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.
