Objective: To investigate the application of heparin-binding haemagglutinin adhesin (HBHA) in tuberculosis (TB) diagnosis.
Methods: We prepared native HBHA from cultivated Mycobacterium Bovis Calmetta Guerin (BCG) in Suton liquid medium. After BCG grew to the stationary status, native HBHA was acquired by specific CL-6B chromatography column binding heparin. At the same time, we cloned hbhA gene from Mycobacterium tuberculosis into PET-32alpha (+) expression vector. Recombinant HBHA from E. coli was obtained. Based on the native HBHA and recombinant HBHA, we chose 4 groups of pulmonary TB, extra-pulmonary TB, PPD (-) and PPD (+) healthy control with 47 in each group and conducted ELISA from serum for specific HBHA antibody level. At last we calculated the sensitivity and specificity in TB diagnosis by detection of anti-HBHA antibody level.
Results: The native HBHA could be diluted and purified with the PBS containing the 375 mmol/L NaCl by specific CL-6B chromatography column binding heparin; There was no significant difference in experimental result based on the natural and recombinant HBHA protein, also no difference between PPD (-) and PPD (+) healthy control groups. Serum antibody level by ELISA could distinguish pulmonary TB and ertra-pulmonary TB (t = 12.224, P< 0.05). The antibody level of the TB groups (pulmonary TB and ertra-pulmonary TB) was higher than the healthy control groups [PPD (+) and PPD (-) healthy control] (t =25.909, P<0.05).
Conclusion: Both recombinant and native HBHA can be used as immunological diagnosis in TB. It can be used in TB and especially extra-pulmonary TB diagnosis.