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EP-4246147-B1 - METHOD FOR MEASURING STEROL IN LIPOPROTEIN, AND REAGENT FOR MEASURING STEROL IN LIPOPROTEIN

EP4246147B1EP 4246147 B1EP4246147 B1EP 4246147B1EP-4246147-B1

Inventors

  • KUBO, TAKUYA
  • NISHIKAWA, YOICHI
  • UNO, SHINNOSUKE
  • HARADA, AMANE
  • MURAKAMI, KATSUHIRO
  • KIM, JEEEUN
  • KIRIYAMA, MARIA
  • MIWA, KEIKO

Dates

Publication Date
20260506
Application Date
20230317

Claims (12)

  1. A method for measuring sterol in lipoprotein, comprising: forming a complex by contacting with each other: lipoprotein in a sample; a tagged sterol; and a first capture body that specifically binds to the tag and comprises a labeling substance, the complex comprising: the lipoprotein comprising the tagged sterol; and the first capture body; and detecting a signal generated by the labeling substance comprised in the complex, wherein in the tagged sterol, the tag is added to C3 position of a sterol skeleton, wherein the tagged sterol is tagged cholesterol and wherein the lipoprotein is high-density lipoprotein, and wherein the tag and the first capture body are a combination selected from an antigen and an antibody that recognizes the antigen, a hapten and an anti-hapten antibody, a peptide or a protein and an aptamer that recognizes them, a ligand and a receptor thereof, an oligonucleotide and an oligonucleotide having a complementary strand thereof, biotin or a biotin analog and a biotin-binding site, a histidine tag and nitrilotriacetic acid that forms a chelate with a nickel ion (Ni-NTA), and glutathione-S-transferase (GST) and glutathione.
  2. The method according to claim 1, wherein the tagged cholesterol is represented by following formula (I): wherein double lines of a solid line and a broken line each independently represent a single bond or a double bond; R 1 is an alkyl group having 1 or more and 6 or less carbon atoms which may have a substituent, or an alkenyl group having 2 or more and 6 or less carbon atoms which may have a substituent; X and Y are identical or different, and are represented by -R 2 -NH-, -NH-R 2 -, -R 2 -(C=O)-NH-, -(C=O)-NH-R 2 -, -R 2 -NH-(C=O)-, -NH-(C=O)-R 2 -, -R 2 -(C=O)-, -(C=O)-R 2 -, - R 2 -(C=O)-O-, -(C=O)-O-R 2 -, -R 2 -O-(C=O)-, -O-(C=O)-R 2 -, -R 2 -(C=S)-NH-, -(C=S)-NH-R 2 -, -R 2 -NH-(C=S)-, -NH-(C=S)-R 2 -, -R 2 -O-, -O-R 2 -, -R 2 -S-, or -S-R 2 -, and each R 2 is independently an atomic bonding, an alkylene group having 1 or more and 10 or less carbon atoms which may have a substituent, an arylene group or heteroarylene group having 6 or more and 12 or less carbon atoms which may have a substituent, or a cycloalkylene group or heterocycloalkylene group having 3 or more and 8 or less carbon atoms which may have a substituent; L is represented by -(CH 2 ) d -[R 3 -(CH 2 ) e ] f - or -[(CH 2 ) e -R 3 ] f -(CH 2 ) d -, and R 3 is an oxygen atom, a sulfur atom, -NH-, -NH-(C=O)-, -(C=O)-NH-, or an atomic bonding; Z is a tag; a and c are identical or different and are an integer of 0 or more and 6 or less; b is 0 or 1; d and e are identical or different and are an integer of 0 or more and 12 or less; and f is an integer of 0 or more and 24 or less.
  3. The method according to claim 2, wherein in the formula (I), a is 0 or 1, b and c are 1, X is represented by -NH-(C=O)-R 2 -, Y is represented by -R 2 -(C=O)-NH-, R 2 is each independently an alkylene group having 1 or more and 6 or less carbon atoms and having no substituent, L is represented by -[(CH 2 ) 2 -O] f -(CH 2 ) d -, d is an integer of 1 or more and 6 or less, and f is an integer of 0 or more and 24 or less.
  4. The method according to any one of claims 1 to 3, wherein the tag is a biotin group.
  5. The method according to any one of claims 1 to 4, wherein the tagged cholesterol is represented by following formula (II): wherein n is an integer of 1 or more and 23 or less, or following formula (III): wherein n is an integer of 1 or more and 7 or less, or following formula (IV): wherein n is an integer of 2 or more and 5 or less.
  6. The method according to any one of claims 1 to 5, wherein the first capture body is a labeled antibody, labeled avidin, or labeled streptavidin, the first capture body specifically binding to the tag.
  7. The method according to any one of claims 1 to 6, wherein in the forming, a second capture body that specifically binds to the lipoprotein is further used for contacting the lipoprotein, the tagged sterol, and the first capture body.
  8. The method according to claim 7, wherein in the forming, the lipoprotein comprising the tagged sterol is contacted with the second capture body, and then a complex of the lipoprotein comprising the tagged sterol and the second capture body is contacted with the first capture body.
  9. The method according to claim 8, wherein in the forming, B/F (bound/free) separation for removing an unreacted free component is performed between i) the contact between the lipoprotein comprising the tagged sterol and the second capture body and ii) the contact between the complex and the first capture body.
  10. The method according to any one of claims 7 to 9, wherein the second capture body is immobilized on a solid phase, and the complex of the lipoprotein comprising the tagged sterol and the second capture body is formed on the solid phase.
  11. The method according to any one of claims 7 to 10, wherein the second capture body comprises an antibody that specifically binds to the lipoprotein.
  12. The method according to any one of claims 1 to 11, wherein B/F (bound/free) separation for removing an unreacted free component is performed between the forming and the detecting.

Description

FIELD OF THE INVENTION The present invention relates to a method for measuring cholesterol in high-density lipoprotein. BACKGROUND In recent years, an index reflecting function of lipoprotein has attracted attention. As a method for examining the function of lipoprotein, for example, methods described in U.S. Patent Application Publication Nos. 2016/0109469 and 2017/0315112 are known. The method measures the ability to uptake cholesterol that is qualitative activity of lipoprotein. These documents have disclose that a lipoprotein's ability to uptake cholesterol can be measured by contacting the lipoprotein and a tagged cholesterol in a sample to form a lipoprotein incorporating the tagged cholesterol esterified by lecithin-cholesterol acyltransferase (LCAT) in the sample and detecting a signal derived from the incorporated tagged cholesterol. SUMMARY OF THE INVENTION In the tagged cholesterol described in U.S. Patent Application Publication Nos. 2016/0109469 and 2017/0315112, a tag is added via a hydrocarbon chain at C17 position of a sterol skeleton, and a hydroxy group at C3 position can be esterified by LCAT. In the methods described in U.S. Patent Application Publication Nos. 2016/0109469 and 2017/0315112, cholesterol in which a tag is added to other sites of the sterol skeleton is not used. As a lipoprotein functional analysis method, there are still few methods for measuring sterol in lipoprotein, and development of a further measurement method is of interest. The present invention provides a method for measuring sterol in lipoprotein, comprising: forming a complex by contacting with each other: a lipoprotein in a sample; a tagged sterol; and a first capture body that specifically binds to the tag and comprises a labeling substance, the complex comprising: the lipoprotein comprising the tagged sterol; and the first capture body; and detecting a signal generated by the labeling substance comprised in the complex, wherein in the tagged sterol, the tag is added to C3 position of a sterol skeleton, wherein the tagged sterol is tagged cholesterol and wherein the lipoprotein is high-density lipoprotein, and wherein the tag and the first capture body are a combination selected from an antigen and an antibody that recognizes the antigen, a hapten and an anti-hapten antibody, a peptide or a protein and an aptamer that recognizes them, a ligand and a receptor thereof, an oligonucleotide and an oligonucleotide having a complementary strand thereof, biotin or a biotin analog and a biotin-binding site, a histidine tag and nitrilotriacetic acid that forms a chelate with a nickel ion (Ni-NTA), and glutathione-S-transferase (GST) and glutathione. In certain embodiments, the tagged cholesterol is represented by the following formula (I): wherein a double line consisting of a solid line and a broken line represents that the bond can be a single bond or a double bond; R1 is an alkyl group having 1 or more and 6 or less carbon atoms which may have a substituent, or an alkenyl group having 2 or more and 6 or less carbon atoms which may have a substituent; X and Y are identical or different, and are represented by -R2-NH-, -NH-R2-, -R2-(C=O)-NH-, -(C=O)-NH-R2-, -R2-NH-(C=O)-, -NH-(C=O)-R2-, -R2-(C=O)-, -(C=O)-R2-, -R2-(C=O)-O-,-(C=O)-O-R2-, -R2-O-(C=O)-, -O-(C=O)-R2-, -R2-(C=S)-NH-, -(C=S)-NH-R2-, -R2-NH-(C=S)-, -NH-(C=S)-R2-, -R2-O-, -O-R2-, -R2-S-, or -S-R2-, and each R2 is independently an atomic bonding, an alkylene group having 1 or more and 10 or less carbon atoms which may have a substituent, an arylene group or heteroarylene group having 6 or more and 12 or less carbon atoms which may have a substituent, or a cycloalkylene group or heterocycloalkylene group having 3 or more and 8 or less carbon atoms which may have a substituent; L is represented by -(CH2)d-[R3-(CH2)e]f- or -[(CH2)e-R3]f-(CH2)d-, and R3 is an oxygen atom, a sulfur atom, -NH-, -NH-(C=O)-, -(C=O)-NH-, or an atomic bonding; Z is a tag; a and c are identical or different and are an integer of 0 or more and 6 or less; b is 0 or 1; d and e are identical or different and are an integer of 0 or more and 12 or less; and f is an integer of 0 or more and 24 or less. In certain embodiments, in the formula (I), a is 0 or 1, b and c are 1, X is represented by -NH-(C=O)-R2-, Y is represented by -R2-(C=O)-NH-, R2 is each independently an alkylene group having 1 or more and 6 or less carbon atoms and having no substituent, L is represented by -[(CH2)2-O]f-(CH2)d-, d is an integer of 1 or more and 6 or less, and f is an integer of 0 or more and 24 or less. In certain embodiments, the tag is a biotin group. In certain embodiments, the tagged cholesterol is represented by following formula (II): wherein n is an integer of 1 or more and 23 or less, or following formula (III): wherein n is an integer of 1 or more and 7 or less,or following formula (IV): wherein n is an integer of 2 or more and 5 or less. In certain embodiments, the first capture body is a labeled an