ABSTRACT
Tuberculosis (TB) represents a major public health threat, with millions of new cases reported worldwide each year. A major hurdle to curtailing the spread of this disease is the need for low-cost, point-of-care (PoC) diagnostics. Mannose-capped lipoarabinomannan, a significant component of the Mycobacterium tuberculosis bacillus, has been heavily studied as a biomarker for TB, but with little success due to its complexation with endogenous components of body fluids in a manner that sterically interferes with its detection by ELISA and other immunoassays. Recent work by our group and others has shown that complexation can be disrupted with protein-denaturing protocols. By way of followup, we recently described an enzymatic digestion (Proteinase K) sample pretreatment that enables quantitative recovery of ManLAM spiked into healthy human control serum. Herein, we report on the transfer of our benchtop sample pretreatment methodology to an automated microfluidic platform. We show that this platform can be configured to: (1) carry out the pretreatment process with very little user interaction and, (2) yield recoveries for ManLAm spiked into control serum which are statistically indistinguishable from those achieved by the benchtop process. Plans to integrate this device with a portable sample reader as a possible basis for a PoC TB diagnostic system and analyze patient samples are briefly discussed.
ABSTRACT
This paper reports on an investigation of an enzymatic pretreatment protocol using proteinase K (ProK) for the analysis of human serum samples spiked with mannose-capped lipoarabinomannan (ManLAM). ManLAM is an antigenic biomarker found in the serum, urine, and other body fluids of individuals infected with tuberculosis (TB). Immunometric measurements of ManLAM are compromised by steric effects due to its complexation with high-molecular-weight components in these matrices that interfere with its capture and/or labeling. Recent work has shown that deproteinization of these types of samples by perchloric acid acidification or ProK digestion releases ManLAM from complexation. Releasing ManLAM greatly improves its detectability and, as a result, its utility as a TB biomarker. The work detailed herein examined how different ProK reaction conditions (e.g., enzyme concentration and digestion time and temperature) affect the recovery and detectability of ManLAM in human serum. As measured by enzyme-linked immunosorbent assay (ELISA), we show that using the optimal set of digestion conditions to free ManLAM, which also yield a small, quantitatively reproducible level of sample concentration, it is possible to achieve a spiked ManLAM recovery of 98 ± 13% and a limit of detection of 10 pg/mL (0.6 pM). Experiments also demonstrated that the ELISA responses measured for a given ManLAM concentration in serum after pretreatment were statistically indistinguishable from those directly determined for the same amounts of ManLAM added to an innocuous buffered solution. Possible adaptations of the digestion protocol for use in point-of-care TB testing are also briefly discussed.
