Zeptomole Detection of an Enzyme by a Simple Colorimetric Method

Abstract

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.

Ultrasensitive Detection of SARS-CoV-2 Spike Proteins Using the Thio-NAD Cycling Reaction: A Preliminary Study before Clinical Trials

“…Therefore, the value of 10 −17 moles/assay might be a challenging detection sensitivity. Our own ELISA attempts to detect proteins at zeptomolar detection limits, i.e., 10 −21 moles/assay [26]. We believe that our system can be called an "ultrasensitive" ELISA.…”

“…Although similar experiments for the SARS-CoV-2 recombinant S1 protein were achieved using an ultrasensitive thio-NAD cycling ELISA [17], in the present study we attempted to improve the detection sensitivity of the recombinant protein by applying our method to viruses. For this purpose, we used another 3α-HSD from Asahi Kasei Pharma [26] and a new combination of antibodies for the anti-S1 protein from Hakarel, as described in Section 2.1. Our ultrasensitive thio-NAD cycling ELISA was based on a sandwich ELISA and developed by Watabe and Ito [23,27].…”

Modified ELISA for Ultrasensitive Diagnosis

“…Thus, 10 −13 moles/L is equivalent to 10 −17 moles/assay, which might be challenging to achieve. Our own ELISA method, however, can detect proteins at zeptomolar detection limits, i.e., 10 −21 moles/assay [ 32 ], and thus, we believe that our system can be classified as an ‘ultrasensitive’ ELISA.…”

“…In 2014, Watabe and Ito attempted to realize an ultrasensitive ELISA by combining a sandwich ELISA and thio-NAD cycling to achieve ultrasensitive, quantitative detection of proteins [ 66 ]. According to the interpretation of the term ‘ultrasensitive’ in Section 2 , we called this system an ultrasensitive ELISA because ALP bound to the secondary antibody in a sandwich ELISA can be detected at the zeptomole (i.e., 10 −21 moles) level [ 32 ]. A sandwich ELISA is used for the quantitative detection of a trace amount of proteins using two different antibodies specific to the target protein.…”

Improved Detection Sensitivity of an Antigen Test for SARS-CoV-2 Nucleocapsid Proteins with Thio-NAD Cycling

“…ELISA with thio-NAD cycling An ELISA with thio-NAD cycling was performed according to the procedures reported previously with slight modification. 17,30) Briefly, a 100-μL solution of primary antibody, which was adjusted to 1 μg/mL in 50 mM Na 2 CO 3 (pH 9.6), was added to each well of the microplates and incubated at room temperature for 1 h. The microplates were incubated with 1% bovine serum albumin (BSA) in Tris-buffered saline (TBS) at room temperature for 1 h. Nucleocapsid protein (100 µL) was added to each well and incubated at room temperature for 1 h. The antigen samples were diluted with TBS containing 0.1% BSA. A 100-μL solution of secondary antibody conjugated with ALP and adjusted to 100 ng/mL in TBS including 0.05% Tween 20 and 0.1% BSA was then added to the wells and incubated at room temperature for 1 h. A 100-μL thio-NAD cycling solution was added to each well.…”