Detection of the tuberculosis biomarker mannose-capped lipoarabinomannan in human serum: Impact of sample pretreatment with perchloric acid

Abstract

The development of an accurate and rapid diagnostic test for tuberculosis (TB) to use at point of need is vital to efforts aimed at reducing the global burden from this disease. This paper builds on our previous studies of mannose-capped lipoarabinomannan (ManLAM) as a serum biomarker for active TB infection by means of a heterogeneous immunoassay. That work found that complexation with components in serum (e.g., proteins) sterically hindered the capture and/or labeling of ManLAM in an immunoassay at levels <10 ng mL−1, compromising the clinical utility of this biomarker for detection of active TB infection. We also showed that the acidification of ManLAM-containing serum samples with perchloric acid improved the detectability of ManLAM by 250× by complex disruption when compared to measurements of untreated serum. The present study examined what effects the PCA treatment of serum samples may have on the recovery and structural integrity of ManLAM, owing to its potential susceptibility to acid hydrolysis. Recovery was assessed with an enzyme-linked immunosorbent assay (ELISA). The possible impact of acid hydrolysis on the ManLAM structure was investigated by gas chromatography-mass spectrometry and carbohydrate chemical degradation methods. The ELISA study indicated that while the signal strength for ManLAM in the serum spike-in experiments was significantly stronger after PCA pretreatment when compared to untreated human serum, it was only ∼20% of the ManLAM measured in physiological buffer. This loss in detectability was shown by structural analysis to arise mainly from the acid-induced degradation of the arabinan domains of ManLAM that are targeted by antibodies used for antigen capture and/or tagging. The implications of these findings in terms of the detection of this important biomarker for TB are also discussed.

Interaction of amphiphilic lipoarabinomannan with host carrier lipoproteins in tuberculosis patients: Implications for blood-based diagnostics

“…In aqueous blood, HDL is a stable lipidic assembly comprised of a core lipid nanodisc stabilized by coat apolipoproteins [15][16][17]. While LAM has been extracted from blood of TB patients [9], direct measurement of LAM in blood or serum has proved to be more elusive, and achieved mainly in individuals with advanced HIV disease [9,18,19]. We hypothesized that sequestration of LAM in host lipoprotein assemblies may contribute to the difficulty in detecting the antigen in blood.…”

Raman spectroscopy for glutathione measurements in Mycobacterium tuberculosis strains with different antibiotic resistance

“…As a result of research, the hypothesis was not justified, since the authors failed to achieve a sufficient degree of reproducibility. In the work, [ 17 ] studies of a biomarker, a component of the cell wall of mycobacterium lipoarabinogalactan, were performed. One such biomarkers can be a GSH—a tripeptide, which consists of L‐cysteine, glycine, and L‐glutamate.…”

Isolation and Purification of Lipoarabinomannan from Urine of Adults with Active Tuberculosis

“…The copyright holder for this preprint this version posted March 25, 2021. ; https://doi.org/10.1101/2021.03.24.436904 doi: bioRxiv preprint on urinary antigen immunoassays targeting urinary LAM (uLAM) have been described, but none are sensitive enough to reliably detect uLAM for active TB (3,20). Efforts are ongoing to improve the sensitivity of rapid uLAM tests via additive methods such as a larger sample volume (3), sampling first pass morning collection versus later urine sampling (13), enrichment of LAM prior to testing (26) or with pretreatment prior to testing (12,17,25).…”