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Keywords = thio-NAD cycling

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8 pages, 817 KiB  
Communication
Advanced Detection Method for Dengue NS1 Protein Using Ultrasensitive ELISA with Thio-NAD Cycling
by Po-Kai Chen, Jyun-Hao Chang, Liang-Yin Ke, Jun-Kai Kao, Chang-Hua Chen, Rei-Cheng Yang, Teruki Yoshimura, Etsuro Ito and Jih-Jin Tsai
Viruses 2023, 15(9), 1894; https://doi.org/10.3390/v15091894 - 8 Sep 2023
Cited by 6 | Viewed by 3455
Abstract
Dengue fever, a mosquito-borne disease in tropical and subtropical climates caused by the dengue virus (DENV), has become a major social and economic burden in recent years. However, current primary detection methods are inadequate for early diagnosis of DENV because they are either [...] Read more.
Dengue fever, a mosquito-borne disease in tropical and subtropical climates caused by the dengue virus (DENV), has become a major social and economic burden in recent years. However, current primary detection methods are inadequate for early diagnosis of DENV because they are either time-consuming, expensive, or require training. Non-structural protein 1 (NS1) is secreted during DENV infection and is thus considered a suitable biomarker for the development of an early detection method. In the present study, we developed a detection method for the NS1 protein based on a previously reported thio-NAD cycling ELISA (i.e., ultrasensitive ELISA) and successfully achieved a LOD of 1.152 pg/mL. The clinical diagnosis potential of the detection system was also evaluated by using 85 patient specimens, inclusive of 60 DENV-positive and 25 DENV-negative specimens confirmed by the NAAT method. The results revealed 98.3% (59/60) sensitivity and 100% (25/25) specificity, which was in almost perfect agreement with the NAAT data with a kappa coefficient of 0.972. The present study demonstrates the diagnostic potential of using an ultrasensitive ELISA as a low-cost, easy-to-use method for the detection of DENV compared with NAAT and could be of great benefit in low-income countries. Full article
(This article belongs to the Section Human Virology and Viral Diseases)
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13 pages, 2314 KiB  
Article
Ultrasensitive Detection of GRP78 in Exosomes and Observation of Migration and Proliferation of Cancer Cells by Application of GRP78-Containing Exosomes
by Naoko Tsurusawa, Kanako Iha, Akane Sato, Hsin-Yi Tsai, Hikaru Sonoda, Satoshi Watabe, Teruki Yoshimura, Deng-Chyang Wu, Ming-Wei Lin and Etsuro Ito
Cancers 2022, 14(16), 3887; https://doi.org/10.3390/cancers14163887 - 11 Aug 2022
Cited by 13 | Viewed by 3065
Abstract
Cancer cells communicate with each other via exosomes in the tumor microenvironment. However, measuring trace amounts of proteins in exosomes is difficult, and thus the cancer stemness-promoting mechanisms of exosomal proteins have not been elucidated. In the present study, we attempted to quantify [...] Read more.
Cancer cells communicate with each other via exosomes in the tumor microenvironment. However, measuring trace amounts of proteins in exosomes is difficult, and thus the cancer stemness-promoting mechanisms of exosomal proteins have not been elucidated. In the present study, we attempted to quantify trace amounts of 78-kDa glucose-regulated protein (GRP78), which is involved in cancer progression, in exosomes released from cultured gastric cancer cells using an ultrasensitive ELISA combined with thio-NAD cycling. We also evaluated the cancer stemness-promoting effects by the application of high-GRP78-containing exosomes to cultured gastric cancer cells. The ultrasensitive ELISA enabled the detection of GRP78 at a limit of detection of 0.16 pg/mL. The stemness of cancer cultured cells incubated with high-GRP78-containing exosomes obtained from GRP78-overexpressed cells was increased on the basis of both an MTT assay and a wound healing assay. Our results demonstrated that the ultrasensitive ELISA has strong potential to measure trace amounts of proteins in exosomes. Further, exosomes with a high concentration of GRP78 promote the cancer stemness of surrounding cells. The technique for quantifying proteins in exosomes described here will advance our understanding of cancer stemness progression via exosomes. Full article
(This article belongs to the Section Methods and Technologies Development)
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13 pages, 1247 KiB  
Review
Modified ELISA for Ultrasensitive Diagnosis
by Naoko Tsurusawa, Jyunhao Chang, Mayuri Namba, Daiki Makioka, Sou Yamura, Kanako Iha, Yuta Kyosei, Satoshi Watabe, Teruki Yoshimura and Etsuro Ito
J. Clin. Med. 2021, 10(21), 5197; https://doi.org/10.3390/jcm10215197 - 7 Nov 2021
Cited by 35 | Viewed by 9274
Abstract
An enzyme-linked immunosorbent assay (ELISA) can be used for quantitative measurement of proteins, and improving the detection sensitivity to the ultrasensitive level would facilitate the diagnosis of various diseases. In the present review article, we first define the term ‘ultrasensitive’. We follow this [...] Read more.
An enzyme-linked immunosorbent assay (ELISA) can be used for quantitative measurement of proteins, and improving the detection sensitivity to the ultrasensitive level would facilitate the diagnosis of various diseases. In the present review article, we first define the term ‘ultrasensitive’. We follow this with a survey and discussion of the current literature regarding modified ELISA methods with ultrasensitive detection and their application for diagnosis. Finally, we introduce our own newly devised system for ultrasensitive ELISA combined with thionicotinamide adenine dinucleotide cycling and its application for the diagnosis of infectious diseases and lifestyle-related diseases. The aim of the present article is to expand the application of ultrasensitive ELISAs in the medical and biological fields. Full article
(This article belongs to the Special Issue Role of Enzyme-Linked Immunosorbent Assay (ELISA))
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9 pages, 921 KiB  
Communication
Ultrasensitive Detection of SARS-CoV-2 Spike Proteins Using the Thio-NAD Cycling Reaction: A Preliminary Study before Clinical Trials
by Yuta Kyosei, Mayuri Namba, Daiki Makioka, Ayumi Kokubun, Satoshi Watabe, Teruki Yoshimura, Tadahiro Sasaki, Tatsuo Shioda and Etsuro Ito
Microorganisms 2021, 9(11), 2214; https://doi.org/10.3390/microorganisms9112214 - 25 Oct 2021
Cited by 11 | Viewed by 3575
Abstract
To help control the global pandemic of coronavirus disease 2019 (COVID-19), we developed a diagnostic method targeting the spike protein of the virus that causes the infection, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We applied an ultrasensitive method by combining a sandwich [...] Read more.
To help control the global pandemic of coronavirus disease 2019 (COVID-19), we developed a diagnostic method targeting the spike protein of the virus that causes the infection, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We applied an ultrasensitive method by combining a sandwich enzyme-linked immunosorbent assay (ELISA) and the thio-nicotinamide adenine dinucleotide (thio-NAD) cycling reaction to quantify spike S1 proteins. The limit of detection (LOD) was 2.62 × 10−19 moles/assay for recombinant S1 proteins and 2.6 × 106 RNA copies/assay for ultraviolet B-inactivated viruses. We have already shown that the ultrasensitive ELISA for nucleocapsid proteins can detect ultraviolet B-inactivated viruses at the 104 RNA copies/assay level, whereas the nucleocapsid proteins of SARS-CoV-2 are difficult to distinguish from those in conventional coronaviruses and SARS-CoV. Thus, an antigen test for only the nucleocapsid proteins is insufficient for virus specificity. Therefore, the use of a combination of tests against both spike and nucleocapsid proteins is recommended to increase both the detection sensitivity and testing accuracy of the COVID-19 antigen test. Taken together, our present study, in which we incorporate S1 detection by combining the ultrasensitive ELISA for nucleocapsid proteins, offers an ultrasensitive, antigen-specific test for COVID-19. Full article
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8 pages, 1053 KiB  
Communication
Proposal of De Novo Antigen Test for COVID-19: Ultrasensitive Detection of Spike Proteins of SARS-CoV-2
by Yuta Kyosei, Mayuri Namba, Sou Yamura, Rikiya Takeuchi, Noriko Aoki, Kazunari Nakaishi, Satoshi Watabe and Etsuro Ito
Diagnostics 2020, 10(8), 594; https://doi.org/10.3390/diagnostics10080594 - 14 Aug 2020
Cited by 42 | Viewed by 11564
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Section Diagnostic Microbiology and Infectious Disease)
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9 pages, 779 KiB  
Review
Ultrasensitive ELISA Developed for Diagnosis
by Kanako Iha, Mikio Inada, Naoki Kawada, Kazunari Nakaishi, Satoshi Watabe, Yong Hong Tan, Chieh Shen, Liang-Yin Ke, Teruki Yoshimura and Etsuro Ito
Diagnostics 2019, 9(3), 78; https://doi.org/10.3390/diagnostics9030078 - 18 Jul 2019
Cited by 48 | Viewed by 8979
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Section Point-of-Care Diagnostics and Devices)
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