1 Ultrasensitive ELISA Developed for Diagnosis 2

For the diagnosis of disease, the ability to quantitatively detect trace amounts of the causal 20 proteins from bacteria/viruses as biomarkers in patient specimens is highly desirable. Here we 21 introduce a simple, rapid, and colorimetric assay as a de novo, ultrasensitive detection method. This 22 ultrasensitive assay consists of sandwich enzyme-linked immunosorbent assay (ELISA) and 23 thionicotinamide-adenine dinucleotide (thio-NAD) cycling, forming an ultrasensitive ELISA, in 24 which the signal substrate (i.e., thio-NADH) accumulates in a triangular manner, and the 25 accumulated thio-NADH is measured at its maximum absorption wavelength of 400 nm. We have 26 successfully achieved a limit of detection of ca. 10–18 moles/assay for a target protein. As an example 27 of infectious disease detection, HIV-1 p24 could be measured at 0.0065 IU/assay (i.e., 10−18 28 moles/assay), and as a marker for a lifestyle-related disease, adiponectin could be detected at 2.3 × 29 10−19 moles/assay. In particular, despite the long-held belief that the trace amounts of adiponectin in 30 urine can only be detected using a radioisotope, our ultrasensitive ELISA was able to detect urinary 31 adiponectin. This method is highly versatile, because simply changing the antibody enables the 32 detection of various proteins. This assay system requires only the measurement of absorbance, thus 33 it requires equipment that is easily obtained by medical facilities, which facilitates diagnosis in 34 hospitals and clinics. Moreover, we describe an expansion of our ultrasensitive ELISA to a non35 amplification nucleic acid detection method for nucleic acids using hybridization. These de novo 36 methods will enable simple, rapid, and accurate diagnosis. 37