Detection reagents and kit for identifying oxidized state and glycated state of low-density lipoproteins

Patent metadata

Field	Value
Publication number	US-11725002-B2
Application number	US-201816609028-A
Country	US
Kind code	B2
Filing date	Apr 27, 2018
Priority date	Apr 28, 2017
Publication date	Aug 15, 2023
Grant date	Aug 15, 2023

Abstract

A detection reagent for comprehensively detecting an oxidized state and a glycated state of a low-density lipoprotein. While an oxidized low-density lipoprotein and a glycated low-density lipoprotein are detected with a fluolophore-labelled antibody, a lipid radical is detected with a fluorescent nitroxide 2,2,6-trimethyl-4-(4-nitrobenzo[1,2,5]oxadiazol-7-ylamino)-6-pentylpiperadine-1-oxyl (NBD-Pen).

First claim

The invention claimed is:

1. A detection reagent containing
a primary detection antibody recognizing an oxidized low-density lipoprotein,
a primary detection antibody recognizing a glycated low-density lipoprotein,
a secondary antibody recognizing the primary detection antibody recognizing the oxidized low-density lipoprotein,
a secondary antibody recognizing the primary detection antibody recognizing the glycated low-density lipoprotein,
wherein the secondary antibody recognizing the primary detection antibody recognizing the oxidized low-density lipoprotein and the secondary antibody recognizing the primary detection antibody recognizing glycated low-density lipoprotein are fluorescently labelled with fluorophores, and
a fluorescent nitroxide represented by the chemical structure (1):

2. The detection reagent according to claim 1 , wherein the primary detection antibody recognizing the oxidized low-density lipoprotein is a mixture containing at least an antibody recognizing malondialdehyde lysine, an antibody recognizing 4-hydroxy-2-nonenal lysine, and an antibody recognizing acrolein lysine.

3. The detection reagent according to claim 1 , wherein the primary detection antibody recognizing the glycated low-density lipoprotein is a mixture containing at least an antibody recognizing pentosidin, an antibody recognizing crossline, an antibody recognizing (Nε-carboxymethyl)lysine, an antibody recognizing (Nε-carboxyethyl)lysine, and an antibody recognizing pyrraline.

4. A kit for identifying an oxidized state and a glycated state of a low-density lipoprotein, which includes
a microplate on which an antibody recognizing a low-density lipoprotein is immobilized, and
the detection reagent according to claim 1 .

5. The kit according to claim 4 , wherein at least one of the fluorophores is a 7-nitrobenzofurazan derivative.

6. The kit according to claim 4 , wherein the fluorophores have emission wavelengths different from each other.

7. The kit according to claim 4 , wherein the secondary antibody recognizing the primary detection antibody recognizing the oxidized low-density lipoprotein and the secondary antibody recognizing the primary detection antibody recognizing the glycated low-density lipoprotein are each fluorescently labelled with a fluorophore which is excited with an excitation wavelength of 485 nm and observed fluorescent intensity at 528 nm.

8. The detection reagent according to claim 1 , wherein the fluorophores have emission wavelengths different from each other.

9. The detection reagent according to claim 1 , wherein the secondary antibody recognizing the primary detection antibody recognizing the oxidized low-density lipoprotein and the secondary antibody recognizing the primary detection antibody recognizing the glycated low-density lipoprotein are each fluorescently labelled with a fluorophore which can be excited with an excitation wavelength at 485 nm and observed fluorescent intensity at 528 nm.

Assignees

• Fuso Pharmaceutical Ind
• Yamada Ken Ichi

Inventors

• Yamada Ken-Ichi
• Ide Tomomi
• Ishida Yuma
• Ichien Go
• Yamamoto Keiichi

Classifications

• C07D413/12 Primary

  linked by a chain containing hetero atoms as chain links · CPC title

• C07K16/18

  against material from animals or humans · CPC title

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• G01N21/78

  producing a change of colour · CPC title

• G01N33/533

  with fluorescent label · CPC title