U.S. patent application number 12/933818 was filed with the patent office on 2011-04-21 for method for quantification of soluble lr11.
This patent application is currently assigned to Sekisui Medical Co., Ltd.. Invention is credited to Hideaki Bujo, Hiroyuki Ebinuma, Isamu Fukamachi, Masanao Matsuo, Yasushi Saito.
Application Number | 20110091993 12/933818 |
Document ID | / |
Family ID | 41090683 |
Filed Date | 2011-04-21 |
United States Patent
Application |
20110091993 |
Kind Code |
A1 |
Matsuo; Masanao ; et
al. |
April 21, 2011 |
METHOD FOR QUANTIFICATION OF SOLUBLE LR11
Abstract
Provided is a method for quantifying soluble LR11 in a
biological sample such as serum by an immunological means
conveniently and accurately without the need of carrying out any
complicated separation manipulation. An immunological
quantification method for soluble LR11 in a sample derived from a
mammal, including a step of treating the sample with at least one
surfactant selected from a group consisting of a polyoxyalkylene
alkyl ether, a polyoxyalkylene alkyl phenyl ether, an alkyl
glycoside, an alkylthio glycoside, an acyl-N-methylglucamide and a
salt of cholic acid.
Inventors: |
Matsuo; Masanao; (Ibaraki,
JP) ; Ebinuma; Hiroyuki; (Ibaraki, JP) ;
Fukamachi; Isamu; (Ibaraki, JP) ; Bujo; Hideaki;
(Chiba, JP) ; Saito; Yasushi; (Chiba, JP) |
Assignee: |
Sekisui Medical Co., Ltd.
Tokyo
JP
|
Family ID: |
41090683 |
Appl. No.: |
12/933818 |
Filed: |
March 17, 2009 |
PCT Filed: |
March 17, 2009 |
PCT NO: |
PCT/JP2009/001176 |
371 Date: |
September 21, 2010 |
Current U.S.
Class: |
436/501 |
Current CPC
Class: |
C07K 16/28 20130101;
G01N 33/92 20130101; G01N 1/34 20130101 |
Class at
Publication: |
436/501 |
International
Class: |
G01N 33/566 20060101
G01N033/566; G01N 33/68 20060101 G01N033/68 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 21, 2008 |
JP |
2008-073047 |
Claims
1. An immunological method for quantifying soluble LR11 in a sample
derived from a mammal, comprising a step of treating the sample
with at least one surfactant selected from a group consisting of a
polyoxyalkylene alkyl ether, a polyoxyalkylene alkyl phenyl ether,
an alkyl glycoside, an alkyl thioglycoside, an
acyl-N-methylglucamine and a salt of cholic acid.
2. The immunological method for quantifying according to claim 1,
wherein the sample is blood, plasma, serum, cerebrospinal fluid or
urine.
3. The immunological method for quantifying according to claim 1 or
2, wherein the immunological method for quantifying further
comprises a step of forming an immunocomplex by using at least two
kinds of antibodies each having an antigen recognition site which
is different from each other.
4. The immunological method for quantifying according to any one of
claims 1 to 3, wherein the surfactant is
acyl-N-methylglucamine.
5. A kit for immunological quantification of soluble LR11,
comprising an anti-soluble LR11 antibody and at least one
surfactant selected from a group consisting of a polyoxyalkylene
alkyl ether, a polyoxyalkylene alkyl phenyl ether, an alkyl
glycoside, an alkyl thioglycoside, an acyl-N-methylglucamine and a
salt of cholic acid.
6. A use of a combination comprising an anti-soluble LR11 antibody
and at least one surfactant selected from a group consisting of a
polyoxyalkylene alkyl ether, a polyoxyalkylene alkyl phenyl ether,
an alkyl glycoside, an alkyl thioglycoside, an
acyl-N-methylglucamine and a salt of cholic acid for manufacturing
a kit for immunological quantification of soluble LR11.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method for quantifying
soluble LR11 in a sample derived from a mammal.
BACKGROUND ART
[0002] LDL receptor relative with 11 ligand-binding repeats (LR11)
is a LDL receptor-analogous protein having a structure specific to
LDL receptor family (Patent Document 1 and Non-Patent Document 1).
It is known that, although hardly expressed in cells of normal
blood vessel wall, the protein is recognized to be specifically
expressed in thickened intimal smooth muscle cells (Non-Patent
Document 2). It is also known that, according to the study using
cultured smooth muscle cells, expression amount of LR11 increases
in accordance with the proliferation of the smooth muscle cells, so
that secretion of LR11 into a culture medium is recognized, and
when LR11 gene is functionally impaired in a mouse model having
defective cuff by using development engineering method, thickening
of vascular intima caused by migration and proliferation of smooth
muscle cells is inhibited (Non-Patent Document 3). Furthermore, it
is recently found that, in addition to membrane-bound type LR11, a
soluble LR11 which is fragmentized by a protease exists (Non-Patent
Document 4). Still furthermore, it is reported that the soluble
LR11 also exists in human blood (Non-Patent Document 5).
[0003] As a method of quantifying soluble LR11, a method in which
soluble LR11 in a sample is separated by using an insoluble carrier
to which a chaperone molecule RAP (receptor associated protein)
having an affinity for LR11 is bonded and then the protein is
subjected to SDS-PAGE and Western blot followed by detection based
on immunostaining using an anti-LR11 antibody, etc. is known
(Non-Patent Documents 5 and 6). However, the method requires a
process of separating soluble LR11 from a sample, and therefore
operation of the process is complicated.
Prior Art Document
[0004] Patent Document 1: Japanese Patent Application Laid-Open
(JP-A) No. H9-163988
[0005] Non-Patent Document 1: J. Biol. Chem. 1996; 271, p
24761-24768
[0006] Non-Patent Document 2: Arterioscler. Thromb. Vasc. Biol.
1999; 19, p 2687-2695
[0007] Non-Patent Document 3: Circ. Res. 2004; 94; p 752-758
[0008] Non-Patent Document 4: IGAKU NO AYUMI, Vol. 221, No. 13, p
1200-1203
[0009] Non-Patent Document 5: J Clin Invest. 2008; 118, p
2733-2746
[0010] Non-Patent Document 6: The 39.sup.th Annual Scientific
Meeting of the Japan Atherosclerosis Society--Abstract, General
lecture subject (poster) 189, p 264
DISCLOSURE OF THE INVENTION
Problem to be Solved by the Invention
[0011] Furthermore, the inventors of the present invention found
that blood concentration of soluble LR11 is significantly higher in
a patient suffering from arteriosclerotic disorder compared to a
normal person, and reported that the measurement of the blood
concentration of soluble LR11 could be a new marker for an
arteriosclerotic disorder (Non-Patent Document 5 and Japanese
Patent Application No. 2007-160225). For such diagnosis,
development of a method for quantification of soluble LR11 by
simple manipulation has been desired.
[0012] However, when an immunological measurement is carried out by
using an antibody which reacts with soluble LR11 in a body fluid,
for example in a system wherein a serum component is present,
specifically, in serum or plasma as a subject, it was found that
accurate quantification of soluble LR11 cannot be achieved due to
influence of certain interfering substances.
[0013] Thus, object of the present invention is to provide a method
for quantifying soluble LR11 in a biological sample such as serum
by an immunological means conveniently and accurately without any
complicated separating manipulation.
Means for Solving the Problem
[0014] Therefore, in order to accurately quantify soluble LR11 in
body fluid, for example, even in a case where serum or plasma is
taken as a sample for direct measurement, the inventors of the
present invention studied various means for preventing the
influence of impeding substances. As a result, it was found that,
when a sample is treated with a certain kind of surfactant and then
measurement is carried out based on an immunological measurement
method, soluble LR11 in the sample can be quantified conveniently
and accurately, and therefore the present invention was
completed.
[0015] Specifically, the present invention provides an
immunological method for measuring soluble LR11 in a sample derived
from a mammal, including a step of treating the sample with at
least one surfactant selected from a group consisting of a
polyoxyalkylene alkyl ether, a polyoxyalkylene alkyl phenyl ether,
an alkyl glycoside, an alkyl thioglycoside, an
acyl-N-methylglucamide and a salt of cholic acid.
[0016] Furthermore, the present invention provides a kit for
immunological measurement of soluble LR11, including an
anti-soluble LR11 antibody and at least one surfactant selected
from a group consisting of a polyoxyalkylene alkyl ether, a
polyoxyalkylene alkyl phenyl ether, an alkyl glycoside, an
alkylthio glycoside, an acyl-N-methylglucamine and a salt of cholic
acid.
[0017] Furthermore, the present invention provides a use of a
combination of an anti-soluble LR11 antibody and the specific
surfactant described above for the manufacture of a kit for
immunological measurement of soluble LR11.
Effect of the Invention
[0018] Based on the quantification method and with the
immunological measurement kit of the present invention,
concentration of soluble LR11 present in body fluid, for example,
blood, can be conveniently measured with high sensitivity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 shows a reaction of each monoclonal antibody against
immobilized soluble LR11.
[0020] FIG. 2 shows a measurement of purified human soluble LR11 by
sandwich ELISA.
[0021] FIG. 3 shows a measurement of purified rabbit soluble LR11
by sandwich ELISA.
[0022] FIG. 4 shows an influence of serum in ELISA system.
[0023] FIG. 5 shows avoidance of influence of serum components by
treatment with surfactant.
[0024] FIG. 6 shows a concentration of the surfactant (MEGA-9)
effective for removing influence of the serum components.
[0025] FIG. 7 shows a sandwich ELISA using the immobilized
monoclonal antibody A2-2-3.
[0026] FIG. 8 shows a sandwich ELISA using the immobilized
monoclonal antibody M3.
[0027] FIG. 9 shows a sandwich ELISA using the immobilized
monoclonal antibody M5.
[0028] FIG. 10 shows a sandwich ELISA using the immobilized
monoclonal antibody R14.
[0029] FIG. 11 shows a sandwich ELISA using the immobilized
monoclonal antibody R23.
[0030] FIG. 12 shows a measurement of soluble LR11 inhuman
serum.
[0031] FIG. 13 shows a measurement of soluble LR11 in serum of
various mammals (A: treated with MEGA-9, B: not treated).
[0032] FIG. 14 shows a measurement of soluble LR11 in human
cerebrospinal fluid (A: treated with MEGA-9, B: not treated).
BEST MODE FOR CARRYING OUT THE INVENTION
[0033] The quantification method of the present invention is a
method of immunological quantification of soluble LR11 in a sample
derived from a mammal. As for the mammal, a mammal including human
such as human, monkey, horse, cow, pig, rabbit, rat, guinea pig,
mouse and the like can be mentioned. As for the sample, a sample
from a living body containing therein impeding substances (such as
serum components) can be mentioned. Examples of the sample include
blood, plasma and serum, as well as spinal fluid and urine.
[0034] The immunological quantification method of the present
invention is not specifically limited as long as it is a method of
quantifying soluble LR11 based on the reaction between an antigen
and an antibody. However, from the viewpoint of avoiding influence
of impeding substances in body fluid such as serum components, a
quantification method using at least two kinds of antibody is
preferable. In particular, a quantification method including a step
of forming an immunocomplex by using at least two kinds of
antibodies each having an antigen recognition site which is
different from each other is preferable. According to the present
invention, examples of the quantification method including a step
of forming an immunocomplex by using at least two kinds of
antibodies each having an antigen recognition site which is
different from each other include sandwich ELISA or latex
turbidimetric immunoassay (LTIA), etc.
[0035] Any kind of antibody including a monoclonal antibody and a
polyclonal antibody may be used as the anti-soluble LR11 antibody,
as long as it reacts with a soluble LR11 purified from serum.
Particularly, the monoclonal antibody is preferably used.
Preparation of the antibody may be carried out according the method
known per se in the art.
[0036] For the preparation of a polyclonal antibody, a mouse, a
rat, a hamster, a rabbit, a goat, a sheep, a chicken and the like
are used as an animal to be immunized. The antiserum may be
obtained after subcutaneously, intradermally or intraperitoneally
administering an antigen to the animal once or several times. When
a protein or a peptide is used as an antigen, immunization with a
mixture containing therein fluid replacement which has an
immunostimulating effect is more preferable.
[0037] Furthermore, preparation of a monoclonal antibody may be
carried out according to a known method of preparing a monoclonal
antibody, for example, in view of "Monoclonal Antibody" (written by
Nagamune Hideaki and Terada Hiroshi, Hirokawa, 1990) or "Monoclonal
Antibody" (James W. Golding, 3rd edition, Academic Press, 1996). In
addition, the monoclonal antibody may be also prepared by DNA
immunization method, and may be prepared in view of the literatures
(Nature 1992 March 12; 356, p 152-154 or J. Immunol Methods March
1; 249, p 147-154).
[0038] As for the antigen that is used for the preparation of an
antibody, LR11 protein or its partial fragment (i.e., peptide), or
a vector containing cDNA which encodes LR11 protein or its partial
fragment may be used. Examples of the partial fragment include the
amino acid sequence that is represented by SEQ ID No. 1 or SEQ ID
No. 2 described below can be mentioned. The amino acid sequence may
include one or several deletions, substitutions or additions of the
amino acids.
[0039] In order to obtain the monoclonal antibody which recognizes
the conformation of LR11, a full-length LR11 vector, which is a
construct including the full-length human LR11 gene, is the most
appropriate antigen gene for immunization. However, other than
that, a construct into which a gene encoding a partial fragment
(i.e., peptide) of the LR11 protein is inserted may be also used as
an antigen gene for immunization. DNA immunization may be carried
out by subcutaneously injecting the gene construct described above
singly or as a mixture to an animal (mouse or rat, etc.) based on
any method of various gene transfer methods for immunizing an
animal (for example, muscle injection, electroporation, or a method
using gene gun, etc.) and by allowing the construct to get
intracellularly transferred.
[0040] An example of preparing a monoclonal antibody using a
peptide antigen is shown below as a reference.
[0041] (1) Production of a Hybridoma
[0042] To the C-terminal of a synthetic peptide containing a
partial amino sequence [432-447] of LR11 (SMNEENMRSVITFDKG) (SEQ ID
No. 1), a cysteine residue is introduced. By using a cross-linking
agent NBS (manufactured by Pierce Company), the synthetic peptide
and KLH are linked to each other via lysine residue of KLH (Keyhole
lympet hemocyanin) to provide an immunogen. This immunogen was
mixed and emulsified with Complete Freund's Adjuvant (manufactured
by GIBCO) in the ratio of 1:1 to give an emulsion of 0.1 mg/0.1 mL,
and the emulsion was then subcutaneously administered 10 times with
1 week interval to a 6-week old female BALB/C mouse. Three days
after the final immunization, the spleen was harvested. The spleen
cells obtained from the harvested spleen and the SP2/O-Ag14 myeloma
cells were admixed with each other with the ratio of 6:1. Then, in
the presence of cell fusion reagent such as PEG/DMSO Solution
(manufactured by Sigma), etc., cell fusion was carried out. The
fused cells were suspended in HAT medium to have the spleen cell
concentration of 2.5.times.10.sup.6/mL, and then aliquoted (0.2 mL
each) to a 96-well culture plate (manufactured by Corning). The
cells were cultured in an incubator under 5% CO.sub.2 at 37.degree.
C. Approximately two weeks later, the culture supernatant
containing the grown hybridoma was subjected to ELISA shown below
to determine the presence or absence of the antibody against the
synthetic peptide (SEQ ID No. 1) described above. Consequently, a
hybridoma cell line having higher possibility of producing the
antibody was selected.
[0043] Specifically, the synthetic peptide (SEQ ID No. 1) described
above was immobilized onto a micro plate (manufactured by NUNC).
IgG contained in each culture supernatant was added to the plate
for the reaction, and consequently allowed to react with anti-mouse
IgG goat antibody labeled with peroxidase. Subsequently, a
peroxidase substrate solution containing ortho-phenylene diamine
(manufactured by Tokyo Chemical industry, Co., Ltd.) was added for
a color development reaction, and the color development was then
stopped by adding 1.5N sulfuric acid. The absorbance was measured
by using micro plate reader (Abs. 492 nm). Accordingly, the
hybridoma cell line showing the reactivity to the synthetic peptide
was selected and the cloning was carried out based on a limiting
dilution method. The final reactivity for LR11 was confirmed by
reaction with the soluble LR11 which has been purified from serum
as described below. As a result, anti-human soluble LR11 monoclonal
antibody-producing hybridoma (A2-2-3) was established.
[0044] (2) Preparation of a Monoclonal Antibody
[0045] To a 12-week old female BALB/C mouse which had been
intraperitoneally injected with 0.5 mL pristane two weeks before,
the hybridoma cells obtained above were intraperitoneally
administered (0.5.times.10.sup.6 cells). About 14 days later,
ascites was collected and the supernatant was obtained by
centrifuge. The supernatant was admixed with the same volume of the
buffer solution for adsorption (3 mol/L NaCl-1.5 mol/L
Glycine-NaOH, pH 8.5), and then filtered. The filtered solution was
applied to the Protein A column (manufactured by GE Healthcare Bio
Science), which had been equilibrated with the buffer solution for
adsorption, to allow the adsorption of the antibody to the column.
Thereafter, the antibody was eluted from the column by using 0.1
mol/L citric acid buffer (pH 3.0) to purify the anti-LR11
monoclonal antibody (A2-2-3 antibody).
[0046] In addition, an example of preparing a monoclonal antibody
based on DNA immunization is given below as a reference.
[0047] (1) Construction of an Expression Vector
[0048] A partial gene fragment consisting of the full length LR11
gene (Q92673) (i.e., a gene encoding the peptide consisting of the
amino acid sequence of LR11 [1000-1550] (SEQ ID No. 2)) was
incorporated to a mammalian expression vector tagged with FLAG
(pcDNA3.1, manufactured by Invitrogen). The expression vector
contains a DNA which encodes the peptide consisting of GPI anchor
sequence originating from human alkaline phosphatase. The resulting
vector was named LR11 [1000-1550] vector.
[0049] (2) Determination of the Expression Product from CHO
[0050] By using the LR11 [1000-1550] vector constructed in the
above, expression of a target gene product on the surface of cell
membrane as intended was determined based on transient transfection
expression test using CHO cells (originating from Chinese hamster
ovary) before immunization. Specifically, one day before the test,
the cells were plated to a 6-well plate (1.times.10.sup.6 cells per
well). Under 5% CO.sub.2, 37.degree. C. condition, the cells were
incubated overnight. On the transfection day, in a polystyrene
round tube, a plasmid dilution solution (3 .mu.g plasmid DNA+500
.mu.L Dulbecco's Modified Eagle Medium (D-MEM)) and a diluted
solution of lipofectamine2000 (9 .mu.L Lipofectamine2000+500 .mu.L
D-MEM) were mixed well with each other. After the incubation at
room temperature for 20 minutes, the culture supernatant of the
cells plated a day before was discarded and the above mixture was
gently added to the cells while being careful not to separate the
cells from the plate. After the incubation under 5% CO.sub.2, at
37.degree. C. for 5 hours, the supernatant was removed and D-MEM
medium containing 5% FCS was added. Then, the cells were cultured
again at 5% CO.sub.2, 37.degree. C. condition for 24 hours.
[0051] Next day, the cells were removed from the plate by using a
dissociation buffer (manufactured by Invitrogen) and used for the
analysis by flow cytometry (FCM). FCM analysis was carried out as
follows. Specifically, for primary antibody reaction, the cells
were reacted with ANTI-FLAG (trade name) M2 antigen (SIGMA) in PBS
buffer containing 3% FCS for 30 minutes at 4.degree. C. The
secondary antibody was washed with PBS buffer containing 3% FCS,
and reacted with the PE-labeled anti-mouse IgG antibody
(manufactured by Beckman) in PBS buffer containing 3% FCS for 30
minutes at 4.degree. C. The cells were then washed with PBS buffer
containing 3% FCS and suspended in an appropriate amount of PBS
buffer containing 3% FCS. The resultant was supplied to a flow
cytometer.
[0052] As a result, it was confirmed that, with the LR11
[1000-1550] vector that has been established as described above,
the target gene product is indeed expressed on the cell
surface.
[0053] (3) Antibody Production Based on DNA Immunization
[0054] Regarding the DNA immunization, gold particle-sensitized
LR11 [1000-1550] vector described in section (1), either alone or
in combinations thereof, was subcutaneously introduced to an animal
to be immunized (mouse or rat) using a gene gun to transfer the
vector into cells. Specifically, by using Helios (trade name) Gene
Gun Optimization Kit (manufactured by Bio-Rad), gold
particle-sensitized LR11 [1000-1550] vector (200 .mu.g vector per
25 mg gold particles) was administered according to the
manufacturer's instructions enclosed in the kit. The immunization
was carried out for 4 times with 2-week intervals. At the time of
the 4th immunization, a small amount of antiserum was sampled. The
antiserum which had been diluted 1,000-fold with PBS containing 3%
FCS was used as a primary antibody for the FCM analysis. In this
case, the CHO cells which transiently express the target gene
product described in the above section (2) (herein below, also
referred to as cells for forced expression) were used for the FCM
analysis. As a result, an increase in the antibody value was
confirmed. Further, the production of monoclonal antibody was
carried out according to a general cell fusion method.
Specifically, after performing twice the final boost, the immunized
animal was sacrificed and the antibody-producing cells were
isolated according to a standard protocol, and the cells were
subjected to the cell-fusion with mouse myeloma cells. As a result,
the antibody-producing hybridoma cell line was prepared. After
culturing this hybridoma cells, part of the culture supernatant was
taken and an enzyme immunoassay using the cells for forced
expression and FCM analysis were carried out. Consequently, the
hybridoma cell lines which have a response to the antigen protein
but not to the non-antigenic protein were selected.
[0055] In addition, the enzyme immunoassay using the cells for
forced expression was carried out as follows. The cells for forced
expression were coated on a 96-well plate and reacted with the
culture supernatant of the hybridoma as a primary antibody. After
the reaction with the primary antibody, the plate was washed and
added with a secondary antibody. Herein, the secondary antibody
indicates an antibody which can recognize the mouse immunoglobulin
or the rat immunoglobulin of the primary antibody and corresponds
to an antibody that is labeled with horse radish peroxidase (HRP).
After the reaction, a fluorescent substrate for the enzyme that is
labeled on the secondary antibody was added and the analysis was
carried out using a fluorescence measurement plate reader.
[0056] Subsequently, the cloning was carried out for the selected
hybridoma cell lines based on a limiting dilution method. As a
result, the cell line recognized to be stable and to have high
antibody value was selected as a hybridoma cell line for producing
a monoclonal antibody.
[0057] Next, mass production of the monoclonal antibody from the
ascites was carried out as follows. To a nude mouse which had been
previously treated with 0.5 mL pristane, 0.5 mL phosphate buffered
physiological saline (pH 7.4) containing the cloned hybridoma cells
(1.times.10.sup.6 to 3.times.10.sup.6 cells) was intraperitoneally
injected. Approximately two weeks later, ascites was collected and
the monoclonal antibody was purified by affinity column using
Protein A.
[0058] Accordingly, the anti-soluble LR11 mouse monoclonal antibody
(M3) and (M5) and the anti-LR11 rat monoclonal antibody (R14) and
(R23) were obtained by DNA immunization. The reactivity for LR11
was finally confirmed based on the reactivity for the soluble LR11
that had been purified from the serum as described below. The above
describes one example showing the production of a monoclonal
antibody by DNA immunization.
[0059] According to the method of the present invention,
immunological quantification is carried out after treating a sample
with a specific kind of surfactant. With such treatment, influence
of the substances which have a negative effect on quantification of
soluble LR11 contained in a sample can be avoided.
[0060] The surfactant should be at least one substance selected
from a group consisting of polyoxyalkylene alkyl ether, a
polyoxyalkylene alkyl phenyl ether, an alkyl glycoside, an
alkylthio glycoside, an acyl-N-methylglucamine and a salt of cholic
acid.
[0061] Among these, polyoxyalkylene alkyl ether, polyoxyalkylene
alkyl phenyl ether, alkyl glycoside, alkyl thioglycoside and
acyl-N-methylglucamine are more preferable. Acyl-N-methylglucamine
is particularly preferable.
[0062] As for the polyoxyalkylene alkyl ether and polyoxyalkylene
alkylphenyl ether, polyoxyethylene C.sub.8-C.sub.24 alkyl ether and
polyoxyethylene C.sub.8-C.sub.24 alkylphenyl ether are preferable.
Specific examples include polyoxyethylene octyl ether,
polyoxyethylene decyl ether, polyoxyethylene dodecyl ether,
polyoxyethylene octyl phenyl ether, polyoxyethylene decyl phenyl
ether, and polyoxyethylene dodecyl phenyl ether. Herein, the number
of polyoxyethylene is preferably from 4 to 40. As a commercially
available product of polyoxyethylene (10) octylphenyl ether, Triton
X-100 (trade name) can be mentioned.
[0063] As for the alkylglycoside and alkylthioglycoside,
C.sub.8-C.sub.24 alkylglucoside, C.sub.8-C.sub.24 alkylmaltoside,
C.sub.8-C.sub.24 alkylthioglucoside and C.sub.8.sup.-C.sub.24
alkylthiomaltoside are preferable. Specific examples include
n-octyl-.beta.-D-glucoside, n-octyl-.alpha.-D-glucoside,
n-octyl-.beta.-D-maltoside, n-decyl-.beta.-D-maltoside,
n-decyl-.beta.-D-maltoside, n-heptyl-.beta.-D-thioglucoside,
n-octyl-.beta.-D-thioglucoside and n-nonyl-.beta.-D-thiomaltoside.
Among these, n-octyl-.beta.-D-glucoside is preferable. As a
commercially available product, n-octyl-.beta.-D-glucoside
(manufactured by Dojindo Laboratories), etc. can be mentioned. As
for the cholic acid salt, examples include sodium cholate and
potassium cholate.
[0064] As for the acyl-N-methylglucamine, C.sub.8-C.sub.24
alkanoyl-N-methylglucamine is preferable. Specific examples include
octanoyl-N-methylglucamine, nonanoyl-N-methylglucamine and
decanoyl-N-methylglucamine. Among these, octanoyl-N-methylglucamine
or nonanoyl-N-methylglucamine is preferable. As a commercially
available product of octanoyl-N-methylglucamine and
nonanoyl-N-methylglucamine, MEGA-8 (manufactured by Dojindo
Laboratories) and MEGA-9 (manufactured by Dojindo Laboratories,
etc. can be mentioned, respectively.
[0065] As a means to treat a sample with a surfactant, the specific
surfactant described above maybe added to the sample before or
during the immunological measurement. Preferably, the surfactant is
added before the immunological measurement.
[0066] The use concentration of the surfactant may be appropriately
decided depending on the type of the surfactant used. For example,
it is 0.1 to 15% by mass, or preferably 2 to 10% by mass in a
sample. The treatment temperature is 5 to 40.degree. C., or
preferably 10 to 30.degree. C. The reaction time is 1 to 24 hrs, or
preferably 6 to 20 hrs including the time for the treatment with
the surfactant and the reaction between the antibody and the
treated sample.
[0067] In addition, the sample is preferably diluted 1 (undiluted
solution) to 50-fold, or preferably 4 to 30-fold. In this case, the
dilution is preferably carried out by using a neutral to alkaline
buffer solution having pH 7 to 10 of phosphate buffer, glycine
buffer, carbonate buffer, Tris buffer and the like.
[0068] With a sample added with the specific surfactant described
above, the immunological quantification using an anti-soluble LR11
antibody may be carried out using a general means.
[0069] As for the means of the immunological quantification,
examples include immunostaining (Western blot), enzyme linked
immunosorbent assay (ELISA), sandwich ELISA, immunoturbidimetry
(TIA or LTIA), enzyme immunoassay, chemiluminescence immunoassay
and fluorescence immunoassay.
[0070] When the soluble LR11 which reacts with the anti-soluble
LR11 antibody is measured quantitatively or semi-quantitatively, it
is preferable that results are compared to LR11 as a standard. As
for the LR11 which may be used as a standard, for example, the
serum-soluble LR11 with known concentration, the LR11 recovered
from cultured cells or culture supernatant of smooth muscle cell or
neuroblast cell line, a recombinant LR11, or a synthetic peptide
used as an immunogen for the antibody production, etc. may be
preferably used.
[0071] As an example of the immunological quantification described
above, quantification based on sandwich ELISA is given below. For
example, in an appropriate buffer solution, one type of the
anti-soluble LR11 monoclonal antibody is immobilized on an
insoluble carrier to give an immobilized antibody. As a secondary
antibody, another anti-soluble LR11 monoclonal antibody which
recognizes a recognition site different from the site that the
aforementioned anti-soluble LR11 monoclonal antibody recognizes is
labeled with an enzyme. By reacting them with a tested sample and
measuring the activity of the enzyme that is linked to the
secondary antibody, the soluble LR11 contained in the sample may be
measured. In addition, by using biotinylated anti-soluble LR11
monoclonal antibody as a secondary antibody, performing the
reaction with a tested sample and the reaction with enzyme-labeled
avidin, and measuring the activity of the labeled enzyme, the LR11
contained in the sample may be also measured.
[0072] Examples of the insoluble carrier used above are preferably
various synthetic polymers (e.g. polystyrene, polyethylene, and
polypropylene), glass, silicone and insoluble polysaccharides
(cross-linked dextran, polysaccharide), etc. These carriers may be
used in the form of a sphere, a rod, fine particles, a test tube, a
micro plate, etc. As for the condition for preparing an immobilized
antibody, when a carrier in the form of a sphere, a rod or in a
test tube or a micro plate is used, the concentration of the
antibody is 1 to 10 .mu.g/mL. When a carrier in the form of fine
particles is used, the concentration of the antibody is 1 to 10
mg/mL. Further, it is preferable that a neutral to alkaline buffer
solution having pH 7 to 10 of phosphate buffer, glycine buffer,
carbonate buffer, Tris buffer, etc. is used and sensitization is
performed at 4.degree. C. to 25.degree. C. for 1 to 72 hrs.
[0073] The enzyme-labeled antibody used may be prepared by a method
known per se in the art. For example, according to the method by
Nakane, et al, (Nakane P. K. et al, J. Histochem Cytochem, 22, p
1084-1089, 1974) or the method by Ishikawa, et al, (maleimide
method: "Enzyme Immunoassay, 3.sup.rd edition", IGAKU-SHOIN Ltd.),
etc., a non-fragmentized immunoglobulin molecule as it is, which
may be limitedly hydrolyzed by a suitable protease to give
F(ab').sub.2 or Fab' if necessary, is labeled with an enzyme, by
which the enzyme-labeled antibody may be prepared. Examples of the
enzyme which may be used for labeling include peroxidase, alkaline
phosphatase, .beta.-D-galactosidase and glucose oxidase.
[0074] Biotinylated antibody may be also prepared by a method known
per se in the art. However, by using a commercially available
reagent for biotinylation (for example, Sulfo-NHS-Biotinylation Kit
manufactured by Pierce Company), it may be more easily
prepared.
[0075] The enzyme-labeled avidin may be also prepared by a method
known per se in the art. However, a commercially available product
maybe also used (for example, StreptAvidin, Horseradish Peroxidase
Conjugated manufactured by Pierce Company).
[0076] Furthermore, when the labeling material is an enzyme, a
substrate and, if necessary, a color developing agent are used for
measuring the activity of the enzyme. When peroxidase is used as an
enzyme, hydrogen peroxide is used as a substrate, and as a color
developing agent, o-phenylenediamine, 3,3',5,
5'-tetramthylbenzidine, 2,2'-azinodi-[3-ethylbenzthiazolinesulfonic
acid] ammonium salt and the like may be used; when alkaline
phosphatase is used as an enzyme, p-nitrophenyl phosphate,
3-(4-methoxyspiro{1,2-dioxetane-3,2'-tricyclo[3.3.1.1.sup.3,7]decane}-4-y-
l)phenylphosphate: AMPPD, etc. may be used as a substrate; when
.beta.-D-galactosidase is used as an enzyme,
.beta.-D-galactopyranoside,
4-methylumbelliferyl-.beta.-D-galactopyranoside, etc. may be used
as a substrate, and; when glucose oxidase is used as an enzyme,
.beta.-D-glucose as a substrate in the co-presence of peroxidase
and a color developing agent for peroxidase as a color developing
agent may be used.
[0077] As shown in the Examples below, when a biological sample
containing impeding substances that are same as the serum
components is treated with the specific surfactant described above
and immunological measurement is carried out by using the antibody
which reacts with the soluble LR11, the soluble LR11 contained in
the sample can be conveniently and accurately quantified. Thus, for
carrying out the immunological measurement described above, the
present invention may provide a kit in which the anti-soluble LR11
antibody and the above-described specific surfactant are contained
in advance, i.e., a kit for the immunological measurement of the
soluble LR11 including the antibody and the surfactant. Further,
the anti-soluble LR11 antibody and the specific surfactant may be
used for the manufacture of a kit for the immunological measurement
of the soluble LR11.
[0078] The kit for the immunological measurement includes the
anti-soluble LR11 antibody and the specific surfactant. These
components may be present separately as a first reagent and a
second reagent, or present in a mixture state. Furthermore, the kit
may also include an optional component that is used for the
detection of the soluble LR11, for example, a buffer solution, a
stabilizing agent, or a reaction vessel, etc.
[0079] As described before, measurement of the soluble LR11 in
blood may be a marker for an arteriosclerotic disorder. Thus, the
kit may be used for evaluation of the presence or severity of the
arteriosclerotic disorder.
[0080] Herein, the term "arteriosclerotic disorder" is a concept
including disorders that are accompanied by progress of conditions
caused by an arteriosclerotic lesion. Specific examples of the
disorder include myocardial infarction, restenosis after
percutaneous transluminal coronary angioplasty, cerebral
infarction, cerebral hemorrhage, aneurysm, and arteriosclerosis
obliterans. However, the disorder is not limited to them.
[0081] Furthermore, the term "evaluation of the presence or
severity" is a concept that is not limited to a case in which an
arteriosclerotic disorder has been already developed, and it is a
concept also including a case in which possibility of future
development into the disorder is determined.
[0082] As for the method for the evaluation of the presence or
severity of the arteriosclerotic disorder, for example, it may be
carried out by obtaining a biological sample from a mammal with
suspected arteriosclerotic disorder, measuring the concentration of
the soluble LR11 therefrom as described above while separately
obtaining the concentration of the soluble LR11 in the same manner
from a normal mammal group having no suspected arteriosclerotic
disorder, and comparing those concentrations to evaluate the
presence or severity of an arteriosclerotic disorder.
[0083] Herein, the term "normal" indicates an individual having no
arteriosclerotic disorder. Regarding an objective indicator for an
arteriosclerotic disorder, in human, it indicates no previous
history of suffering from an arteriosclerotic disorder such as
coronary occlusion, etc. and, for example, a normal blood pressure
according to the "Year 2004 Guidelines for Treating Hypertension"
by the Japanese Hypertension Society (i.e., systolic blood pressure
of less than 130 mmHg and diastolic blood pressure of less than 85
mmHg); a normal type based on "Committee report regarding the
criteria for classification and diagnosis of diabetes (1999)" by
Japan Diabetes Society (i.e., fasting blood sugar level of less
than 110 mg/dl) and; no relation with the "Diagnostic criteria for
hyperlipidaemia (2002)" by Japan Atherosclerosis Society (i.e.,
regarding the lipid value obtained from serum taken when fasting,
total cholesterol is less than 220 mg/dl, LDL cholesterol is less
than 140 mg/dl, HDL cholesterol is 40 mg/dl or above, and
triglyceride is less than 150 mg/dl).
[0084] In addition, by monitoring a change in the concentration of
the soluble LR11 with time by using the kit of the present
invention, effectiveness of a treatment using a pharmaceutical
agent, etc. may be also determined.
[0085] The determination may be carried out based on a change in
the concentration of the soluble LR11. For example, when there is
an increasing tendency, it is determined that the disorder (for
example, an arteriosclerotic disorder) has progressed. On the other
hand, when there is a tendency showing a decrease or a flat
response, it is determined that the progress of the disorder has
been inhibited.
EXAMPLES
[0086] Herein below, the present invention will be explained in
greater detail in view of the examples. However, it is evident that
the present invention is not limited to them.
Reference Example 1
[0087] Purification of the soluble LR11 from rabbit serum or human
serum was carried out as follows.
[0088] (Purification of the Soluble LR11)
[0089] E. coli DH5.alpha. transformed with the vector pGEX2T
(manufactured by GE Healthcare Bio Science) to which human RAP gene
is transferred was cultured and the cells were collected by
centrifuge. The cells collected from the 3 L culture medium were
suspended in phosphate buffer (pH 7.2) containing lysozyme and the
surfactant TritonX-100 (trade name), and then disrupted by
ultrasonication. The RAP/GST fusion protein contained in the
supernatant obtained through centrifugation of the disrupt solution
was passed through 10 mL Glutathione Sepharose 4 FF (manufactured
by GE Healthcare Bio Science) for adsorption. After washing with
phosphate buffer (PBS, pH 7.2), RAP-Sepharose resin was
prepared.
[0090] The resulting RAP-Sepharose resin (10 mL) was mixed with
either the rabbit serum or the human serum (1 L). After the
overnight reaction under mild stirring at 4.degree. C., the
RAP-Sepharose resin was recovered and washed with phosphate buffer
(pH 7.2) for each case. Next, the rabbit soluble LR11 or the human
soluble LR11 was eluted by using citrate buffer (pH 5.0) followed
by concentration. Then, the dialysis against phosphate buffer (pH
7.2) was performed, and the product was obtained as purified rabbit
soluble LR11 or purified human soluble LR11.
Example 1
[0091] 1) Reactivity of the Soluble LR11 Purified from Serum with
the Monoclonal Antibody
[0092] Reactivity of each monoclonal antibody with the immobilized
soluble LR11 was confirmed as follows. First, to a micro plate
(manufactured by NUNC), a solution in which the purified rabbit
soluble LR11 (Lot. 061208) was diluted 100-fold with 20 mM
phosphate buffer having pH 7.2 (PBS) or a solution in which the
purified human soluble LR11 (Lot. 061208) was diluted 20-fold with
PBS was added in an amount of 50 .mu.L/well to perform overnight
immobilization of each soluble LR11. The resultant was washed with
20 mM phosphate buffer having pH 7.2 (BSA-PBST) and containing
0.05% Tween20 (trade name), followed by addition of 20 mM phosphate
buffer having pH 7.2 (BSA-PBST) and containing 1% BSA and 0.05%
Tween20 (trade name) (100 .mu.L per well) to perform blocking at
room temperature for 1 hr. After washing with PBST, 50 .mu.L of the
antibody solution in which each of the monoclonal antibody A2-2-3,
M3 and R14 labeled with biotin using the biotinylating agent
(manufactured by Pierce Company) had been diluted with BSA-PBST to
have concentration of 10 .mu.g/mL was added, and then the reaction
was carried out at room temperature for 2 hrs. After washing with
PBST, 50 .mu.L solution in which peroxidase-labeled streptavidin
(manufactured by Pierce Company) had been diluted with BSA-PBST to
have concentration of 0.2 .mu.g/mL was added, and the reaction was
carried out at room temperature for 2 hrs. After washing with PBST,
50 .mu.L of OPD substrate solution (0.1 M citrate buffer having pH
5.0 and containing 20 mM ortho-phenylene diamine and 12 mM hydrogen
peroxide) was added and the reaction was carried out at room
temperature for 10 minutes. Then, 50 .mu.L of 1.5 N sulfuric acid
was added to stop the color development. Then, the measurement was
carried out by using a micro plate reader (Abs. 492 nm).
[0093] In addition, as a negative control for measuring the
reactivity of the antibody, mouse IgG was used.
[0094] As a result (FIG. 1), while the mouse IgG as a negative
control hardly reacted to both the immobilized rabbit soluble LR11
and the immobilized human soluble LR11, the monoclonal antibody
A2-2-3, M3, M5, R14 and R23 reacted to them.
[0095] 2) ELISA Measurement System
[0096] Each of the monoclonal antibody A2-2-3, M3, M5, R14 or R23
was diluted with PBS to 5 .mu.g/mL, added to a micro plate
(manufactured by NUNC) to have 50 .mu.L per well, and then
immobilized overnight. In addition, as a negative control, mouse
IgG was also immobilized. After washing with PBST, BSA-PBST was
added in an amount of 100 .mu.L per well to perform blocking at
room temperature for 1 hr. After washing with PBST, a solution in
which the purified rabbit soluble LR11 (Lot. 061208) had been
diluted 25-fold with BSA-PBST or a solution in which the purified
human soluble LR11 (Lot. 061208) had been diluted 25-fold with
BSA-PBST was added in an amount of 50 .mu.L/well followed by
reaction at room temperature for 2 hrs. After washing with PBST, 50
.mu.L of the antibody solution in which each of the monoclonal
antibody A2-2-3, M3, M5, R14 or R23 which had been labeled with
biotin and diluted with BSA-PBST to have concentration of 10
.mu.g/mL was added, and the reaction was carried out at room
temperature for 2 hrs (as a negative control, biotinylated mouse
IgG was used). After washing with PBST, 50 .mu.L of the solution in
which peroxidase-labeled streptavidin (manufactured by Pierce
Company) had been diluted with BSA-PBST to have concentration of
0.2 .mu.g/mL was added, and the reaction was carried at room
temperature for 2 hrs. After washing with PBST, 50 .mu.L of the OPD
substrate solution was added and the reaction was carried out at
room temperature for 10 minutes. Then, 50 .mu.L of 1.5 N sulfuric
acid was added to stop the color development. Then, the measurement
was carried out by using a micro plate reader (Abs. 492 nm).
[0097] As a result, as shown in Table 1 and Table 2, it was found
that the purified rabbit soluble LR11 and human soluble LR11 in
liquid phase are detected from the sandwich ELISA system having the
combination of monoclonal antibodies shown below.
TABLE-US-00001 TABLE 1 Monoclonal antibody-monoclonal antibody
sandwich ELISA (antigen: purified human LR11) Antibody in liquid
phase Mouse IgG A2-2-3 M3 M5 R14 R23 Im- Mouse -0.003 0.015 0.024
0.007 -0.017 -0.020 mobilized IgG antibody A2-2-3 0.003 0.110 0.432
0.449 0.693 0.690 M3 0.001 1.075 0.029 1.017 2.061 1.915 M5 -0.001
0.150 0.158 0.079 0.308 0.309 R14 -0.004 1.130 0.861 0.870 -0.012
0.422 R23 -0.002 0.878 1.264 1.165 -0.005 0.007 Absorbance (492
nm)
TABLE-US-00002 TABLE 2 Monoclonal antibody-monoclonal antibody
sandwich ELISA (antigen: purified rabbit LR11) Antibody in liquid
phase Mouse IgG A2-2-3 M3 M5 R14 R23 Im- Mouse 0.043 -0.017 -0.017
-0.017 -0.019 -0.026 mobi- IgG lized A2-2-3 -0.004 0.007 1.898
0.856 1.822 1.212 anti- M3 -0.001 2.552 0.003 1.915 2.867 2.895
body M5 -0.007 0.229 0.300 0.144 0.535 0.361 R14 -0.009 2.032 1.119
1.029 0.002 0.058 R23 -0.009 1.579 2.475 1.833 0.005 0.041
Absorbance (492 nm) Immobilized antibody A2-2-3-antibody in liquid
phase M3, M5, R14, R23 Immobilized antibody M3 -antibody in liquid
phase A2-2-3, M5, R14, R23 Immobilized antibody M5 -antibody in
liquid phase A2-2-3, M3, R14, R23 Immobilized antibody R14
-antibody in liquidphase A2-2-3, M3, M5, R23 Immobilized antibody
R23 -antibody in liquidphase A2-2-3, M3, M5
[0098] 3) Measurement of the Purified LR11 Based on Sandwich
ELISA
[0099] To a micro plate (manufactured by NUNC), monoclonal antibody
M3 which has been diluted with PBS to have 5 .mu.g/mL was added (50
.mu.L per well), and the immobilization was carried out at room
temperature for 2 hrs. After washing with PBST, BSA-PBST was added
in an amount of 100 .mu.L per well for blocking at room temperature
for 1 hr. After washing with PBST, a solution in which the rabbit
soluble LR11 or the human soluble LR11 purified though the method
described in the above-Reference example 1 was diluted with
BSA-PBST to perform reaction. After washing with PBST, biotinylated
monoclonal antibody R14 which had been diluted with BSA-PBST to
have 5 .mu.g/mL was added thereto to perform the reaction. After
washing with PBST, peroxidase-labeled streptavidin (manufactured by
Pierce Company) which had been diluted with BSA-PBST to have
concentration of 0.2 .mu.g/mL was reacted with the antibody. After
washing with PEST, TMB substrate solution (0.1 M citrate buffer
having pH 3.7 and containing 0.3 mg/mL of
3,3'-5,5'-tetramethyl-benzidine dihydrochloride (manufactured by
Sigma) and 12 mM hydrogen peroxide) was added thereto in an amount
of 50 .mu.L to allow the color development for 30 min at room
temperature. Then, 50 .mu.L of 1.5 N sulfuric acid was added to
stop the color development. Then, the measurement was carried out
by using a micro plate reader (Abs. 450 nm).
[0100] As shown in FIG. 2 and FIG. 3, it was confirmed that, by
using the purified soluble LR11, the antigen
concentration-dependent reaction can be obtained according to the
variation in concentration of the antigen, i.e., human soluble LR11
or rabbit soluble LR11.
[0101] 4) Effect of the Serum on ELISA System
[0102] First, to a micro plate (manufactured by NUNC), monoclonal
antibody M3 which had been diluted with PBS to have 5 .mu.g/mL was
added (50 .mu.L per well), and the immobilization was carried out
at room temperature for 2 hrs. After washing with PEST, BSA-PBST
was added in an amount of 100 .mu.L per well to perform blocking at
room temperature for 1 hr.
[0103] After washing with PBST, a solution in which the purified
rabbit soluble LR11 (Lot. 070601) had been diluted 100-, 200- or
400-fold with BSA-PBST or a solution in which the purified rabbit
soluble LR11 (Lot. 070601) had been diluted 100-, 200- or 400-fold
with human serum was added in an amount of 50 .mu.L, respectively,
followed by reaction at room temperature for 2 hrs.
[0104] After washing with PBST, 50 .mu.L of biotinylated monoclonal
antibody R14, which had been diluted with BSA-PBST to have 5
.mu.g/mL, was added thereto for the reaction for 2 hrs at room
temperature. After washing with PBST, peroxidase-labeled
streptavidin (manufactured by Pierce Company) which had been
diluted with BSA-PBST to have concentration of 0.2 .mu.g/mL was
added in an amount of 50 .mu.L and the reaction was carried out at
room temperature for 2 hrs. Subsequently, 50 .mu.L of the TMB
substrate solution was added to allow the color development for 30
min at room temperature.
[0105] Then, 50 .mu.L of 1.5 N sulfuric acid was added to stop the
color development. Then, the measurement was carried out by using a
micro plate reader (Abs. 450 nm).
[0106] As shown in FIG. 4, concentration-dependent reaction was
observed for the rabbit soluble LR11 diluted in the buffer
solution, while reaction of the rabbit soluble LR11 diluted in the
human serum was inhibited under the influence of the serum
components, and therefore no detection can be made.
[0107] Further, even for any of the combination of the antibodies
of the above section 2) from which purified rabbit soluble LR11 and
human soluble LR11 in liquid phase were detected, no detection can
be made due to the influence of the serum components. Thus, without
being limited to a combination of specific kinds of antibodies, the
influence of the serum components was recognized.
[0108] 5) Avoidance of the Influence of the Serum Components Based
on Treatment with Surfactant
[0109] To a micro plate (manufactured by NUNC), monoclonal antibody
M3 which had been diluted with PBS to have 5 .mu.g/mL was added (50
.mu.L per well), and the immobilization was carried out at room
temperature for 2 hrs.
[0110] After washing with PBST, BSA-PBST was added in an amount of
100 .mu.L per well to perform blocking at room temperature for 1
hr. After washing with PBST, human serum in which the purified
rabbit soluble LR11 (Lot. 070601) had been added so as to obtain
dilution of 50-, 100- or 200-fold at the time of the reaction
(human serum was diluted to obtain dilution of 10-fold at the time
of the reaction) was reacted in an amount of 50 .mu.L per well (PBS
for blank) at room temperature (15.degree. C. to 25.degree. C.) for
2 hrs in the presence of each of the surfactants of Tween20 (trade
name), Triton-X100 (trade name), octanoyl-N-methylglucamine
(MEGA-8), nonanoyl-N-methylglucamine (MEGA-9), sodium cholate and
n-octyl-.beta.-D-glucopyranoside (3.6% concentration for each).
[0111] After washing with PBST, 50 .mu.L of a standard antibody
solution in which biotinylated anti-human soluble LR11 monoclonal
antibody R14 is diluted with BSA-PBST to have 5 .mu.g/mL was added
to the above (50 .mu.L per well) for the reaction for 4 hrs at room
temperature. After washing with PBST, peroxidase-labeled
streptavidin (manufactured by Pierce Company) which has been
diluted with BSA-PBST to have concentration of 0.2 .mu.g/mL was
added to the well (50 .mu.L per well) and the reaction was carried
out for 2 hrs at room temperature.
[0112] Subsequently, after washing with PBST, the TMB substrate
solution was added to the well (50 .mu.L per well) to allow the
color development for 30 min at room temperature. Then, 1.5 N
sulfuric acid was added in an amount of 50 .mu.L per well to stop
the color development. Then, the measurement was carried out by
using a micro plate reader (Abs. 450 nm).
[0113] As shown in FIG. 5, in case where the surfactant Tween20
(trade name) was used, influence of the serum components could not
be avoided. However, in case where Triton-X100 (trade name),
MEGA-8, MEGA-9, sodium cholate or n-octyl-.beta.-D-glucopyranoside
was used, even in the presence of the serum components, a reaction
which is dependent on the concentration of the added rabbit soluble
LR11 (Lot. 070601) was obtained. Such effect was particularly
significant for MEGA-8 and MEGA-9. Meanwhile, FIG. 5 was obtained
after subtracting the reagent blank value.
Example 2
[0114] (Concentration Variation of MEGA-9)
[0115] In the same manner as Example 1-5), human serum to which a
solution containing the purified rabbit soluble LR11 (Lot. 070601)
diluted 50-or 100-fold with PBS (PBS for blank) was added to
perform reaction (the serum was diluted to obtain dilution of
8-fold at the time of the reaction) in the presence of the
surfactant MEGA-9 (0%, 2.5%, 5% or 10%). As a result (see FIG. 6),
almost the equivalent reaction was obtained from 2.5% to 10%
MEGA-9, and it was found that the measurement of the soluble LR11
could be achievedwhile the influence of the serum components was
avoided.
Example 3
[0116] For the combination of the monoclonal antibodies other than
the combination of immobilized monoclonal antibody M3 and
monoclonal antibody in liquid phase R14 in Example 2, effect of
MEGA-9 (0%, 2%, 4%, 5% or 7.5%) was determined. The sample was
human serum (the serum was diluted 8-fold at the time of the
reaction). As a result (FIGS. 7 to 11), from all of the
combinations determined, an increase in the measurement value was
recognized in accordance with the addition of MEGA-9. In cases of
2% to 7.5% addition group, almost the constant value was obtained.
Thus, it was found that, without being limited to a combination of
specific kinds of antibodies, the measurement of the soluble LR11
might be achieved by the addition of the surfactant while the
influence of the serum components was avoided.
Example 4
[0117] (Measurement of the Soluble LR11 from Human Serum) Purified
rabbit soluble LR11 (Lot. 071120) and highly pure bovine serum
albumin with known protein concentration were subjected to
SDS-polyacrylamide gel electrophoresis followed by silver staining.
Thereafter, the stained image was analyzed by densitometry. As a
result, the concentration of the LR11 in the rabbit soluble LR11
(Lot. 071120) was calculated to be 3.0 .mu.g/mL. Thus-obtained
purified rabbit soluble LR11 with known concentration value was
used as a standard material for obtaining the concentration in
human serum according to the following method.
[0118] To a micro plate (manufactured by NUNC), monoclonal antibody
M3 which had been diluted with PBS to have 5 .mu.g/mL was added
(100 .mu.L per well), and the immobilization was carried out at
room temperature for 2 hrs. After washing with PBST, BSA-PBST was
added (200 .mu.L per well) to perform blocking at room temperature
for 1 hr.
[0119] After washing with PBST, three samples of human serum which
had been diluted 32-, 16-, 8- or 4-fold with PBS were added (100
.mu.L per well) in the presence of 5-% MEGA-9 to the overnight
reaction at room temperature (15.degree. C. to 25.degree. C.).
[0120] Thereafter, a standard antibody solution in which
biotinylated monoclonal antibody R14 had been diluted with BSA-PBST
to have 0.4 .mu.g/mL was added (100 .mu.L per well) for the
reaction for 4 hrs at room temperature. After washing with PBST,
peroxidase-labeled streptavidin (manufactured by Pierce Company)
which had been diluted with BSA-PBST to have concentration of 0.2
.mu.g/mL was added to the above (100 .mu.L per well) for the
reaction at room temperature for 1 hr.
[0121] Subsequently, after washing with PBST, the TMB substrate
solution was added (100 .mu.L per well) to allow the color
development for 30 min at room temperature. Then, 1.5 N sulfuric
acid was added in an amount of 100 .mu.L per well to stop the color
development. Then, the measurement was carried out by using a micro
plate reader (Abs. 450 nm).
[0122] As a result (FIG. 12), in the case of the treatment with
MEGA-9, a reaction which is dependent on the diluted concentration
of the serum, i.e., a reaction which is dependent on the LR 11
concentration in the serum, was obtained from all of the three
serum samples. With respect to the value converted in terms of the
LR11 concentration obtained, good dilution linearity was observed
in the serum dilution range between 32-fold and 8-fold.
Example 5
[0123] (Measurement of the Soluble LR11 from Serum of a Mammal
Other Than Human)
[0124] In view of the measurement method of Example 4, each serum
obtained from rabbit, monkey, goat and pig was treated with MEGA-9
and subjected to the measurement by sandwich ELISA.
[0125] As a result, from any of the cases above, good dilution
linearity was observed like the human serum (FIG. 13A). Meanwhile,
when the treatment with MEGA-9 was not carried out, basically no
absorbance increase was observed from the serum of the monkey, goat
and pig. In the case of the rabbit serum, the absorbance increase
was observed. However, the absorbance increase ratio was
significantly lower than the case wherein MEGA-9 treatment was
carried out (FIG. 13B.
Example 6
[0126] (Measurement of the Soluble LR11 from Human Cerebrospinal
Fluid)
[0127] Having the purified rabbit soluble LR11 with the previously
calculated value of Example 4 as a standard material, concentration
of the soluble LR11 in diluted human cerebrospinal fluid was
obtained according to the method shown below.
[0128] To a micro plate (manufactured by NUNC), monoclonal antibody
M3 which had been diluted with PBS to have 10 .mu.g/mL was added
(100 .mu.L per well), and the immobilization was carried out at
room temperature for 2 hrs. After washing with PBST, BSA-PBST
containing 10% sucrose was added in an amount of 200 .mu.L per well
to blocking at room temperature for 1 hr. After aspirating off the
blocking solution, the plate was dried.
[0129] To the above, three samples of human cerebrospinal fluid
which had been diluted 32-, 16-, 8-, 4- or 2-fold with PBS were
added (100 pi per well) in the presence of 5% MEGA-9 and reacted
overnight at room temperature (15.degree. C. to 25.degree. C.).
[0130] Thereafter, the labeled antibody solution containing
biotinylated monoclonal antibody R14 obtained by dilution with
BSA-PBST to have 0.4 .mu.g/mL was added (100 .mu.L per well) and
reacted for 4 hrs at room temperature. After washing with PBST,
peroxidase-labeled streptavidin (manufactured by Pierce Company)
which had been diluted with BSA-PBST to have concentration of 0.2
.mu.g/mL was added to the above (100 .mu.L per well) for the
reaction at room temperature for 1 hr.
[0131] Subsequently, after washing with PBST, the TMB substrate
solution was added (100 .mu.L per well) to allow the color
development for 30 min at room temperature. Then, 1.5 N sulfuric
acid was added in an amount of 100 .mu.L per well to stop the color
development. Then, the measurement was carried out by using a micro
plate reader (Abs. 450 nm).
[0132] As a result, in the case of the treatment with MEGA-9, a
reaction which is dependent on the diluted concentration of the
cerebrospinal fluid, i.e., a reaction which is dependent on the LR
11 concentration in the cerebrospinal fluid, was obtained from all
of the three cerebrospinal fluid samples. With respect to the value
converted in terms of the LR11 concentration obtained, good
dilution linearity was observed in the cerebrospinal fluid dilution
ratio range between 32-fold and 2-fold (FIG. 14A).
[0133] In contrast, since no sufficient absorbance increase was
observed for any of the cerebrospinal fluid samples when the
treatment with MEGA-9 was not carried out, it is believed that the
value converted in terms of the LR11 concentration becomes a
significantly small value (FIG. 14B), and therefore the accurate
measurement can not be carried out.
Sequence CWU 1
1
2116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Ser Met Asn Glu Glu Asn Met Arg Ser Val Ile Thr
Phe Asp Lys Gly1 5 10 152551PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 2Gln Leu Thr Gly Leu Met
Asp Met Lys Ile Phe Tyr Lys Gly Lys Asn1 5 10 15Thr Gly Ser Asn Ala
Cys Val Pro Arg Pro Cys Ser Leu Leu Cys Leu 20 25 30Pro Lys Ala Asn
Asn Ser Arg Ser Cys Arg Cys Pro Glu Asp Val Ser 35 40 45Ser Ser Val
Leu Pro Ser Gly Asp Leu Met Cys Asp Cys Pro Gln Gly 50 55 60Tyr Gln
Leu Lys Asn Asn Thr Cys Val Lys Glu Glu Asn Thr Cys Leu65 70 75
80Arg Asn Gln Tyr Arg Cys Ser Asn Gly Asn Cys Ile Asn Ser Ile Trp
85 90 95Trp Cys Asp Phe Asp Asn Asp Cys Gly Asp Met Ser Asp Glu Arg
Asn 100 105 110Cys Pro Thr Thr Ile Cys Asp Leu Asp Thr Gln Phe Arg
Cys Gln Glu 115 120 125Ser Gly Thr Cys Ile Pro Leu Ser Tyr Lys Cys
Asp Leu Glu Asp Asp 130 135 140Cys Gly Asp Asn Ser Asp Glu Ser His
Cys Glu Met His Gln Cys Arg145 150 155 160Ser Asp Glu Tyr Asn Cys
Ser Ser Gly Met Cys Ile Arg Ser Ser Trp 165 170 175Val Cys Asp Gly
Asp Asn Asp Cys Arg Asp Trp Ser Asp Glu Ala Asn 180 185 190Cys Thr
Ala Ile Tyr His Thr Cys Glu Ala Ser Asn Phe Gln Cys Arg 195 200
205Asn Gly His Cys Ile Pro Gln Arg Trp Ala Cys Asp Gly Asp Thr Asp
210 215 220Cys Gln Asp Gly Ser Asp Glu Asp Pro Val Asn Cys Glu Lys
Lys Cys225 230 235 240Asn Gly Phe Arg Cys Pro Asn Gly Thr Cys Ile
Pro Ser Ser Lys His 245 250 255Cys Asp Gly Leu Arg Asp Cys Ser Asp
Gly Ser Asp Glu Gln His Cys 260 265 270Glu Pro Leu Cys Thr His Phe
Met Asp Phe Val Cys Lys Asn Arg Gln 275 280 285Gln Cys Leu Phe His
Ser Met Val Cys Asp Gly Ile Ile Gln Cys Arg 290 295 300Asp Gly Ser
Asp Glu Asp Ala Ala Phe Ala Gly Cys Ser Gln Asp Pro305 310 315
320Glu Phe His Lys Val Cys Asp Glu Phe Gly Phe Gln Cys Gln Asn Gly
325 330 335Val Cys Ile Ser Leu Ile Trp Lys Cys Asp Gly Met Asp Asp
Cys Gly 340 345 350Asp Tyr Ser Asp Glu Ala Asn Cys Glu Asn Pro Thr
Glu Ala Pro Asn 355 360 365Cys Ser Arg Tyr Phe Gln Phe Arg Cys Glu
Asn Gly His Cys Ile Pro 370 375 380Asn Arg Trp Lys Cys Asp Arg Glu
Asn Asp Cys Gly Asp Trp Ser Asp385 390 395 400Glu Lys Asp Cys Gly
Asp Ser His Ile Leu Pro Phe Ser Thr Pro Gly 405 410 415Pro Ser Thr
Cys Leu Pro Asn Tyr Tyr Arg Cys Ser Ser Gly Thr Cys 420 425 430Val
Met Asp Thr Trp Val Cys Asp Gly Tyr Arg Asp Cys Ala Asp Gly 435 440
445Ser Asp Glu Glu Ala Cys Pro Leu Leu Ala Asn Val Thr Ala Ala Ser
450 455 460Thr Pro Thr Gln Leu Gly Arg Cys Asp Arg Phe Glu Phe Glu
Cys His465 470 475 480Gln Pro Lys Thr Cys Ile Pro Asn Trp Lys Arg
Cys Asp Gly His Gln 485 490 495Asp Cys Gln Asp Gly Arg Asp Glu Ala
Asn Cys Pro Thr His Ser Thr 500 505 510Leu Thr Cys Met Ser Arg Glu
Phe Gln Cys Glu Asp Gly Glu Ala Cys 515 520 525Ile Val Leu Ser Glu
Arg Cys Asp Gly Phe Leu Asp Cys Ser Asp Glu 530 535 540Ser Asp Glu
Lys Ala Cys Ser545 550
* * * * *