U.S. patent application number 12/490216 was filed with the patent office on 2009-12-31 for affinity particle and affinity separation method.
This patent application is currently assigned to Shiseido Company, Ltd.. Invention is credited to Kazuhiko Ishihara, Akira Ishikubo, Katsuyuki Maeno, Kazuyuki Miyazawa.
Application Number | 20090321357 12/490216 |
Document ID | / |
Family ID | 35428284 |
Filed Date | 2009-12-31 |
United States Patent
Application |
20090321357 |
Kind Code |
A1 |
Maeno; Katsuyuki ; et
al. |
December 31, 2009 |
Affinity Particle And Affinity Separation Method
Abstract
The present invention is affinity particles that are
characterized by having phosphorylcholine groups represented by the
following formula (1) covalently bonded onto the surface of
inorganic powder and also by having ligands having specific
affinity with a certain target substance covalently bonded or
adsorbed onto the surface of inorganic powder. The object of the
present invention is to provide an affinity separation method that
uses affinity particles utilizing inexpensive inorganic particles
and is capable of separating the target substance easily and with
high accuracy. ##STR00001##
Inventors: |
Maeno; Katsuyuki;
(Yokohama-shi, JP) ; Miyazawa; Kazuyuki;
(Yokohama-shi, JP) ; Ishikubo; Akira;
(Yokohama-shi, JP) ; Ishihara; Kazuhiko;
(Yokohama-shi, JP) |
Correspondence
Address: |
TOWNSEND & BANTA;c/o PORTFOLIO IP
PO BOX 52050
MINNEAPOLIS
MN
55402
US
|
Assignee: |
Shiseido Company, Ltd.
Tokyo
JP
|
Family ID: |
35428284 |
Appl. No.: |
12/490216 |
Filed: |
June 23, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11580000 |
Nov 9, 2006 |
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PCT/JP2005/009088 |
May 18, 2005 |
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12490216 |
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Current U.S.
Class: |
210/656 ;
428/402; 435/188; 530/345; 530/391.1; 530/402; 530/403; 556/26;
558/166 |
Current CPC
Class: |
B01J 20/3285 20130101;
B01J 20/3248 20130101; B01J 20/28004 20130101; B01J 2220/58
20130101; B01J 20/286 20130101; B01D 15/3804 20130101; G01N
33/54393 20130101; B01J 20/3242 20130101; B01J 20/3274 20130101;
C07F 9/091 20130101; B01J 20/3259 20130101; B01J 20/3204 20130101;
Y10T 428/2982 20150115; B01J 20/3219 20130101 |
Class at
Publication: |
210/656 ;
558/166; 556/26; 530/391.1; 530/403; 435/188; 530/345; 530/402;
428/402 |
International
Class: |
B01D 15/36 20060101
B01D015/36; C07F 9/02 20060101 C07F009/02; C07F 9/09 20060101
C07F009/09; C07K 17/14 20060101 C07K017/14; C12N 9/96 20060101
C12N009/96; B32B 5/16 20060101 B32B005/16 |
Foreign Application Data
Date |
Code |
Application Number |
May 24, 2004 |
JP |
2004-153253 |
May 11, 2005 |
JP |
2005-138560 |
Claims
1-7. (canceled)
8. A method of affinity separation of a target substance using
affinity particles comprising: (1) a first step whereby ligands are
bonded onto the affinity particles, said affinity particles
comprising: (a) inorganic particles; (b) reactive groups or
adsorptive groups bound onto the surface of the inorganic
particles; (c) phosphorylcholine groups represented by the
following formula (1): ##STR00018## covalently bonded onto the
surface of said inorganic particles; (2) a second step whereby the
affinity particles having the ligands bonded thereto prepared in
the first step are either dispersed in a solution sample containing
the target substance selectively, so as to capture the target
substance by the ligands, or are disposed with a column as
carriers, so as to provide an affinity column for recovering the
target substance; and (3) a third step whereby the captured target
substance is recovered from the affinity particles.
9. The method of affinity separation of a target substance using
affinity particles of claim 8, wherein the inorganic particles
comprise silica, titanium oxide, zinc flower, alumina, iron oxide,
talc, mica, sericite, and gold colloid particles.
10. The method of affinity separation of a target substance using
affinity particles of claim 8, wherein said inorganic particles
have an average particle size of 20 nm to 500 .mu.m, and a specific
gravity of 1.0 g/cm.sup.3 or higher.
11. The method of affinity separation of a target substance using
affinity particles of claim 8, wherein, in the first step, the
reactive groups or adsorptive groups comprise amino groups, said
amino groups being primary or secondary amines.
12. A method of manufacturing affinity particles capable of bonding
with ligands, said method comprising: (a) introducing reactive
and/or adsorptive groups onto the surface of inorganic particles;
and (b) covalently bonding phosphorylcholine groups represented by
the following chemical formula (1): ##STR00019## via the reactive
groups onto the surface of the inorganic particles.
13. The method of manufacturing affinity particles capable of
bonding with ligands of claim 12, wherein the reactive and/or
adsorptive groups are selected from the group consisting of amino
groups, hydroxyl groups, carboxyl groups, thiol groups, and
aldehyde groups.
14. The method of manufacturing affinity particles capable of
bonding with ligands of claim 12, wherein the reactive and/or
adsorptive groups onto the surface of inorganic particles are
introduced onto the surface of the inorganic particles by: (a)
introducing the inorganic particles into a solution of
3-aminopropyl trimethoxysilane; and (b) heating the resultant
solution for 5-7 hours at about 100.degree. C., according to the
following scheme: ##STR00020##
15. The method of manufacturing affinity particles capable of
bonding with ligands of claim 12, comprising forming a chemical
bond between silica particles and phosphorylcholine groups by
dehydrating the hydroxyl groups on the surface of the silica
particles and the Si--OMe portion of the compound of following
formula (3) or formula (4): ##STR00021## wherein m=2-6.
16. The method of manufacturing affinity particles capable of
bonding with ligands of claim 12, wherein the the ligands are
covalently bonded or adsorbed onto remaining amino groups not bound
to phosphorycholine groups.
17. The method of manufacturing affinity particles capable of
bonding with ligands of claim 12, wherein said ligands are selected
from the group consisting of antibodies, antigens, enzymes,
substrates, receptors, lectin, peptides, DNA, RNA, aptamers,
protein A, protein G, avidin, biotin, chelating compounds, and
metal ions.
18. A method of manufacturing affinity particles having ligands
bound thereto, said method comprising: (a) introducing reactive
and/or adsorptive groups onto the surface of inorganic particles;
(b) covalently bonding phosphorylcholine groups represented by the
following chemical formula (1): ##STR00022## via the reactive
groups onto the surface of the inorganic particles; and (c)
covalently bonding or adsorbing ligands having specific affinity
with a target substance onto the surface of said inorganic
particles.
19. The method of manufacturing affinity particles having ligands
bound thereto of claim 18, wherein the reactive and/or adsorptive
groups are selected from the group consisting of amino groups,
hydroxyl groups, carboxyl groups, thiol groups, and aldehyde
groups.
20. The method of manufacturing affinity particles having ligands
bound thereto of claim 18, wherein the reactive and/or adsorptive
groups onto the surface of inorganic particles are introduced onto
the surface of the inorganic particles by: (a) introducing the
inorganic particles into a solution of 3-aminopropyl
trimethoxysilane; and (b) heating the resultant solution for 5-7
hours at about 100.degree. C., according to the following scheme:
##STR00023##
21. The method of manufacturing affinity particles having ligands
bound thereto of claim 18, comprising forming a chemical bond
between silica particles and phosphorylcholine groups by
dehydrating the hydroxyl groups on the surface of the silica
particles and the Si--OMe portion of the compound of following
formula (3) or formula (4): ##STR00024## wherein m=2-6.
22. The method of manufacturing affinity particles having ligands
bound thereto of claim 18, wherein the ligands are covalently
bonded or adsorbed onto remaining amino groups not bound to
phosphorycholine groups.
23. The method of manufacturing affinity particles having ligands
bound thereto of claim 18, wherein said ligands are selected from
the group consisting of antibodies, antigens, enzymes, substrates,
receptors, lectin, peptides, DNA, RNA, aptamers, protein A, protein
G, avidin, biotin, chelating compounds, and metal ions.
Description
TECHNICAL FIELD
[0001] The present invention relates to affinity particles and an
affinity separation method. More specifically, it relates to
affinity particles utilizing inorganic particles and an affinity
separation method that allows easy and highly precise separation of
the target substance. The present invention is very useful in
various separation, purification, and testing methods including
latex agglutination methods and immunoprecipitation methods that
allow easy and highly sensitive detection of the target
substance.
BACKGROUND ART
[0002] Conventionally, column chromatography has been used for
separation and purification of biological substances. However,
column separation has some fatal problems as described in the
following (1) to (3): [0003] (1) Many kinds of columns have to be
used to obtain the target substance, resulting in a poor
purification efficiency. [0004] (2) A verification test is required
to make sure the target substance is contained in the fractionated
ingredients, which means purification is time consuming. [0005] (3)
Because of the large purification loss, a large quantity of the
sample is required.
[0006] On the other hand, for separation and purification of the
target substances, affinity particles and affinity columns
supporting ligands are used (Patent Document 1 and Patent Document
2).
[0007] However, separation and purification using affinity columns
have the following problems: [0008] (1) The desired target
substance is not selectively separated. That is, in addition to the
target substance captured by the ligand, unwanted substances are
also adsorbed onto the column. [0009] (2) The capture efficiency is
low, which means a large quantity of the liquid sample is
required.
[0010] The affinity separation method in which affinity particles
are dispersed in a liquid sample for separation uses agarose and
such (Non-patent Document 1), but this method has the following
problems: [0011] (1) The desired target substance is not
selectively separated. That is, in addition to the target substance
captured by the ligand, unwanted target substances are also
adsorbed onto the affinity particles. [0012] (2) The specific
gravity is small, which makes the separation of the affinity
particles difficult. [0013] (3) The carrier is easily
disintegrated, which leads to poor durability.
[0014] On the other hand, inorganic particles adsorb more
substances than organic particles do, therefore those skilled in
the art didn't think of using inorganic particles for affinity
particles.
[0015] Patent Document 1: Japanese Patent Publication H8-26076
[0016] Patent Document 2: Japanese Patent Laid-Open 2002-511141
bulletin
[0017] Non-patent Document 1: Bioconjugate Chem.; 2002; 13(2);
163-166
DISCLOSURE OF INVENTION
Problem That the Present Invention Aims to Solve
[0018] The object of the present invention is to provide a
groundbreaking affinity separation method that uses affinity
particles utilizing inexpensive inorganic particles and is capable
of separating the target substance easily and with high accuracy.
The present invention is very useful in various separation,
purification, and testing methods including latex agglutination
methods and immunoprecipitation methods that allow easy and highly
sensitive detection of the target substance.
Means to Solve the Problem
[0019] That is, the present invention provides affinity particles
that are characterized by having phosphorylcholine groups
represented by the following formula (1) covalently bonded onto the
surface of inorganic particles.
##STR00002##
[0020] Also, the present invention provides affinity particles that
are characterized by having phosphorylcholine groups represented by
the following formula (1) covalently bonded onto the surface of
inorganic particles and also by having reactive groups or
adsorptive groups, which are capable of bonding with ligands having
specific affinity with a certain target substance, covalently
bonded or adsorbed onto the surface of inorganic particles.
##STR00003##
[0021] Furthermore, the present invention provides affinity
particles that are characterized by having phosphorylcholine groups
represented by the following formula (1) covalently bonded onto the
surface of inorganic particles and also by having ligands having
specific affinity with a certain target substance covalently bonded
or adsorbed onto the surface of inorganic particles.
##STR00004##
[0022] Also, the present invention provides the aforementioned
affinity particles wherein said inorganic particles are selected
from a group consisting of silica, titanium oxide, zinc flower,
alumina, iron oxide, talc, mica, sericite, and gold colloid, and
having an average particle size of 20 nm to 500 .mu.m and a
specific gravity of 1.0 g/cm.sup.3 or higher.
[0023] Furthermore, the present invention provides the
aforementioned affinity particles wherein said ligands are one,
two, or more types of ligands chosen from a group consisting of
various antibodies, antigens, enzymes, substrates, lectin,
receptors, peptides, DNA, RNA, aptamers, protein A, protein G,
avidin, biotin, chelating compounds, and various metal ions.
[0024] Also, the present invention provides a method of affinity
separation of a target substance by using inorganic particles that
includes (1) a first process whereby arbitrary ligands are bonded
to the affinity particles of claim 1 or 2, (2) a second process
whereby the affinity particles prepared in the first process are
dispersed in a liquid sample containing a target substance
selectively captured by the arbitrary ligands, and (3) a third
process whereby the target substance captured is recovered from the
affinity particles.
[0025] Furthermore, the present invention provides a method of
affinity separation of a target substance by using inorganic
particles that includes (1) a first process whereby the affinity
particles of claim 3 are dispersed in a liquid sample containing a
target substance selectively captured by the arbitrary ligands, and
(2) a second process whereby the target substance captured is
recovered from the affinity particles. When the affinity particles
of the present invention are used for detection of antibodies and
protein, such as in the immunoprecipitation method and the latex
agglutination method, the recovery process (2) is not required;
detection can be done easily by visually observing changes in the
dispersion state.
EFFECTS OF THE INVENTION
[0026] The affinity particles of the present invention use ligands
to capture only a certain target substance (the substance desired
to be separated) and suppresses adsorption of other substances onto
the particles, resulting in a very high separation selectivity.
They also exhibit superior dispersion properties and make
separation from liquid samples very easy, which makes it possible
to separate the target substance easily and with high accuracy by
using inexpensive inorganic powder particles for affinity
particles.
[0027] That is, the target substance separation method of the
present invention can effectively and easily separate the target
substance to be separated in a short amount of time. Since
substances have a tendency to adsorb onto foreign substances,
conventional affinity particles have difficulties efficiently
isolating only the target substance; however, it is possible to
very efficiently prevent non-specific adsorption of other
substances to the affinity particles and thus increase the
purification yield by modifying the particle surface with
phosphorylcholine groups.
[0028] Also, phosphorylcholine groups are extremely hydrophilic and
they also improve the dispersion properties of the affinity
particles in a liquid sample containing water.
[0029] Furthermore, conventional particles tend to aggregate in the
presence of salts and therefore the purification efficiency
decreases when the target substance is to be isolated from serum
because of aggregation due to various salts in serum; however, the
affinity particles of the present invention don't aggregate
significantly even under the presence of salts, which makes it
possible to recover the target substance efficiently.
[0030] The affinity particles used in the present invention are
composed of inorganic particles and therefore their specific
gravity is high, which makes it easy to recover them by leaving the
mixture at rest or by light centrifugation. These particles can be
filled in a column as carriers and such a column can be used as an
affinity column to recover a target substance.
BRIEF DESCRIPTION OF DRAWINGS
[0031] FIG. 1 is a schematic showing the difference between the
protein capture selectivity of the affinity particles of the
present invention and conventional affinity particles.
[0032] FIG. 2 shows a chemical structure formula and an NMR
spectrum of the chemical compound prepared in Synthesis example
1.
[0033] FIG. 3 shows a chemical structure formula and an NMR
spectrum of the chemical compound prepared in Synthesis example
2.
[0034] FIG. 4 shows the result of the P quantification using the PC
particles (A), (B), and (C) prepared in Synthetic example 5 and the
Af particles (A), (B), and (C) prepared in Synthetic example 6.
[0035] FIG. 5 is a 31P-CPMAS spectrum of the PC particles (A)
prepared in Reference example 1.
[0036] FIG. 6 is a FT-IR spectrum of the PC particles (A) prepared
in Reference example 1.
[0037] FIG. 7 is a 13C-CPMAS spectrum of the PC particles (A)
prepared in Reference example 3.
[0038] FIG. 8 is an evaluation of suppression of non-specific
adsorption of proteins on the PC particles (A), (B), and (C)
prepared in Reference example 1, 2, and 3.
[0039] FIG. 9 is a selectivity evaluation of the Af particles (A)
on the anti-bovine albumin antibody and anti-human hemoglobin
antibody that was conducted in Example 1.
[0040] FIG. 10 is a selectivity evaluation of the Af particles (B)
on the anti-bovine albumin antibody and anti-human hemoglobin
antibody that was conducted in Example 2.
[0041] FIG. 11 is a selectivity evaluation of the Af particles (C)
on the anti-bovine albumin antibody and anti-human hemoglobin
antibody that was conducted in Example 3.
[0042] FIG. 12 is a selectivity evaluation of the Af particles (A)
and (C) on goat anti-serum that was conducted in Examples 1 and
3.
[0043] FIG. 13 is a selectivity evaluation of the Af particles (A)
and (C) on anti-human hemoglobin in goat serum that was conducted
in Examples 1 and 3.
[0044] FIG. 14 is a selectivity evaluation of the Af particles (A)
on the anti-bovine albumin antibody and anti-human hemoglobin
antibody that was conducted in Comparative example 1.
BEST MODE FOR CARRYING OUT THE INVENTION
[0045] The present invention is described in detail below.
"Inorganic Particles"
[0046] The selection of the inorganic particles that constitute the
affinity particles is not limited in particular in the present
invention. "An inorganic particle" generally means any inorganic
object having an average particle size of about 20 nm to 500 .mu.m.
for example, talc, kaolin, mica, sericite, muscovite, phlogopite,
synthetic mica, lepidolite, biotite, vermiculite, magnesium
carbonate, calcium carbonate, aluminum silicate, barium silicate,
calcium silicate, magnesium silicate, strontium silicate, tungstic
acid metal salt, magnesium, silica, zeolite, barium sulfate, firing
calcium sulfate (calcined gypsum), calcium phosphate,
fluorine-apatite, hydroxy apatite, ceramic powder, metallic soaps
(for example, myristic acid zinc, calcium palmitate, and aluminum
stearate), boron nitride, cerium oxide, and gold colloid.
[0047] Particularly preferable particles include silica, titanium
oxide, zinc flower, alumina, iron oxide, talc, mica, sericite, and
gold colloid. Nonporous inorganic particles are more preferable
than porous inorganic particles.
[0048] Since the phosphorylcholine group represented by the
aforementioned formula (1) and reactive groups or adsorptive groups
that are capable of bonding with ligands are to be introduced onto
the particle surface by means of covalent bonding, the surface
should preferably have reactive groups such as amino groups.
[0049] Preferable affinity particles are those whose inorganic
particles have an average particle size of 20 nm to 500 .mu.m and a
specific gravity of 1.0 g/cm.sup.3 or higher.
[0050] Examples include silica, titanium oxide, zinc flower,
alumina, iron oxide, talc, mica, sericite, and gold colloid.
"Reactive Groups or Adsorptive Broups to Which the Ligand Can
Bind"
[0051] The selection is not limited as long as bonding with the
ligand is possible. Preferable examples of the covalent bond form
include an amide, ester, urethane, ether, secondary amine, urea
bond, and disulfide bond. Therefore, reactive groups for which
ligands can take the corresponding covalent bond forms are
preferable; examples include amino groups, hydroxyl groups,
carboxyl groups, thiol groups, and aldehyde groups. Also, for the
adsorption form, preferable are an avidin-biotin, metal-chelating
compound, etc. Therefore, adsorptive groups for which ligands can
take the corresponding adsorptive forms are preferable; examples
include avidin, biotin, and chelating compounds.
"Ligands"
[0052] In the present invention, a "ligand" means a substance that
binds specifically to a certain target substance; examples include
various antibodies, antigens, enzymes, substrates, receptors,
peptides, DNA, RNA, aptamers, protein A, protein G, avidin, biotin,
chelating compounds, and various metal ions. Examples of the
various antibodies include IgG, IgM, IgA, IgD, IgE, and IgY,
examples of antigens include protein and polysaccharides, examples
of enzymes include glutathione-S-transferase, examples of
substrates include glutathione, examples of receptors include
hormone receptors and cytokine receptors, examples of chelating
compounds include nitrile triacetate, and examples of various metal
ions include Ni.sup.2+, Co.sup.2+, Cu.sup.2+, Zn.sup.2+, and
Fe.sup.3+.
"A Method of Preparing the Affinity Particles of the Present
Invention"
[0053] Since the essence of the present invention is to have the
phosphorylcholine group represented by formula (1) covalently
bonded onto the surface of inorganic particles and also for the
inorganic particles to have reactive groups or adsorptive groups
capable of bonding to ligands having specific affinity to a certain
target substance that are directly present on their surface by
means of covalent bonding or adsorption, there is no limitation on
the selection of the preparation method; bonding can be done by any
means.
[0054] However, as mentioned earlier, this does not include methods
in which a polymer already having the phosphorylcholine group and
reactive groups or adsorptive groups capable of bonding to ligands
is used to simply coat the particle surface without chemical
bonding. This is because the coating polymer can peel off. and/or
there may be an influence from the coating polymer.
[0055] The affinity particles of the present invention can be
prepared with the following method, for example.
[0056] Step 1: The phosphorylcholine group represented by the
following formula (1) and reactive groups or adsorptive groups
capable of bonding to ligands are introduced onto the particles.
The selection of the reactive group or adsorptive group is not
limited; examples include an amino group, hydroxyl group, carboxyl
group, and aldehyde group.
[0057] Step 2: The phosphorylcholine group represented by formula
(1) and the ligand are bonded to the reactive group or adsorptive
group introduced onto the particles. Any chemical structure
(spacer) can exist between the phosphorylcholine group or ligand
and the reactive group or adsorptive group. Examples of such
arbitrary spacers include a methylene chain, oxyethylene chain, as
well as an alkylene chain containing one or a plurality of amino
groups.
"When the Reactive Group or Adsorptive Group on the Particle
Surface is an Amino Group"
[0058] Step 1: Amino groups are introduced to any particle by using
a prior art method or a method that will be developed in the
future. Amino groups are directly introduced onto the particle
surface. The amino group can be a primary amine or a secondary
amine.
[0059] Step 2: An aldehyde derivative or hydrate derivative
obtained by the oxidative cleavage reaction of
glycerophosphorylcholine is used in a reductive amination reaction
to directly add phosphorylcholine groups to the surface of the
particle having amino groups.
[0060] Not all the amino groups are bonded with the
phosphorylcholine group (the reaction level is controlled) so that
the remaining amino groups are available as substituents for the
ligand to bind to.
[0061] Or, a carboxyl derivative obtained by the oxidative cleavage
reaction of glycerophosphorylcholine is used in an amidation
reaction to directly add phosphorylcholine groups to the surface of
the particles having amino groups.
[0062] Not all the amino groups are bonded with the
phosphorylcholine group (the reaction level is controlled) so that
the remaining amino groups are available as substituents for the
ligand to bind to.
"A Method of Introducing Amino Groups Onto the Particle
Surface"
[0063] Examples of a prior art method for introducing amino groups
to the particles (step 1) follow:
1. Introduction of Amino Groups By Means of a Surface Reaction Via
a Plasma Treatment
[0064] Amino groups are introduced to the particle surface by means
of a low temperature plasma in a nitrogen gas atmosphere.
Specifically, the particles are put into a plasma reactor vessel
and, after a vacuum pump is used to form a vacuum in the reactor
vessel, nitrogen gas is introduced. Amino groups can be then
introduced onto the particle surface by means of glow discharge. It
is also possible to mechanically turn the plasma-treated inorganic
material into particles.
References Related to the Plasma Treatment are Shown Below:
[0065] 1. M. Muller, C. oehr [0066] Plasma aminofunctionalisation
of PVDF microfiltration membranes: comparison of the in plasma
modifications with a grafting method using ESCA and an
amino-selective fluorescent probe Surface and Coatings Technology
116-119 (1999) 802-807 [0067] 2. Lidija Tusek, Mirko Nitschke,
Carsten Werner, Karin Stana-Kleinschek, Volker Ribitsch Surface
characterization of NH3 plasma treated polyamide 6 foils [0068]
Colloids and Surfaces A: Physicochem. Eng. Aspects 195 (2001) 81-95
[0069] 3. Fabienne Poncin-Epaillard, Jean-Claude Brosse, Thierry
Falher [0070] Reactivity of surface groups formed onto a plasma
treated poly (propylene) film Macromol. Chem. Phys. 200. 989-996
(1999)
2. Introduction of Amino Groups By Means of a Surface Modifier
[0071] The surface of the inorganic particles such as
silanol-containing particles is treated with a surface modifier
having amino groups, such as alkoxysilane, chlorosilane, and
silazane.
[0072] For example, silica particles are treated with
3-aminopropyltrimethoxysilane, which has a primary amino group, to
introduce amino groups. Specifically, silica is soaked in a mixed
solution of water and 2-propanol, and, after adding
3-aminopropyltrimethoxysilane, the temperature is raised to
100.degree. C. and the reaction is carried out for six hours. After
cooling down to room temperature, the silica is rinsed with
methanol and dried to obtain particles that have amino groups
directly introduced onto the silica surface. In addition to silica,
examples of the particles preferably treated with this method
include particles composed of glass, alumina, talc, clay, aluminum,
iron, mica, asbestos, titanium oxide, zinc flower, and iron
oxide.
3. Introduction of Amino Groups By Means of the Silicone Vapor
Phase Treatment (Refer to Japanese Patent Publication No. H1-54379,
Japanese Patent Publication No. H1-54380 Bulletin, and Japanese
Patent Publication No. H1-54381 Bulletin.)
[0073] The particle surface is treated with
1.3.5.7-tetramethylcyclotetrasiloxane and then Si--H groups
introduced onto the surface are reacted with monomers having an
amino group to obtain an aminated surface. For example, mica and
1.3.5.7-tetramethylcyclotetrasiloxane are put into a desiccator and
an aspirator is used to deaerate it. The reaction is carried out
for 16 hours at 80.degree. C., and the mica is taken out and dried
at 120.degree. C. The obtained mica is dispersed in ethanol, to
which allylamine is added, and an ethanol solution of
chloroplatinic acid is added, followed by two hours of stirring at
60.degree. C. After the reaction is completed, filtration, ethanol
rinsing, and reduced-pressure drying is carried out to obtain
aminated mica. Various inorganic particles (mica, talc, kaolin,
alumina, titanium oxide, zinc oxide, iron oxide, various inorganic
pigments, etc.) are preferably treated.
[0074] For the monomer to be used in this method, an amine-type
monomer can be used. The amine-type monomer is not limited to
allylamine as long as it has a reactive site such as polymerizable
vinyl and acrylate, and an amino group. The amino group can be
protected by a butoxycarbonyl group, benzyloxycarbonyl group or the
like.
[0075] In addition to an amine-type monomer, a monomer having a
functional group such as an epoxy group, to which an amino group
can be easily introduced by means of, for example, a reaction with
diamine, can be used as well.
"A Method for Introducing Phosphorylcholine Groups Onto the
Particles Having Amino Groups"
[0076] Next, a method for introducing phosphorylcholine groups onto
the aminated particle surface (step 2) is described below.
[0077] The particles are soaked in methanol, to which
phosphatidylglyceroaldehyde is added, and the mixture is left alone
for six hours at room temperature. Sodium cyanoborate is then added
at 0.degree. C., followed by overnight heating and stirring, to add
a phosphorylcholine group to an amino group. The particles are
rinsed with methanol and dried to obtain particles that have
phosphorylcholine groups directly on the surface. For the reaction
solvent, protic solvents such as water, ethanol, and 2-propanol can
be used in addition to methanol; the introduction rate tends to be
higher when methanol is used.
[0078] Shown below is a scheme in which amino groups are introduced
onto silica by using 3-aminopropyl trimethoxysilane as the surface
modifier and then phosphorylcholine groups (abbreviated as PC) are
introduced.
##STR00005##
[0079] As described above, the particles directly having
phosphorylcholine groups on the surface can be obtained by a method
in which particles having amino groups are prepared and then a
reductive amination reaction with a hydrate derivative or aldehyde
derivative obtained by the oxidative cleavage reaction of
glycerophosphorylcholine is used to directly add phosphorylcholine
groups to the particle surface.
[0080] This method has the following great advantages: the
introduction rate of the phosphorylcholine group is high, and the
surface of various inorganic particles can be modified.
[0081] The aforementioned compound containing aldehyde is obtained
by oxidative cleavage of the prior art glycerophosphorylcholine
group by means of a prior art method, which is a very easy step.
For example, 1,2-diol is oxidized with an oxidant such as periodic
acid, periodate, or bismuth trioxide to cleave the bond and obtain
an aldehyde derivative. The reaction is usually carried out in
water or an organic solvent containing water at a reaction
temperature between 0.degree. C. and room temperature. The aldehyde
derivative may go through an equilibrium reaction in water to
become a hydrate, but this does not affect the subsequent reaction
with the amine. An example of a scheme for preparing a
monofunctional aldehyde derivative containing a phosphorylcholine
group is described below.
##STR00006##
[0082] The reductive amination reaction for bonding the aldehyde
derivative (or hydrate derivative) obtained by the oxidative
cleavage reaction of glycerophosphorylcholine to the amino groups
of the particles can be carried out easily by stirring both of them
in a solvent. This reaction is carried out by dissolving or
dispersing these two in water or alcohol (a third organic solvent
ingredient can be mixed in, too) to form an imine and reducing it
with a reducing agent to obtain a secondary amine. For the reducing
agent, a mild reducing agent such as sodium cyanoboronate is
preferable, but other reducing agents can be used as long as the
phosphorylcholine is stable. The reaction is usually carried out at
0.degree. C. to room temperature, but heating may be done depending
on the situation.
[0083] It is also possible to react any amount of the compound
represented by formula (2) to the aforementioned amino groups and
leave the remaining amino groups as reactive groups or adsorptive
groups to which ligands can bind.
##STR00007##
[0084] n denotes an integer 1-12.
[0085] Examples of the specific methods include a method in which
the compound of formula (2) is reacted with thionyl chloride in
N,N'-dimethylformamide to obtain an acid chloride, which is then
reacted with particles having amino groups in
N,N'-dimethylformamide to introduce the phosphorylcholine group
represented by formula (1) by means of amide bonding.
[0086] The compound of formula (2) can be synthesized by using the
following scheme.
"Reactive Groups or Adsorptive Groups to Which Ligands Can
Bind"
[0087] Not all the amino groups are bonded with the
phosphorylcholine group (the reaction level is controlled) so that
the remaining amino groups are available as reactive groups or
adsorptive groups for the ligand to bind to. These particles are
the affinity particles described in claim 2, i.e. the particles
that are inorganic particles directly having on their surface the
phosphorylcholine groups represented by formula (1) and reactive
groups or adsorptive groups to which the ligand can bond. When the
ligand is bonded to these remaining amino groups, the affinity
particles described in claim 3, i.e. the inorganic particles
directly having the phosphorylcholine group represented by formula
(1) and the ligand on their surface, are obtained.
[0088] The product form of the affinity particles described in
claim 2 is such that the user can bond any ligand to them depending
on the substance to be captured (target substance). The product
form of the affinity particles described in claim 3 is such that
the ligand is already bonded. The affinity particles described in
claim 1 are affinity particles having at least the
phosphorylcholine group of formula (1) on the particle surface and
their product form is such that the user can bind any ligand to
them depending on the substance to be captured (target substance),
regardless of the presence or absence of the ligand or reactive
group or adsorptive group that can bind to it. Affinity particles
of any form are included as long as the phosphorylcholine group of
formula (1) is present on the particle surface; for example, the
forms described in claim 2 and claim 3 are included as well.
[0089] In the aforementioned reaction, leaving some amino groups as
reactive groups or adsorptive groups to which the ligand can bind
can be made possible, for example, by adjusting the reaction
quantity or by a competitive reaction of 3-aminopropyl
trimethoxysilane and 3-aminopropyl trimethoxysilane to which the
phosphorylcholine group is introduced.
[0090] It is also possible to react this amino group with a
compound having any functional group and use this functional group
as the reactive group or adsorptive group to which the ligand can
bind. Examples include glutaraldehyde, alkyl diimidate, acyl
azides, and isocyanates.
[0091] In a scheme in which 3-aminopropyl trimethoxysilane is used
for the aforementioned surface modifier, it is also possible to
adjust the reaction quantity of the surface modifier to leave some
hydroxyl groups (OH) on the particle surface and use these
remaining OH groups as reactive groups or adsorptive groups to
which the ligand can bind.
"A Method of Binding the Ligand to the Particles Having Amino
Groups"
[0092] When the ligand is a protein, one aldehyde group of
glutaraldehyde is reacted with an amino group on the inorganic
particle and the other aldehyde group is reacted with an amino
group in the protein, thus binding the protein.
"When the Reactive Group or Adsorptive Group on the Particle
Surface is a Hydroxyl Group"
[0093] Since most inorganic particles have hydroxyl groups on their
surface, no reactive group or adsorptive group to which the ligand
can bind, such as amino groups as mentioned above, needs to be
introduced; the hydroxyl groups (OH) present on the particle
surface are used as they are to introduce the phosphorylcholine
group and the ligand or reactive groups or adsorptive groups to
which the ligand can bind. The affinity particles of the present
invention are preferably prepared with this method.
[0094] "A Method for Introducing Phosphorylcholine Groups Onto the
Particles Having Hydroxyl Groups"
[0095] A chemical bond is formed by dehydration of the hydroxyl
group on the particle surface and Si--OMe of the compound of the
following formula (3) or (4). This chemical reaction proceeds very
easily and quantitatively in most organic solvents if heating and
refluxing are provided. Chemically and physically very stable
phosphorylcholine groups can be introduced by means of this
dehydration reaction, which is preferable. The phosphorylcholine
group-containing compound represented by the following formula (3)
or (4) is a new compound.
##STR00008##
[0096] In this formula, m denotes 2-6 and n denotes 1-4. OMe can be
replaced by OEt or Cl. Up to two of the OMe's, OEt's, or Cl's to be
bonded to Si can be replaced by a methyl group, ethyl group, propyl
group, isopropyl group, or isobutyl group.
"A Method of Preparing the Phosphorylcholine Group-Containing
Chemical Compound of Formula (3)"
[0097] The phosphorylcholine derivative shown in the following
formula (5) is dissolved in distilled water. The phosphorylcholine
derivative of the following formula (5) is a prior art chemical
compound and commercially available.
##STR00009##
[0098] An aqueous solution of the chemical compound of formula (5)
is cooled in an ice water bath; then sodium periodate is added,
followed by five hours of stirring. The reaction fluid is
concentrated under reduced pressure and dried under reduced
pressure; methanol is used to extract a phosphorylcholine
derivative having an aldehyde group shown in the following formula
(6).
##STR00010##
[0099] 0.5 equivalents of 3-aminopropyltrimethoxysilane is added to
the methanol solution of formula (6). This mixed solution is
stirred for a prescribed amount of time at room temperature and
cooled with ice; an appropriate amount of sodium cyanohydroborate
is then added and the temperature is returned back to room
temperature, followed by 16 hours of stirring. During this time dry
nitrogen is continued to be fed through the reaction vessel. After
filtering the precipitate, a methanol solution of formula (3)
and/or (4) is obtained.
[0100] The procedure described above can be carried out in the
exact same way even when m and n in the chemical compounds
represented by formula (3) or (4) change. The procedure shown here
is for m=3 and n=2. The reaction solvent is not limited in
particular; in addition to methanol, which was mentioned above,
water, alcohols such as ethanol, propanol, and butanol, and aprotic
solvents such as DMF and DMSO can be used. Dehydrated solvents are
preferable to prevent polymerization during the reaction; of these,
dehydrated methanol is particularly preferable.
[0101] If a methoxy group (OMe) in formula (3) or (4) is replaced
by an ethoxy group (OEt), then the reaction is carried out by using
ethanol instead of methanol; if it is replaced by Cl, then
dimethylformamide or dimethylsulfoxide is used instead.
[0102] Furthermore, even when one or two of the OMe groups, OEt, or
Cl's to be bonded to Si are replaced by a methyl group, ethyl
group, propyl group, butyl group, isopropyl group, or isobutyl
group, the preparation can be carried out in exactly the same
manner as described above.
"A Method of Preparing the Phosphorylcholine Group-Containing
Chemical Compound of Formula (4)"
[0103] The phosphorylcholine derivative shown in the following
formula (5) is dissolved in a mixture of distilled water and
acetonitrile. The phosphorylcholine derivative of the following
formula (5) is a prior art chemical compound and commercially
available.
##STR00011##
[0104] An aqueous solution of the chemical compound of formula (5)
is cooled in an ice water bath; sodium periodate and ruthenium
trichloride are added, followed by three hours of stirring. The
reaction fluid is concentrated under reduced pressure and dried
under reduced pressure; methanol is used to extract a
phosphorylcholine derivative having an carboxyl group shown in the
following formula (7).
##STR00012##
[0105] Thionyl chloride is then added to formula (7) in
N,N'-dimethylformamide to turn it into an acid chloride, to which
0.5 equivalents of 3-aminopropyltrimethoxysilane and 2 equivalents
of triethylamine are added. This mixed solution is stirred at room
temperature for a prescribed amount of time to obtain an
N,N'-dimethylformamide solution of formula (4).
[0106] The procedure described above can be carried out in the same
way even when m and n in the chemical compounds represented by
formula (4) change. The procedure shown here is for m=3 and n=2.
The reaction solvent is not limited in particular; in addition to
N,N'-dimethylformamide, which was mentioned above, aprotic solvents
such as acetonitrile, tetrahydrofuran, and dimethylsulfoxide can be
used. It is preferable to use a dehydrated solvent to prevent
polymerization during the reaction.
[0107] Furthermore, even when one or two of the OMe groups, OEt, or
Cl's to be bonded to Si are replaced by a methyl group, ethyl
group, propyl group, isopropyl group, butyl group, or isobutyl
group, the preparation can be carried out in exactly the same
manner as described above.
"Reactive Groups or Adsorptive Groups to Which Ligands Can
Bind"
[0108] Not all the hydroxyl groups are bonded with the
phosphorylcholine group (the reaction level is controlled) so that
the remaining hydroxyl groups are available as reactive groups or
adsorptive groups for the ligand to bind to. These particles are
the affinity particles described in claim 2, i.e. the particles
that are inorganic particles directly having on their surface the
phosphorylcholine groups represented by formula (1) and reactive
groups or adsorptive groups to which the ligand can bond. When the
ligand is bonded to these remaining hydroxyl groups, the affinity
particles described in claim 3, i.e. the inorganic particles
directly having the phosphorylcholine group represented by formula
(1) and the ligand on their surface, are obtained.
[0109] The product form of the affinity particles described in
claim 2 is such that the user can bond any ligand to them depending
on the substance to be captured (target substance). The product
form of the affinity particles described in claim 3 is such that
the ligand is already bonded. The affinity particles described in
claim 1 are affinity particles having at least the
phosphorylcholine group of formula (1) on the particle surface and
their product form is such that the user can bind any ligand to
them depending on the protein to be captured (target substance),
regardless of the presence or absence of the ligand or reactive
group or adsorptive group that can bind to the ligand. Affinity
particles of any form are included as long as the phosphorylcholine
group of formula (1) is present on the particle surface; for
example, the forms described in claim 2 and claim 3 are included as
well.
"A Method of Binding the Ligand to the Particles Having Hydroxyl
Groups"
[0110] When the ligand is a protein, hydroxyl groups on the
particles are activated by using cyanogen bromide. Amino groups in
the protein are reacted with these to bind the protein.
[0111] It is also possible to react this hydroxyl group with a
compound having any functional group and use this functional group
as the reactive group or adsorptive group to which the ligand can
bind.
"When the Reactive Group or Adsorptive Group on the Particle
Surface is a Carboxyl Group"
[0112] Step 1: Carboxyl groups are introduced to any particle by
using a prior art method or a method that will be developed in the
future. Carboxyl groups are directly introduced onto the particle
surface.
[0113] Step 2: It is also possible to react the
phosphorylcholine-containing compound represented by formula (2)
with the particles having carboxyl groups so as to form an acid
amide bonding with the phosphorylcholine group and use the
remaining carboxyl groups as reactive groups or adsorptive groups
to which ligands can bind.
[0114] Not all the carboxyl groups are bonded with the
phosphorylcholine group (the reaction level is controlled) so that
the remaining carboxyl groups are available as reactive groups or
adsorptive groups for the ligand to bind to.
##STR00013##
"A Method of Introducing Carboxyl Groups Onto the Particle
Surface"
[0115] Examples of a prior art method for introducing carboxyl
groups to the particles (step 1) follow:
1. Introduction of Carboxyl Groups By Means of a Surface
Modifier
[0116] The surface of the inorganic particles such as
silanol-containing particles is treated with a surface modifier
having carboxyl groups, such as alkoxysilane, chlorosilane, and
silazane.
[0117] For example, silica particles are treated with
triethoxysilylpropyl succinate anhydrate to introduce carboxyl
groups. Specifically, triethoxysilylpropyl succinate anhydrate is
dissolved in N,N-dimethylformamide, to which distilled water and
4-dimethylaminopyridine is added, followed by stirring at room
temperature for 16 hours to obtain a silane coupling agent having
carboxylic acid represented in the following formula (3). This
reaction is a hydrolysis reaction of succinic acid anhydrate using
4-dimethylaminopyridine.
[0118] Silica particles are treated with the silane coupling agent
having carboxyl groups to introduce carboxyl groups. Specifically,
silica is soaked in a mixed solution of water and 2-propanol, and,
after adding the silane coupling agent having carboxyl groups, the
temperature is raised to 100.degree. C. and the reaction is carried
out for six hours. After cooling down to room temperature, the
silica is rinsed with methanol and dried to obtain particles that
have carboxyl groups directly introduced onto the silica surface.
In addition to silica, examples of the particles preferably treated
with this method include particles composed of glass, alumina,
talc, clay, aluminum, iron, mica, asbestos, titanium oxide, zinc
flower, and iron oxide.
2. Introduction of Carboxyl Groups By Means of the Silicone Vapor
Phase Treatment (Refer to Japanese Patent Publication No. H1-54379,
Japanese Patent Publication No. H1-54380 Bulletin, and Japanese
Patent Publication No. H1-54381 Bulletin.)
[0119] The particle surface is treated with
1.3.5.7-tetramethylcyclotetrasiloxane and then Si--H groups
introduced onto the surface are reacted with monomers having a
carboxyl group to obtain a carboxylated surface. Various inorganic
particles (mica, talc, kaolin, alumina, titanium oxide, zinc oxide,
iron oxide, various inorganic pigments, etc.) are preferably
treated.
[0120] For the monomer to be used in this method, a carboxyl-type
monomer can be used. The selection of the carboxyl-type monomer is
not limited as long as it has a reactive site such as a carboxyl
group, polymerizable vinyl and acryl.
"A Method for Introducing Phosphorylcholine Groups Onto the
Particles Having Carboxyl Groups"
[0121] Next, a method for introducing phosphorylcholine groups onto
the carboxylated particle surface (step 2) is described below.
[0122] When particles having carboxyl groups on the surface are
soaked in a solution of N-hydroxysuccinimide and
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, the particle
surface is coated with active ester groups. A solution of the
phosphorylcholine derivative having an amino group represented by
formula (7) is added to this to introduce phosphorylcholine
groups.
"Reactive Groups or Adsorptive Groups to Which Ligands Can
Bind"
[0123] Not all the carboxyl groups are bonded with the
phosphorylcholine group (the reaction level is controlled) so that
the remaining carboxyl groups are available as reactive groups or
adsorptive groups for the ligand to bind to. These particles are
the affinity particles described in claim 2, i.e. the particles
that are inorganic particles directly having on their surface the
phosphorylcholine groups represented by formula (1) and reactive
groups or adsorptive groups to which the ligand can bond. When the
ligand is bonded to these reactive or adsorptive groups to which
the ligand can bind, the affinity particles described in claim 3,
i.e. the inorganic particles directly having the phosphorylcholine
group represented by formula (1) and the ligand on their surface,
are obtained.
[0124] The product form of the affinity particles described in
claim 2 is such that the user can bond any ligand to them depending
on the substance to be captured (target substance). The product
form of the affinity particles described in claim 3 is such that
the ligand is already bonded. The affinity particles described in
claim 1 are affinity particles having at least the
phosphorylcholine group of formula (1) on the particle surface and
their product form is such that the user can bind any ligand to
them depending on the substance to be captured (target substance),
regardless of the presence or absence of the ligand or reactive
group or adsorptive group that can bind to the ligand. Affinity
particles of any form are included as long as the phosphorylcholine
group of formula (1) is present on the particle surface; for
example, the forms described in claim 2 and claim 3 are included as
well.
[0125] In the aforementioned reaction, leaving some carboxyl groups
as reactive groups or adsorptive groups to which the ligand can
bind can be made possible, for example, by adjusting the reaction
quantity of the silane coupling agent having a carboxylic acid to
which the phosphorylcholine group is introduced.
[0126] It is also possible to react this carboxyl group with a
compound having any functional group and use this functional group
as the reactive group or adsorptive group to which the ligand can
bind.
"A Method of Binding the Ligand to the Particles Having Carboxyl
Groups"
[0127] When the ligand is a protein, inorganic particles having
carboxyl groups on the surface are soaked in a solution of
N-hydroxysuccinimide and
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to esterify the
particle surface. Amino groups in the protein are reacted with
these to bind the protein. It is also possible to react this
hydroxyl group with a compound having any functional group and use
this functional group as the reactive group or adsorptive group to
which the ligand can bind.
"A Method of Affinity Separation of a Target Substance"
[0128] Using the affinity particles of the present invention
obtained as described above, the affinity separation of a target
substance of the present invention is carried out.
[0129] The method of the present invention is a groundbreaking
separation/purification method for a target substance in that high
precision separation can be easily done by using inorganic
particles.
[0130] The method of the present invention contains the following 3
processes. The first process is omitted for the affinity particles
to which the ligand is already bonded (claim 2) since this process
has already been done for such particles. [0131] 1. The first
process in which any ligand is chemically bonded to affinity
particles that are characterized by having phosphorylcholine groups
represented by the following formula (1) covalently bonded onto the
surface of inorganic particles or affinity particles that are
characterized by having phosphorylcholine groups represented by the
following formula (1) covalently bonded onto the surface of
inorganic particles and also by having reactive groups or
adsorptive groups, that are capable of bonding with ligands having
specific affinity with a certain target substance, covalently
bonded or adsorbed onto the surface of inorganic particles.
[0132] For example, 1 ml of a PBS solution of any ligand and
affinity particles that are inorganic particles having the
phosphorylcholine group represented by formula (1) covalently
bonded onto their surface and reactive groups or adsorptive groups
to which the ligand can bind covalently bonded or adsorbed on their
surface are put into a 2 ml eppen tube, followed by gentle shaking
at 4.degree. C. for 30 minutes. This is centrifuged for 5 minutes
at 5000 rpm and the supernatant is discarded. The sample is cleaned
by adding 1 ml of a PBS solution to it, gently shaking it,
centrifuging it for 5 minutes at 5000 rpm, and discarding the
supernatant. This cleaning operation is repeated 3 times. [0133] 2.
The second process in which the affinity particles prepared in the
first process are dispersed in a liquid sample containing the
target substance that is selectively captured by any ligand.
[0134] For example, the affinity particles prepared in the first
process are dispersed in a liquid sample containing the target
substance that is selectively captured by any ligand, followed by
gentle shaking for 30 minutes at 4.degree. C. This is centrifuged
for 5 minutes at 5000 rpm and the supernatant is discarded. The
sample is cleaned by adding 1 ml of a PBS solution to it, gently
shaking it, centrifuging it for 5 minutes at 5000 rpm, and
discarding the supernatant. This cleaning operation is repeated 3
times. [0135] 3. The third process in which the captured target
substance is recovered from the separated affinity particles.
[0136] For example, for the purpose of recovering the captured
target substance from the affinity particles, 1 ml of an elution
buffer is added, followed by gentle shaking for 30 minutes at
4.degree. C. to elute the target substance from the particles, and
the supernatant is recovered. 1 ml of a PBS solution is added to
it, followed by gentle shaking and centrifugation for 5 minutes at
5000 rpm, and the supernatant is recovered. This operation is
repeated twice.
[0137] FIG. 1 is a schematic showing the differences between the
target substance capture selectivity of the affinity particles of
the present invention and conventional affinity particles.
Examples
[0138] Next, the present invention is described in detail by
referring to Examples. The present invention is not limited to
these Examples. The phosphorylcholine groups introduced onto the
particle surface can be verified and quantified by the FT-IR and
element analysis.
Synthesis Example 1
"An Aldehyde Chemical Compound Containing Phosphorylcholine
Groups"
[0139] 1-alpha-glycerophosphorylcholine (6.29 g) was dissolved in
210 ml of distilled water and cooled in an ice water bath. Sodium
periodate (10.23 g) was added, followed by five hours of stirring.
The reaction fluid was concentrated under reduced pressure and
dried under reduced pressure; methanol was then used to extract the
target substance. The structure is shown in the following chemical
formula (6).
[0140] A 1H NMR spectrum of the compound of formula (6) is shown in
FIG. 2. Since the compound of formula (6) is in equilibrium with
formula (9) in water, the actual spectrum reflects both formula (6)
and formula (9).
##STR00014##
Synthesis Example 2
"A Carboxylic Acid Chemical Compound Containing Phosphorylcholine
Groups"
[0141] 5 g of glycerophosphorylcholine, 17 g of sodium periodate,
81 mg of ruthenium trichloride n-hydrate, 70 g of ion-exchanged
water and 30 g of acetonitrile were put into a 200 ml flask. After
stirring for two hours at room temperature, filtering was carried
out and the solvent was removed from the filtrate. Methanol was
used to extract the target compound from the obtained solid
product; methanol was then removed to obtain the target compound
(7).
[0142] A 1H NMR spectrum of the compound of formula (7) is shown in
FIG. 3.
##STR00015##
Synthesis Example 3
"Compound of Formula (10)"
[0143] 9.0 g of the compound of Synthesis example 1 was dissolved
in 55 mL of dehydrated methanol, and the air inside the vessel was
replaced by dry nitrogen. Next, 2.84 g of
3-aminopropyltrimethoxysilane was added to the methanol solution of
chemical compound 1. This mixed solution was stirred overnight at
room temperature and cooled with ice; 1.39 g of sodium
cyanohydroborate was then added and the temperature was returned
back to room temperature, followed by 5 hours of stirring. During
this time dry nitrogen was continued to be fed through the reaction
vessel. After filtering the precipitation, a methanol solution of
the target substance, i.e. the compound of the following formula
(10), was obtained.
##STR00016##
Synthesis Example 4
"Compound of Formula (11)"
[0144] 5.0 g of the compound of Synthesis example 4 was dissolved
in 300 mL of N,N'-dimethylformamide, and the air inside the vessel
was replaced by dry nitrogen. 4.5 g of thionyl chloride was then
added, followed by 15 minutes of stirring, after which 2.84 g of
3-aminopropyltrimethoxysilane and 9.5 g of triethylamine were
added. This mixed solution was stirred at room temperature
overnight and the precipitate was filtered to obtain an
N,N'-dimethylformamide solution containing the target substance,
i.e. the compound of the following formula (11).
##STR00017##
Reference Example 1
"Phosphorylcholine Particles (PC Particles (A)) That are Inorganic
Particles Having Phosphorylcholine Groups Covalently Bonded Onto
Their Surface"
[0145] 97.7 .mu.l of a methanol solution containing 50 .mu.mol of
the compound of formula (10) prepared in Synthesis example 3 was
sampled, to which 47.5 ml of methanol and 2.5 ml of distilled water
were added, and then 5 g of silica gel having an average particle
size of 1.5 .mu.m and a specific surface area of 6 m.sup.2/g was
added. This particle dispersion was refluxed at 80.degree. C.
overnight for coupling. After the refluxing, the particles were
cleaned by means of centrifugation using methanol to obtain the PC
particles of claim 1 (hereafter "PC particles (A)"). FIG. 4 shows
the P quantification measurements of the PC-treated particles (A)
prepared in the aforementioned procedure using the surface modifier
of Synthetic example 3. The PC introduction level thus determined
was 3.1 .mu.mol/g-particles, which confirmed the introduction of PC
groups onto the particle surface.
Reference Example 2
"Phosphorylcholine Particles (PC Particles (B)) That are Inorganic
Particles Having Phosphorylcholine Groups Covalently Bonded Onto
Their Surface"
[0146] 278 .mu.l of a dimethylformamide solution containing 50
.mu.mol of the compound of formula (11) prepared in Synthesis
example 4 was sampled, to which 50 ml of dimethylformamide was
added, and then 5 g of silica gel having an average particle size
of 1.5 .mu.m and a specific surface area of 6 m.sup.2/g was added.
This particle dispersion was refluxed at 160.degree. C. overnight
for coupling. After the refluxing, the particles were cleaned by
means of centrifugation using methanol to obtain the PC particles
of claim 1 (hereafter "PC particles (B)"). FIG. 4 shows the P
quantification measurements of the PC-treated particles (B)
prepared in the aforementioned procedure using the surface modifier
of Synthetic example 4. The PC introduction level thus determined
was 3.4 .mu.mol/g-particles, which confirmed the introduction of PC
groups onto the particle surface.
Reference Example 3
"Phosphorylcholine Particles (PC Particles (C)) That are Inorganic
Particles Having Phosphorylcholine Groups Covalently Bonded Onto
Their Surface"
[0147] 278 .mu.l of a dimethylformamide solution containing 50
.mu.mol of the compound of formula (11) prepared in Synthesis
example 4 was sampled, to which 47.5 ml of dimethylformamide and
2.5 ml of distilled water were added, and then 5 g of silica gel
having an average particle size of 1.5 .mu.m and a specific surface
area of 6 m.sup.2/g was added. This particle dispersion was
refluxed at 160.degree. C. overnight for coupling. After the
refluxing, the particles were cleaned by means of centrifugation
using methanol to obtain the PC particles of claim 1 (hereafter "PC
particles (C)"). FIG. 4 shows the P quantification measurements of
the PC-treated particles (C) prepared in the aforementioned
procedure using the surface modifier of Synthetic example 4. The PC
introduction level thus determined was 7.3 .mu.mol/g-particles,
which confirmed the introduction of PC groups onto the particle
surface.
[0148] A 13C-CPMAS spectrum and a 13C-PSTMAS spectrum of the PC
particles (A) of Reference example 1 are shown in FIG. 5. The
PSTMAS spectrum selectively captures a spectrum of free moving
molecular chains; this method is widely used for analysis of
modifying chains on the particle surface. In FIG. 5, a spectrum due
to carbons in the phosphorylcholine group is observed at 54.2
ppm.
[0149] In the 31P-CPMAS spectrum of the PC particles (A) of
Reference example 1 in FIG. 6, peaks are detected at almost the
same chemical shifts as NaH.sub.2PO.sub.4, which is measured as a
control; this verifies the presence of phosphate groups. The
results shown above indicate that phosphorylcholine groups were
introduced onto the carrier silica gel surface.
[0150] In FIG. 5, spectra due to propyl groups, the spacer, are
observed near 9 ppm and 23 ppm, and spectra due to ethyls in
phosphorylcholine are observed near 60 ppm and 69 ppm. What is
described above indicates that the structures of formulas (10) and
(11) are introduced onto the silica gel without being
destroyed.
[0151] FIG. 7 shows a FT-IR spectrum of the PC particles (C) of
Reference example 3. Absorption specific to amide bonding is
observed near 1650 cm.sup.-1.
"Evaluation of Non-Specific Protein Adsorption on Phosphorylcholine
Particles"
[0152] 25 mg each of untreated silica gel particles having no
phosphorylcholine group introduced used in Reference example 1
(abbreviated as "untreated particles") and the PC particles (A),
(B), and (C) prepared in Reference examples 1, 2, and 3 were
sampled, to each of which 1 ml of distilled water was added,
followed by one minute of ultrasonic treatment. After removing the
distilled water by means of centrifugation, 1 ml of albumin (100
.mu.g/ml) or lysozyme (100 .mu.g/ml) was added, followed by a 1
hour reaction at room temperature and cleaning by means of
centrifugation/purification (5,000 g) 5 times with PBS. 1 ml of SDS
(1%) was then added, followed by a 1 hour reaction at room
temperature and centrifugation (5,000 g); the supernatant was
quantified with the Micro BCA method. The results are shown in FIG.
8. PC particles (A), which had been treated with phosphorylcholine
groups, showed significantly suppressed adsorption of both albumin
and lysozyme compared with the agarose particles. PC particles (B)
and (C) showed even more suppressed adsorption of both albumin and
lysozyme compared with untreated particles or PC particles (A).
Example 1
"Affinity Particles (Af Particles (A)) That are Inorganic Particles
Having Phosphorylcholine Groups and Amino Groups Covalently Bonded
Onto Their Surface"
[0153] 87.9 .mu.l of a methanol solution containing 45 .mu.mol of
the compound of formula (10) prepared in Synthesis example 3 and 50
.mu.l of a methanol solution containing 5 .mu.mol of
3-aminotrimethoxysilane were sampled, to which 47.5 ml of methanol
and 2.5 ml of distilled water were added, and then 5 g of silica
gel having an average particle size of 1.5 .mu.m and a specific
surface area of 6 m.sup.2/g was added. This particle dispersion was
refluxed at 80.degree. C. overnight for coupling. After the
refluxing, the particles were cleaned by means of centrifugation
using methanol to obtain the Af particles of claim 1 (hereafter "Af
particles (A)"). FIG. 4 shows the P quantification measurements of
the Af particles (A) prepared in the aforementioned procedure using
the surface modifier of Synthetic example 3. The PC introduction
level thus determined was 2.7 .mu.mol/g-particles, which confirmed
the introduction of PC groups onto the particle surface.
Example 2
"Affinity Particles (Af Particles (B)) That are Inorganic Particles
Having Phosphorylcholine Groups and Amino Groups Covalently Bonded
Onto Their Surface"
[0154] 250 .mu.l of a dimethylformamide solution containing 45
.mu.mol of the compound of formula (11) prepared in Synthesis
example 4 and 50 .mu.l of a dimethylformamide solution containing 5
.mu.mol of 3-aminotrimethoxysilane were sampled, to which 5 g of
silica gel having an average particle size of 1.5 .mu.m and a
specific surface area of 6 m.sup.2/g was added. This particle
dispersion was refluxed at 160.degree. C. overnight for coupling.
After the refluxing, the particles were cleaned by means of
centrifugation using methanol to obtain the affinity particles of
claim 1 (hereafter "Af particles (B)"). FIG. 4 shows the P
quantification measurements of the Af particles (B) prepared in the
aforementioned procedure using the surface modifier of Synthetic
example 4. The PC introduction level thus determined was 3.3
.mu.mol/g-particles, which confirmed the introduction of PC groups
onto the particle surface.
Example 3
"Affinity Particles (Af Particles (C)) That are Inorganic Particles
Having Phosphorylcholine Groups and Amino Groups Covalently Bonded
Onto Their Surface"
[0155] 250 .mu.l of a methanol solution containing 45 .mu.mol of
the compound of formula (11) prepared in Synthesis example 4 and 50
.mu.l of a dimethylformamide solution containing 5 .mu.mol of
3-aminotrimethoxysilane were sampled, to which 47.5 ml of
dimethylformamide and 2.5 ml of distilled water were added, and
then 5 g of silica gel having an average particle size of 1.5 .mu.m
and a specific surface area of 6 m.sup.2/g was added. This particle
dispersion was refluxed at 160.degree. C. overnight for coupling.
After the refluxing, the particles were cleaned by means of
centrifugation using methanol to obtain the affinity particles of
claim 1 (hereafter "Af particles (C)"). FIG. 4 shows the P
quantification measurements of the Af particles (C) prepared in the
aforementioned procedure using the surface modifier of Synthetic
example 4. The PC introduction level thus determined was 6.3
.mu.mol/g-particles, which confirmed the introduction of PC groups
onto the particle surface.
"Evaluation of the Selectivity of Affinity Particles, Part 1"
[0156] Next, the affinity separation method shown in claim 6 is
described. 25 mg each of Af particles (A), (B), and (C) prepared in
Examples 1, 2, and 3 were sampled, to each of which 1 ml of
distilled water was added, followed by one minute of ultrasonic
treatment. After removing the distilled water by means of
centrifugation, 1 ml of a glutaraldehyde solution (8%) and 10 mg of
sodium cyanotrihydroborate, for stabilizing the Schiff base, were
added to these affinity particles and the reaction was carried out
for 5 hours at room temperature, followed by a
centrifugation/purification (5,000 g) operation 5 times using MQ
water for cleaning. The affinity particles of claim 2 that have
glutaraldehyde as reactive groups or adsorptive groups to which the
ligand can bind were thus obtained. 1 ml of bovine albumin (1
mg/ml) or human hemoglobin (1 mg/ml) and 10 mg of sodium
trihydroborate were added and the reaction was carried out for 1
day at room temperature, followed by 4 times of a
centrifugation/purification (5,000 g) with PBS. This bovine albumin
or human hemoglobin is the ligand. After this is the affinity
separation method shown in claim 7. 1 ml of ethanolamine
hydrochloride (0.5 M, pH 7.1) and 10 mg of sodium trihydroborate
were added and the reaction was carried out for 1 hour at room
temperature to deactivate the remaining glutaraldehyde, followed by
4 times of a centrifugation/purification (5,000 g) with PBS to
obtain the affinity particles of claim 3. 1 ml of HRP-conjugated
anti-bovine albumin antibody (10 .mu.g/ml) or HRP-conjugated human
hemoglobin antibody (10 .mu.g/ml) was added and the reaction was
carried out for 1 hour at room temperature, followed by 5 times of
a centrifugation/purification (5,000 g) with PBS. An additional 1
ml of PBS was added, followed by stirring; 10 .mu.l each was
transferred onto a 96-well plate and a color development test was
conducted using substrate TMBZ; the measurement was done at 450 nm.
The results are shown in FIG. 9, FIG. 10, and FIG. 11. Af particles
(A) exhibited selectivity for human hemoglobin-HRP conjugated
anti-human hemoglobin antibody. Also, Af particles (B) and Af
particles (C) exhibited selectivity for both bovine albumin-HRP
conjugated anti-bovine albumin antibody and human hemoglobin-HRP
conjugated anti-human hemoglobin antibody.
"Evaluation of the Selectivity of Affinity Particles, Part 2"
[0157] Goat anti-serum for human hemoglobin was used to conduct a
selectivity test. 25 mg each of Af particles (A) and (C) prepared
in Examples 1 and 3 were sampled, to each of which 1 ml of
distilled water was added, followed by one minute of ultrasonic
treatment. After removing the distilled water by means of
centrifugation, 1 ml of a glutaraldehyde solution (8%) and 10 mg of
sodium cyanotrihydroborate, for stabilizing the Schiff base, were
added to these affinity particles and the reaction was carried out
for 5 hours at room temperature, followed by a
centrifugation/purification (5,000 g) operation 5 times using MQ
water for cleaning. The affinity particles of claim 2 that have
glutaraldehyde as reactive groups or adsorptive groups to which the
ligand can bind were thus obtained. 1 ml human hemoglobin (1 mg/ml)
and 10 mg of sodium trihydroborate were added and the reaction was
carried out for 1 day at room temperature, followed by
centrifugation/purification (5,000 g) 4 times with PBS. This human
hemoglobin is the ligand. After this is the affinity separation
method shown in claim 7. 1 ml of ethanolamine hydrochloride (0.5 M,
pH 7.1) and 10 mg of sodium trihydroborate were added and the
reaction was carried out for 1 hour at room temperature to
deactivate the remaining glutaraldehyde, followed by a
centrifugation/purification (5,000 g) 4 times with PBS to obtain
the affinity particles of claim 3. 1 ml of goat anti-serum, diluted
100 times, was then added, followed by a one hour reaction at room
temperature. Centrifugation (5,000 g) was then conducted and the
supernatant was sampled (supernatant fraction).
[0158] Centrifugation/purification (5,000 g) was conducted 5 times
using PBS. 1 ml of a Gly-HCl buffer (0.2M, pH 2.5) was then added
and the reaction was carried out for one hour at room temperature
to elute the anti-human hemoglobin antibody and centrifugation
(5,000 g) was conducted to obtain the supernatant (elution
fraction). This supernatant fraction and elution fraction were fed
to SDS-PAGE, followed by silver staining; the results are shown in
FIG. 12. Both Af particles (A) and (C) exhibit a dark band of the
heavy chain of the antibody but no other bands in the elution
fraction, which indicates that the antibody was captured highly
selectively.
"Evaluation of the Selectivity of Affinity Particles, Part 3"
[0159] The goat anti-serum mixed with anti-human hemoglobin was
used to conduct a selectivity test. 25 mg each of Af particles (A)
and (C) prepared in Examples 1 and 3 were sampled, to each of which
1 ml of distilled water was added, followed by one minute of
ultrasonic treatment. After removing distilled water by means of
centrifugation, 1 ml of a glutaraldehyde solution (8%) and 10 mg of
sodium cyanotrihydroborate, for stabilizing the Schiff base, were
added to these affinity particles and the reaction was carried out
for 5 hours at room temperature, followed by a
centrifugation/purification (5,000 g) operation 5 times using MQ
water for cleaning. The affinity particles of claim 2 that have
glutaraldehyde as reactive groups or adsorptive groups to which the
ligand can bind were thus obtained. 1 ml human hemoglobin (1 mg/ml)
and 10 mg of sodium trihydroborate were added and the reaction was
carried out for 1 day at room temperature, followed by a
centrifugation/purification (5,000 g) 4 times with PBS. This human
hemoglobin is the ligand. After this is the affinity separation
method shown in claim 7. 1 ml of ethanolamine hydrochloride (0.5 M,
pH 7.1) and 10 mg of sodium trihydroborate were added and the
reaction was carried out for 1 hour at room temperature to
deactivate the remaining glutaraldehyde, followed by a
centrifugation/purification (5,000 g) 4 times with PBS to obtain
the affinity particles of claim 3. 1 ml of goat anti-serum, diluted
100 times and mixed with 50 .mu.g of anti-human hemoglobin, was
then added, followed by a one hour reaction at room temperature.
Centrifugation (5,000 g) was then conducted and the supernatant was
sampled (supernatant fraction).
[0160] Centrifugation/purification (5,000 g) was conducted 5 times
using PBS. 1 ml of a Gly-HCl buffer (0.2M, pH 2.5) was then added
and the reaction was carried out for one hour at room temperature
to elute the anti-human hemoglobin antibody and centrifugation
(5,000 g) was conducted to obtain the supernatant (elution
fraction). This supernatant fraction and elution fraction were fed
to SDS-PAGE, followed by silver staining; the results are shown in
FIG. 13. Both Af particles (A) and (C) exhibit a dark band of the
heavy chain of the antibody but no other bands are faint, which
indicates that the antibody was captured highly selectively. The
band darkness indicates that the antibody capture level is 10-20
.mu.g. The antibody activity of the elution fractions were
confirmed by means of sandwich ELISA; the activity was equivalent
to 13 .mu.g for Af particles (A) and 10.1 .mu.g for Af particles
(C).
Comparative Example 1
"Affinity Particles (Amino Particles) That are Inorganic Particles
Having Amino Groups Covalently Bonded Onto Their Surface"
[0161] 500 .mu.l of a methanol solution containing 50 .mu.mol of
3-aminopropyltrimethoxysilane was sampled, to which 47.5 ml of
methanol and 2.5 ml of distilled water were added, and then 5 g of
silica gel having an average particle size of 1.5 .mu.m and a
specific surface area of 6 m.sup.2/g was added. This particle
dispersion was refluxed at 80.degree. C. overnight for coupling.
After refluxing and then cleaning by means of centrifugation using
methanol, the amino particles were obtained. 1 ml of distilled
water was added to 25 mg of these amino particles, followed by one
minute of ultrasonic treatment. After removing distilled water by
means of centrifugation, 1 ml of a glutaraldehyde solution (8%) and
10 mg of sodium cyanotrihydroborate, for stabilizing the Schiff
base, were added to these affinity particles and the reaction was
carried out for 5 hours at room temperature, followed by a
centrifugation/purification (5,000 g) operation 5 times using MQ
water for cleaning. Glutaraldehyde is the reactive group or
adsorptive group that can bind to the ligand. 1 ml of bovine
albumin (1 mg/ml) or human hemoglobin (1 mg/ml) and 10 mg of sodium
trihydroborate were added and the reaction was carried out for 1
day at room temperature, followed by centrifugation/purification
(5,000 g) 4 times with PBS. This bovine albumin or human hemoglobin
is the ligand. 1 ml of ethanolamine hydrochloride (0.5 M, pH 7.1)
and 10 mg of sodium trihydroborate were added and the reaction was
carried out for 1 hour at room temperature to deactivate the
remaining glutaraldehyde, followed by centrifugation/purification
(5,000 g) 4 times with PBS to obtain the affinity particles of
claim 3. 1 ml of HRP-conjugated anti-bovine albumin antibody (10
.mu.g/ml) or HRP-conjugated human hemoglobin antibody (10 .mu.g/ml)
was added and the reaction was carried out for 1 hour at room
temperature, followed by centrifugation/purification (5,000 g) 5
times with PBS. An additional 1 ml of PBS was added, followed by
stirring; 10 .mu.l each was transferred onto a 96-well plate and a
color development test was conducted using substrate TMBZ; the
measurement was done at 450 nm. The results are shown in FIG. 14.
There is a high level of non-specific adsorption of proteins and
the selectivity is low.
INDUSTRIAL APPLICABILITY
[0162] The affinity particles of the present invention capture only
the target protein that is desired to be separated and therefore
they exhibit very high selectivity. They also exhibit superior
dispersion properties and make separation from liquid samples very
easy. Since a target substance can be separated easily with high
accuracy using affinity particles made from inexpensive inorganic
particles, the present invention is useful in bio-industries where
a highly accurate separation and detection of the target substance
is required.
* * * * *