US 11,702,684 B2
Method for measuring protein and nucleic acid with ultrahigh sensitivity
Etsuro Ito, Hokkaido (JP); Toshiaki Miura, Sapporo (JP); Teruki Yoshimura, Sapporo (JP); and Satoshi Watabe, Ito (JP)
Assigned to BioPhenoMA Inc., Tokyo (JP)
Appl. No. 16/89,488
Filed by BioPhenoMA Inc, Tokyo (JP)
PCT Filed Mar. 7, 2017, PCT No. PCT/JP2017/008982
§ 371(c)(1), (2) Date Sep. 28, 2018,
PCT Pub. No. WO2017/169565, PCT Pub. Date Oct. 5, 2017.
Claims priority of application No. 2016-068686 (JP), filed on Mar. 30, 2016.
Prior Publication US 2019/0106727 A1, Apr. 11, 2019
Int. Cl. C12Q 1/32 (2006.01); C12Q 1/00 (2006.01); G01N 21/78 (2006.01); G01N 33/569 (2006.01); G01N 33/58 (2006.01); G01N 33/53 (2006.01); G01N 33/535 (2006.01); C12Q 1/68 (2018.01); G01N 21/25 (2006.01)
CPC C12Q 1/32 (2013.01) [C12Q 1/008 (2013.01); C12Q 1/68 (2013.01); G01N 21/78 (2013.01); G01N 33/53 (2013.01); G01N 33/535 (2013.01); G01N 33/5695 (2013.01); G01N 33/581 (2013.01); G01N 21/25 (2013.01); G01N 2333/35 (2013.01); G01N 2333/90209 (2013.01)] 3 Claims
 
1. An enzymatic measurement method using an antibody-enzyme complex, the method being implemented in which an enzyme reaction product of the antibody-enzyme complex is assayed by generating thio-NADH and/or thio-NADPH by an enzymatic cycling reaction using NADH and/or NADPH, thio-NAD and/or thio-NADP and a dehydrogenase (DH), followed by:
i) measuring the generated thio-NADH and/or thio-NADPH, or
ii) measuring a color change caused by the generated thio-NADH and/or thio-NADPH, wherein
an enzyme reaction system that selectively reduces the NAD and/or NADP generated from NADH and/or NADPH by the enzymatic cycling reaction is included in the enzymatic cycling reaction system,
wherein the enzyme reaction system that selectively reduces the NAD and/or NADP uses a substrate that does not become a substrate for the enzyme of the antibody-enzyme complex and a substrate for the enzyme of the enzymatic cycling reaction, and an enzyme that does not react with the substrate for the enzyme of the antibody-enzyme complex and the substrate of the enzyme of the enzymatic cycling reaction; and
wherein the combination of enzyme and substrate used in the enzyme reaction system that selectively reduces NAD and/or NADP is selected from the group consisting of:
(i) glutamate dehydrogenase and glutamic acid;
(ii) leucine dehydrogenase and leucine;
(iii) alanine dehydrogenase and alanine;
(iv) phenylalanine dehydrogenase and phenylalanine;
(v) serine dehydrogenase and serine;
(vi) valine dehydrogenase and valine;
(vii) tryptophan dehydrogenase and tryptophan; and
(viii) aspartate dehydrogenase and aspartic acid.