Ultrasensitive ELISA Developed for Diagnosis

Iha, Kanako; Inada, Mikio; Kawada, Naoki; Nakaishi, Kazunari; Watabe, Shigeo; Tan, Yong; Shen, Chieh; Ke, Liang‐Yin; Yoshimura, Teizo; Ito, Etsuro

doi:10.20944/preprints201905.0328.v1

Cited by 10 publications

(9 citation statements)

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“…The experimental protocol was as follows [ 20 , 21 , 22 ]. A 100-μL solution of primary antibody, adjusted to 10 μg/mL in 50 mM Na 2 CO 3 (pH 9.6), was added to microplate wells and incubated at room temperature for 1 h. The microplates were incubated with 1% bovine serum albumin (BSA) in Tris-buffered saline (TBS) at room temperature for 1 h. Spike protein (100 µL, i.e., antigen) was added to each well and incubated at room temperature for 1 h with shaking.…”

Section: Methodsmentioning

confidence: 99%

Proposal of De Novo Antigen Test for COVID-19: Ultrasensitive Detection of Spike Proteins of SARS-CoV-2

et al. 2020

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Polymerase chain reaction (PCR)-based antigen tests are technically difficult, time-consuming, and expensive, and may produce false negative results requiring follow-up confirmation with computed tomography. The global coronavirus disease 2019 (COVID-19) pandemic has increased the demand for accurate, easy-to-use, rapid, and cost-effective antigen tests for clinical application. We propose a de novo antigen test for diagnosing COVID-19 using the combination of sandwich enzyme-linked immunosorbent assay and thio-nicotinamide adenine dinucleotide (thio-NAD) cycling. Our test takes advantage of the spike proteins specific to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The limit of detection of our test was 2.3 × 10−18 moles/assay. If the virus has ~25 spike proteins on its surface, our method should detect on the order of 10−20 moles of virus/assay, corresponding to ~104 copies of the virus RNA/assay. The detection sensitivity approaches that of PCR-based assays because the average virus RNA load used for PCR-based assays is ~105 copies per oro- or naso-pharyngeal swab specimen. To our knowledge, this is the first ultrasensitive antigen test for SARS-CoV-2 spike proteins that can be performed with an easy-to-use microplate reader. Sufficient sensitivity can be achieved within 10 min of thio-NAD cycling. Our antigen test allows for rapid, cost-effective, specific, ultrasensitive, and simultaneous multiple measurements of SARS-CoV-2, and has broad application for the diagnosis for COVID-19.

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Section: Methodsmentioning

confidence: 99%

Proposal of De Novo Antigen Test for COVID-19: Ultrasensitive Detection of Spike Proteins of SARS-CoV-2

et al. 2020

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“…These approaches include, for example, digital PCR and clustered regularly interspaced short palindromic repeats (CRISPR). We ourselves began to develop a new method without the amplification of nucleic acids to overcome the limitations of PCR [ 23 , 24 ]. This non-amplification method for the detection of nucleic acids will be described elsewhere; however, in the present review, the limitations of PCR are discussed in detail.…”

Section: Introductionmentioning

confidence: 99%

Antigen tests for COVID-19

et al. 2021

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PCR diagnosis has been considered as the gold standard for coronavirus disease 2019 (COVID-19) and other many diseases. However, there are many problems in using PCR, such as non-specific (i.e., false-positive) and false-negative amplifications, the limits of a target sample volume, deactivation of the enzymes used, complicated techniques, difficulty in designing probe sequences, and the expense. We, thus, need an alternative to PCR, for example an ultrasensitive antigen test. In the present review, we summarize the following three topics. (1) The problems of PCR are outlined. (2) The antigen tests are surveyed in the literature that was published in 2020, and their pros and cons are discussed for commercially available antigen tests. (3) Our own antigen test on the basis of an ultrasensitive enzyme-linked immunosorbent assay (ELISA) is introduced. Finally, we discuss the possibility that our antigen test by an ultrasensitive ELISA technique will become the gold standard for diagnosis of COVID-19 and other diseases.

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“…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] ]. For example, see the supplementary data for the detailed methods of our culture-free ultrasensitive ELISA.…”

Section: Methodsmentioning

confidence: 99%

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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Ultrasensitive ELISA Developed for Diagnosis

Cited by 10 publications

References 0 publications

Proposal of De Novo Antigen Test for COVID-19: Ultrasensitive Detection of Spike Proteins of SARS-CoV-2

Proposal of De Novo Antigen Test for COVID-19: Ultrasensitive Detection of Spike Proteins of SARS-CoV-2

Antigen tests for COVID-19

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

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