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.

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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Objective The 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 methods We 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. Results The 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. Conclusions Our 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.

We aimed to study Mycobacterium tuberculosis (MTB)-secreted MPT64 as a surrogate of bacterial viability for the diagnosis of active pulmonary tuberculosis (PTB) and for follow-up treatment. In our proof-of-concept prospective study, 50 PTB patients in Tokyo metropolitan region between 2017 and 2018 were included consecutively and 30 healthy individuals were included. Each PTB patient submitted sputum on Days 0, 14, and 28 for diagnosis and follow-up, and each healthy individual submitted one sputum sample. We performed smear microscopy, Xpert MTB/RIF, MGIT and solid culture, and MPT64 detection on the sputum samples. Ultrasensitive ELISA (usELISA) was used to detect MPT64. The receiver operating characteristic analyses for diagnosis and follow-up revealed the optimal cutoff value of MPT64 absorbance for detecting culture positivity at multiple intervals. The sensitivity of MPT64 for diagnosing PTB was 88.0% [95% CI: 75.7-95.5] and the specificity was 96.7% [95% CI: 82.8-99.9]. The specificity of MPT64 for predicting negative culture results on Day 14 was 89.5% [95% CI: 66.9-98.7]. The sensitivity of MPT64 for predicting positive culture results on Day 28 was 81.0% [95% CI: 58.1-94.6]. This study revealed that MPT64 is useful for diagnosing active PTB in patients and predicting treatment efficacy at follow-up.

To reduce the window period between HIV-1 infection and the ability to diagnose it, a fourth-generation immunoassay including the detection of HIV-1 p24 antigen has been developed. However, because the commercially available systems for this assay use special, high-cost instruments to measure, for example, chemiluminescence, it is performed only by diagnostics companies and hub hospitals. To overcome this limitation, we applied an ultrasensitive ELISA coupled with a thio-NAD cycling, which is based on a usual enzyme immunoassay without special instruments, to detect HIV-1 p24. The p24 detection limit by our ultrasensitive ELISA was 0.0065 IU/assay (i.e., ca. 10^-18 moles/assay). Because HIV-1 p24 antigen is thought to be present in the virion in much greater numbers than viral RNA copies, the value of 10^-18 moles of the p24/assay corresponds to ca. 10³ copies of the HIV-1 RNA/assay. That is, our ultrasensitive ELISA is chasing the detection limit (10² copies/assay) obtained by PCR-based nucleic acid testing (NAT) with a margin of only one different order. Further, the detection limit by our ultrasensitive ELISA is less than that mandated for a CE-marked HIV antigen/antibody assay. An additional recovery test using blood supported the reliability of our ultrasensitive ELISA.