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Review
. 2021 Nov 7;10(21):5197.
doi: 10.3390/jcm10215197.

Modified ELISA for Ultrasensitive Diagnosis

Naoko Tsurusawa  1 , Jyunhao Chang  1 , Mayuri Namba  1 , Daiki Makioka  1 , Sou Yamura  1 , Kanako Iha  1 , Yuta Kyosei  1 , Satoshi Watabe  2 , Teruki Yoshimura  3 , Etsuro Ito  1   2   4
Affiliations
Review

Modified ELISA for Ultrasensitive Diagnosis

Naoko Tsurusawa et al. J Clin Med. .

Abstract

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.

Keywords: diagnosis; infectious disease; lifestyle-related disease; thio-NAD cycling; ultrasensitive ELISA.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Scheme of a digital ELISA. The left panel shows that antibody-conjugated magnetic beads are first used to capture single molecules of the target protein (i.e., antigen), and the protein-bead complexes are labeled with a second antibody-conjugated enzyme. The right panel shows a case of low antigen concentration and a case of high antigen concentration. In the tube, first, antibody-conjugated magnetic beads, an antigen, and a second antibody-conjugated enzyme are sequentially mixed. Then, the bead complexes are assessed using a precision-fabricated, femtoliter-volume microwell array capable of capturing one bead per well. After adding the substrate as a fluorophore, fluorescence images of a small section of the well array are obtained. The concentration of protein in bulk solution is correlated with the percentage of beads that carry a protein molecule [33].
Figure 2
Figure 2
Scheme of ELISA using gold nanoparticles (AuNPs). This ELISA has unique structural, electronic, magnetic, optical, and catalytic properties. The use of AuNPs allows the attachment of a multiple enzyme system that can generate an amplified optical signal while keeping low background signals [40]. TMB is tetramethylbenzidine.
Figure 3
Figure 3
Scheme of an ultrasensitive ELISA with thio-NAD cycling. Both a conventional sandwich ELISA and a thio-NAD cycling assay produce signals as a linear function against time. When both assays are combined, the signals can be amplified in a quadratic-function-like response (e.g., triangle number) over time. This system comprises ALP linked with a secondary antibody against the target protein, an androsterone derivative as the first substrate, 3α-HSD as the enzyme for a thio-NAD cycling, and their coenzymes, such as NADH and thio-NAD. Thio-NADH accumulation is measured as absorbance at 405 nm [66].
See this image and copyright information in PMC

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