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Ultrasensitive ELISA Developed for Diagnosis

  • Iha, Kanako1
  • Inada, Mikio1
  • Kawada, Naoki1
  • Nakaishi, Kazunari2, 3
  • Watabe, Satoshi2, 3
  • Tan, Yong Hong4
  • Shen, Chieh4
  • Ke, Liang-Yin4
  • Yoshimura, Teruki5
  • Ito, Etsuro1, 3, 6
  • 1 Department of Biology, Waseda University, Tokyo 162-8480, Japan
  • 2 R&D Headquarters, TAUNS Laboratories, Inc., Shizuoka 410-2325, Japan
  • 3 Waseda Research Institute for Science and Engineering, Waseda University, Tokyo 162-8480, Japan
  • 4 Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung 80756, Taiwan
  • 5 School of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Hokkaido 061-0293, Japan
  • 6 Graduate Institute of Medicine, School of Medicine, Kaohsiung Medical University, Kaohsiung 80756, Taiwan
Published Article
Publication Date
Jul 18, 2019
DOI: 10.3390/diagnostics9030078
PMID: 31323782
PMCID: PMC6787603
PubMed Central


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.

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