Non-amplification nucleic acid detection with thio-NAD cycling

Yamura, Sou; Kawada, Naoki; Yamakado, Shinnosuke; Kyosei, Yuta; Watabe, Shigeo; Yoshimura, Teizo; Murase, Yoshiro; Mitarai, Satoshi; Ito, Etsuro

doi:10.1016/j.mimet.2022.106647

Cited by 2 publications

(2 citation statements)

References 34 publications

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“…This thio-NAD cycling solution contained 1.0 mM NADH, 2.0 mM thio-NAD, 0.4 mM 17β-methoxy-5β-androstan-3α-ol 3-phosphate, and 10 U/mL 3α-HSD in 100 mM Tris-HCl (pH 9.0). 14,15) In thio-NAD cycling, thio-NADH was measured with a microplate reader at 405 nm.…”

Section: Biological and Pharmaceutical Bulletin Advance Publicationmentioning

confidence: 99%

Removal of Soluble ACE2 in VeroE6 Cells by 17β-Estradiol Reduces SARS-CoV-2 Infectivity

Kyosei,

Yoshimura,

Ito

2023

Biological & Pharmaceutical Bulletin

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Women are less susceptible than men to coronavirus disease 2019 , which might be due to the female steroid hormone 17β-estradiol. We hypothesized that 17β-estradiol removes the soluble portion of angiotensin-converting enzyme 2 (sACE2) to which SARS-CoV-2 binds in host cells and that sACE2 then binds to the virus, thereby reducing the infectivity. In the present study, VeroE6/TMPRSS2 cells were infected with pseudo SARS-CoV-2 viruses and used as our model system. This infectivity was reduced by the application of 17β-estradiol. After applying 17β-estradiol to the VeroE6/TMPRSS2 cells, we used a sandwich enzyme-linked immunosorbent assay to measure the sACE2 concentration in the culture medium. Our findings revealed that 17β-estradiol removes sACE2 from VeroE6/TMPRSS2 cells. Furthermore, the pseudo SARS-CoV-2 viruses were incubated in culture medium with ACE2 collected from 17β-estradiol-treated VeroE6/TMPRSS2 cells, and the viruses were measured with an ultrasensitive ELISA using anti-spike protein antibodies. The amount of spike proteins decreased according to the concentration of 17β-estradiol applied. These results clearly demonstrated that the soluble portion of ACE2, which was removed from 17β-estradiol-treated VeroE6/TMPRSS2 cells, bound to the spike proteins of SARS-CoV-2, thereby reducing COVID-19 infectivity.

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Section: Biological and Pharmaceutical Bulletin Advance Publicationmentioning

confidence: 99%

Removal of Soluble ACE2 in VeroE6 Cells by 17β-Estradiol Reduces SARS-CoV-2 Infectivity

Kyosei,

Yoshimura,

Ito

2023

Biological & Pharmaceutical Bulletin

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“…In the present study, we developed an ultrasensitive ELISA coupled with the thionicotinamide-adenine dinucleotide (thio-NAD) cycling as the signal amplification system, which has already been applied for the detection of several different pathogens, to detect the NS1 protein [ 17 , 18 , 19 ]. The results obtained by our method were compared with the clinical diagnostic values obtained by NAAT using clinical patient specimens.…”

Section: Introductionmentioning

confidence: 99%

Advanced Detection Method for Dengue NS1 Protein Using Ultrasensitive ELISA with Thio-NAD Cycling

Chen,

Chang,

et al. 2023

Viruses

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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.

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Non-amplification nucleic acid detection with thio-NAD cycling

Cited by 2 publications

References 34 publications

Removal of Soluble ACE2 in VeroE6 Cells by 17β-Estradiol Reduces SARS-CoV-2 Infectivity

Removal of Soluble ACE2 in VeroE6 Cells by 17β-Estradiol Reduces SARS-CoV-2 Infectivity

Advanced Detection Method for Dengue NS1 Protein Using Ultrasensitive ELISA with Thio-NAD Cycling

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