
This study included three groups: group I: 175 tuberculous patients with pulmonary TB proves with sputum Ziehl-Neelsen (ZN) for acid-fast bacilli and sputum culture (all cases were culture positive for MTB); Group II: 65 patients with diseases other than pulmonary TB as bronchial carcinoma (17 patients), bronchial asthma (29 patients) and chronic obstructive pulmonary disease (19 patients); group III: 50 healthy individuals. Groups II and III served as negative control groups. The target mycobacterium antigen was identified in both crude mycobacterium antigens extract and serum of patients with pulmonary TB, using western blotting technique and anti-TB monoclonal antibody (TB20-mAb) and then it was estimated in the serum samples of all studied groups as an index of tuberculosis, using a newly developed FD-ELISA.


RESULTS
The target mycobacterium antigen was identified at 20 kDa molecular mass in crude mycobacterium antigens extract as well as in serum of patients with pulmonary TB. The developed FD-ELISA detected the mycobacterium antigen in the sera of 159 out of 175 pulmonary TB patients with a sensitivity of 90.8% and 93.0% positive predictive value (PPV). In addition, it identified 12 false weakly positive cases out of 115 samples of negative control groups (7 out of 65 non-TB patients and 5 out of 50 healthy individuals) with a specificity of 89.6% and 86.6% negative predictive value (NPV). Standardization of the FD-ELISA using a serial dilution of the purified mycobacterium antigen indicated that the assay was able to detect 1.8 microg/ml as a lowest detectable antigen concentration.


CONCLUSIONS
The newly developed FD-ELISA is a simple, rapid and highly sensitive assay for detection of mycobacterium antigen in patients with pulmonary TB. Moreover, all steps were performed at room temperature and without the need to use expensive equipment, and this may enhance the application of this assay in tuberculosis screening programs. Further study is needed for confirmation of FD-ELISA reproducibility in light infected pulmonary TB patients and in a large population.
El-Masry
Samir
S
Molecular and Cellular Biology Department, Genetic Engineering and Biotechnology Research Institute, Minufiya University, Sadat City, P.O. 79, Minufiya, Egypt. samirelmasry@yahoo.com
El-Kady
Ibrahim
I
Zaghloul
Mohamed Hosam
MH
Al-Badrawey
Mohamed Khairy
MK
D000293:Adolescent; D000328:Adult; D000368:Aged; D000911:Antibodies, Monoclonal / Q000032:analysis; D000942:Antigens, Bacterial / Q000097:blood*; D015153:Blotting, Western / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D008297:Male; D008875:Middle Aged; D009161:Mycobacterium / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology; D014397:Tuberculosis, Pulmonary / Q000097:blood* / Q000382:microbiology*
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; C072681:Mycobacterium tuberculosis antigens