Diagnosis of pulmonary and extrapulmonary tuberculosis based on detection of mycobacterial antigen 85B by immuno-PCR
We developed a novel indirect sandwich immuno-polymerase chain reaction (I-PCR) assay for the detection of mycobacterial antigen 85B (Ag85B, 30kDa, Rv1886c) in pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis (EPTB) patients. The amino-modified reporter DNA was covalently attached with the antidetection antibody through a heterobifunctional cross-linking agent succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate. The detection limit of Ag85B by I-PCR was found to be 1 femtogram (fg)/mL, which was 10(6)-fold lower than an analogous enzyme-linked immunosorbent assay (ELISA). The sensitivities of 85% and 77% with I-PCR and 77.6% and 62.5% with ELISA were observed in smear-positive and smear-negative PTB patients, respectively, with high specificity. On the other hand, sensitivities of 84% and 63.7% with I-PCR and 68% and 47.5% with ELISA were observed in confirmed and clinically suspected EPTB cases, respectively, with high specificity.