Detection of the tuberculosis antigenic marker mannose-capped lipoarabinomannan in pretreated serum by surface-enhanced Raman scattering

Abstract

The ability to detect tuberculosis (TB) continues to be a global health care priority. This paper describes the development and preliminary assessment of the clinical accuracy of a heterogeneous immunoassay that integrates a serum pretreatment process with readout by surface-enhanced Raman scattering (SERS) for the low-level detection of mannose-capped lipoarabinomannan (ManLAM). ManLAM is a major virulence factor in the infectious pathology of Mycobacterium tuberculosis (Mtb) that has been found in the serum and other body fluids of infected patients. The effectiveness of ManLAM as a TB diagnostic marker, however, remains unproven for reasons not yet well understood. As reported herein, we have found that (1) ManLAM complexes with proteins and possibly other components in serum; (2) these complexes have a strongly detrimental impact on the ability to detect ManLAM using an immunoassay; (3) a simple pretreatment step can disrupt this complexation; and (4) disruption by pretreatment improves detection by 250×. We also describe the results from a preliminary assessment on the utility of serum pretreatment by running immunoassays on archived specimens from 24 TB-positive patients and 10 healthy controls. ManLAM was measurable in 21 of the 24 TB-positive specimens, but not in any of the 10 control specimens. These findings, albeit for a very small specimen set, translate to a clinical sensitivity of 87.5% and a clinical specificity of 100%. Together, these results both provide much needed evidence for the clinical utility of ManLAM as a TB marker, and demonstrate the potential utility of our overall approach to serve as a new strategy for the development of diagnostic tests for this disease.

Surface‐enhanced resonance Raman scattering for the sensitive detection of a tuberculosis biomarker in human serum

“…As before, the spectral intensities increase with the concentration of ManLAM in the sample but below the levels found for the buffer matrix in Figure . This is consistent with recent observations made by our laboratory and is in large part attributed to the labile nature of the arabinan domains of ManLAM with respect to acid hydrolysis …”

“…The next set of experiments contrasted the two types of immunoassays using human serum as a more complex sample matrix . These samples were prepared by spiking ManLAM into human serum and analyzing the same samples using both immunoassay formats.…”

“…These samples were prepared by spiking ManLAM into human serum and analyzing the same samples using both immunoassay formats. Previous work in our laboratory has described the initial development of a PCA sample treatment procedure to liberate ManLAM from complexation with proteins and potentially other serum components so it can be detected effectively in such matrices . Figure shows the spectra collected from the SERRS and SERS assays for these samples.…”

“…A list of reagents, materials, and procedures for the pretreatment of the serum samples and the SERS immunoassay have been reported in detail previously and are presented in the Supporting Information . As discussed in the Supporting Information, this work utilized a commercially available polyclonal antibody as the capture reagent.…”

Incipient and Subclinical Tuberculosis: a Clinical Review of Early Stages and Progression of Infection

“…Urinary LAM has been shown to detect subclinical TB in 25% (7/28) of HIV-infected ambulatory patients in South Africa (205). Despite these limitations, there is the potential to improve diagnostic sensitivity in next-generation urinary LAM assays (206)(207)(208)(209).…”