| Object Tuberculosis is one kind of chronic infective diease which is harmful to the health of human beings.The rising incidence of tuberculosis worldwide means an increasing burden on diagnostic facilities, so tests simpler than Ziehl-Neelsen staining are heed. It is an important study to develop sero-immunological assays which are more rapid, simple, sensitive and specific. But the specificity of antigen is associated with coincidence of diagnosing tuberculosis. It is necessary to find some sensitive and specific M.tuberculosis antigens for the serodiagnosis of tuberculosis.For above reasons, We sought to purify sensitive and specific polypeptide antigen from M.tuberculosis H37RV strain by sonication, SDS-PAGE and Western-blot. Then We will develop some serological assays with more rapid and simple to hadle for diagnosis and epidemiological investigation of tuberculosis.Materials and Methods 1. Bacterial strains and preparation of bacterial antigens: M. tuberculosis H37RV strains were provided by National Institute for the Control of Pharmaceutical and Biological Products. M. tuberculosis H37RV strains were cultured on Lowenstein - Jensen solid media and Sauton liquid media for 1~3 weeks at 37℃, then were harvested, inactivated and stored at -20℃ until used. 2. Preparation of crude antigens: Inactivated M. tuberculosis H37RV were suspended in phosphate - buffered saline and ultrasonic antigens were obtained from supernatants of M. tuberculosis cells that had been sonicated on ice. 3. SDS-PAGE analysis: Sample were analyzed with SDS-PAGE using a 5% stacking gel and 12% separatinggel for 2h at 80V and 120V. 4. Western-blot: Following separation by SDS-PAGE, proteins were transferred to nitrocellulose membrane (NC) by electroblotting for 2h at 75mA. After blocked with 3% BSA, the NC membrane were incubated with 1:100 dilutions of human sera for 2h at 37°C. After washed, NC membrane were incubated with colloidal gold-conjugated staphylococcal protein A for 1h. 5. Elution from gel: After separated with SDS-PAGE, the needed protein band was localized, cut out and extracted for 24h with 0.05mol/L NH4HCO3 and 0.05% SDS. The extract was dialyzed against 0.05 mol/L NH4HCO3 and subsequently concentrated by lyophilization. 6. Polypeptide (TB4) and Lipoarabinomannan(LAM) of M. tuberculosis were used as antigens to detect anti-TB-antibodies in sera by dot immunogold filtration assay (DIGFA). 78 sera from patients with TB, 15 sera from patients with tracheitis and 50 sera from healthy controls were tested by respectively using TB4 and LAM as antigens. 7. TB4 of M. tuberculosis were used as antigen to detect antibodies by ELISA and DIGFA. 69 patients with TB, including 26 patients with sputum smear positive and 43 with sputum smear negative, 45 with tracheitis and 70 healthy controls were tested with two methods. Sensitivity and negative coincidence of two methods were evaluated.Results 1. Analysis with SDS-PAGE and Western-blot of the sonicated antigens from M. tuberculosis H37RV strains, 34~40kD polypeptide showed good antigenicity was a specific antigen of M. tuberculosis. 2. Sensitivity of TB4 and LAM for detection of anti-TB-antibodies were 84.6% and 73.1%, negative coincidence of them were 94.0% and 94.0%. there was no significant difference between TB4 and LAM (x2=3.11, 0.18) (P>0.05). It suggest that TB4 may be useful for diagnosis of tuberculosis. 3. Sensitivity of ELISA and DIGFA were 76.8% and 84.1%, negative coincidence of them were 94.3% and 94.3%. The coincident rates of antibodies with sputum smear positive were 88.5% and 92.3%. There was no significant difference between two assays (x2=1.15, 0.13)(P>0.05). This results indicated that each diagnostic index of two assays with TB4 antigen were similar.Conclusion 1. Polypeptide (TB4) could be purified from cells of M.tuberculosis H37RV strains by sonication, SDS-PAGE and elution from gel. Themolecular weight of purified protein was 34~40kD. 2. TB4 was an available antigen for serodiagnosis of tuberculosis. 3. DIGFA was a simple and rapid assay in comp...
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