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.

A new flavanone, shisoflavanone A (1), and several flavonoids were purified from the ethyl acetate-soluble fraction of green perilla leaves (Perilla frutescens Britton var. crispa form viridis), and their structures were identified. Shisoflavanone A was elucidated as 8-hydroxy-6,7-dimethoxyflavanone based on its spectral data. Other constituents of the ethyl acetate-soluble fraction, i.e. 5,8-dihydroxy-7-methoxyflavanone (2), negletein (5,6-dihydroxy-7-methoxyflavone) (3), luteolin (4), apigenin (5), esculetin (6), and protocatechuic acid (7), were identified. This is the first time that constituents 2, 3, and 6 have been found in green perilla. Shisoflavanone A and the other constituents (except 7) significantly inhibited nitric oxide production in interleukin 1β-stimulated rat hepatocytes, which have been used to monitor the anti-inflammatory effects of herbal constituents. The present findings suggest that these constituents, including shisoflavanone A, may be involved in the anti-inflammatory effects of green perilla leaves.

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.

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.

An enzyme immunoassay, in which an enzyme (e.g., alkaline phosphatase, ALP) is conjugated with an antibody, is a precise and simple protein detection method. Precise measurements of enzymes at low concentrations allow for ultrasensitive protein detection. The application of a phosphorylated substrate to ALP, followed by using a dephosphorylated substrate in thionicotinamide-adenine dinucleotide cycling, provides a simple and precise quantification of ALP. We describe a protocol for detecting ALP at the zeptomole level using a simple colorimetric method.