Immunoreactive Insulin in Diabetes Mellitus Patient Sera Detected by Ultrasensitive ELISA with Thio-NAD Cycling

Ito, Etsuro; Kaneda, Mugiho; Kodama, Hiromi; Morikawa, Mika; Tai, Momoko; Aoki, Kana; Watabe, Shigeo; Nakaishi, Kazunari; Hashida, Seiichi; Tada, Satoshi; Kuroda, Noriyuki; Imachi, Hitomi; Murao, Koji; Yamashita, Masakane; Yoshimura, Teizo; Miura, Toshiaki

doi:10.2144/000114355

Cited by 18 publications

(16 citation statements)

References 42 publications

(26 reference statements)

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“…For insulin, a recent study using MALDI-TOF-MS reported that 10 6 insulin molecules could be detected in this manner, that is, 1.7×10 −18 moles [ 38 ]. Although the comparison between our ultrasensitive ELISA [ 21 , 23 ] and the MS is not straightforward, in general our method can be considered easier, because we can detect trace amounts of proteins simply by applying thio-NAD cycling regents to the usual ELISA system.…”

Section: Discussionmentioning

confidence: 99%

Subattomole detection of adiponectin in urine by ultrasensitive ELISA coupled with thio-NAD cycling

et al. 2015

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Adiponectin is a hormone secreted from adipocytes, and it demonstrates antidiabetic, anti-atherosclerotic, antiobesity and anti-inflammatory effects. However, the patterns of change in urinary adiponectin levels in various diseases remain unknown, because only trace amounts of the hormone are present in urine. In the present study, we applied an ultrasensitive ELISA coupled with thio-NAD cycling to measure urinary adiponectin levels. Spikeand-recovery tests using urine confirmed the reliability of our ultrasensitive ELISA. The limit of detection for adiponectin in urine was 2.3×10−19 moles/assay (1.4 pg/mL). The urinary adiponectin concentration ranged between 0.04 and 5.82 ng/mL in healthy subjects. The pilot study showed that the urinary adiponectin levels, which were corrected by the creatinine concentration, were 0.73±0.50 (ng/mg creatinine, N=6) for healthy subjects, versus 12.02±3.85 (ng/mg creatinine, N=3) for patients with diabetes mellitus (DM). That is, the urinary adiponectin levels were higher (P<0.05) in DM patients than in healthy subjects. Further, these urinary adiponectin levels tended to increase with the progression of DM accompanied with nephropathy. Our method is thus expected to provide a simple, rapid and reasonably priced test for noninvasive monitoring of the progression of DM without the requirement of special tools.

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Section: Discussionmentioning

confidence: 99%

Subattomole detection of adiponectin in urine by ultrasensitive ELISA coupled with thio-NAD cycling

et al. 2015

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“…03359123001; Roche, Mannheim, Germany) by using an Alkaline Phosphate Labeling Kit-SH (Dojindo Molecular Technologies, Kumamoto, Japan). As the primary substrate, we used 17β-methoxy-5β-androstan-3α-ol 3-phosphate [16,17]. For the thio-NAD cycling, 3α-hydroxysteroid dehydrogenase (3α-HSD, EC.…”

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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“…An ultrasensitive ELISA coupled with thionicotinamide-adenine dinucleotide (thio-NAD) cycling was originally developed by Watabe and Ito 11 23–26. Shortly, a sandwich method using a primary and a secondary antibody for antigens is employed in an ELISA.…”

Section: Methodsmentioning

confidence: 99%

Urinary adiponectin as a new diagnostic index for chronic kidney disease due to diabetic nephropathy

Yamakado

Cho

Inada

et al. 2019

BMJ Open Diab Res Care

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ObjectiveThe chronic kidney disease (CKD) is widely diagnosed on the basis of albuminuria and the glomerular filtration rate. A more precise diagnosis of CKD, however, requires the assessment of other factors. Urinary adiponectin recently attracted attention for CKD assessment, but evaluation is difficult due to the very low concentration of urinary adiponectin in normal subjects.Research design and methodsWe developed an ultrasensitive ELISA coupled with thionicotinamide-adenine dinucleotide cycling to detect trace amounts of proteins, which allows us to measure urinary adiponectin at the subattomole level. We measured urinary adiponectin levels in 59 patients with diabetes mellitus (DM) and 24 subjects without DM (normal) to test our hypothesis that urinary adiponectin levels increase with progression of CKD due to DM.ResultsThe urinary adiponectin levels were 14.88±3.16 (ng/mg creatinine, mean±SEM) for patients with DM, and 3.06±0.33 (ng/mg creatinine) for normal subjects. The threshold between them was 4.0 ng/mg creatinine. The urinary adiponectin levels increased with an increase in the CKD risk. Furthermore, urinary adiponectin mainly formed a medium-molecular weight multimer (a hexamer) in patients with DM, whereas it formed only a low-molecular weight multimer (a trimer) in normal subjects. That is, the increase in urinary adiponectin in patients with DM led to the emergence of a medium-molecular weight form in urine.ConclusionsOur new assay showed that urinary adiponectin could be a new diagnostic index for CKD. This assay is a non-invasive test using only urine, thus reducing the patient burden.

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Immunoreactive Insulin in Diabetes Mellitus Patient Sera Detected by Ultrasensitive ELISA with Thio-NAD Cycling

Cited by 18 publications

References 42 publications

Subattomole detection of adiponectin in urine by ultrasensitive ELISA coupled with thio-NAD cycling

Subattomole detection of adiponectin in urine by ultrasensitive ELISA coupled with thio-NAD cycling

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

Urinary adiponectin as a new diagnostic index for chronic kidney disease due to diabetic nephropathy

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