JPH07128333A - Immunoassay for cholesterol ester transfer protein - Google Patents

Abstract

PURPOSE:To conduct highly accurate high sensitivity determination of CETP in a specimen by a simple and convenient operation with no influence of interfering substance, e.g. albumin or globulin, using a sample containing a trace of cholesterol ester transfer protein(CETP), e.g. a clinical blood sample, as the specimen. CONSTITUTION:In the immunoassay for CETP in a specimen, and a kit therefor, the specimen is treated with alcohol and then caused to react with an anti- cholesterol ester transfer protein antibody.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to cholesteryl ester transfer protein.
nsfer protein: hereinafter abbreviated as CETP), and more specifically, to a method for accurately and simply measuring CETP, which is capable of removing interfering substances affecting the CETP measurement system, and a kit using the same.

[0002]

2. Description of the Related Art In the metabolism of cholesterol in serum,
Low-density lipoprotein (LDL) plays a major role in the cholesterol supply pathway to peripheral tissues. On the other hand, high-density lipoprotein (HDL) is considered to be an important lipoprotein in a pathway that extracts cholesterol from peripheral tissues and reversely transfers it to the liver. Furthermore, many epidemiological studies have revealed that blood HDL levels have a negative correlation with the occurrence frequency of ischemic heart disease, and it is known that HDL acts as a protective factor for arteriosclerosis. Lecithin-cholesterol acyltransferase (L) has an important function in the reverse transfer system that extracts cholesterol in HDL.
In addition to enzymes such as CAT), hepatic lipase (HTGL), and lipoprotein lipase (LPL), there is CETP. In the living body, CETP is a very hydrophobic protein, which is first synthesized as a peptide consisting of 476 amino acids, and undergoes glycosylation in the cell to have a molecular weight of 74.
It is known to be a mature protein of 000 [Hesl
er, C.I. B. et al. J. Biol. Che
m. , 263 , 5020 (1988)]. Although there are many unclear points about the mechanism of action of CETP, it is said that it promotes lipid exchange / transfer between lipoproteins. That is, LCA
By the T reaction, free cholesterol esterified on HDL is transferred to triglyceride-rich lipoproteins such as VLDL (TGrich lipoprotein), but in exchange for this, TG is reversely transferred at a molar ratio of 1: 1. It HDL which became TG rich is HTG
The action of L causes the TG part to be hydrolyzed, resulting in a smaller HD
It is considered to be L particles. That is, CETP is considered to be a cholesterol transfer protein having a function of transferring cholesteryl ester (CE) to VLDL or LDL. As an abnormality of the human CETP activation system, CETP deficiency has been reported when HDL is elevated [Yamashita S. et al. et al. , Ather
Osclerossis, 70, 7 (1988)]. In addition, it has been reported that chronic drinking alcohol patients have decreased ETP activity and increased CETP activity associated with primary biliary cirrhosis (PBC). Thus, the measurement of CETP activity or CETP amount is performed in arteriosclerosis or liver. It is extremely important in the diagnosis of diseases.

As a method for measuring the lipid transfer activity of CETP, for example, serum or plasma obtained by fasting blood sampling is separated by ultracentrifugation from serum (plasma) to obtain a fraction having a specific gravity of 1.21 or more, Get as. Next, in advance, the above-mentioned sample was added to radiolabeled radioactive CE-labeled HDL (20 to 50 μg total cholesterol (TC)) and LDL (100 to 150 μg (TC)) to make 100 μl, and incubated at 37 ° C. for 2 to 3 hours. , 400 mg of a fraction of serum having a specific gravity of 1.21 or more of 50 mg / ml was added, and further 50 μl of heparin / MnCl ₂ (2250 IU heparin / 1M
Method for calculating CE transfer activity by measuring the radioactivity of HDL in the supernatant by precipitating LDL with (MnCl ₂ ) and changing the radioactivity before and after incubation [Norihiro Sasaki, Nihon Clinic, 47 , 534 (1989)] There is. However, such a method for measuring the lipid transfer activity of CETP has drawbacks in that the operation such as using ultracentrifugation is complicated and the accuracy is low.

On the other hand, as a method for measuring the amount of CETP, both a method using a monoclonal antibody and a method using a polyclonal antibody have been reported. A method using a monoclonal antibody is toll (Tall,
A. ) Et al., A monoclonal antibody TP against CETP
-2 is used to report a competitive RIA method for immobilizing an antigen. That is, a lipoprotein fraction was obtained by an ultracentrifugation method, an antigen in the lipoprotein fraction was immobilized, and then reacted with a TP-2 antibody, and the reaction product and ¹²⁵ I-anti-mouse I as a second antibody.
It is a method of reacting with a gG antibody and measuring with a γ-counter [Marcel, Y. L. et al. J. C
lin. Invest. , 85 , 10-17 (199
0)]. However, using this monoclonal antibody, Brown et al. Reported that plasma CETP could not be detected in some of the hyperalphalipoproteinemic families [Brown, M. et al. L. et al. ，
Nature, 342 , 448-451 (198).
9)]. Thus, when a monoclonal antibody is used, the protein structure is partially changed when a gene mutation occurs in the epitope recognized by the antibody, and the monoclonal antibody recognizing a single epitope is disadvantageous in this case. Further, when the CETP fragment shows catalytic activity, the epitope from the protein may be deleted, and in this case, it will not be recognized by the monoclonal antibody. Further, in the case of CETP deficiency, the expression level of CETP protein is different between the homozygote and the heterozygote, and the type of heterozygote may not be recognized depending on the monoclonal antibody. Furthermore, since this method is an RIA method, it is difficult to make it into a kit.

Regarding the measurement of the amount of CETP using a polyclonal antibody, the enzyme immunoassay method of Yamashita et al. Has been reported [Yamashita S. et al. et
al. , Clin. Chim. Acta. , 194 , 1
45-160 (1990)]. The method is first CETP
Partially purified and immobilized with polyclonal antibody
Using a TP antibody-binding affinity gel, this is reacted with serum, then the above partially purified CETP is added, and the CE transfer activity of unreacted CETP in the supernatant is measured.
This is a method of displaying the CETP amount as a relative percentage when the normal control is 100%. In this method, a large amount of partially purified CETP is required, and CETP directly in blood is required.
Finally, the CETP activity will be measured. Moreover, since an isotope is required to measure the CE transfer activity, it is difficult to prepare a kit, and it takes a long time to perform the measurement.

On the basis of these backgrounds, the present inventors have conducted a heat treatment on a serum sample at 100 ° C., and cooled and centrifuged the obtained serum to obtain the following:
After reacting with the immobilized anti-CETP polyclonal antibody,
A method of reacting a peroxidase-labeled enzyme-labeled antibody with an anti-human CETP antibody purified in advance to Fab ′ and measuring the amount of CETP by the enzyme reaction was previously reported [Takamitsu Nakano et al., Atherosclerosis Society, December 1991. 5th (Tokyo)]. In addition, in this report, the method of applying this heat treatment made it possible to measure the amount of CETP that could not be measured by the method without sample treatment. However, serum albumin was recognized as having an effect on the measurement system. It was shown that As a result of further research, in addition to albumin, Ig
Since G was found to affect the CETP amount measurement system, a method was described in which plasma was once separated and removed by treatment with anhydrous sodium sulfate, fractionated by a molecular sieve, and then reacted with the immobilized anti-CETP polyclonal antibody. [Kenmitsu Kenya et al., Arteriosclerosis Society, June 9, 1992 (Osaka)]. However, in this method, the pretreatment of the sample becomes extremely complicated with the anhydrous sodium sulfate treatment and the molecular sieve fractionation treatment, and it cannot be adopted as the inspection means in a normal clinical laboratory.

In the conventional method, substances such as lipoprotein transfer inhibitory protein (LTIP) are said to affect the CETP measurement value.

[0008]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a method for measuring the amount of CETP in a sample such as serum or plasma with a simple operation and high accuracy.

[0009]

Therefore, the present inventors have
Various studies have been conducted to develop a method for removing factors that affect the CETP measurement value in blood by a simple operation. As a result, a sample such as plasma pretreated with alcohol is treated with anti-C.
The present inventors have found that CETP in a sample can be quantified with extremely high accuracy by a simple operation if it is measured by an immunological method using an ETP antibody, and the present invention has been completed.

That is, the present invention provides an immunoassay method for CETP in a sample, which comprises treating the sample with alcohol and then reacting it with an anti-CETP antibody. The present invention also provides a CETP immunoassay kit containing alcohol and an anti-CETP antibody.

The alcohol used in the present invention includes methanol, ethanol, n-propyl alcohol, i-propyl alcohol, n-butyl alcohol,
Lower alcohols such as i-butyl alcohol and t-butyl alcohol are exemplified, and these may be used alone or in combination of two or more. Ethanol is the most preferred.

The anti-CETP antibody used in the assay method of the present invention may be a polyclonal antibody or a monoclonal antibody, but a polyclonal antibody is preferred.

The anti-CETP polyclonal antibody used in the assay method of the present invention is obtained by sensitizing purified CETP as an immunogen to animals such as mouse, rat, guinea pig, rabbit, sheep, goat, horse and cow. Antisera,
Immunoglobulins separated from these (eg Ig
G, IgM, etc.), and these processed products are F (ab ') ₂ , F
(Ab '), F (ab) and the like.

Purified CET used here as an immunogen
P is, for example, a fraction obtained by fractionating human plasma into fractions having a specific gravity of 1.25 or more by ultracentrifugation such as concentration gradient ultracentrifugation or continuous isopycnic ultracentrifugation, affinity chromatography, ion exchange chromatography, It can be obtained by purification by ordinary means such as gel filtration, ammonium sulfate fractionation, salting out and the like. Purification of such CETP is carried out by the method of Yamashita et al. [Yamashita, S. et al. , Et al. , C
linica Chimica Acta, 194 , 1
45-160 (1990)] is preferred.
More specifically, human plasma was subjected to ultracentrifugation at 46000rp.
Ultracentrifugation at a specific gravity of 1.21 g / ml or more for 48 hours at 4 ° C at 4 m, and a fraction of the obtained plasma with a specific gravity of 1.21 g / ml or more was equilibrated with 4 mol / l NaCl Phenyl Sepharose CL-4B column It can be chromatographically separated using a Sepharose column (2.5 × 20 cm) or the like. The column was 50 mmol / l Tris-hydrochloric acid, 150 mmol / l NaCl, 0.02% NaN ₃ ,
After washing and extraction with Tris-hydrochloric acid buffer of pH 7.4, the protein bound to the column was extracted with water, and the fraction containing the active fraction was equilibrated with 50 mmol / l sodium acetate (pH 7.5) CM-cellulose. Column (CM-52) (2.
5 × 20 cm) and separated, 50 mmol / l sodium acetate, 0.02% NaN ₃ , 1 mg / ml EDTA-2Na
It is possible to elute with a 600 ml linear gradient of NaCl in (pH 4.5). The eluted fractions were collected in a tube containing 1 ml of 1 mmol / l Tris-hydrochloric acid (pH 8.5) and neutralized, and the fractions containing the active fraction were adsorbed and dialyzed against Tris / NaCl buffer. After that, 100% by ultrafiltration with Amicon PM30 membrane
It is possible to obtain 0-fold purified CETP.

To obtain an anti-CETP polyclonal antibody using purified CETP, for example, purified CETP is added to PBS.
After adding a buffer such as (phosphate buffered saline) to adjust the concentration to an appropriate level, add an equal amount of an adjuvant such as Freund's complete adjuvant to make a suspension. A suitable amount of the liquid is added to the above mammal, for example, CET.
As the amount of P, the dose is 20 to 1000 μg / animal,
Immunize with subcutaneous administration every two weeks. This can be performed by repeating this 6 times and immunizing, and then collecting whole blood from the animal to obtain antiserum. The antiserum thus obtained can be purified by conventional means such as affinity chromatography, ion exchange chromatography, gel filtration, ammonium sulfate fractionation and salting out.

In the measuring method of the present invention, examples of the sample include blood and cell tissue fluid.
In particular, fasted serum or plasma is preferable, and these can be prepared from a subject after blood collection according to a conventional method.

To carry out the measuring method of the present invention, the sample is first treated with alcohol. The alcohol treatment is performed, for example, by mixing a sample and alcohol, and then removing insoluble matter and alcohol. Here, it is preferable to remove the insoluble matter by centrifugation, filtration or the like and then remove the alcohol by concentration under reduced pressure. More specifically, first, a predetermined amount of test serum (plasma) was placed in a test tube, chilled in ice, and cold ethanol chilled in ice gave a final ethanol concentration (v / v) of 50 to 55%. And mix well while cooling in ice so that it does not generate heat, and leave it in ice for a while (about 10 minutes), then centrifuge at low temperature and medium speed (0 ° C, 4000 rpm) to insoluble matter. Is removed to obtain a supernatant, and ethanol is removed from the supernatant by decompression or the like.

Next, from the obtained alcohol-treated sample, C
To detect ETP, conventional immunological means using anti-CETP antibody, such as competitive method, radioimmunoassay (RIA) method by sandwich method, immunoassay method (ELI).
SA), an aggregation method or the like. this house,
The above sample is brought into contact with an insoluble carrier on which an anti-CETP antibody is immobilized, and then a labeled anti-CETP antibody is reacted therewith,
It is preferably carried out by measuring the labeled amount of the reaction product in the carrier.

Immobilization of the anti-CETP antibody is carried out by physically or chemically binding the antibody to an insoluble carrier according to a conventional method. As the insoluble carrier for immobilization, for example, polystyrene, Sephadex, ion exchange resin, plastic tube, amino copolymer and the like can be used, and insolubilization is carried out by diazo method as covalent bond method, peptide method, alkylation method, crosslinking. Carrier-bound method with reagents, Ugi
It can also be carried out by a chemical reaction such as a carrier-binding method by a reaction, an ionic-bonding method using a carrier such as an ion exchange resin, a physical adsorption method using a porous glass such as glass beads as a carrier, and the like. It is preferable to use a 96-well plastic plate from the viewpoint of operability and the like when simultaneously treating the sample of 1.

The labeling agent used for labeling the anti-CETP antibody is a conventional labeling agent such as iodine isotopes (eg ¹²⁵ I, ¹³¹ I), ¹⁴ C, radioactive isotopes such as tritium, fluorescein isocyanate, Examples include fluorescent substances such as fluorescein isothiocyanate and rhodamine; enzymes such as peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, and penicillinase. Enzymes are preferable for preparing a kit. As the enzyme, commercially available peroxidase (POD), β-D-galactosidase, acid phosphatase, alkaline phosphatase and the like can be used. With these labeling agents, anti-CET
A method known per se is used to label the P antibody.

As the solvent used in the measurement system, various ordinary ones which do not adversely affect the reaction can be used, and examples thereof include a citrate buffer solution, a phosphate buffer solution, a tris acid buffer solution, an acetic acid buffer solution and the like. It is preferable to use a buffer solution having a pH of about 5.0 to 9.0. In the present invention, the above solvent contains about 0.1 to 30 w / v% serum (without CETP to be measured) and / or about 0.1%.
It is preferable to include about 2 M of NaCl, which is more suitable for the purpose of the measuring method of the present invention.

The immunoreaction conditions for CETP detection are not particularly limited, and may be the same as those used in ordinary measuring methods of this type. Generally 45 ° C or less, preferably about 4 to
The reaction may be performed under a temperature condition of about 40 ° C. for about 1 to 80 hours. Solid phase-liquid phase (complex of the reaction complex and labeled antibody in the sandwich method-unbound labeled antibody) after completion of the immune reaction is usually separated by, for example, centrifugation, filtration, decantation, washing, etc. It can be performed by the method of.

To measure the amount of the reaction product labeled in the carrier thus separated, it depends on the kind of the labeling agent.
When the labeling agent is an enzyme, it can also be carried out by coexisting a coloring reagent to induce a coloring reaction and measuring the amount of the dye. Examples of the coloring solution used at that time include o-phenylenediamine and o-nitrophenyl-β-
D-galactoside, tyramine, 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (AB
TS), N-ethyl-N-sulfopropyl-m-anisidine (ADPS), p-nitrophenylphosphoric acid and the like. For example, when peroxidase is used as the enzyme, o-phenylenediamine (OPD) and the like can be used, and the color reaction can be stopped according to a conventional method. It can be carried out by adding an agent. To obtain the CETP amount in the sample from the obtained labeled amount, a calibration curve is prepared in advance,
It is preferable to do this in contrast to this.

A particularly convenient method for carrying out the above-mentioned CETP measurement method is to use a kit for detecting CETP. The kit includes alcohol and anti-CET
A kit in which reagents containing P antibody are combined is preferable. As the latter reagent, anti-CETP immobilized on an insoluble carrier is used.
More preferably, the antibody and labeled anti-CETP antibody are supplied. In addition, a stabilizer and / or a preservative such as sucrose, glycerol, bovine serum protein and the like can be added to the kit if necessary, as long as it does not adversely affect the measured value. The antibody reagent may be lyophilized, and the kit may contain a water-soluble or water-miscible solvent. Further, the antibody reagent may be blended with a buffer solution for keeping the reconstituted reagent system at a constant pH at the time of measurement and / or a preservative and / or stabilizer for preventing deterioration of the sample. . Although the buffer solution is not an essential component of the kit reagent, it is preferable to use a buffer solution having a pH of about 5.0 to 9.0 when carrying out the assay method of the present invention.
Further, the reconstitution agent preferably contains water,
It is also possible to replace part or all of the water with a solvent miscible with water. Examples of the solvent miscible with water include glycerin, alcohols, glycol ethers and the like.

[0025]

According to the method of the present invention, a sample containing a small amount of CETP such as a clinical blood sample is used as a sample, and CETP in the sample is highly accurate without being affected by interfering substances such as albumin and globulin. It can be quantified with high sensitivity and simple operation.

[0026]

The present invention will be described in more detail below with reference to Reference Examples and Examples.

Reference Example 1 Purification of human CETP Normal human plasma was subjected to ultracentrifugation at 46000 rpm at 4 ° C.
48 hours at a specific gravity of 1.21 g / ml or more for continuous density ultracentrifugation, and the obtained plasma fraction having a specific gravity of 1.21 g / ml or more was previously equilibrated with 4 mol / l NaCl Phenyl Sepharose column CL-4B Column (2.5 x
Chromatography was performed using a Sepharose column (20 cm: manufactured by Pharmacia). Next, the column is 50 mm
ol / l Tris-hydrochloric acid, 150 mmol / l NaCl, 0.
After washing and extraction with 02% NaN ₃ , pH 7.4 Tris-HCl buffer, the protein bound to the column was extracted with water, and the fraction containing the active fraction was equilibrated with 50 mmol / l sodium acetate (pH 7.5). CM-Cellulose column (CM-52) (2.5 x 20 cm: Pharmacia)
50 mmol / l sodium acetate, 50 mm
ol NaCl, 0.02% NaN ₃ , 1 mg / ml EDTA
-2Na (pH 4.5) was eluted. The elution fraction was collected in a tube containing 1 ml of 1 mol / l Tris-hydrochloric acid (pH 8.5) for neutralization, dialyzed against a Tris / NaCl buffer, and then concentrated with an Amicon PM30 membrane. Human CET purified 1000 times by the above operation
P was obtained.

Reference Example 2 Preparation of anti-human CETP polyclonal antibody 2 mg / ml of purified human CETP obtained in Reference Example 1 and Freund's complete adjuvant (Freud's Compl)
An equal amount of ete adjuvant (manufactured by Nacalai Tesque, Inc.) was mixed and injected into several tens of skin on the limbs and back of a rabbit to immunize. It was similarly administered 5 to 6 times during 2 to 3 months. Two weeks after the final administration, whole blood was collected and its serum was obtained.

Reference Example 3 Preparation of ELISA Plate Immobilized with Anti-Human CETP Polyclonal Antibody The serum obtained in Reference Example 2 was salted out with 50% saturated ammonium sulfate, and the precipitate was dissolved in a small amount of PBS buffer.
Dialysis was performed against the same PBS buffer solution at 4 ° C. for 24 to 28 hours while exchanging the external solution several times. After dialysis, 4 ℃, 3000
The supernatant obtained by centrifugation at rpm for 10 minutes was passed through a prefilter and added to DEAE cellulose equilibrated with 0.0175M PBS (pH 6.3). The eluted fraction was measured at an absorbance of 280 nm, and the protein fractions were collected and added to 0.1%.
1M PBS was added in an amount of 1/9 and the mixture was stored at 4 ° C. Next, the above IgG was added to 0.01M P containing 0.02% thimerosal.
Adjust the concentration to 0.1 mg / ml with BS, dispense 100 μl each into a 96-well plate, leave at 4 ° C for 24 hours, then 0.01M
Wash with PBS, 0.01M PBS containing 1% BSA
The plate was blocked by standing at 4 ° C. for 24 hours to obtain an antibody-bound ELISA plate.

Reference Example 4 Preparation of Peroxidase-Labeled Anti-CETP Polyclonal Antibody a) Preparation of Anti-CETP Polyclonal Antibody F (ab ′) Fragment The purified antibody obtained in Reference Example 2 was added with 0.1M NaCl in 0.1M sodium acetate buffer. It was dialyzed against (pH 4.5). Pig stomach and pepsin (manufactured by Sigma) in this solution
Was dissolved in 4% of the amount of IgG, reacted at 37 ° C. for about 18 hours, and then equilibrated with 0.1 M sodium phosphate buffer (pH 6.0) containing 5 mM EDTA-2Na to Sephacryl S-200HR (1.6 × 100 cm: manufactured by Pharmacia). After collecting the F (ab ') ₂ peak, it was concentrated by ultrafiltration to about 5 mg / ml,
0.1M 2-mercaptoethylamine, and 5 mM EDT
A 0.1 M phosphate buffer (pH 6.0) containing A was added in a volume of 1/10, and the mixture was reacted at 37 ° C. for 90 minutes. Then, this was added with 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA.
Sephacryl S-200HR (1.6 x 1
00cm: Made by Pharmacia Co., Ltd., and attached to a column of F (a
b ') Fractions were collected. The recovered F (ab ') fraction was concentrated by ultrafiltration.

The fragment obtained above was added to 0.1% S
DS (sodium dodecyl sulfate: manufactured by Nacalai Tesque)
3-20% PAGE (polyacrylamide gel electrophoresis) analysis was carried out in the presence of to confirm the molecular weight of the purified antibody fragment. As a result of the above SDS-PAGE,
Since a band was confirmed at a position of a molecular weight of about 50 kD,
The antibody fragment was confirmed to have the expected molecular weight of approximately 50 kD.

B) Anti-CETP polyclonal antibody Fa
Peroxidase labeling of b'fragment N-succinimidyl-4- (N-maleimidomethyl)
Cyclohexane-1-carboxylate 2.5 mg N,
A solution of N-dimethylformamide dissolved in 150 μl was added with 0.1 M sodium phosphate buffer (pH 7.0) 1.
5 mg to 10 mg horseradish peroxidase (HR
P) was added to the dissolved solution and reacted at 30 ° C. for 60 minutes. The precipitate present in the reaction solution is then removed by centrifugation,
It was applied to a Sephadex G-25 gel column equilibrated with a 0.1 M sodium phosphate buffer (pH 6.0) to collect a maleimide / HRP fraction, which was then concentrated by ultrafiltration.

Next, 3.0 mg of F (ab ') obtained in a)
And maleimide / HRP (3.0 mg) obtained in step b) were mixed, reacted at 30 ° C. for 1 hour, and then equilibrated with 0.1 M phosphate buffer (pH 6.5), Sephacryl S-200HR.
Attached to (1.6 × 100 cm: Pharmacia), HR
The PF (ab ') fraction was collected. Recovered HRP-
F (ab ') fraction contains 3 mg / ml thimerosal and 10%
BSA was added at a volume of 1/100, dispensed and stored at -20 ° C.

Example 1 (1) Fasting EDTA blood was collected from a patient or a healthy person and centrifuged (2500 to 3000 rpm) to obtain plasma. Take 2.0 ml of the obtained plasma in a test tube and add 2M NaCl.
(Ionic strength about 0.12) was added (135 μl), sufficiently cooled in water, and then 2.5 ml of cold ethanol sufficiently cooled in ice was slowly added so as not to generate heat, and lightly mixed in ice, and then a mixer was used. And thoroughly mixed (ethanol final concentration 55% (v / v)). After standing in ice for about 10 minutes, centrifuge at 4000 rpm, 0 ° C. for 20 minutes,
The insoluble material was removed. The supernatant was concentrated under reduced pressure to remove ethanol and concentrated to 500 μl.
50 μl is further concentrated to about 50 μl and then SDS-
PAGE was performed. 250 μl of the remaining concentrate is 0.
50 mM phosphate buffer containing 1 M NaCl, 1% BSA, 0.03% thimerosal and 2 mM EDTA-2Na (pH
7.3) (hereinafter referred to as EIA buffer solution) was added to the anti-CETP polyclonal antibody-bound ELISA plate at a volume of 1.0 ml, and the mixture was incubated at 37 ° C for 2 hours.
After completion of incubation, 0.0 containing 0.1M NaCl
After washing three times with 1M phosphate buffer (pH 7.2) (hereinafter referred to as "washing solution"), peroxidase-labeled anti-CETPF
Ab 'was adjusted to 2 µg / ml with EIA buffer, 100 µl of this preparation was added, and the mixture was incubated at 37 ° C for 1 hour. Then, after washing with the washing solution 3 to 5 times, 2,2-azino-
3-ethylbenzothiazoline sulfonate (ABTS)
0.05M phosphate-sodium citrate buffer pH 4.
The solution was dissolved in 2.5 mM at 2, and 100 μl was added and preincubated at 37 ° C. for 2 minutes, 50 μl of 0.02% hydrogen peroxide solution was added, and the mixture was incubated at 37 ° C. for 5 to 10 minutes. Next, 50 μl of 0.05% sodium azide solution was added to stop the reaction, and the absorbance at a wavelength of 405 nm was measured. The result of SDS-PAGE immunoblotting in the assay method of the present invention is shown in FIG.

From FIG. 1, according to the measuring method of the present invention, a band was observed at a molecular weight of 74 kD. The band is anti-C
Reacted with ETP antibody. Further, FIG. 2 shows the result of comparison between the standard curve of CETP purified using the assay method of the present invention and the plasma dilution curve. From FIG. 2, according to the measuring method of the present invention, the plasma dilution curve showed almost parallel to the CETP standard curve.

[Brief description of drawings]

FIG. 1 shows the results of SDS-PAGE immunoblotting.

FIG. 2 is a diagram showing a plasma dilution curve of CETP as a standard curve of CETP.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shoichi Adachi 9349-393 Ishihara-cho, Takasaki-shi, Gunma Prefecture (72) Inventor Kenya Kanemitsu 330-328 Bodhiji, Kosai-cho, Koga-gun, Shiga Prefecture (72) Kazuyoshi Nishikawa 1078-34 Fukagawa, Konan Town, Koga District, Shiga Prefecture

Claims (4)

[Claims] 1. An immunoassay method for cholesteryl ester transfer protein in a sample, which comprises treating the sample with alcohol and then reacting it with an anti-cholesteryl ester transfer protein antibody. 2. The immunoassay method according to claim 1, wherein the alcohol treatment mixes a sample with alcohol to remove insoluble matter and alcohol. 3. A reaction product in the insoluble carrier, which is obtained by treating a sample with alcohol, bringing the sample into contact with an insoluble carrier on which an anti-cholesteryl ester transfer protein antibody is immobilized, and then reacting with a labeled anti-cholesteryl ester transfer protein antibody. An immunoassay method for cholesteryl ester transfer protein in a sample, which comprises measuring the labeled amount of cholesteryl ester. 4. A kit for immunoassay of cholesteryl ester transfer protein, which comprises alcohol and an anti-cholesteryl ester transfer protein antibody.