Immunochromatographic Detection of MPB64 Secreted from Active BCG by Heating: Toward Same-Day Diagnosis of Tuberculosis

Nakaishi, Kazunari; Watabe, Shigeo; Kitagawa, Toshiyuki; Ito, Etsuro

doi:10.2144/btn-2019-0020

Cited by 7 publications

(10 citation statements)

References 13 publications

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“…The precipitate obtained was washed again and suspended in 200 μL of heat treatment buffer comprising phosphate buffer (0.033 M, pH 6.8) and 2% Tween 20. This solution was heated in an aluminum block heater at 46 °C for 1 h, resulting in the secretion of MPT64 from live bacilli [18] .…”

Section: Methodsmentioning

confidence: 99%

“…In the culture-free ultrasensitive ELISA tests, we detected a specific protein for M. tuberculosis , MPT64 [19] , which is secreted from only live M. tuberculosis in heated sputum specimens [18] . An ultrasensitive ELISA coupled with thio-NAD cycling was originally developed by Watabe and Ito [ 16 , [20] , [21] , [22] , [23] , [24] , [25] , [26] ].…”

Section: Methodsmentioning

confidence: 99%

“…Thus, MPT64 is an important component for live M. tuberculosis . The feasibility of detecting MPT64 has already been confirmed using immunochromatography [18] , which is less sensitive than the ultrasensitive ELISA.…”

Section: Introductionmentioning

confidence: 99%

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A novel, rapid (within hours) culture-free diagnostic method for detecting live Mycobacterium tuberculosis with high sensitivity

Wang

Takeuchi²,

Jain

et al. 2020

EBioMedicine

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Background Nucleic acid amplification tests (NAATs) are widely used to diagnose tuberculosis (TB), but cannot discriminate live bacilli from dead bacilli. Live bacilli can be isolated by culture methods, but this is time-consuming. We developed a de novo TB diagnostic method that detects only live bacilli with high sensitivity within hours. Methods A prospective study was performed in Taiwan from 2017 to 2018. Sputum was collected consecutively from 1102 patients with suspected TB infection. The sputum was pretreated and heated at 46°C for 1 h to induce the secretion of MPT64 protein from live Mycobacterium tuberculosis . MPT64 was detected with our ultrasensitive enzyme-linked immunosorbent assay (ELISA) coupled with thionicotinamide-adenine dinucleotide (thio-NAD) cycling. We compared our data with those obtained using a culture test (MGIT), a smear test (Kinyoun staining), and a NAAT (Xpert). Findings The limit of detection for MPT64 in our culture-free ultrasensitive ELISA was 2.0 × 10 −19 moles/assay. When the criterion for a positive response was set as an absorbance value ≥17 mAbs, this value corresponded to ca . 330 CFU/mL in the culture method – almost the same high-detection sensitivity as the culture method. To confirm that MPT64 is secreted from only live bacilli, M. bovis BCG was killed using 8 μg/mL rifampicin and then heated. Following this procedure, our method detected no MPT64. Our rapid ultra-sensitive ELISA-based method required only 5 h to complete. Comparing the results of our method with those of culture tests for 944 specimens revealed a sensitivity of 86.9% (93/107, 95% CI: 79.0–92.7%) and a specificity of 92.0% (770/837, 95% CI: 89.9–93.7%). The performance data were not significantly different (McNemar's test, P = 0.887) from those of the Xpert tests. In addition, at a ≥1+ titer in the smear test, the positive predictive value of our culture-free ultrasensitive ELISA tests was in a good agreement with that of the culture tests. Furthermore, our culture-free ultrasensitive ELISA test had better validity for drug effectiveness examination than Xpert tests because our test detected only live bacilli. Interpretation Our culture-free ultrasensitive ELISA method detects only live TB bacilli with high sensitivity within hours, allowing for rapid diagnosis of TB and monitoring drug efficacy. Funding Matching Planner Program from JST (VP29117939087), the A-STEP Program from JST (AS3015096U), Waseda University grants for Specific Research Projects (2017A-015 and 2019C-123), the Precise Measurement Technology Promotion Foundation to E.I.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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A novel, rapid (within hours) culture-free diagnostic method for detecting live Mycobacterium tuberculosis with high sensitivity

Wang

Takeuchi²,

Jain

et al. 2020

EBioMedicine

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“…We are now applying this ultrasensitive ELISA to detect specific proteins from active bacilli [2] and pathogenic viruses as well as proteins that can be used as cancer markers.…”

Section: Detection Of Trace Amount Of Proteinsmentioning

confidence: 99%

“…Examining patient specimens for the presence of proteins from pathogenic bacteria and viruses or as biomarkers enables rapid and accurate diagnosis, resulting in early treatment. Immunochromatography and enzyme-linked immunosorbent assay (ELISA) are commonly used methods for this purpose [1,2]. The sensitivity of these assays, however, requires further improvement.…”

Section: Introductionmentioning

confidence: 99%

Ultrasensitive ELISA Developed for Diagnosis

et al. 2019

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For the diagnosis of disease, the ability to quantitatively detect trace amounts of the causal proteins from bacteria/viruses as biomarkers in patient specimens is highly desirable. Here we introduce a simple, rapid, and colorimetric assay as a de novo, ultrasensitive detection method. This ultrasensitive assay consists of a sandwich enzyme-linked immunosorbent assay (ELISA) and thionicotinamide-adenine dinucleotide (thio-NAD) cycling, forming an ultrasensitive ELISA, in which the signal substrate (i.e., thio-NADH) accumulates in a triangular manner, and the accumulated thio-NADH is measured at its maximum absorption wavelength of 405 nm. We have successfully achieved a limit of detection of ca. 10−18 moles/assay for a target protein. As an example of infectious disease detection, HIV-1 p24 could be measured at 0.0065 IU/assay (i.e., 10−18 moles/assay), and as a marker for a lifestyle-related disease, adiponectin could be detected at 2.3 × 10−19 moles/assay. In particular, despite the long-held belief that the trace amounts of adiponectin in urine can only be detected using a radioisotope, our ultrasensitive ELISA was able to detect urinary adiponectin. This method is highly versatile because simply changing the antibody enables the detection of various proteins. This assay system requires only the measurement of absorbance, thus it requires equipment that is easily obtained by medical facilities, which facilitates diagnosis in hospitals and clinics. Moreover, we describe an expansion of our ultrasensitive ELISA to a non-amplification nucleic acid detection method for nucleic acids using hybridization. These de novo methods will enable simple, rapid, and accurate diagnosis.

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