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The M. tuberculosis Antigen ELISA, is an enzyme linked immunsorbent assay intended for the detection and quantitative determination of specific antigen of M. tuberculosis secreted in culture broth, human blood or pleural effusion. This kit may also be used for monitoring the efficiency of tuberculosis antibiotics therapy.
| Code | Description | Tests | |
 | MB-N001 | Tuberculosis Antigen ELISA | 96 |
| MB-N002 | Tuberculosis Antigen ELISA | 5 x 96 |
PRINCIPLES OF THE ASSAY
1. Capture of specific antigen in the sample:
Individual with activated M. tuberculosis infection produces secreted antigens in body fluids. Our ELISA is to quantify the antigens in the any sample containing the specific TB antigens. This is accomplished by incubating sample in a microtiter plate coated with primary anti-TB antibody. The antigens will bind specifically to the antibody on the microtiter plate.
2. Detection of bound antigen:
After incubation for the specified time at the specified temperature, unbound antigens are removed by aspiration and washing. The presence of bound antigen is then disclosed by using an anti-TB antibody 2 conjugated with of horse-radish peroxidase (HRP) and the colorimetric reagent, TMB. The colorimetric result can be determined by the microplate reader. The positivity and the concentration of antigen of the unknown samples are then calculated through an equation and a standard curve.
Performance Characteristics
18 mycobacterial species culture broths were tested. The result showed all strains of BCG and in enviroment isolates with the exception of M. kansasli and M.marinum are obtained negative results
| Species | Results |
| A) TB complex | |
| i) M tuberculosis | positive |
| ii) M africanum | positive |
| iii) M bovis | positive |
| B) BCG sub-strains | |
| i) danish | negative |
| ii) gothenburg | negative |
| iii) moreau | negative |
| iv) pasteur | negative |
| v) tokyo | negative |
| C) Atypical Strains | |
| i) M.abcessus | negative |
| ii) M avium | negative |
| iii) M celatum | negative |
| iv) M chelonae | negative |
| v) M intracellulare | negative |
| vi) M kansasii | negative |
| vii) M malmoense | negative |
| viii) M marinum | negative |
| ix) M smegmatis | negative |
| x) M xenopi | negative |
Evaluation Reports
Results from the comparison of the 128 cases of tuberculosis between utilizing M. tuberculosis Ag ELISA (Method A) and acid-fast stain (Method B) are:
92% of the 128 cases of TB (118/128) were found to be positive of M. tuberculosis specific proteins in the serum by the ELISA method. The acid-fast stain method found 35% (45/128) of the cases to be positive.
| Groups | Cases | Method A (Detection Rate (%) | Method B (Detection Rate (%) |
| TB Group | 128 | 92（118/128） | 35（45/128） |
| Control Group | 120 | 0 | 0 |
With X2 examination, (there is a marked difference between the two different methods)（P〈0.01〉）.the difference between the two methods is significant.
Comparison of the specificity and sensitivity between the two methods:
| Method | Number of Cases | Sensitivity (%) | Specificity (%) |
| A | 248 | 92 | 100 |
| B | 248 | 35 | 100 |
The results demonstrate that the M. tuberculosis Ag ELISA has a sensitivity of 92% and a specificity of 100%, and has a marked difference in comparison with the acid-fast stain method.
Comparison of TB Antigen ELISA/Rapid Test with other commercial Tests
| Detection by | Antigen | Interferon | Antibody | Phage Amplification | PCR |
| Product | |||||
| Specificity | 87.50% | >95% | 80-90% | 99.00% | >90% |
| Sensitivity | 100% | >95% | 60-85% | 70.30% | 70-90% |
| Convenience | simple procedures | requires high technical skills and needs special detector | simple procedures | requires high technical skills needs special detector | requires high technical skills |
| Safety | No risk; apply on serum but some risk apply on culture broth | Safe | Safe | high risk apply on sputum | high risk apply on sputum and culture medium |
| Efficiency | 2 hours | 24 hours | 2 hours | 48 hours | 2-3 hours |
| Application | can be used for diagnosis of active lung or extrapulmonary tuberculosis | hard to distinguish from active TB and latent TB | hard to distinguish from active TB and latent TB | only detect active tuberculosis | hard to distinguish from active TB and latent TB |
Limitations of use
1. The M. tuberculosis Antigen ELISA is not used for individual who has taken antibiotics for the TB therapy.
2. The user of this kit is advised to carefully read and understand the package insert. Strict adherence to the protocol is necessary to obtain reliable test results. In particular, correct sample and reagent pipetting, along with careful washing and timing of the incubation steps are essential for accurate results.
3. The results of ELISA immunoassays performed on sample from immunosuppressed patients must be interpreted with caution.
4. Samples that remain equivocal after repeat testing should be retested by an alternate method. If results remain equivocal upon further testing, an additional sample should be taken.
5. Results of this test should be interpreted by the physician in the light of other clinical findings and diagnostic procedures.
6. Icteric, lipemic, hemolyzed, or heat inactivated sera may cause erroneous results and should be avoided.
7. Kit procedures or practices outside those in this package insert may yield questionable results.
8. The performance characteristics have not been established for matrices other than culture broth, human serum and pleural effusion.
Literatures
1. Andersen P, Andersen AB, Sorensen AL, Nagai S. Recall of long-lived immunity to Mycobacterium tuberculosis infection in mice. J Immunol 1995: 154; 3359-3372
2. Chaparas SD, Maloney CJ, Hedrick SR. Specificity of tuberculins and antigens from various species of mycobacteria. Am Rev Respir Dis 1970: 101; 74-83
3. Colangeli R, Spencer JS, Bifani P, Williams A, Lyashchenko K, Keen MA, Hill PJ, Belisle J, Gennaro ML. MTSA-10, the product of the Rv3874 gene of Mycobacterium tuberculosis, elicits tuberculosis-specific, delayed-type hypersensitivity in guinea pigs. Infect Immunol 2000; 68: 990-993
4. Dalovisio JR, Montenegro-James S, Kemmerly SA, Genre CF, Chambers R, Greer D, Pankey GA, Failla DM, Haydel KG, Hutchinson L. Comparison of the amplified Mycobacterium tuberculosis direct test, Amplicor MTB PCR, and IS6110-PCR for detection of MTB in respiratory specimens. Clin Infect Dis 1996; 23: 1099-1106
5. Dannenberg AM. Immunopathogenesis of pulmonary tuberculosis. Hosp Pract 1993: 28; 51-58
6. Harboe M, Oettinger T, Wiker HG, Rosenkrands I, Andersen P. Evidence for occurrence of the ESAT-6 protein in Mycobacterium tuberculosis and virulent Mycobacterium bovis and for its absence in Mycobacterium bovis BCG. Infect Immunol 1996; 40: 908-912
7. Huebner RE, Schein MF, Bass JB The tuberculin skin test. Clin Infect Dis 1993; 17: 968-975
8. Mustafa AS, Amoudy HA, Wiker HG, Abal AT, Ravin P, Oftung F, Andersen P. Comparison of antigen-specific T cell responses of tuberculosis patients using complex or single antigens of Mycobacterium tuberculosis. Scand J Immunol 1998; 48, 535-543
9. Noordhoek GT, Kolk AH, Bjune G, Catty D, Dale JW, Fine PE, Godfrey-Faussett P, Cho SN, Shinnick T, Svenson SB. Sensitivity and specificity of PCR for detection of Mycobacterium tuberculosis: a blind comparison study among seven laboratories. J Clin Microbiol 1994; 32: 277-284
10. North RJ, Jung YJ. Immunity to tuberculosis. Annu Rev Immunol 2004; 22: 599-623
11. Snider DE. The tuberculin skin test. Am Rev Respir Dis 1982: 125; 108-118
12. Trajkovic V, Natarajan K, Sharma P. Immunomodulatory action of mycobacterial secretory proteins. Microbes Infect 2004; 6: 513-519
