Publications (38) View all
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Article: Association of lipoarabinomannan with high density lipoprotein in blood: Implications for diagnostics.
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ABSTRACT: Understanding the pathophysiology of tuberculosis, and the bio-distribution of pathogen-associated molecules in the host is essential for the development of efficient methods of intervention. One of the key virulence factors in the pathology of tuberculosis infection is Lipoarabinomannan (LAM). Previously, we have demonstrated the reliable detection of LAM in urine from tuberculosis patients in a sandwich immunoassay format. We have also applied an ultra-sensitive detection strategy developed for amphiphilic biomarkers, membrane insertion, to the detection of LAM with a limit of detection of 10 fM. Herein, we evaluate the application of membrane insertion to the detection of LAM in patient serum, and demonstrate that the circulating concentrations of 'monomeric' LAM in serum are very low, despite significantly higher concentrations in the urine. Using spiked samples, we demonstrate that this discrepancy is due to the association of LAM with high-density lipoprotein (HDL) nanodiscs in human serum. Indeed, pull-down of HDL nanodiscs from human serum allows for the recovery of HDL-associated LAM. These studies suggest that LAM is likely associated with carrier molecules such as HDL in the blood of patients infected with tuberculosis. This phenomenon may not be limited to LAM in that many pathogen-associated molecular patterns like LAM are amphiphilic in nature and may also be associated with host lipid carriers. Such interactions are likely to affect host-pathogen interactions, pathogen bio-distribution and clearance in the host, and must be thoroughly understood for the effective design of vaccines and diagnostics.Tuberculosis (Edinburgh, Scotland) 03/2013; · 2.54 Impact Factor -
Article: Taste of sugar at the membrane: thermodynamics and kinetics of the interaction of a disaccharide with lipid bilayers.
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ABSTRACT: Sugar recognition at the membrane is critical in various physiological processes. Many aspects of sugar-membrane interaction are still unknown. We take an integrated approach by combining conventional molecular-dynamics simulations with enhanced sampling methods and analytical models to understand the thermodynamics and kinetics of a di-mannose molecule in a phospholipid bilayer system. We observe that di-mannose has a slight preference to localize at the water-phospholipid interface. Using umbrella sampling, we show the free energy bias for this preferred location to be just -0.42 kcal/mol, which explains the coexistence of attraction and exclusion mechanisms of sugar-membrane interaction. Accurate estimation of absolute entropy change of water molecules with a two-phase model indicates that the small energy bias is the result of a favorable entropy change of water molecules. Then, we incorporate results from molecular-dynamics simulation in two different ways to an analytical diffusion-reaction model to obtain association and dissociation constants for di-mannose interaction with membrane. Finally, we verify our approach by predicting concentration dependence of di-mannose recognition at the membrane that is consistent with experiment. In conclusion, we provide a combined approach for the thermodynamics and kinetics of a weak ligand-binding system, which has broad implications across many different fields.Biophysical Journal 02/2013; 104(3):622-32. · 3.65 Impact Factor -
Article: Rapid detection of Mycobacterium tuberculosis biomarkers in a sandwich immunoassay format using a waveguide-based optical biosensor.
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ABSTRACT: Early diagnosis of active tuberculosis (TB) remains an elusive challenge, especially in individuals with disseminated TB and HIV co-infection. Recent studies have shown a promise for the direct detection of pathogen-specific biomarkers such as lipoarabinomannan (LAM) for the diagnosis of TB in HIV-positive individuals. Currently, traditional immunoassay platforms that suffer from poor sensitivity and high non-specific interactions are used for the detection of such biomarkers. In this manuscript, we demonstrate the development of sandwich immunoassays for the direct detection of three TB-specific biomarkers, namely LAM, early secretory antigenic target 6 (ESAT6) and antigen 85 complex (Ag85), using a waveguide-based optical biosensor platform. Combining detection within the evanescent field of a planar optical waveguide with functional surfaces that reduce non-specific interactions allows for the ultra-sensitive and quantitative detection of biomarkers (an order of magnitude enhanced sensitivity, as compared to plate-based ELISA) in complex patient samples (urine, serum) within a short time. We also demonstrate the detection of LAM in urine from a small sample of subjects being treated for TB using this approach with excellent sensitivity and 100% corroboration with disease status. These results suggest that pathogen-specific biomarkers can be applied for the rapid and effective diagnosis of disease. It is likely that detection of a combination of biomarkers offers greater reliability of diagnosis, rather than detection of any single pathogen biomarker. NCT00341601.Tuberculosis (Edinburgh, Scotland) 06/2012; 92(5):407-16. · 2.54 Impact Factor -
Article: Understanding the interaction of Lipoarabinomannan with membrane mimetic architectures.
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ABSTRACT: Lipoarabinomannan (LAM) is a critical virulence factor in the pathogenesis of Mycobacterium tuberculosis, the causative agent of tuberculosis. LAM is secreted in urine and serum from infected patients and is being studied as a potential diagnostic indicator for the disease. Herein, we present a novel ultra-sensitive and specific detection strategy for monomeric LAM based on its amphiphilic nature and consequent interaction with supported lipid bilayers. Our strategy involves the capture of LAM on waveguides functionalized with membrane mimetic architectures, followed by detection with a fluorescently labeled polyclonal antibody. This approach offers ultra-sensitive detection of lipoarabinomannan (10 fM, within 15 min) and may be extended to other amphiphilic markers. We also show that chemical deacylation of LAM completely abrogates its association with the supported lipid bilayers. The loss of signal using the waveguide assay for deacylated LAM, as well as atomic force microscopy (AFM) images that show no change in height upon addition of deacylated LAM support this hypothesis. Mass spectrometry of chemically deacylated LAM indicates the presence of LAM-specific carbohydrate chains, which maintain antigenicity in immunoassays. Further, we have developed the first three-dimensional structural model of mannose-capped LAM that provides insights into the orientation of LAM on supported lipid bilayers.Tuberculosis (Edinburgh, Scotland) 01/2012; 92(1):38-47. · 2.54 Impact Factor -
Article: Quantitative multiplex detection of pathogen biomarkers on multichannel waveguides.
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ABSTRACT: No single biomarker can accurately predict disease. An ideal biodetection technology should be capable of the quantitative, reproducible, and sensitive detection of a limited suite of such molecules. To this end, we have developed a multiplex biomarker assay for protective antigen and lethal factor of the Bacillus anthracis lethal toxin using semiconductor quantum dots as the fluorescence reporters on our waveguide-based biosensor platform. The platform is extendable to a wide array of biomarkers, facilitating rapid, quantitative, sensitive, and multiplex detection, better than achievable by conventional immunoassay. Our assay allows for the sensitive (limit of detection 1 pM each), specific (minimal nonspecific binding), and rapid (15 min) detection of these biomarkers in complex biological samples (e.g., serum). To address the issue of reproducibility in measurement and to increase our sample throughput, we have incorporated multichannel waveguides capable of simultaneous multiplex detection of biomarkers in three samples in quadruplicate. In this paper, we present the design, fabrication, and development of multichannel waveguides for the simultaneous detection of lethal factor and protective antigen in serum. Evaluation of the multichannel waveguide shows an excellent concordance with single-channel data and effective, simultaneous, and reproducible measurement of lethal toxins in three samples.Analytical Chemistry 12/2009; 82(1):136-44. · 5.86 Impact Factor
