Proteinase K-pretreated ConA-based ELISA assay: a novel urine LAM detection strategy for TB diagnosis

Huan Huang; Rong Qu; Kang Wu; Jinchuan Xu; Jianhui Li; Shuihua Lu; Guodong Sui; Xiao-Yong Fan

doi:10.3389/fmicb.2023.1236599

Proteinase K-pretreated ConA-based ELISA assay: a novel urine LAM detection strategy for TB diagnosis

Front Microbiol. 2023 Aug 25:14:1236599. doi: 10.3389/fmicb.2023.1236599. eCollection 2023.

Authors

Huan Huang^#^{1

2}, Rong Qu^#^{2

3}, Kang Wu², Jinchuan Xu², Jianhui Li², Shuihua Lu^{2

4}, Guodong Sui^{1

5}, Xiao-Yong Fan^{2

3

5}

Affiliations

¹ Shanghai Key Laboratory of Atmospheric Particle Pollution Prevention (LAP3), Department of Environmental Science and Engineering, Fudan University, Shanghai, China.
² Shanghai Public Health Clinical Center, Fudan University, Shanghai, China.
³ School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, China.
⁴ National Clinical Research Center for Infectious Disease, Shenzhen Third People's Hospital, Shenzhen, China.
⁵ Shanghai Institute of Infectious Disease and Biosecurity, Fudan University, Shanghai, China.

^# Contributed equally.

Abstract

Objectives: Lipoarabinomannan (LAM), an abundant cell wall glycolipid of mycobacteria including Mycobacterium tuberculosis (Mtb), is a promising TB diagnostic marker. The current commercially available urine LAM assays are not sufficiently sensitive, and more novel detection strategies are urgently needed to fill the current diagnostic gap.

Methods: A proteinase K-pretreated Concanavalin A (ConA)-based ELISA assay was developed. Diagnostic performance was assessed by several bacterial strains and clinical urine samples.

Results: The limit of detection (LoD) of the assay against ManLAM was 6 ng/ml. The assay reacted strongly to Mtb H37Rv and M. bovis BCG, intermediately to M. smegmatis mc²155, and weakly to four non-mycobacteria pathogens. This method could distinguish TB patients from healthy controls (HCs) and close contacts (CCs) in 71 urine samples treated with proteinase K, which increases urine LAM antibody reactiveness. In TB⁺HIV⁺ and TB⁺HIV^- patients, the sensitivity was 43.8 and 37.5%, respectively, while the specificity was 100.0%. The areas under ROC curves (AUCs) were 0.74 and 0.82, respectively.

Conclusion: This study implies that ConA can be paired with antibodies to detect LAM. Proteinase K treatment could effectively enhance the sensitivity by restoring the reactiveness of antibodies to LAM.

Keywords: ConA; ELISA; LAM; Mycobacterium tuberculosis; proteinase K; urine.

Grants and funding

This study was supported by grants from the National Key Research and Development Program of China (2022YFC2302900 and 2021YFC2301503), the National Natural and Science Foundation of China (82171815 and 82171739), the Shanghai Hygiene and Health Outstanding Leader Project (2022XD060), and the Shanghai Science and Technology Commission (20Y11903400).