Development of Gold-Nanoparticle-Based Lateral Flow Immunoassays for Rapid Detection of TB ESAT-6 and CFP-10

Abstract

The current study reports on the development of a rapid and cost-effective TB-antigen diagnostic test for the detection of Mycobacterium biomarkers from non-sputum-based samples. Two gold nanoparticle (AuNP)-based rapid diagnostic tests (RDTs) in the form of lateral flow immunoassays (LFIAs) were developed for detection of immunodominant TB antigens, the 6 kDa early secreted antigen target EsxA (ESAT-6) and the 10 kDa culture filtrate protein EsxB (CFP-10). AuNPs were synthesized using the Turkevich method and characterized by UV-vis spectrophotometer and transmission electron microscope (TEM). The AuNP–detection probe conjugation conditions were determined by comparing the stability of 14 nm AuNPs at different pH conditions, following salt challenge. Thereafter, ESAT-6 and CFP-10 antibodies were conjugated to the AuNPs and used for the colorimetric detection of TB antigens. Selection of the best detection and capture antibody pairs was determined by Dot spotting. The limits of detection (LODs) for the LFIAs were evaluated by dry testing. TEM results showed that the 14 nm AuNPs were mostly spherical and well dispersed. The ESAT-6 LFIA prototype had an LOD of 0.0625 ng/mL versus the CFP-10 with an LOD of 7.69 ng/mL. Compared to other studies in the literature, the LOD was either similar or lower, outperforming them. Moreover, in some of the previous studies, an enrichment/extraction step was required to improve on the LOD. In this study, the LFIAs produced results within 15 min and could be suitable for use at PoCs either in clinics, mobile clinics, hospitals or at home by the end user. However, further studies need to be conducted to validate their use in clinical samples.

Development of a Multiplex HIV/TB Diagnostic Assay Based on the Microarray Technology

“…The above being said, similar ELISA-based technologies, such as the electrochemical impedimetric ESAT6 immunosensor [ 52 ], gold-nanoparticle-based lateral flow TB immunoassay [ 53 ] and HIV p24 detection using combined immunoprecipitation and digital ELISA [ 54 ], have generally reported limits of detection less than ours of 0.000954 µg/mL ( Table A1 ); however, our assay still has the main advantage of being able to detect HIV and TB at the same time. In addition, the combination of the selected three TB antigens is likely to enable our assay to detect active TB [ 32 , 33 , 34 , 35 ].…”

Establishment of an indirect ELISA for Mycobacterium tuberculosis MTB39A protein antibody

“…To date, the diagnostic markers for MTB include the Ag85 complex (Barbier et al 2023 ), ESAT-6 (Dewi et al 2019 ; Zhang et al 2022 ), CFP10 (Seele et al 2023 ), MPT64 (Wang et al 2020 ), and PPE17. Among them, the vaccine MVA85A with Ag85A as the antigen was found to be ineffective in enhancing the protective effect of BCG-immunized infants against MTB infection (Tameris et al 2013 ).…”