JP3043528B2 - Assay method for cholesteryl ester transfer protein - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cholesteryl ester transfer protein (CETP: Cholesterylester).
The present invention relates to a method for measuring transfer protein, and more particularly, to CET which is a factor in diagnosing arteriosclerosis.
The present invention relates to a method for simply and accurately measuring P and a kit used for the method.

[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 the pathway of extracting cholesterol from peripheral tissues and transferring it back to the liver. Further, many epidemiological studies have revealed that blood HDL levels are negatively correlated with the frequency of occurrence of ischemic heart disease, and it is known that HDL acts as a protective factor against arteriosclerosis. An important function of the reverse transfer system for extracting HDL cholesterol is lecithin / cholesterol acyltransferase (L
In addition to enzymes such as CAT), hepatic lipase (HTGL) and lipoprotein lipase (LPL), there is CETP. In the living body, CETP is first synthesized as a peptide of 476 amino acids as a very hydrophobic protein, and is glycosylated in cells to have a molecular weight of 74.
000 mature proteins [Hesl
er, C.E. B. et al. , J. et al. 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 and transfer between lipoproteins. That is, LCA
By the T reaction, free cholesterol esterified on HDL is transferred to triglyceride-rich lipoprotein (TGrich lipoprotein) such as VLDL. In exchange for this, TG is reversely transferred at a molar ratio of 1: 1. You. HDL that became TG rich is HTG
The TG part is hydrolyzed by the action of L, and a smaller HD
It is thought 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 and LDL. As an abnormality of the CETP activity system, CETP deficiency has been reported as a case where HDL is elevated [Ya
mashita S .; et al. , Atherosc
lerosis, 70, 7 (1988)]. Also CE
There have been reports of hyper-HDLemia in chronic alcoholic patients with decreased TP activity and hyper-HDL-emia associated with primary biliary cirrhosis. Thus, measurement of CETP activity or CETP amount is extremely important for diagnosis of arteriosclerosis and liver disease.

[0003] As a method for measuring the lipid transfer activity of CETP, for example, serum or plasma obtained by fasting blood sampling is subjected to ultracentrifugation to separate a fraction having a specific gravity of 1.21 or more from serum (plasma). Get as. Next, the above sample was added to radioactively labeled radioactive CE-labeled HDL (20 to 50 µg total cholesterol (TC)) and LDL (100 to 150 µg (TC)) to make 100 µl, followed by incubation at 37 ° C for 2 to 3 hours. , 50 μl of a fraction with a specific gravity of serum of 1.21 or more was added, and 50 μl of heparin / MnCl ₂ (2250 IU heparin / 1M) was added.
LDL is precipitated with MnCl ₂ ), the radioactivity of HDL in the supernatant is measured, and the CE transfer activity is calculated based on the change in radioactivity before and after the incubation [Norihiro Sasaki, Japan Clinic, 47 , 534 (1989)]. There is. However, such a method for measuring the lipid transfer activity of CETP has the disadvantage that the operation is complicated and the accuracy is low, such as using ultracentrifugation.

[0004] 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. Methods using monoclonal antibodies include Tall (Tall,
A. ) Et al., A monoclonal antibody TP against CETP.
-2, a competitive RIA method for immobilizing an antigen has been reported. That is, a lipoprotein fraction is obtained by ultracentrifugation, the antigen in the lipoprotein fraction is immobilized, and then reacted with the TP-2 antibody. The reaction product is reacted with ¹²⁵ I-anti-mouse I as a second antibody.
This is a method of reacting with a gG antibody and measuring with a γ-counter [Marcel, Y. et al. L. et al. , J. et al. C
lin. Invest. , 85 , 10-17 (199
0)]. However, using this monoclonal antibody, Brown et al. Report that they could not detect plasma CETP in some members of a family of certain hyperalphalipoproteinemias [Brown, M., et al. L. et al. ,
Nature, 342 , 448-451 (198
9)]. As described above, when a monoclonal antibody is used, if a gene mutation occurs partially in an epitope recognized by the antibody, the protein structure becomes different, and a monoclonal antibody that recognizes a single epitope is disadvantageous in this case. If the CETP fragment exhibits catalytic activity, the epitope from the protein may be missing, and will not be recognized by the monoclonal antibody. In the case of CETP deficiency, the expression level of CETP protein differs between homozygotes and heterozygotes, and depending on the monoclonal antibody, the type of heterozygotes may not be recognized. Furthermore, since this method is an RIA method, it is difficult to make a kit.

As for the measurement of the amount of CETP using a polyclonal antibody, an enzyme immunoassay by Yamashita et al. Has been reported [Yamashita S. et al. et
al. , Clin. Chim. Acta. , 194 , 1
45-160 (1990)]. The method begins with CETP
Was partially purified and polyclonal antibody was immobilized on anti-CE
Using a TP antibody binding affinity gel, reacting it with serum, then adding the partially purified CETP, and measuring the CE transfer activity of unreacted CETP in the supernatant,
In this method, the amount of CETP is expressed as a relative% when the normal control is set to 100%. This method requires a large amount of partially purified CETP, and directly
Is not measured, but the CETP activity is ultimately measured. In addition, the measurement of CE transfer activity requires an isotope, which makes it difficult to make a kit, and that the measurement takes time.

[0006] Based on these backgrounds, the inventors of the present invention have performed a heat treatment of a serum sample at 100 ° C.
After reacting the immobilized anti-CETP polyclonal antibody,
A method of measuring the amount of CETP by reacting an enzyme-labeled antibody labeled with peroxidase with an anti-human CETP antibody purified in advance to Fab 'was previously reported [Takamitsu Nakano et al., The Arteriosclerosis Society December, 1991. 5 (Tokyo)]. In addition, in this report, this method of heat treatment enabled quantitative measurement of CETP, which could not be measured by the sample-free method, but serum albumin was recognized as affecting the measurement system. Was shown. As a result of further study, Ig
Since G was found to affect the CETP amount measurement system, a method was described in which plasma was once separated by an anhydrous sodium sulfate treatment to separate and remove IgG, followed by fractionation with a molecular sieve, and then reaction with the above-mentioned immobilized anti-CETP polyclonal antibody. [Kenmitsu Kenke et al., Atherosclerosis Society, June 9, 1992 (Osaka)]. However, in this method, the pretreatment operation of the specimen becomes extremely complicated with the anhydrous sodium sulfate treatment and the molecular sieve fractionation treatment, and thus cannot be adopted as a test means in a normal clinical laboratory.

[0007] In the conventional method, it is said that substances such as lipoprotein transfer inhibitory protein (LTIP) affect CETP measurement values.

[0008]

SUMMARY OF THE INVENTION Accordingly, 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 with high accuracy.

[0009]

Means for Solving the Problems Accordingly, the present inventors have:
Various studies have been conducted to develop a means for removing factors affecting the CETP measurement value in blood by a simple operation, and a sample such as plasma is pretreated with an anti-apoprotein AI antibody,
The present inventors have found that CETP in the obtained apoprotein AI can be quantified with high precision by a simple operation if the CETP in the obtained apoprotein AI is measured by an immunological technique using an anti-CETP polyclonal antibody, thereby completing the present invention. .

That is, the present invention relates to a method for preparing an anti-apoprotein A-
Reacting the antibody I to obtain apoprotein AI, and then detecting a lipoprotein that binds to the anti-CETP polyclonal antibody from the apoprotein AI;
It provides a method for measuring TP. The present invention also provides a kit for measuring CETP containing an anti-apoprotein AI antibody and an anti-CETP polyclonal antibody.

In the method of the present invention, the anti-apoprotein AI used for obtaining the apoprotein AI from the sample is used.
The antibody may be either a monoclonal antibody or a polyclonal antibody, but a monoclonal antibody is preferred. Preparation of anti-apoprotein AI antibody is carried out by a conventional method, for example, polyclonal antibody is obtained by ingesting apoprotein AI to obtain antiserum, and monoclonal antibody is obtained by using purified apoprotein AI as immunogen. It can be performed by a fusion method. Here, purified apoprotein AI as an immunogen
Is known and is prepared according to a conventional method [Advanced
Lipid Research, 6 , 1-68 (19
68)], the human serum was subjected to ultracentrifugation to obtain a specific gravity of 1.063 to
An HDL having a molecular weight of about 1.210 is collected, fractionated by gel filtration to obtain a fraction having a molecular weight of about 28,000, and further purified by gel filtration. A method for producing an anti-apoprotein AI monoclonal antibody is described in Hanland, P .; Chem. Ph
ys. Lipids, 15 , 105 (1975); Ha
nland, P .; Chem. Phys. Lipid
s, 10 , 201 (1976); Koskielak,
J. , Eur. J. Biochem. , 37 , 214
(1978) and JP-A-60-253871. JP-A-60-253871
Anti apoprotein A-I monoclonal antibody obtained by the method described in JP an immunoglobulin subclasses belonging to IgG _1, reacted strongly with HDL, has a characteristic that does not substantially react with VLDL and LDL, Particularly preferred.

The anti-CETP polyclonal antibody used in the assay of the present invention can be obtained by sensitizing purified CETP as an immunogen to animals such as mice, rats, guinea pigs, rabbits, sheep, goats, horses, cows, etc. Antiserum,
Immunoglobulins separated therefrom (for example, Ig
G, IgM, etc.), and F (ab ') ₂ , F
(Ab '), F (ab) and the like.

The purified CET used here as an immunogen
P is 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, continuous isopycnic ultracentrifugation, affinity chromatography, ion exchange chromatography, It is obtained by purification by usual means such as gel filtration, ammonium sulfate fractionation and salting out. Purification of such CETP is performed according to the method of Yamashita et al. [Yamashita, S .; , Et al. , C
linica Chimica Acta, 194 , 1
45-160 (1990)].
More specifically, human plasma was subjected to 46,000 rp in an ultracentrifuge.
Ultracentrifugation at a specific gravity of 1.21 g / ml or more at 48 ° C. for 48 hours at 4 ° C., and a phenyl sepharose CL-4B column equilibrated with a fraction having a specific gravity of 1.21 g / ml or more with 4 mol / l NaCl. (2.5 × 20 cm) and can be separated by chromatography using a Sepharose column. The column 50 mmol / l Tris - HCl, 150mmol / l NaCl, 0.02% NaN 3,
After washing and extraction with a pH 7.4 Tris-HCl buffer, the protein bound to the column was extracted with water, and the fraction containing the active fraction was CM-cellulose equilibrated with 50 mmol / l sodium acetate (pH 7.5). Column (CM-52) (2.
5 × 20 cm), 50 mmol / l sodium acetate, 0.02% NaN ₃ , 1 mg / ml EDTA-2N
a (pH 4.5) can be eluted with a linear gradient of 600 ml of NaCl. Elution fraction is 1 ml
Collected in a tube containing 1 mmol / l Tris-hydrochloric acid (pH 8.5), neutralized, adsorbed the fraction containing the active fraction, dialyzed against Tris / NaCl buffer, and then treated with Amicon PM30 membrane. 10 by ultrafiltration
It is possible to obtain a CETP that has been purified by a factor of 00.

In order to obtain an anti-CETP polyclonal antibody using purified CETP, for example, PBS can be added to purified CETP.
(Phosphate-buffered saline) and the like to adjust to an appropriate concentration, and then add an equal amount of an adjuvant such as Freund's complete adjuvant solution to prepare a suspension. The solution is added to the above mammal in an appropriate amount, for example, CET.
The amount of P is 20 to 1000 μg / animal per dose.
Immunization is performed by subcutaneous administration every two weeks. This can be performed by repeating the procedure six times and immunizing, and then collecting whole blood from the animal to obtain an antiserum. The antiserum thus obtained can be purified by usual means such as affinity chromatography, ion exchange chromatography, gel filtration, ammonium sulfate fractionation and salting out.

In the measurement method of the present invention, examples of the specimen include blood, cell tissue fluid, and the like.
In particular, fasting serum or plasma is preferable, and these can be prepared from a subject by collecting blood from a subject according to a conventional method.

In order to carry out the measurement method of the present invention, first, an anti-apoprotein AI antibody is allowed to act on a sample to obtain apoprotein AI from the sample. The means for obtaining the apoprotein AI includes:
Although not particularly limited, a method using an affinity gel in which an anti-apoprotein AI antibody is immobilized on an insoluble carrier is preferable. More specifically, a sample is added to the affinity gel mixture to which the anti-apoprotein A-I antibody has been bound and reacted, to form an apoprotein A-I insolubilized antibody complex, and the unbound substance is separated with an appropriate washing solution. After that, the apoprotein AI and the anti-apoprotein AI antibody may be separated from the apoprotein AI insolubilized antibody complex.

The affinity gel used here can be prepared by a usual method. For example, an anti-apoprotein AI antibody is coupled to an affinity gel activated with bromocyan or the like, and then both are mixed and equilibrated with an appropriate buffer. As a typical example of the affinity gel used here, for example, Sepharose 4B (manufactured by Pharmacia LKB) activated with bromocyan or the like can be exemplified. The coupling reaction is commonly used for the coupling reaction between the gel and the protein. The pH is usually 7-7.
The reaction can be performed within 2 hours at room temperature (about 20 to 25 ° C.) using about 10 to 100 μmol of the anti-apoprotein AI antibody per ml of gel in the range of 5. The affinity gel mixture thus prepared can be used as it is or in a tube that can be shaken and centrifuged beforehand with an appropriate buffer solution, for example, 0.1 mL.
01M Tris-HCl buffer (pH 7.4), 0.15M
It is preferable to equilibrate the gel with a buffer such as NaCl. Further, the buffer may contain commonly used preservatives such as, for example, sodium azide.

The reaction between the obtained affinity gel mixture and the sample is carried out, for example, by placing the affinity gel mixture in a container capable of shaking and centrifuging, equilibrating with an appropriate buffer, and then adding a predetermined amount of This is performed by adding a sample and allowing the mixture to stand or shake at room temperature. After the reaction, the supernatant is removed by centrifugation at a relatively low speed (800 to 1000 rpm), and the apoprotein AI-insolubilized antibody complex is obtained by collecting the precipitate. The apoprotein AI can be separated from the obtained complex by subjecting the residue to solid phase-liquid phase separation which is usually used, for example, centrifugation, filtration, decantation, washing and the like. it can. More specifically,
After washing with PBS (pH 7.2) or the like to remove non-antibody-bound substances, the anti-apoprotein AI antibody-bound complex was
3M for separation into I and antibody binding affinity gel
Apoprotein AI can be separated by elution with an antibody separation solution such as sodium thiocyanate (pH 7.4). The apoprotein AI obtained as described above can be further purified by usual means such as salting out, gel filtration, affinity chromatography and the like. By the above operation, apoprotein A containing the substance to be measured CETP
-I is obtained with high recovery.

Next, anti-CET was obtained from the obtained apoprotein AI.
In order to detect a lipoprotein that binds to the P polyclonal antibody, usual immunological means using an anti-CETP polyclonal antibody, for example, a competition method, a radioimmunoassay (RIA) method by a sandwich method, an immunoassay method (ELIS
A), which can be performed by an immunological technique such as an agglutination method. The lipoprotein molecule is brought into contact with an insoluble carrier on which an anti-CETP polyclonal antibody is immobilized, and then a labeled anti-CETP polyclonal antibody is reacted therewith. It is preferable to carry out the measurement by measuring the labeling amount of the reaction product in the carrier.

Here, the immobilization of the anti-CETP polyclonal antibody is performed 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, etc. can be used, and the insolubilization is performed by a diazo method, a peptide method, an alkylation method, a cross-linking method as a covalent bond method. Carrier binding method using reagents, chemical reaction such as carrier binding method by Ugi reaction, or ion binding method using a carrier such as ion exchange resin, physical adsorption method using porous glass such as glass beads as a carrier However, from the viewpoint of operability when washing and simultaneously processing a large number of samples, 96
Preferably, a well plastic plate is used.

The labeling agent used for labeling the above-mentioned anti-CETP polyclonal antibody includes a conventional labeling agent,
For example, iodine isotopes (eg, ¹²⁵ I, ¹³¹ I), ¹⁴
C, radioactive isotopes such as tritium; fluorescent substances such as fluorescein isocyanate, fluorescein isothiocyanate and rhodamine; peroxidase, alkaline phosphatase, β-D-galactosidase,
Enzymes such as glucose oxidase and penicillinase can be mentioned, but enzymes are preferred for kitting reagents.
The enzyme is already commercially available peroxidase (POD), β
-D-galactosidase, acid phosphatase, alkaline phosphatase and the like can be used. For labeling the anti-CETP polyclonal antibody with these labeling agents, a method known per se is employed.

As the solvent used in the measurement system, any of various ordinary solvents which do not adversely affect the reaction can be used. For example, citrate buffer, phosphate buffer, tris acid buffer, acetate buffer, etc. It is preferable to use a buffer having a pH of about 5.0 to 9.0. In the present invention, the solvent contains about 0.1 to 30% w / v of serum (containing no CETP to be measured) and / or about 0.1% w / v.
It is preferable to include about 2 M of NaCl, because it is more consistent with the purpose of the measurement method of the present invention.

The immune reaction conditions for detecting CETP are not particularly limited, and may be the same as those of a usual measurement method of this type. Generally below 45 ° C, preferably about 4 to
The reaction may be performed at a temperature of about 40 ° C. for about 1 to 80 hours. Separation of the solid-liquid phase (combination of the reaction complex and the labeled antibody in the sandwich method-unbound labeled antibody) after the completion of the immune reaction is performed by, for example, centrifugation, filtration, decantation, washing, and the like. It can be performed by the method of.

In order to measure the amount of the reaction product in the carrier thus separated, the amount varies depending on the type of the labeling agent.
When the labeling agent is an enzyme, it can be carried out by further coexisting a coloring reagent to lead to a coloring reaction and measuring the amount of the dye. Examples of the coloring solution used in this case include o-phenylenediamine, 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) or the like can be used, and the coloration reaction can be stopped according to a conventional method. It can be carried out by adding an agent. In order to determine the amount of CETP in the sample from the obtained amount of label, a calibration curve is created in advance,
It is preferable to carry out by contrasting this.

As a particularly convenient method for carrying out the above CETP measurement method, there is a method using a kit for detecting CETP. The kit is preferably a kit in which a reagent containing an anti-apoprotein AI antibody and a reagent containing an anti-CETP polyclonal antibody are combined. It is preferable that the kit is supplied with the anti-apoprotein AI antibody as an affinity gel as a first reagent and a reaction buffer at the same time. As the second reagent, an anti-CETP polyclonal antibody immobilized on an insoluble carrier and a labeled anti-CETP
More preferably, a polyclonal antibody is provided.
In addition, as long as the measured value is not adversely affected,
Further, if necessary, a stabilizer and / or a preservative such as saccharose, glycerol, bovine serum protein and the like can be added. The antibody reagent may be freeze-dried, and the kit may contain a water-soluble or water-miscible solvent. Further, the antibody reagent may contain a buffer solution for maintaining the reagent system reconstituted at the time of measurement at a constant pH and / or a preservative and / or a stabilizer for preventing deterioration of the sample. . Although the buffer is not an essential component of the kit reagent, when performing the measurement method of the present invention, the pH is adjusted to about 5.
It is preferable to use one having a value of about 0 to 9.0. The reconstituting agent preferably contains water, but part or all of the water can be replaced with a solvent miscible with water. Examples of the water-miscible solvent include glycerin, alcohols, glycol ethers and the like.

[0026]

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

[0027]

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

Reference Example 1 Preparation of affinity gel for anti-human apoprotein A-I monoclonal antibody
The purified anti-human apoprotein A-I monoclonal antibody (IgG subclass is IgG ₁ ) obtained in Examples of 0-253871 was used. Sepharose 4B (Pharmacia LKB) was used as the affinity gel for antibody binding. Sepharose 4 previously activated with Bromcian
B was added to an anti-human apoprotein AI monoclonal antibody and a gel in 0.01M Tris-HCl buffer (pH 7.4) at a pH of 7.5 by adding 5 mg of each antibody protein / ml to the gel.
The mixture was subjected to a coupling reaction at room temperature for 2 hours to obtain 20 ml of a mixture of affinity gels binding anti-human apoprotein AI monoclonal antibody. This mixture is placed in a 50 ml centrifuge tube in 0.01 M Tris-HCl buffer (pH
Equilibrated in buffer of 7.4) and 0.15 M NaCl and stored at 4 ° C.

Reference Example 2 Purification of human CETP 300 ml of normal human plasma was subjected to an ultracentrifuge (model name: 55P-7).
2, Hitachi, Ltd.) at 46000 rpm at 4 ° C
Phenyl sepharose CL-4B column which was subjected to continuous isopycnic ultracentrifugation at a specific gravity of 1.21 g / ml or more for 48 hours, and a fraction of the obtained plasma having a specific gravity of 1.21 g / ml or more equilibrated with 4 mol / l NaCl in advance. (2.5 × 20c
m; manufactured by Pharmacia LKB) and chromatographed on a Sepharose column. Then add 50 columns
mmol / l Tris-HCl, 150 mmol / l NaCl,
0.02% NaN _3, Tris pH 7.4 - washed extracted with HCl buffer, the protein bound to the column was extracted with water, the active fraction fraction 50 mmol / l sodium acetate containing (pH 7.5) -Cellulose column (CM-52) equilibrated with (2.5 × 20 cm; Pharmacia LKB)
50 mmol / l sodium acetate,
0.02% NaN ₃ , 1 mg / ml EDTA-2Na (pH
The mixture was eluted with a linear gradient of 600 ml of NaCl in 4.5). The eluted fraction was collected in a tube containing 1 ml of 1 mmol / l Tris-hydrochloric acid (pH 8.5) and neutralized, the fraction containing the active fraction was adsorbed, and dialyzed against Tris / NaCl buffer. Later, Amicon P
Ultrafiltered through a M30 membrane. By the above operation, 1000 times purified CETP was obtained.

Reference Example 3 Preparation of anti-human CETP polyclonal antibody Purified CETP (2 mg / ml) obtained in Reference Example 2 and Freund's complete adjuvant (Freud's Complete)
Eadjuvant (manufactured by Nacalai Tesque, Inc.) was mixed in equal amounts, and the mixture was injected into dozens of rabbit limbs and back skin to immunize. The same administration was performed 5 to 6 times during 2 to 3 months. Two weeks after the final administration, whole blood was collected, and the serum was obtained.

Reference Example 4 Preparation of Immunoplate Immobilized with Anti-Human CETP Polyclonal Antibody The serum obtained in Reference Example 3 was subjected to salting out and fractionation with 50% saturated ammonium sulfate, and the precipitate was dissolved in a small amount of PBS buffer.
Dialysis was performed against the same buffer at 4 ° C. for 24 to 28 hours while exchanging the external solution several times. After dialysis, 4 ° C, 3000 rpm,
The supernatant after centrifugation for 10 minutes was passed through a prefilter and added to DEAE cellulose equilibrated with 0.0175 M PBS (pH 6.3). The eluted fraction was measured at an absorbance of 280 nm, and the protein fraction was collected and 0.1 M
1/9 volume of PBS was added and stored at 4 ° C. IgG0.
0.01M P containing 1mg / ml 0.05% thimerosal
Put 100 μl / well of BS into immunoplate,
Immobilized for 0.01 days, 0.01M PBS containing 0.1% BSA
And washed three times. 1% BSA, 5% D-sorbitol,
0.01M PBS200 containing 0.05% thimerosal
Incubate at 4 ° C. for 24 hours at μl / well and block. Further, the plate was used after freeze-drying.

Reference Example 5 Preparation of peroxidase-labeled human CETP polyclonal antibody a) Preparation of anti-CETP polyclonal antibody F (ab ') fragment The purified antibody obtained in Reference Example 3 was replaced with 0.1 M Na
0.1 M sodium acetate buffer containing Cl (pH 4.5)
Was dialyzed against. This solution was dissolved by adding pig stomach / pepsin (manufactured by Sigma) at 4% of the IgG amount, and the solution was added at 37 ° C. for about 1 hour.
8 hours, then adjusted to pH 8.0 with 1N NaOH, and equilibrated with 0.1 M sodium borate buffer (pH 8.0) containing 0.1 M NaCl.
It was applied to a column of cA44 (1.5 × 45 cm; manufactured by IBF Biotechnics). After collecting the F (ab ') ₂ peak, it was concentrated to about 5 mg / ml by ultrafiltration,
Dialysis was performed against a 0.1 M phosphate buffer (pH 6.0) containing mM EDTA. After dialysis, one-ninth volume of 0.1 M phosphate buffer (pH 6.0) containing 0.1 M 2-mercaptoethylamine and 5 mM EDTA was added, and the mixture was added at 37 ° C., 9
The reaction was performed for 0 minutes. Then, this was applied to a column of Sephadex G-25 gel (1 × 30 cm; manufactured by Pharmacia LKB) equilibrated with a 0.1 M phosphate buffer containing 50 mM EDTA and 0.1 M NaCl, and F (ab ′) Fractions were collected. The collected F (ab ') fraction was concentrated by ultrafiltration, and dialyzed against 0.1 M phosphate buffer containing 2.5 mM EDTA. 0.1% SD of the obtained fragment
7.5% PAGE (polyacrylamide gel electrophoresis) analysis was performed in the presence of S (sodium dodecyl sulfate: manufactured by Nacalai Tesque) to confirm the molecular weight of the purified antibody fragment. As a result of SDS-PAGE, a band was confirmed at a position having a molecular weight of about 75 kd, which confirmed that the antibody fragment had the expected molecular weight.

B) Peroxidase labeling of anti-CETP polyclonal antibody fragment 2.5 mg of N-succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate was added to N, N
A solution of dimethylformamide dissolved in 150 μl of 0.1 M sodium phosphate buffer (pH 7.0)
10 mg of horseradish peroxidase (HR in 5 ml)
P) was dissolved in the solution and reacted at 30 ° C. for 60 minutes. Then, the precipitate present in the reaction solution was removed by centrifugation,
The mixture was subjected to Sephadex G-25 gel equilibrated with 0.1 M sodium phosphate buffer (pH 7.0),
After collecting the HRP fraction, it was concentrated and concentrated to 2.5 mM EDT.
A was dialyzed against 0.1 M sodium phosphate buffer containing A.
Next, 2.0 mg of F (ab ') obtained in a) and 1.8 mg of maleimide HRP obtained in b) were mixed and mixed with 2.5 mM E.
0.1 M sodium phosphate buffer containing DTA was added to make up to 1.0 ml, reacted at 30 ° C. for 1 hour, and then reacted with 50 nM N 2.
20 μl of ethylmaleimide was added. Then 0.1M
It was applied to Ultrogel AcA44 (1.5 × 45 cm; IBF Biotechnics) equilibrated with a 0.1 M phosphate buffer containing NaCl, and the HRP F (ab ′) fraction was collected. The recovered HRP F (ab ') fraction is
After dialysis with S (-), 2 mg / ml thimerosal was added at 1/100, dispensed and stored at -20 ° C.

Example 1 (1) Fasting EDTA blood was collected from a patient or a healthy subject, and centrifuged (2,500 to 3000 rpm) to obtain plasma. For each of the obtained plasmas, the amount of triglyceride was determined by the enzyme method (T
G-555, manufactured by Kyowa Medex Co., Ltd.), and the amount of cholesterol is determined by an enzyme method (Mercko Test CH).
O, manufactured by Kanto Chemical Co .; 2 ml / 10 μl plasma, 37 ° C., 10
(Incubation for 3 minutes) (absorbance at 365 nm). Apoprotein AI was measured using an anti-human apoprotein AI monoclonal antibody (manufactured by Japan Antibody Research Institute, Inc.).

(2) 0.25 ml of serum obtained by removing fibrinogen such as fibrinogen from the above plasma was added to a test tube, and the anti-human apoprotein AI monoclonal antibody-bound affinity gel obtained in Reference Example 1 was contained. 300 μl of the reaction solution to be prepared was dispensed, shaken for 30 minutes, and then centrifuged at a low speed (800 rpm) to remove the supernatant.
Next, the precipitate was washed with 20 ml of a PBS buffer (pH 7.2), and then 1.5 ml of 3M sodium thiocyanate (pH 7.0).
The antibody separation solution of 4) was added to elute 1.5 ml of Apoprotein AI. The volume of the eluate obtained above was adjusted to 2 ml, and 100 μl / well was placed in the immunoplate immobilized with the anti-human CETP polyclonal antibody obtained in Reference Example 4,
Incubated at 7 ° C. for 2 hours. The reaction solution was removed.
0.01M PBS (pH 7.0) containing 1% BSA 300
Washed three times with μl. Then, peroxidase-labeled anti-C
0.01M of ETP-F (ab ') containing 0.1% BSA
100 µl / well of PBS (pH 7.0) was added, and 37 ° C
For 1 hour. After removing the reaction solution, the immunoplate was washed three times with 300 μl of the above washing solution. 2.5
Buffer containing mM 2,2'-azino-3-ethylbenzothiazoline sulfonate (ABTS) (0.01 M trisodium citrate and 0.01 M phosphate buffer, pH 4.0).
0) was added at 100 μl / well, and the mixture was pre-incubated at 37 ° C. for 2 minutes. 50 μl of 0.02% hydrogen peroxide solution /
After adding a well and incubating at 37 ° C for 6 minutes, 0.05
The reaction was stopped by adding 50 μl / well of a 50% sodium azide solution, and the absorbance at a wavelength of 405 nm was measured using an immunoreader. C purified using the assay of the present invention
The result of comparing the standard curve of ETP and the CETP dilution curve is shown in FIG. As shown in FIG. 1, according to the measuring method of the present invention, CE
The TP dilution curve was almost parallel to the CETP standard curve. Further, the measurement method of the present invention was applied to 5 healthy subjects and high HD.
Table 1 shows the results obtained by using blood of five persons with abnormal lipid metabolism showing L. From Table 1, the quantification value by the measurement method of the present invention showed a high negative correlation with Apoprotein AI.

[0036]

[Table 1]

[0037]

[Brief description of the drawings]

FIG. 1 shows a standard curve of CETP and a dilution curve of CETP.

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Claims (4)

(57) [Claims] 1. An anti-apoprotein AI antibody is allowed to act on a sample to obtain an apoprotein AI, and then a lipoprotein that binds to the anti-cholesteryl ester transfer protein polyclonal antibody from the apoprotein AI is detected. A method for measuring cholesteryl ester transfer protein in a sample, which is characterized in that: 2. The method according to claim 1, wherein the means for obtaining Apoprotein AI uses an affinity gel in which an anti-Apoprotein AI antibody is immobilized on an insoluble carrier. 3. Detection of lipoprotein binding to an anti-cholesteryl ester transfer protein polyclonal antibody is performed by contacting apoprotein A-I with an insoluble carrier having an anti-cholesteryl ester transfer protein polyclonal antibody immobilized thereon, and then labeling it with a labeled anti-cholesteryl ester transfer protein polyclonal antibody. 2. The method according to claim 1, wherein the reaction is carried out with an ester transfer protein polyclonal antibody, and the amount of the labeled reaction product in the carrier is measured. 4. A kit for measuring cholesteryl ester transfer protein, comprising an anti-apoprotein AI antibody and an anti-cholesteryl ester transfer protein polyclonal antibody.