Performance of novel antibodies for lipoarabinomannan to develop diagnostic tests for Mycobacterium tuberculosis

Abstract

Lipoarabinomannan (LAM), a component of the Mycobacterium tuberculosis (MTB) cell wall, is detectable in the urine of MTB infected patients with active tuberculosis (TB). LAM-specific antibodies (Igs) have been developed by a variety of traditional and recombinant methods for potential use in a rapid diagnostic test (RDT). We evaluated the analytical performance of the TB LAM Igs to identify pairs that offer superior performance over existing urine LAM tests. We assessed 25 new and 4 existing Igs in a matrixed format using a multiplex electrochemiluminescence-based liquid immunoassay. A total of 841 paired Ig combinations were challenged with in vitro cultured LAM (cLAM) derived from MTB strains representing diverse phylogenetic lineages, alongside urinary LAM (uLAM) from the urine of adults with active pulmonary TB. Analytical sensitivity of down-selected Ig pairs was determined using MTB Aoyama-B cLAM, while diagnostic accuracy was determined using clinical samples. When testing cLAM, the reactivity of Ig pairs was similar across MTB lineages 1–4 but lineage 5:6 had significantly more reactivity among Ig pairs. Overall, 41 Ig pairs had a strong binding affinity to cLAM, as compared to the reference pair of S4-20/A194-01, and 28 Ig pairs therein exhibited a strong affinity for both cLAM and uLAM. Retrospective testing on clinical urine specimens demonstrated varying sensitivities (12–80%) and specificities (14–100%). The five top pairs had a similar analytical limit of detection to the reference pair but in four instances, the sensitivity and specificity with clinical uLAM samples was poor. Overall, epitopes presented by uLAM are different from cLAM, which may affect antibody performance when testing uLAM in patient samples. Several new Ig pairs had similar ranges of high sensitivity to cLAM but overall, there were no new candidate Ig pairs identified in this round of screening with increased performance with uLAM as compared to an existing optimal pair.

Urine lipoarabinomannan concentrations among HIV-uninfected adults with pulmonary or extrapulmonary tuberculosis disease in Vietnam

“…The enrichment of uLAM concentration in urine specimens has been achieved via a variety of methods using either the direct capture of uLAM via specific antibodies [35,36], a uLAM specific chemical ligand or via proteolysis, chemical treatment and ultrafiltration [27,37]. Others have focused on improving high sensitivity assays via platform improvement, novel antibodies, and the application of machine learning algorithms to improve diagnostic accuracy [38][39][40]. The application of multiple antibodies, each targeting different LAM epitopes, has also been demonstrated to increase diagnostic performance [25].…”

“…The application of multiple antibodies, each targeting different LAM epitopes, has also been demonstrated to increase diagnostic performance [25]. A key component to this work is to ensure that clinical samples are used early in the product development phase and that clinical evaluation should encompass multiple populations as evidenced in this and other work where the detection of uLAM in the urine of TB positive HIV negative patients varies [19,28,38].…”

Emerging Extracellular Molecular Targets for Innovative Pharmacological Approaches to Resistant Mtb Infection

“…Among the first-line treatments for TB, ethambutol (n.26, Table 4) acts by inhibiting the LAM synthesis during the static phase of the infection, affecting its immunomodulatory function, and modulating the structure of the Mtb cell wall [17]. LAM is also useful as a marker to test the presence of Mtb infection in urine using different types of antibodies against LAM; for example, U-LAM-based diagnostics in urine is a sensitive, non-invasive and rapid diagnostic test using an anti-LAM monoclonal antibody [58,59]. First-line drug…”

“…against LAM; for example, U-LAM-based diagnostics in urine is a sensitive, non-invas and rapid diagnostic test using an anti-LAM monoclonal antibody[58,59].…”