US2014323324A1PendingUtilityA1
Method for Enrichment and Separation of Spinal Fluid Glycoprotein, Method for Searching for Marker for Central Nervous System Diseases Which Utilizes the Aforementioned Method, and Marker for Central Nervous System Diseases
Est. expiryDec 16, 2030(~4.4 yrs left)· nominal 20-yr term from priority
Inventors:Hisashi NarimatsuJun HirabayashiAtsushi KunoHideki MatsuzakiYuzuru IkeharaHiromi ItoYasuhiro HashimotoKeiro ShirotaniSatoshi FutakawaHajime AraiMasakazu MiyajimaKazuo Fujihara
G01N 2400/40G01N 2333/4728G01N 33/6896G01N 2333/47G01N 2800/285H01J 49/0036G01N 33/6842
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Claims
Abstract
The purpose of the present invention is to develop: a method for selectively separating a glycoprotein derived from the central nervous system from a body fluid or a central nervous system cell; and a method for searching for an index marker for central nervous system diseases, which utilizes the aforementioned method. A protein derived from the central nervous system, which occurs in a trace amount in a body fluid or a central nervous system cell, can be selectively enriched by a two-stage separation procedure comprising removing a glycoprotein having sialic acid at a non-reducing terminal thereof from the body fluid or the central nervous system cell and then separating a glycoprotein having N-acetylglucosamine at a non-reducing terminal thereof.
Claims
exact text as granted — not AI-modifiedWe claim:
1 . A method for enriching or isolating a terminal N-acetylglucosamine-containing glycoprotein from a body fluid or a central neural cell derived from a test subject, comprising the steps of:
(1) removing a terminal sialic acid-containing glycoprotein from the body fluid or the central neural cell using a sialic acid-binding substance; and (2) binding a terminal N-acetylglucosamine-containing glycoprotein present in a sample obtained in the removal step to an N-acetylglucosamine-binding substance and isolating a formed complex.
2 . The method according to claim 1 , further comprising the step of dissociating the complex and eluting the terminal N-acetylglucosamine-containing glycoprotein.
3 . The method according to claim 1 , further comprising, prior to the removal step, the step of removing albumin from the body fluid or the central neural cell using an albumin-binding substance.
4 . The method according to claim 1 , wherein the sialic acid-binding substance is an anti-sialic acid antibody or an active fragment thereof, or a sialic acid-binding lectin.
5 . The method according to claim 1 , wherein the sialic acid is α2,6 sialic acid.
6 . The method according to claim 5 , wherein the α2,6 sialic acid-binding lectin is selected from the group consisting of SSA lectin, SNA lectin, and TJA-I lectin.
7 . The method according to claim 1 , wherein the N-acetylglucosamine-binding substance is an anti-N-acetylglucosamine antibody or an active fragment thereof, or an N-acetylglucosamine-binding lectin.
8 . The method according to claim 7 , wherein the N-acetylglucosamine-binding lectin is PVL lectin or WGA lectin.
9 . The method according to claim 1 , wherein the albumin-binding substance is Blue Sepharose, or an anti-albumin antibody or an active fragment thereof.
10 . The method according to claim 1 , wherein the body fluid is spinal fluid, blood (including serum, plasma, and interstitial fluid), lymph, periradicular fluid, or a tissue or cell extract.
11 . A method for selecting an index marker for central nervous system disease, comprising the steps of:
enriching or isolating terminal N-acetylglucosamine-containing glycoproteins from body fluids or central neural cells derived from a control subject and an individual affected with a particular central nervous system disease, respectively, using a method according to claim 1 ; measuring the proportions of the glycoproteins obtained in the enrichment or isolation step; and comparing the measured proportions of the corresponding glycoproteins derived from the control subject and the individual affected with a particular central nervous system disease and selecting the glycoproteins that exhibit a statistically significant quantitative difference therebetween, as an index marker for the particular central nervous system disease.
12 . A method for selecting an index marker for central nervous system disease, comprising the steps of:
measuring the proportions of at least two or more of the glycoproteins shown in Table 1 in respective body fluids or central neural cells of a control subject and an individual affected with a particular central nervous system disease; and comparing the proportions of the corresponding glycoproteins derived from the control subject and the individual affected with a particular central nervous system disease and selecting the glycoproteins that exhibit a statistically significant quantitative difference therebetween, as an index marker for the particular central nervous system disease:
TABLE 1
Accession
Molecular
No.
Glycoprotein name
#
weight (Da)
1
Acetyl-CoA carboxylase 2
O00763
279696.3
2
Multiple EGF-like domain protein 4
Q7Z7M0
254575.3
3
α2-macroglobulin
P01023
163279.4
4
Plasma protease C1 inhibitor
P05155
55154.7
5
Transferrin 1
P02787
77050.6
6
Glutamate carboxylase-like protein
Q96KN2
56779.8
7
α1-antichymotrypsin
P01011
47651.3
8
Zinc-α2-glycoprotein
P25311
33872.5
9
Inhibin βA chain
P08476
47442.7
10
Prostaglandin-H2 D-isomerase
P41222
21029.0
(prostaglandin-D2 synthase)
11
Transthyretin
P02766
15887.2
12
Cathepsin D
P07339
44553.0
13
Procollagen C-endopeptidase
Q15113
47973.0
enhancer 2
13 . A kit for enriching or isolating a terminal N-acetylglucosamine-containing glycoprotein, comprising a sialic acid-binding substance and an N-acetylglucosamine-binding substance.
14 . The kit according to claim 13 , wherein the sialic acid-binding substance is an anti-sialic acid antibody or a sialic acid-binding lectin.
15 . The kit according to claim 14 , wherein the sialic acid is α2,6 sialic acid.
16 . The kit according to claim 15 , wherein the α2,6 sialic acid-binding lectin as the sialic acid-binding lectin is SSA lectin, SNA lectin, and/or TJA-I lectin.
17 . The kit according to claim 13 , wherein the N-acetylglucosamine-binding substance is an anti-N-acetylglucosamine antibody and/or an N-acetylglucosamine-binding lectin.
18 . The kit according to claim 17 , wherein the N-acetylglucosamine-binding lectin is PVL lectin and/or WGA lectin.
19 . The kit according to claim 13 , further comprising a buffer for complex dissociation.
20 . The kit according to claim 19 , wherein the buffer for complex dissociation comprises N-acetylglucosamine.
21 . The kit according to claim 13 , further comprising an albumin-binding substance.
22 . The kit according to claim 21 , wherein the albumin-binding substance is Blue Sepharose, or an anti-albumin antibody or an active fragment thereof.
23 . An index marker for central nervous system disease, consisting of one or more glycoprotein(s) set forth in Table 1,
TABLE 1
Accession
Molecular
No.
Glycoprotein name
#
weight (Da)
1
Acetyl-CoA carboxylase 2
O00763
279696.3
2
Multiple EGF-like domain protein 4
Q7Z7M0
254575.3
3
α2-macroglobulin
P01023
163279.4
4
Plasma protease C1 inhibitor
P05155
55154.7
5
Transferrin 1
P02787
77050.6
6
Glutamate carboxylase-like protein
Q96KN2
56779.8
7
α1-antichymotrypsin
P01011
47651.3
8
Zinc-α2-glycoprotein
P25311
33872.5
9
Inhibin βA chain
P08476
47442.7
10
Prostaglandin-H2 D-isomerase
P41222
21029.0
(prostaglandin-D2 synthase)
11
Transthyretin
P02766
15887.2
12
Cathepsin D
P07339
44553.0
13
Procollagen C-endopeptidase
Q15113
47973.0
enhancer 2
provided the glycoprotein is not transferrin 1 when the central nervous system disease is idiopathic normal pressure hydrocephalus, or fragment(s) thereof, the glycoproteins each comprising a N-acetylglucosamine residue at a non-reducing terminus and having an α2,6 sialic acid-free sugar chain.
24 . The index marker for central nervous system disease according to claim 23 , wherein the central nervous system disease is neuromyelitis optica.
25 . The index marker for central nervous system disease according to claim 23 , wherein the central nervous system disease is Guillain-Barre syndrome.
26 . The index marker for central nervous system disease according to claim 23 , wherein the central nervous system disease is acute disseminated encephalomyelitis.
27 . The index marker for central nervous system disease according to claim 23 , wherein the central nervous system disease is encephalopathy.
28 . The index marker for central nervous system disease according to claim 24 , wherein the index marker for central nervous system disease is α2-macroglobulin set forth in Table 1.
29 . A method comprising detecting an one or more index marker(s) for central nervous system disease according to claim 23 from a body fluid or a central neural cell derived from a test subject and determining the presence or absence of a particular central nervous system disease developed in the test subject on the basis of the detection results.
30 . The method according to claim 29 , wherein the particular central nervous system disease is neuromyelitis optica.
31 . The method according to claim 30 , wherein the index marker for central nervous system disease to be detected is α2-macroglobulin set forth in Table 1.Cited by (0)
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