Mycobacterium tuberculosis lipoarabinomannan (LAM) is a biomarker for active tuberculosis (TB) disease. The presence of LAM in the urine of TB patients, whether HIV positive or negative, has been validated by a gas chromatography/mass spectral method with good specificity (84%) and sensitivity (99%). However, point-of-care (POC) methods to detect TB LAM in urine using immunoassays have poor sensitivity and are limited to only HIV coinfected TB diagnosis. We hypothesized that these disappointing results with the POC methods may be due to the antibodies used in the immunoassays as there could be structural differences between LAM in vivo and LAM in vitro. To address this issue, we infected C3HeB/FeJ mice with M.tb W. Beijing SA161 and purified LAM from the lung. Analysis of these sources of LAM using a panel of existing mAbs revealed differences in epitope patterns. Conventionally, the non-reducing termini of LAM are identified by their release with endoarabinanase. These epitopes correspond to linear tetra-(Ara4), branched hexa-(Ara6) arabinofuranosides, and their mannose-capped versions. We discovered two distinct epitopes. In the first case, it was found that the non-reducing termini of LAM from M.tb strain SA161 are highly succinylated, especially when the LAM was isolated from the mouse lungs. In the second case, it was found that Cellulomonas endoarabinanase digestion of LAM from both SA161 and LAM from a TB+ HIV- patient's urine yielded epitopes based on 5 arabinoses as major components and a profound lack of Ara6. The epitopes based on 5 arabinoses from M.tb SA161 and from the LAM in human urine must result from underlying structural and thus epitope differences. These results suggest approaches to develop specific antibodies for POC tests for LAM in the urine of suspected TB patients.
Keywords: C3HeB/FeJ mice; Cul-LAM; LAM epitopes; LAM structure; LC/MS-MS; Mlung-LAM; urinary LAM.