Rapid detection of Mycobacterium tuberculosis biomarkers in a sandwich immunoassay format using a waveguide-based optical biosensor

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.

Figure 1

Representative curves for biomarker detection on the waveguide-based platform. A) LAM (100 pM), B) ESAT6 (200 pM) and C) Ag85 complex (500 pM). RFU measured is plotted as function of wavelength. Black lines indicate waveguide-associated background. Gray lines represent NS-binding associated with binding of reporter antibody to a control sample. Black circles indicate specific signal measured for the corresponding biomarker.

Figure 2

Standard curves constructed for LAM spiked in urine (A) and ESAT6 spiked in serum (B) on the waveguide-based platform. RFU is plotted as a function of concentration for each biomarker. Measurements (n=3/concentration) were made on different waveguides.

Figure 3

Quantitative measurement of urinary LAM in a small cohort of TB patients without HIV co-infection. LAM concentration is plotted as a function of patient ID. LAM was detected only in patients with active TB (1-9) and not in QFG-negative controls (10-14). Each measurement was repeated twice for confirmation.

Figure 4

Quantitative measurement of serum ESAT6 in a small cohort of TB patients without HIV co-infection. ESAT6 concentration is plotted as a function of patient ID. Only samples from patients with active TB were positive for ESAT6 (1-3), whereas it was undetectable in negative controls (4-6). * indicates significant difference from controls.

Scheme 1

Illustration of the sandwich immunoassay on a functionalized waveguide. A) Waveguides functionalized with a lipid bilayer are prepared by binding of capture antibody using biotin-streptavidin chemistry. B) Addition of the sample containing the antigen results in specific binding. C) Finally, addition of a fluorescently labeled reporter antibody results in a specific signal measured via a spectrometer interface. Schematic not drawn to scale, and represents process for all three biomarkers investigated in this report.