Immobilization of Proteinase K for urine pretreatment to improve diagnostic accuracy of active tuberculosis

Abstract

The World Health Organization (WHO) calls for the development of a rapid, biomarker-based, non-sputum test capable of detecting all forms of tuberculosis (TB) at the point-of-care to enable immediate treatment initiation. Lipoarabinomannan (LAM) is the only WHO-endorsed TB biomarker that can be detected in urine, an easily collected sample matrix. For obtaining optimal sensitivity, we and others have shown that some form of sample pretreatment is necessary to remove background from patient urine samples. A number of systems are paper-based often destined for resource limited settings. Our current work presents incorporation of one such sample pretreatment, proteinase K (ProK) immobilized on paper (IPK) and test its performance in comparison to standard proteinase K (SPK) treatment that involves addition and deactivation at high temperature prior to performing a capture ELISA. Herein, a simple and economical method was developed for using ProK immobilized strips to pretreat urine samples. Simplification and cost reduction of the proposed pretreatment strip were achieved by using Whatman no.1 paper and by minimizing the concentration of ProK (an expensive but necessary reagent) used to pretreat the clinical samples prior to ELISA. To test the applicability of IPK, capture ELISA was carried out on either LAM-spiked urine or the clinical samples after pretreatment with ProK at 400 μg/mL for 30 minutes at room temperature. The optimal conditions and stability of the IPK were tested and validation was performed on a set of 25 previously analyzed archived clinical urine samples with known TB and HIV status. The results of IPK and SPK treated samples were in agreement showing that the urine LAM test currently under development has the potential to reach adult and pediatric patients regardless of HIV status or site of infection, and to facilitate global TB control to improve assay performance and ultimately treatment outcomes.

Fig 1

A. Structure of LAM. A major component of the cell envelope of Mtb is lipoarabinomannan (LAM). LAM is a complex of D-mannan and D-arabinan attached to a phosphatidyl-myo-inositol (PI) moiety that anchors in the mycobacterial cell wall [7]. The D-mannan consist of a highly branched structure with an α-(1→6)-linked mannopyranose (Manp) backbone substituted at C-2 by single Manp units. The arabinan consists of a linear α-(1→5)-linked Araf backbone punctuated by branching produced by 3,5-O-linked α-D-Araf residues. The lateral side chains are organized either as a linear tetra-arabinofuranoside or as a biantennary hexa-arabinofuranosides [13, 38]. These chain terminating arabinan can be further capped with short mannose α-(1→2)-linked oligosaccharides and are epitopes for binding to mAbs used in this study. B. Periodic Acid Silver Staining of Mtb CDC1551 LAM. LAM purified from in vitro grown cells showing a tight smear (MW ~ 15–17 kDa). Western blot profile of the CDC1551 LAM with the anti-LAM mouse monoclonal CS35 antibody and anti-LAM human monoclonal A194, the two antibodies used as a pair in our Capture ELISA.

Fig 2. Scheme of immobilizing Proteinase K (IPK).

Schematic representation of the immobilization of Proteinase K on Whatman paper # 1. Lithium chloride in the presence of sodium Periodate was used to change the functional group of the paper from hydroxyl to aldehyde group. Proteinase K was covalently immobilized on the charged paper and treated with sodium cyanoborohydride to preserve the covalent bonds, followed by blocking the non specific binding sites.

Fig 3. Graphical representation showing negligible loss of Proteinase K.

During the immobilization steps where Wash 1, 2 and 3 are from the washes tested in a BCA protein estimation assay from steps following the immobilization of Proteinase K to the filter paper, treatment with NaBH₃CN to preserve the covalent bonding and the final wash before the blocking of the non specific sites respectively. The blue dotted line shows the Proteinase K stock solution used for immobilization.

Fig 4. Indirect ELISA.

A) Shows the concentration curve (0–1000 μg /mL) for IPK that would give optimal results when used for the pretreatment of urine spiked with LAM. The inset shows 400 μg /mL of IPK (red) to be the optimal concentration that gives the best results compared to the urine spiked with LAM that did not see any IPK (dark blue). B) Showing the standard Proteinase K (SPK) treatment of urine from a healthy volunteer spiked with LAM purified from M.tb CDC1551 cells. The blue line with circles shows the effect of SPK in sequestering LAM from the protein/s or other inhibitors present in urine compared to the red line with triangles where the urine spiked with LAM has not been pretreated with SPK. The yellow line shows the background signal obtained with the urine and the green line with diamonds shows that Proteinase K itself does not have any effect on blank urine. C) Shows the optimal time required for the pretreatment, with IPK at 400 μg /mL, of urine spiked with LAM. At 60 min (grey line) and 120 min (yellow line) (see inset) compared to the 0 min (pink line) pretreatment time, more LAM seems to be available for binding to the antibody. D) Shows the optimal temperature needed (55°C) for the IPK pretreatment of urine spiked with LAM for 60 min at 400 μg/mL of IPK.

Fig 5. Capture ELISA.

A) Graph plot showing the concentration curve (0–1000 μg / mL) for IPK pretreatment of NEU spiked with LAM. Optimal results are obtained at 400 μg / mL concentration of IPK as compared to 0 μg /mL (see inset). B) Shows the optimal time and temperature required to obtain the best results in a capture ELISA when using IPK for pretreatment of urine spiked with LAM. The graph shows no significant difference between the various time points and temperatures variations, hence the optimal conditions for C-ELISA taken with IPK is 400 μg / mL for 30 min at room temperature.

Fig 6. Correlation of IPK vs. SPK.

Optical density at 450nm for C-ELISA using SPK is plotted on the x-axis; optical density at 450nm for C-ELISA using IPK is plotted on the y-axis, and a regression line is shown (r² = 0.63). Triangles indicate samples that were non-TB by clinical assignments; circles were TB-positive. The non-TB samples clustered together separately from TB-positive samples in both ELISAs. Sample 7 had an unusually high reading by IPK, as indicated by the circled and labelled point. CS35 IgG is used as the capture mAb and A194-01 IgG1 is used as the detection mAb in the assay.