A Novel Sensitive Immunoassay Targeting the 5-Methylthio-d-Xylofuranose-Lipoarabinomannan Epitope Meets the WHO's Performance Target for Tuberculosis Diagnosis

Abstract

The only currently commercialized point-of-care assay for tuberculosis (TB) that measures lipoarabinomannan (LAM) in urine (Alere LF-LAM) has insufficient sensitivity. We evaluated the potential of 100 novel monoclonal antibody pairs targeting a variety of LAM epitopes on a sensitive electrochemiluminescence platform to improve the diagnostic accuracy. In the screening, many antibody pairs showed high reactivity to purified LAM but performed poorly at detecting urinary LAM in clinical samples, suggesting differences in antigen structure and immunoreactivity of the different LAM sources. The 12 best antibody pairs from the screening were tested in a retrospective case-control study with urine samples from 75 adults with presumptive TB. The best antibody pair reached femtomolar analytical sensitivity for LAM detection and an overall clinical sensitivity of 93% (confidence interval [CI], 80% to 97%) and specificity of 97% (CI, 85% to 100%). Importantly, in HIV-negative subjects positive for TB by sputum smear microscopy, the test achieved a sensitivity of 80% (CI, 55% to 93%). This compares to an overall sensitivity of 33% (CI, 20% to 48%) of the Alere LF-LAM and a sensitivity of 13% (CI, 4% to 38%) in HIV-negative subjects in the same sample set. The capture antibody targets a unique 5-methylthio-d-xylofuranose (MTX)-dependent epitope in LAM that is specific to the Mycobacterium tuberculosis complex and shows no cross-reactivity with fast-growing mycobacteria or other bacteria. The present study provides evidence that improved assay methods and reagents lead to increased diagnostic accuracy. The results of this work have informed the development of a sensitive and specific novel LAM point-of-care assay with the aim to meet the WHO's performance target for TB diagnosis.

Keywords: biomarker; diagnostics; immunoassays; lipoarabinomannan; tuberculosis.

FIG 1

Results of antibody screen to identify antibody pairs for detecting LAM. (a) Schematic of the U-PLEX format used for immunoassay measurements. The U-PLEX plates have an array of binding reagents specific for 10 different U-PLEX “linkers.” Biotin-labeled antibodies are coupled to the linkers and then self-assembled to specific locations on the U-PLEX array. These arrays can then be used to carry out multiplexed sandwich immunoassays using detection antibodies carrying ECL labels. (b) Heat maps that show the ability of each pairwise combination of capture (rows) and detection (columns) antibodies to detect 10 ng/ml of purified LAM from cultured M. tuberculosis (Mtb; left) and a 1:50 dilutions of urine from TB-positive HIV-positive individuals (right). The heat maps display the signal-to-blank (S/B) ratio. The data in the urinary LAM heat map represent the maximum values for urine samples from two individuals. The antibody names are color coded on the basis of the LAM epitopes they target, as determined by binding to glycan arrays, and the epitopes are listed next to the names of the capture antibodies (see Fig. S2 in the supplemental material for details of the epitope mapping results). (c) Schematic of LAM illustrating the different epitopes listed in the heat map. (Adapted from reference with permission of the publisher; kindly provided by Bruce Turnbull.) MTX, 5-methylthio-xylofuranose; MSX, 5-methylsulfoxy-xylofuranose.

FIG 2

Measured LAM concentrations and assay signals in clinical study samples. (a) Heat map showing the measured LAM concentrations for all tested urine samples (columns) for the six different capture antibodies tested in multiplex format in combination with the A194-01 detection antibody. The samples are grouped by the donors' TB and HIV statuses. The bottom row provides the Alere LF-LAM test grade for each sample for comparison (only samples with positive Alere LF-LAM test results are colored). The results from panel a for the FIND 28 (b) and S4-20 (c) capture antibodies in scatter plot format. The plots show the measured signal-to-blank (S/B) ratios (left axes) and LAM concentrations (right axes) for each urine sample as a function of the TB and HIV status of the donor. The dashed orange lines show the assay threshold (S/B = 1.375). Concentration values are only meaningful for points above the assay threshold. The points are colored by the results of the Alere LF-LAM test for the same samples. Scatter plots for the other 4 capture antibodies can be found in Fig. S5 in the supplemental material.

FIG 3

Analysis of LAM assay performance. (a) Correlation of assay signals measured with FIND 28 and S4-20 capture antibodies paired with A194-01 as a detection antibody. Region 1, points that are false positives for FIND 28 but not S4-20; region 2, points that are low true positives for S4-20 but are undetectable with FIND 28; region 3, points with signals above the cutoffs for both FIND 28 and S4-20. Assays signals for TB-positive subjects broken down by HIV status and CD4 count (in cells per μl) (b) and Alere LF-LAM test grade (c). *, P < 0.05 versus the left-most group by Mann-Whitney test. (d) The observed clinical sensitivity and specificity (with 95% confidence intervals) for each candidate capture antibody when paired with the A194-01 detection antibody. The plot also shows the minimal (triangle) and optimal (diamond) target sensitivity and specificity requirements set by the WHO in its target product profile (TPP) requirements document for POC TB tests (42) used for two different use case scenarios: (i) definitive detection/diagnosis of TB (purple symbols) or (ii) triage to identify patients who should undergo further confirmatory testing for TB (green symbols). The marker representing the performance of an assay would ideally be above and to the left of the marker representing the requirement for a use case (the area of interest is highlighted).