JPH11346782A - Monoclonal antibody for human pltp protein, measurement of human pltp using the same and measuring kit for carrying out this measurement - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new monoclonal antibody for human PLTP protein which is composed of a monoclonal antibody for human phospholipid transfer protein(PLTP) and is conveniently with high sensitivity measurable PLTP in blood and useful in the diagnosis or the like of lipid metabolic disorder disease, e.g. arterosclerosis. SOLUTION: This antibody is a new monoclonal antibody for human phospholipid transfer protein (PLTP), enables to conveniently with high sensitivity measure PLTP in blood, gives a judgement more precisely to diseases deeply participating in lipid metabolic disorder, e.g. arterosclerosis or the like, and can suitably decide the therapeutic course. The monoclonal antibody is obtained by initially carrying out PCR (polymerase chain reaction) with a specific primer using cDNA arising from human liver as a template, administrating the recombinant human PLTP prepared by a conventional method using the resultant PLTP gene to the Balb/c mouse together with an adjuvant to immunize, fusing its spleen cells with mouse myeloma cells, cloning the resultant hybridoma followed by introduction into the mouse abdominal cavity and finally separating from ascites 10 days after introduction.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a monoclonal antibody, a hybridoma used in a production process thereof, and a measurement method using the monoclonal antibody. More specifically, human phospholipid transfer protein (PLTP) plays an important role in human lipid metabolism
And a method for measuring a human phospholipid transfer protein using a monoclonal antibody against the human phospholipid transfer protein, which can be an important indicator of a disease closely related to lipid metabolism abnormalities such as arteriosclerosis and a hybridoma used in the production process thereof, and The present invention relates to a measurement kit for performing this measurement method.

[0002]

2. Description of the Related Art Ester cholesterol (hereinafter, also simply referred to as cholesterol) present in plasma together with free fatty acids plays an important role in living organisms as a component of cell membranes and a source component of endocrine hormones. Cholesterol in vivo is supplied in part by uptake from the small intestine upon food intake, but most of it comes from biosynthesis in cells of each tissue (particularly liver).
The following pathways are known as pathways for supplying cholesterol to peripheral cells in vivo by this biosynthesis.

[0003] That is, free cholesterol biosynthesized in the liver and the like is taken up by very low density lipoprotein (VLDL) and secreted into the blood. (HTGL), the intermediate specific gravity lipoprotein (ID)
L) and is metabolized to low density lipoprotein (LDL). Next, it is known that this LDL is taken into cells via LDL receptors present on the peripheral cell membrane surface, thereby supplying free cholesterol to the cells. In contrast to such a system for transferring cholesterol from the liver or the like to peripheral cells, a "reverse cholesterol transfer system"
It is known that there is a pathway for transferring cholesterol from peripheral cells to the liver, which transfers excess cholesterol to the liver again, called cholesterol.

[0004] That is, excess free cholesterol supplied from the liver to peripheral cells is extracted by high density lipoprotein (HDL) in the blood and converted into cholesterol ester (CE) by the action of lecithin cholesterol acyltransferase (LCAT). It is converted and stored in HDL in the blood. The CE stored in HDL extracted from peripheral cells causes cholesterol ester transfer protein (CETP) to act on VLDL,
Cholesterol is reversely transferred to the liver by being indirectly taken into hepatocytes via the LDL receptor in the liver from the VLDL or the like that has been transferred to IDL or LDL and received CE. Also, the HDL that pulled out the CE is
CE can also be transferred by being directly incorporated into hepatocytes via hepatocyte HDL receptors.

[0005] This reverse cholesterol transfer system has been attracting attention as a mechanism for protecting cholesterol from accumulating in peripheral cells and thus preventing arteriosclerosis. In particular, HDL plays an important role in extracting cholesterol from peripheral cells in the reverse cholesterol transfer system.
With regard to, many epidemiological studies have reported the fact that the blood HDL cholesterol concentration is inversely correlated with the development of coronary artery disease. As a result, HDL has been widely recognized as a lipoprotein capable of exerting an anti-atherosclerotic effect.

[0006] In recent years, the importance of the action of CETP in mediating the transfer of cholesterol ester in HDL to blood LDL has been increasing in various diseases such as CETP deficiency, hyperlipidemia, diabetes mellitus and nephrotic syndrome. It is being revealed from the relationship with. For example, in the hyperlipidemic group, the CETP in the blood was higher than that in the normal control group.
It has been experimentally confirmed that the activity is high. Also,
CETP deficient HDL particles exhibiting hyper HDL blood
It is rich in CE and has become large particles.
It has been clarified that DL does not exert an antifoaming / defoaming effect on macrophages.

The role of HDL involved in reverse cholesterol transfer described above has been particularly actively studied in recent years.
Pre β-HDL, which can be said to be a precursor, is a small particle HDL that is poor in lipid components mainly composed of apolipoprotein A1, extracts cholesterol from peripheral cells, and is converted into spherical HDL circulating in the blood. It has been found that this spherical HDL is carried to the liver.

[0008] Further, HDL has a particle size, constituent components,
The HDL subpopulations are considered to have different functions, depending on the particle density or the difference in electrophoretic mobility, divided into several subpopulations.

It is important to elucidate the factors controlling the distribution of HDL subpopulations in order to know the cholesterol metabolism and the pathogenesis of coronary artery disease and to prevent them. In this regard, several plasma factors are known to affect HDL components or particle size.

[0010] For example, LCAT, HTGL, LPL, CETP, and phospholipid transfer protein (PLTP) are present in blood and have the ability to transfer lipids between lipoproteins. Has the function of transferring phospholipids between lipoproteins
llefson, Albers, Methods Enzymol, 129,797-816,1986, Ta
ll, J Lipid Res, 34, 1255-1274, 1993), and it has recently been found to alter the constituents and particle size of HDL (Lusa, Jauhiainen, Metso, Somerharju, Ehnh).
olm, Biochem J, 313, 275-282, 1996).

The transfer of lipids between HDL subpopulations by PLTP is as important as the transfer of lipids between HDL and other lipoproteins. By adding purified PLTP to HDL3 (HDL in a form incorporating cholesterol ester) in a test tube, 2
It has been shown to produce two new subpopulations of different particle sizes (small and large subpopulations different from HDL3 particles).
(Jauhiainen, Metso, Pahlman, Blomqvist, van Tol, Ehnhol
m, J Biol Chem, 268,4032-4036,1993, Tu, Nishida, J Biol
Chem, 268, 23098-23105, 1993). This subpopulation of small particles is pre-β, which is considered to be primitive HDL.
1-HDL, capable of extracting excess cholesterol from cells. Furthermore, PLTP is known to have a large effect of converting HDL particles that have become TG-rich due to the action of CETP in blood into subpopulations of large and small particle diameters (Rye KA, Jauhiaine).
n M, BarterPJ, Ehnholm C, J Lipid Res, 1998, 39: 613-62
2.)

Furthermore, a human PLTP cDNA is incorporated into an adenovirus [a gene encoding PLTP has also been identified (Joseph R, et al., J Biol Chem, 269: 9388-9).
391, 1994.)).
In mice expressing human PLTP, P in blood
With the increase in LTP activity, a decrease in blood HDL and apoA1 was observed, and the decrease in HDL, which is considered to be due to the expression of human PLTP, is known to be due to reverse transfer of HDL to the liver (Foger , Santamaria-Fujo, Shambure
k, Parrot, Talley, Brewer Jr., J Biol Chem, 43,27393-27
400, 1997).

[0013]

As described above, the PL
It is almost clear that TP plays a very important role in the reverse cholesterol transfer system in vivo. As for PLTP, in addition to the above, the following items have already been clarified:

It does not transfer cholesterol esters or triglycerides in lipoproteins. It is not recognized by an antibody against "LTP-I" which is a human cholesterol ester transfer protein. No amino acid sequence homology is observed with this human cholesterol ester transfer protein “LTP-I”.

The apparent molecular weight is Sephacryl S2
In 00, it is about 70000, and in SDS-PAGE, it is about 69000. The apparent isoelectric point by chromatofocusing is
5.0. The heat treatment at 58 ° C. for 1 hour almost eliminates the activity. Has affinity for heparin sepharose.

[0016] The problem to be solved by the present invention is to produce a PLTP monoclonal antibody which plays an important role in such a lipid metabolism system, and to use the monoclonal antibody to convert PLTP in blood in a simple and convenient manner. Establish a means for measuring PLTP that can be measured with high sensitivity and can be used in clinical practice, make more accurate judgments on abnormalities in lipid metabolism, and determine an appropriate treatment policy Is to provide. Note that the above PLTP
Monoclonal antibodies against P
It is considered to be extremely useful as a reagent for separating and purifying LTP itself.

[0017]

Means for Solving the Problems The present inventor has made intensive studies to solve this problem. As a result, the desired P
A monoclonal antibody against LTP, a hybridoma used for producing the monoclonal antibody, and a method for measuring PLTP using the monoclonal antibody were established. In particular, the present inventors have determined that by using a biologically active, recombinant human PLTP as an immunogen, different high binding specificities can be obtained for human-derived PLTPs, especially for PLTPs present in human body fluids. It was possible to successfully produce an anti-human PLTP monoclonal antibody, and to solve the above problem in an extremely accurate manner by using such an anti-human PLTP monoclonal antibody in the above-described method for measuring PLTP. The inventors have found that this is possible and completed the present invention.

That is, the present inventor provides the following invention in the present application. First, a monoclonal antibody against human PLTP, particularly a monoclonal antibody having the following properties (labeled with a labeling substance that produces a detectable signal by reacting the labeling substance alone or the labeling substance with another substance): (Including labeled "monoclonal antibodies" and "immobilized monoclonal antibodies" immobilized on an insoluble carrier). No antigen-antibody reaction is observed with human PLTP; at least no mouse-PLTP antigen-antibody reaction is detected; and these monoclonal antibodies have the mature amino acid sequence of human PLTP of 167-476.
Or a monoclonal antibody recognizing the region 1-165.

Second, the present invention provides a hybridoma which produces the above-mentioned monoclonal antibody against human PLTP [specifically, mouse hybridoma PLTP113 (hereinafter referred to as # 11).
No. 3; accession number: FERM P-16605),
mouse hybridoma PLTP114 (hereinafter also referred to as # 114;
Deposit number: FERM P-16606) and mouse hybr
An example is any hybridoma selected from the group consisting of idoma PLTP110G (hereinafter, also referred to as # 110G; accession number: FERM P-16607)].

Third, one or more of the above-mentioned monoclonal antibodies against human PLTP and an antigen-antibody reaction with human PLTP in the sample, such as an enzyme immunoassay, are used to detect the human phospholipid in the sample. Provided is a method for measuring a human phospholipid transfer protein, which measures a transfer protein.

Fourth, a measurement kit for performing this measurement method is provided.

The present inventor has proposed a recombinant human PL which is preferable as an immunogen when producing the above hybridoma.
A vector capable of efficiently expressing TP in the host and secreting it into the culture supernatant was also created.

[0023]

Embodiments of the present invention will be described below. The monoclonal antibody according to the present invention (hereinafter referred to as the present monoclonal antibody) is a monoclonal antibody against PLTP as described above.

A. Production of the Monoclonal Antibody of the Present Invention The monoclonal antibody of the present invention produces a hybridoma between immunocytes of an animal immunized with recombinant human PLTP and myeloma cells of the animal, and thereby selects a clone producing an antibody that recognizes PLTP. And by culturing this clone.

PLTP as an immunizing antigen can be obtained as follows. Conventionally known separation / fractionation from lipoprotein-deficient serum (density: d> 1.21) obtained from plasma of human or other animal species, if necessary, by a conventional method such as ultracentrifugation Method, for example, gel filtration chromatography, hydrophobic chromatography, ion exchange chromatography, affinity chromatography using heparin agarose, or the like, and may be used in combination as necessary (for example, Jo).
hn H Tollefson et al., J Lipid Res, 29,1593-1602,198
8 and Pirkko Pussinen et al., J Lipid Res, 36, 975-98
5, 1995).

PLT as immunizing antigen used here
P is not particularly limited, and PLTP encoded by the PLTP gene (including a partially modified base sequence thereof) as used in the present invention can be used. PLT encoded by some fragments
A PLTP partial peptide obtained by subjecting a P fragment or PLTP to an enzymatic treatment or the like or chemically synthesized can also be used as an immunizing antigen for producing the monoclonal antibody of the present invention.

The animal to be immunized is not particularly limited, and mice and rats can be widely used. When a monoclonal antibody is produced, compatibility with the myeloma cells used for cell fusion is required. It is desirable to select in consideration of.

Immunization can be carried out by a general method, for example, by administering the above immunizing antigen to an animal to be immunized by intravenous, intradermal, subcutaneous, intraperitoneal injection or the like.

More specifically, the above-mentioned immunizing antigen is administered to an animal to be immunized several times every 2 to 14 days by the above-mentioned means, optionally in combination with a usual adjuvant, to produce the monoclonal antibody of the present invention. For example, spleen cells after immunization can be obtained.

When a monoclonal antibody is produced, the myeloma cell as the other parent cell to be fused with the immune cell is already known, for example, SP2 / 0-Ag.
14, P3-NS1-1-Ag4-1, MPC11-4
5,6. TG1.7 (all derived from mouse); 210. R
CY. Ag 1.2.3 (from rat); SKO-00
7, GM15006TG-A12 (all derived from humans) and the like can be used.

Cell fusion between the above-mentioned immune cells and the myeloma cells is carried out by a generally known method, for example, the method of Kohler and G. and Milstein, C., Nature , 256 , 495.
(1975)).

More specifically, the cell fusion is carried out by a generally known fusion promoter such as polyethylene glycol (PE).
G) or in the presence of Sendai virus (HVJ) or the like, a hybridoma is prepared in a usual culture medium to which an auxiliary agent such as dimethyl sulfoxide is added as necessary to improve the fusion efficiency.

The desired hybridoma can be isolated by culturing it in a usual selection medium, for example, a HAT (hypoxanthine, aminopterin and thymidine) medium. That is, hybridomas can be separated by culturing them in this selection medium for a time sufficient to kill cells other than the target hybridomas. The hybridoma thus obtained can be subjected to a search for a target monoclonal antibody and a single cloning by a usual limiting dilution method.

The target monoclonal antibody producing strain can be searched for, for example, by ELISA, plaque, spot method, or the like.
It can be performed according to a general search method such as an agglutination reaction method, an octalony method, and an RIA method.

The hybridoma producing the desired monoclonal antibody recognizing human PLTP thus obtained can be subcultured in a usual medium,
Furthermore, it can be stored for a long time in liquid nitrogen.

The desired monoclonal antibody can be collected from the hybridoma by culturing the hybridoma according to a conventional method to obtain a culture supernatant, or by administering the hybridoma to an animal that is compatible with the hybridoma and growing it. Then, a method of obtaining ascites and the like can be used.

The immune cells are cultured in vitro in the presence of PLTP or a part thereof, and after a certain period of time, a hybridoma of the immune cells and myeloma cells is prepared using the above-mentioned cell fusion means. And the monoclonal antibody of the present invention can be obtained by screening (Reading, CL, J. Immunol. Meth. , 53, 261 (198
2); Pardue, RL, et al., J. Cell Biol., 96 , 1149 (198
3)).

The polyclonal antibody and the monoclonal antibody obtained as described above can be further purified by ordinary means such as salting out, gel filtration, affinity chromatography and the like.

The thus obtained monoclonal antibody of the present invention is an antibody having specific reactivity to PLTP. This monoclonal antibody of the present invention can be used in clinical practice,
It is extremely useful as a reagent for its own separation and purification.

The specific contents of the monoclonal antibody of the present invention will be described later in Examples. The thus obtained monoclonal antibody of the present invention can be labeled with a labeling substance, if necessary, and used in a measurement method or the like according to the present invention described later.

Such a labeling substance can be used alone or by reacting the labeling substance with another substance.
It is a labeling substance that provides a detectable signal, and specifically, for example, horseradish peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase , Fluorescent substances such as penicillinase, catalase, apoglucose oxidase, urease, luciferase or acetylcholinesterase, fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelates, dansyl chloride or tetramethylrhodamine isothiocyanate, ¹²⁵ I, ¹⁴ C, ³
Radioisotopes such as H; chemical substances such as biotin, avidin and digokesigenin; and chemiluminescent substances.

As the labeling method of the monoclonal antibody of the present invention with these labeling substances, a known labeling method can be appropriately used according to the type of labeling substance to be selected.

Further, the monoclonal antibody of the present invention (including a labeled antibody) may be immobilized on an insoluble carrier and used as an immobilized monoclonal antibody in a measurement method and the like according to the present invention described later.

As such an insoluble carrier, various insoluble carriers already used as an insoluble carrier for an antibody can be used. Specifically, for example, a plate represented by a microplate, a test tube, a tube, a bead,
For affinity chromatography of balls, filters, membranes, or cellulosic carriers, agarose carriers, polyacrylamide carriers, dextran carriers, polystyrene carriers, polyvinyl alcohol carriers, polyamino acid carriers, or porous silica carriers. Examples include the insoluble carrier used.

In the method for immobilizing the monoclonal antibody of the present invention on these insoluble carriers, the method for immobilizing the antibody already established on various insoluble carriers is appropriately selected according to the type of the insoluble carrier to be selected. be able to.

B. Measuring method according to the present invention The measuring method according to the present invention (hereinafter referred to as the measuring method of the present invention) comprises the monoclonal antibody of the present invention and PLTP
This is a method for measuring PLTP in which a PLTP in a sample is measured by utilizing an antigen-antibody reaction with the PLTP.

Examples of the measurement method of the present invention utilizing the antigen-antibody reaction include, for example, enzyme immunoassay, radioimmunoassay, analysis by flow cytometry, Western blotting, and the like. Considering the simplicity of handling and measurement sensitivity, it is preferable to select an enzyme immunoassay.

The enzyme immunoassay is also referred to as an enzyme immunoassay, and is a measurement method using an enzyme as a labeling substance among labeled immunoassays. Enzyme immunoassay requires so-called B / F separation
It is roughly classified into "heterogeneous enzyme immunoassay" and "homogeneous enzyme immunoassay" which does not require B / F separation.

In the present invention, among these, the former "heterogeneous enz" which is generally said to have high measurement sensitivity
It is more preferable to select "yme immunoassay" and use "enzyme-linked immunosorbent a"
More preferably, "ssay (ELISA)" is selected.

As a more specific measurement mode, an enzyme immunoassay method by a so-called sandwich method (hereinafter, also referred to as a sandwich method) can be exemplified. Such a sandwich method is one of the particularly preferable measurement modes, especially in view of simplicity of operation and economic convenience, especially versatility as a clinical test.

When the sandwich method is carried out, the monoclonal antibody of the present invention is immobilized on a microplate having a large number of wells, such as a 96-well microplate, and the above-described Western antibody It is preferable to use the monoclonal antibody of the present invention labeled with an enzyme such as horseradish peroxidase or biotin.

The sandwich method comprises at least the following (a)
And an enzyme immunoassay method comprising the steps of (b). (a) a step of reacting a sample represented by a blood sample such as human plasma with the immobilized monoclonal antibody in which the monoclonal antibody of the present invention is immobilized on an insoluble carrier. In this step (a), usually, after the reaction, the used microplate is washed, and the unreacted sample is removed from the immobilized monoclonal antibody.

(B) a step of reacting the antigen-antibody complex formed by the binding of the immobilized monoclonal antibody and human PLTP in the sample with the monoclonal antibody of the present invention labeled with horseradish peroxidase, biotin, or the like.

In this step (b), usually, after the reaction, the used microplate is washed, and the unreacted labeled monoclonal antibody of the present invention is removed from the immobilized monoclonal antibody.

In this step (b), it is necessary to make the label signal visible by using a means for expressing the label signal according to the type of the label in the reacted second antibody. For example, when biotin is used as the labeling substance, the labeling signal can be made visible using avidin or the like. Also, for example, when horseradish peroxidase is selected as a labeling substance,
The substrate can be added, if necessary, together with a chromogenic substance to make the label signal visible.

The human PLTP in a desired sample can be measured by measuring the color signal that has been exposed in this way by using a signal specifying means corresponding to the type of the color signal. it can.

According to the above-described measurement method of the present invention, PLTP in a sample such as a blood sample such as plasma can be measured simply and with high sensitivity. In this way, the PL in the sample is
By measuring TP, in particular, low HDL blood and high H
Detailed pathological analysis of patients with abnormal HDL levels, such as DL blood disease, becomes possible, and can be used as an index for performing appropriate treatment for patients with arteriosclerosis or the like due to abnormal lipid metabolism.

In the present invention, a kit for measuring PLTP containing the monoclonal antibody of the present invention for using the monoclonal antibody of the present invention in the measuring method of the present invention is also provided (hereinafter referred to as a kit for measuring the present invention).

In the kit for measurement of the present invention, elements for providing the monoclonal antibody of the present invention for the measurement of PLTP in a sample, such as the labeled monoclonal antibody of the present invention and the immobilized monoclonal antibody of the present invention, etc. May be included.

The measuring kit of the present invention also corresponds to the above-described measuring method of the present invention, and has a measuring principle of an enzyme immunoassay, particularly a PLT capable of performing a sandwich method.
It is preferably a P measurement kit.

[0061]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples and the like, but the technical scope of the present invention is not limited to these examples and the like.

[Construction of PLTP Activity Measuring System] In this example, it is essential to establish an accurate PLTP activity (phospholipid transfer activity) measuring system in order to appropriately evaluate experimental results and the like. .

In this example, unless otherwise specified, the measurement of the PLTP activity (phospholipid transfer activity) was carried out in a system constructed as follows. This measurement system was performed according to the method of Cheung et al. (Biochim Biophys Acta, 1303, 103-110, 1996).

That is, first, a donor liposome composed of a liposome (PC liposome) containing radiolabeled phosphatidylcholine (PC), an acceptor lipoprotein composed of high-density lipoprotein (HDL3), and P
Mixing a measurement sample containing LTP and reacting under physiological conditions;

Next, the amount of the labeled PC transferred from the donor liposome to the acceptor lipoprotein was determined by measuring the decrease in the radioactivity of the donor liposome or the increase in the radioactivity of the acceptor lipoprotein.
The PC transfer activity of P was measured and determined as PLTP activity. Hereinafter, the measurement system will be sequentially described.

Preparation of donor liposome PC liposome was 10 μmol egg PC (manufactured by Sigma),
2.5 μmol phosphatidylserine (PS, manufactured by Nacalai Tesque), 1 μCi [ ¹⁴ C] DPPC (dipalmitoylphosphatidylcholine, manufactured by NEN Research Product)
And 100 nM

BHT (manufactured by Wako Pure Chemical Industries, Ltd.) was mixed in a glass test tube, and the solvent was evaporated under a nitrogen stream.
10 mM Tris buffer (pH: 50 mM) containing 50 mM sodium chloride and 1 mM EDTA (ethylenediaminetetraacetic acid).
7.4) Suspended in 1.5 mL. Further, this suspension was sonicated (5 minutes, 3 times), made 10 mL with the same 10 mM Tris buffer (pH 7.4), and centrifuged (150 mL).
(00 rpm, 10 minutes), and the clear layer of the supernatant ([ ¹⁴ C] PC liposome) was used as a donor liposome for PLTP activity measurement.

Preparation of acceptor lipoprotein Potassium bromide (KBr) was added to healthy human plasma (400 mL) to adjust the specific gravity d to 1.125 g / mL, and ultracentrifuged (100,000 × g, 12 ° C, 40 hours),
A fraction with a specific gravity d> 1.125 g was collected. The fraction obtained in this manner was adjusted to a specific gravity d = 1.21 g / mL with KBr
And centrifugation (100,000 × g, 12
° C, 40 hours) and specific gravity 1.125 <d <1.21
Fractions were obtained. The HDL3 fraction was further subjected to specific gravity d = 1.
Ultracentrifugation (100,000 × g, 4 ° C., 40 hours) was performed using physiological saline adjusted to 21 g / mL, and the washed HDL3 fraction was collected. The obtained washed HDL
The three fractions were combined with 150 mM sodium chloride and 1 mM EDT.
Dialysis was performed against 10 mM Tris buffer containing A (pH 7.4).

The washed HDL3 fraction (specific gravity d = 1.125 to 1.21) thus obtained was used as an acceptor lipoprotein. This washed HDL3 fraction contains the following PLT
No PC transfer activity was observed by the P activity assay.

Method for Measuring PLTP Activity The donor liposome ([ ¹⁴ C] PC obtained as described above)
Liposome), acceptor lipoprotein (washed HDL3)
And the measurement sample was prepared using a PC of 75 nm
l. The amount of the acceptor lipoprotein was added to the microtube so that the protein amount was 250 μg, and the total amount was adjusted to 400 μL / tube with 10 mM Tris buffer (pH 7.4) containing 150 mM sodium chloride and 1 mM EDTA, for 2 seconds. After vigorous stirring, the microtube is
Incubate in a 7 ° C. water bath for 90 minutes. Then, after this incubation, transfer the microtube on ice,
The reaction was stopped, 0.3 mL of a 536 mM sodium chloride solution containing 363 mM manganese chloride and 52 units of heparin was added to the microtube, and the mixture was vigorously stirred for 10 seconds.
Leave at room temperature for 10 minutes. Next, this microtube was subjected to centrifugation at 15,000 rpm for 10 minutes at room temperature using a tabletop centrifuge (manufactured by Eppendorf).
The radioactivity of 0.5 mL of the centrifuged supernatant is measured using a liquid scintillation counter. PL included in the measurement sample
The PLTP activity of TP is determined by an increase in the radioactivity based on the difference between the radioactivity of the measurement sample and the radioactivity of the control (blank) operated in the same manner as above except that the same amount of the above Tris buffer was added instead of the measurement sample. The PLTP activity for transferring phospholipids (PC) per unit time is expressed as nmol / ml / hr or% transfer rate.

[Preparation of Purified Human PLTP] The following procedures, such as purification, were carried out according to a conventional method, and all were carried out at 4 ° C. or on an ice bath. Purification by Hydrophobic Chromatography Peripheral blood collected from a plurality of healthy persons is subjected to centrifugal separation according to a conventional method to remove hemocytes and to remove human blood plasma (1).
L) was collected. To the obtained human plasma (1 L), KBr was added.
, And a lower layer (lipoprotein-deficient serum) having a specific gravity d> 1.21 was obtained by ultracentrifugation. This lipoprotein-deficient serum was applied to a butyl toyopearl 650 (5.0 × 15 cm) column previously equilibrated with 10 mM Tris buffer (pH 7.4) containing 2 M NaCl and 1 mM EDTA.
The column was washed with 500 mL of 50 mM Tris buffer (pH 7.4) containing 1 mM EDTA, and further washed with 50-3
Elution was carried out with a concentration gradient of mM Tris buffer (pH 7.4). And this elution pattern was measured by the absorbance at a wavelength of 280 nm. According to this elution pattern, the PLTP activity of each eluted fraction was further measured by the above method, and the fraction containing human PLTP was collected.

CM Sepharose column chromatography
The fraction having PLTP activity obtained by the above-mentioned hydrophobic chromatography is dialyzed against 10 mM sodium citrate buffer (pH 5.5) and equilibrated with 10 mM sodium citrate buffer (pH 5.5) in advance. And applied to a modified CM Toyopearl 650 (2.5 × 24 cm) column. The column is loaded with a 10 mM sodium citrate buffer (pH 5.
5) was added and the mixture was washed, and eluted with a sodium chloride solution having a concentration gradient of 0 to 1.2 M. The elution pattern was measured at a wavelength of 280
Measured by absorbance at nm. According to this elution pattern, the PLTP activity of each elution fraction was measured by the above method,
Fraction containing human PLTP (salt concentration 0.15 to 0.35 M
(A fraction eluted with sodium chloride).

Heparin Sepharose column chromatography
Purification by Filt The fraction having PLTP activity, obtained by the above-described CM Sepharose column chromatography, was dialyzed against a 10 mM Tris buffer (pH 7.4) containing 50 mM sodium chloride, and the mixture was separated into a heparin Sepharose column (1.5 × 8.
5 cm). The column was washed by adding 10 mM Tris buffer (pH 7.4) containing 50 mM sodium chloride, and eluted by adding 10 mM Tris buffer (pH 7.4) containing 0.5 M sodium chloride. This elution pattern was measured by the absorbance at a wavelength of 280 nm. According to this elution pattern, the PLTP activity of each eluted fraction was measured using the method described above, and the fraction containing human PLTP (a 0.5 M salt concentration eluted sodium chloride fraction, fraction N
o. 36-40) were recovered.

For Mono Q column chromatography
The fraction having PLTP activity obtained by the above-mentioned heparin sepharose column chromatography was subjected to 1 mM ED
Dialysis was performed with 25 mM Tris buffer (pH 7.4) containing TA, and a Mono QHR 5/5 column (1.5 × 8.
5 cm). The column was loaded with 2 mM 1 mM EDTA.
After washing with 5 mM Tris buffer (pH 7.4), 0 to 1 M
Elution was carried out with a concentration gradient of sodium chloride. This elution pattern was measured by the absorbance at a wavelength of 280 nm. According to this elution pattern, the PLTP activity of each eluted fraction was measured using the above method, and a fraction containing human PLTP (a sodium chloride eluted fraction with a salt concentration of 200 mM) was collected.

Purification by gel filtration chromatography Obtained by the Mono Q column chromatography described above,
The fraction having PLTP activity collected in the Example was previously purified with a 25 mM Tris buffer (pH 7.0) containing 1 mM EDTA.
The mixture was applied to a Superose 6HR column equilibrated in 4) and eluted with PBS (pH 7.4). This elution pattern was measured by the absorbance at a wavelength of 280 nm. According to this elution pattern, the PLTP activity of each eluted fraction was measured using the above-mentioned method, and a fraction containing human PLTP (fraction Nos. 29 to 34 eluted fraction) was collected and used as purified human PLTP. .

According to these steps, purified human PLTP purified from human plasma has a molecular weight of about 80, by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) using 7.5% polyacrylamide gel. 000 and 4200 nmol / mL
/ Hr PLTP activity (FIG. 1).

[Preparation of Recombinant Human PLTP Protein] Preparation of cDNA Encoding Human PLTP Using human liver-derived cDNA as a template, specific primers [sense primer: 5'-CAGCCTCCACCG
CTGAGCCCGCTC-3 ′ (SEQ ID NO: 1), antisense primer: 5′-ACAGCTGCCAGC
RTTG-PCR was performed using TTGGGGATTGAGG-3 ′ (SEQ ID NO: 2)] to clone the PLTP cDNA.

That is, using a specific primer,
The DNA fragment amplified by T-PCR is
Separated by agarose gel electrophoresis, about 1.5 Kbp of DN
After excising the A fragment together with the gel, QIAEX II gel extract
Extraction was performed with an ion kit (manufactured by QIAGEN) to obtain a desired cDNA fragment.

The thus obtained DNA fragment of about 1.5 Kbp was ligated to the pT7 Blue vector (INVITROGE) using a DNA ligation kit (Takara Shuzo).
N company).

Next, the pT7 Blue vector was transferred to Escherichia coli JM.
109 Competent cells (manufactured by Toyobo). Further, plasmid DNA was extracted from the Escherichia coli, the plasmid DNA was sequenced, and the nucleotide sequence of the target cDNA fragment of about 1.5 Kbp was confirmed. It was confirmed that the thus obtained PCR product of about 1.5 Kbp was a cDNA encoding the target human PLTP.

Construction of Recombinant Human PLTP Protein Expression System The cDNA encoding human PLTP obtained as described above was incorporated into an animal cell expression vector pEF321-3.1.1 vector.

That is, using the obtained cDNA encoding human PLTP as a template, a primer [sense primer: 5'-GGCAGGCGCA CATAGG GAGT]
TCCCAGGCTGCAAGATAC-3 '(SEQ ID NO: 3), antisense primer: 5'-ACAGCTG
PCR was performed using CCAGCTTGGGGGATTGAGG-3 ′ (SEQ ID NO: 2)]. The DNA fragment amplified by this PCR is separated by agarose electrophoresis according to a conventional method, and a DNA fragment of about 1.5 Kbp is cut out together with the gel, and then a QIAEX II gel extraction kit (Q
IAGEN) to obtain the desired cDNA fragment.

The thus obtained DNA fragment of about 1.5 Kbp was ligated to the pBluescript II KS (-) vector (STR) using a DNA ligation kit (Takara Shuzo).
ATAGENE).

Next, the pBluescript II KS (-) vector was introduced into E. coli JM109 competent cells (manufactured by Toyobo). Further, plasmid DNA was extracted from the Escherichia coli, the plasmid DNA was sequenced, and the nucleotide sequence of the target cDNA fragment of about 1.5 Kbp was confirmed.

The cDNA encoding human PLTP obtained in this manner was ligated with the vector pEF321-3.1.1 [human E
The vector pEF321 (Ki
m DGet al, Gene, 91, 217-223, 1990), and an inhibin alpha signal sequence for facilitating extracellular secretion in CHO cells, and a histidine tag for facilitating purification of recombinant proteins. A new expression vector: the construction diagram is shown in FIG. 2] using a DNA ligation kit (Takara Shuzo) to obtain the vector pEF321-3.1.1 / PLTP.

Using a lipofectin reagent (manufactured by Gibco), pEF321-3.1.1 / PLTP and pEF321 / neo containing a drug resistance gene (the neomycin resistance gene was introduced into the pEF321 vector by a conventional method). ),
At the same time, CHO cells (5 × 10 ⁵ cells) were introduced and 10%
The cells were cultured in F12 medium containing fetal calf serum for 3 days. Next, the cultured CHO cells were treated with trypsin / EDTA, the medium was replaced with 12 mL of the above F12 medium containing 1 mg / mL geneticin (manufactured by Sigma), and seeded on a 96-well plate at a rate of 0.1 mL / well. Cultured. Replace with the above F12 medium containing geneticin every 2 days,
The selective cultivation was performed for a day. In such selective culture, RNA was extracted from cells in which proliferation was observed according to a conventional method.

Using the obtained RNA, human PLTPc
A Northern plot using DNA as a probe was performed, and pEF3
21-3.1.1 / Human PLT in CHO cells transfected with PLTP
The specific transcriptional expression of P was confirmed.

The culture medium was replaced with CHO-S-SFMII (manufactured by Gibco) and cultured, and the culture supernatant containing the recombinant human PLTP was collected. The collected recombinant human PLTP culture supernatant was subjected to Ni-NTA agarose (QIAGEN),
Mix by inverting at 4 ° C overnight, and on the next day, Ni-NTA after the reaction.
The column was filled with agarose, washed with PBS (pH 7.4) and PBS (pH 6.0) sequentially, and then washed with 100 mM E.
Purified recombinant human PLTP eluted with PBS containing DTA
A protein fraction was obtained.

Properties of Recombinant Human PLTP The molecular weight of the purified recombinant human PLTP obtained as described above was analyzed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) according to a conventional method. The result is shown in FIG. From these results, the above recombinant human PLTP has a molecular weight of about 80 kDa.
Was confirmed.

The purification of human PLTP has been attempted by many researchers and its molecular weight has been reported [Jauhiainen M et al., J Biol Chem, 268: 4032-4036 (19
93), Tu AY et al., J Biol Chem, 268: 23098-23105 (19
93), Tollefson JH et al., JLipid Res, 29: 1593-1602
(1988) etc.]. The reported values are not completely consistent due to differences in purification method, molecular weight analysis method, etc.
All are approximately 70 to 80 kDa.

From the comparison with these already reported molecular weights, the above-mentioned recombinant human PLTP has the same molecular weight, and the above-mentioned recombinant human PLTP has the same molecular weight as 6500.
Since it had a PLTP activity of nmol / mL / hr, it was shown to have a biological activity as a PLTP protein equivalent to human purified PLTP.

[Preparation of hybridoma according to the present invention]
The above recombinant human PLTP (190 μg / mL) was mixed with an equal volume of Freund's complete adjuvant, and 0.4 mL of the mixture was mixed.
Was immunized into the abdominal cavity of a 7-week-old Balb / c mouse (female, Japan SLC). Two weeks later, the mice were immunized in the same manner using Freund's incomplete adjuvant, and thereafter, once every two weeks, for a total of three immunizations.

3 × 10 4 spleen cells of the mouse 3 days after the final immunization
^Eight cells were used for mouse myeloma cells (SP2 / 0-Ag14 (4 × 10
⁷ )) and 50% polyethylene glycol (average molecular weight 1500: Boehringer Mannheim) PBS
Cell fusion was performed using the solution. The cells after the treatment are transferred to RP
In a MI1640 medium (manufactured by GIBCO), the cells were seeded on a 96-well plate at a rate of 5 × 10 ⁵ cells per well, and from the next day, a HAT reagent (manufactured by Sigma) was added.
When selective culture was performed for one day, hybridoma growth was observed in almost all wells.

Human PL in the culture supernatant of each hybridoma
The presence or absence of a monoclonal antibody specific to TP was screened by ELISA using purified recombinant human PLTP as an antigen. That is, a 96-well plate on which 100 ng of purified recombinant human PLTP protein is immobilized per well (immobilized purified recombinant human PLTP plate)
, 50 μl of the culture supernatant of the hybridoma was added thereto and reacted at room temperature for 2 hours (primary reaction).

Next, the plate was placed in 0.1% Tween2.
After washing 6 times with PBS containing 0, 4000
Peroxidase-labeled rabbit anti-mouse Ig diluted 1: 2
G antibody (Zymed Laboratories) was added and reacted at room temperature for 1 hour (secondary reaction). Plate with PBS 6
After washing twice, 0.25 mg / vol. Containing 0.015% hydrogen peroxide
100 µl of each ml OPD solution (manufactured by Sigma)
In addition, the mixture was reacted at room temperature for 30 minutes (substrate reaction). After the reaction,
Further, the reaction was stopped by adding 50 μl of 2N sulfuric acid to each well. Then, the absorbance at a wavelength of 492 nm was measured with a microplate reader (manufactured by Lab System). As a result, a hybridoma culture supernatant having an absorbance of 1.0 or more was regarded as positive. The hybridoma culture supernatant that was positive in the above screening method was further subjected to high density lipoprotein (H
DL, 1.063 <d <1.210) PLTP in fractionation
Reactivity was confirmed, and clones which showed specificity by ELISA using the above-mentioned recombinant human PLTP as an antigen and Western blotting of human HDL fractions were selected.

[0096] The operation of seeding 0.5 cells per well on a 96-well plate in an RPMI1640 medium supplemented with an HT reagent (Sigma) for the hybridoma whose specificity was confirmed in this manner was performed three times. Repeated cloning yielded monoclonal antibody-producing hybridomas (# 113, # 114, # 110G).

[0097] These hybridomas have been deposited at the Institute of Microbial Industry and Technology, Ministry of Industry and Technology [mouse hy
bridoma PLTP113 (Deposit No .: FERM P-1660
5), mouse hybridoma PLTP114 (deposit number: FERM
P-16606), mousehybridoma PLTP110G (deposit number: FERM P-16607)].

[Preparation of Monoclonal Antibody of the Present Invention] Monoclonal antibody purified in large quantities from Balb / c mice (female, 8 weeks old, 10 mice, Japan SLC
Company), the hybridoma obtained as described above [#
113, # 114, and # 110G (10 ⁶ to 10 ⁷ cells / 0.5 mL / mouse each)] were introduced by intraperitoneal injection. Ten days after the introduction, the mouse was laparotomized under anesthesia, and the monoclonal antibody (# 11
3, # 114, # 110G) were prepared in large amounts, respectively.

Determination of isotype Using a mouse monoclonal antibody isotype determination kit (manufactured by Sangstat Medical), the operation was performed according to the experimental operation protocol attached to the kit, and monoclonal antibodies (# 113, # 114, # 110G) were obtained.
Was determined. As a result, the isotype of monoclonal antibody # 113 was IgG2b,
4 and # 110G were IgG1.

Purification of Monoclonal Antibodies The monoclonal antibodies described above (# 113, # 114, # 1
10G), each centrifuged supernatant (5 mL each) mixed with 5 mL of a saturated ammonium sulfate solution was cooled on ice, allowed to stand for 2 hours, and then centrifuged at 4 ° C., 10,000 × g for 10 minutes. After centrifugation, the precipitate was dissolved in a binding buffer (3 M sodium chloride / 1.5 M glycine solution, pH 8.9), mixed with 1 mL of Protein A Sepharose (Pharmacia), and mixed by inversion at 4 ° C. overnight. . The next day, the column was packed with Protein A Sepharose and washed 6 times with 6 mL of binding buffer.
Elution buffer (0.1 M citric acid solution, pH 4.0)
The solution was eluted in 2 mL portions, and each solution eluted and recovered was neutralized with 0.2 mL of a 2 M Tris solution (pH 10.0). The absorbance (280 nm) of each fraction was measured, and the eluted fraction of the monoclonal antibody was collected and collected in a phosphate buffer (pH 7.0).
Dialysis (4) at 4 ° C. for 24 hours yielded purified monoclonal antibodies (# 113, # 114, # 110G).

Human PLTP Monoclonal of the Present Invention
Characterization of Antibody Reactivity to Human PLTP Purified monoclonal antibody (#
113, # 114, and # 110G) were confirmed by Western blotting using human plasma and purified human PLTP (described above) purified from human plasma.

Human plasma sample: 2 μL / lane, purified human PLTP: 60 ng / lane, human HDL fraction: 15 μg
/ Lane, mouse HDL fraction at a concentration of 60 μg / lane, using 7.5%, 10%, or 5-20% SDS-polyacrylamide gel (manufactured by Atto) for SDS-
PAGE electrophoresis was performed. The gel was then transferred to a PVDF membrane. Further, the transfer membrane was incubated overnight at 4 ° C. in a blocking reagent [PBS containing 5% skim milk (pH 7.4)].

The transfer film was made of P containing 0.1% Tween20.
After washing 5 times with BS (pH 7.4), 0.1% Tween
Each purified monoclonal antibody (# 113, # 11) adjusted to a concentration of 1 μg / mL with PBS containing 20 (pH 7.4)
4, # 110G) was reacted at room temperature for 2 hours. After the reaction,
The membrane was washed with PBS containing 0.1% Tween 20 (pH 7.
4) Wash 5 times with PB containing 0.1% Tween 20
S (pH 7.4) and the secondary antibody (H
(RP-labeled anti-mouse IgG antibody: manufactured by Zymmet) at room temperature for 1 hour. After the reaction, the membrane was washed with 0.1% Tween.
The plate was washed 5 times with n20-containing PBS (pH 7.4), and reacted with a chemilumi reagent (manufactured by NEN Life Science Products) for 1 minute. Next, the chemiluminescence reagent was removed, and after exposure to an X-ray film, the film was developed and a reaction band was detected.

As a result, each purified monoclonal antibody (#
113, # 114, # 110G) purified human PLTP
Was confirmed to react with the purified human PLTP having a molecular weight of about 80 kDa (FIG. 4). In addition, each of these purified monoclonal antibodies (# 113, # 113)
114, # 110G) recognizes a single protein having a molecular weight of about 80 kDa, as in the case of purified human PLTP.
It was shown that the human PLTP protein in the L fraction was specifically recognized (FIGS. 5 and 6). In addition, mouse HD
Reactivity with the L fraction indicated that each of the purified monoclonal antibodies (# 113, # 114, # 110G) did not react with mouse-derived PLTP (FIG. 7).

Reactivity with Immobilized Purified Recombinant Human PLTP Each purified monoclonal antibody (#
113, # 114, # 110G) recombinant human PLTP
Was tested on immobilized purified recombinant human PLTP microplates. That is, the recombinant human PLTP (100 ng / well) purified by the above method was incubated in a microplate at 4 ° C. for 24 hours.
It was immobilized on a microplate.

After the incubation, the microplate was washed four times with a phosphate buffer (200 μL). Discard this washing solution and add blocking reagent [200 μL
And a phosphate buffer (200 μL) containing 1% Block Ace powder (Dainippon Pharmaceutical Co., Ltd.), and incubated at room temperature for 2 hours to block the site where recombinant human PLTP was not bound. Then, each well was filled with 0.1% Tween.
The plate was washed four times with a phosphate buffer containing n20 (200 μl). Further, 100 μl of each concentration of the purified monoclonal antibody (concentration of 0 to 1.25 μg / mL) was added to each well, and reacted at room temperature for 2 hours.

After the reaction, each well was filled with 0.1% Tween.
The plate was washed four times with a phosphate buffer containing 200 (200 μL).
After washing, PBS containing 0.1% Tween 20 (pH 7.
In 4), a 4000-fold diluted secondary antibody (HRP-labeled anti-mouse IgG antibody: manufactured by Zymmet) was reacted at room temperature for 1 hour. After the reaction, each well was placed in 0.1% Tween2.
After washing 5 times with 200 μl of a phosphate buffer solution containing 0.
100 μl of a 0.25 mg / ml OPD solution (manufactured by Sigma) containing 015% hydrogen peroxide was added, and reacted at room temperature for 30 minutes. Further, 2N sulfuric acid (50 μL) was added to each well to stop the reaction.

Then, the absorbance at a wavelength of 492 nm was measured with a microplate reader (manufactured by Lab System).
As a result, the purified monoclonal antibodies (# 113, # 11
4, # 110G) is 0.06 to 100 ng of recombinant human PLTP immobilized on the microplate.
It was shown to react at a concentration of 0.3 μg / mL (No. 8).
Figure).

Epitope Analysis of Monoclonal Antibody Construction of Recombinant PLTP Protein Expression System by Escherichia coli In order to analyze the epitope reacting with the PLTP protein of each purified monoclonal antibody, the following method was used.
The partially recombinant PLTP having the NH ₂ terminus and the partially recombinant PLTP having the COOH terminus of PLTP were expressed using Escherichia coli.

That is, the human P obtained as described above
Using a cDNA encoding LTP as a template, a primer [sense primer: 5'-GCAGGCGC GCAT
GC AGAGTTCCCAGGCTGCAA-3 '(SEQ ID NO: 4), antisense primer: 5'-GACA
GGGCA AAGCTT CTGGTTGAGGAGGA
AGCGCAT-3 ′ (SEQ ID NO: 5)]
R was performed. The DNA fragment amplified by the PCR is separated by agarose electrophoresis according to a conventional method, and the separated DNA fragment is added to about 0.
After cutting out a 5 Kbp DNA fragment together with the gel, QIAEX
The gel was dissolved with a II gel extraction kit (manufactured by QIAGEN) to obtain a desired DNA fragment of about 0.5 Kbp.

The thus obtained DNA fragment of about 0.5 Kbp was inserted into a pQE31 vector (manufactured by QIAGEN) using a DNA ligation kit (manufactured by Takara Shuzo). Then, this pQE31 vector was
E. coli JM109 was introduced into competent cells (manufactured by Toyobo). Further, plasmid DNA was extracted from the Escherichia coli, and the plasmid DNA was sequenced.
The nucleotide sequence of the Kbp cDNA fragment was confirmed.

Thus, about 0.5 Kb for expressing a partially recombinant human PLTP having an NH ₂ terminus was obtained.
p cDNA (human PLTP mature amino acid sequence 1-1)
65 corresponding).

The partially recombinant human PLTP cDNA having a COOH terminus can be obtained by replacing the plasmid containing the human PLTP cDNA obtained as described above with the restriction enzyme B.
glII (manufactured by Takara Shuzo) and Hind III (manufactured by Takara Shuzo), and the digested DNA fragments are separated by agarose electrophoresis to obtain a target DNA fragment of about 1.0 Kbp. After cutting out the whole gel, QIAE
The gel was dissolved with an XII gel extraction kit (manufactured by QIAGEN), and the desired DNA fragment of about 1.0 Kbp was digested with restriction enzymes BglII (manufactured by Takara Shuzo) and HindIII (manufactured by Takara Shuzo). I got

The thus obtained DNA fragment of about 1.0 Kbp was inserted into the pQE32 vector (QIAGEN) using a DNA ligation kit (Takara Shuzo).

Next, this pQE32 vector was introduced into E. coli JM109 competent cells (manufactured by Toyobo). Further, plasmid DNA was extracted from the Escherichia coli, and the plasmid DNA was sequenced.
The nucleotide sequence of the cDNA fragment of p was confirmed.

In this manner, a compound having a COOH terminal
Approximately 1.0 to express partially recombinant human PLTP.
Kbp cDNA (human PLTP mature amino acid sequence 16
7-476).

Expression of partially recombinant human PLTP by Escherichia coli A clone containing a plasmid into which a partially recombinant human PLTP cDNA having an NH ₂ terminus or a COOH terminus obtained by the above-described method was inserted was subjected to a conventional method at 50 μg / ml.
The cells were cultured in mL of TB medium containing ampicillin. Further, IPTG (Isopropyl β) was added so that the final concentration was 0.3 mM.
-D-Thiogalactopyranoside) (manufactured by Sigma) and add 3
After culturing at 7 ° C for 3 hours, the expression of the partially recombinant human PLTP protein was induced.

After the induction culture, the respective cells were collected, subjected to sonication, and then subjected to Ni-NTA agarose (QIA
GEN) using a partially recombinant human PLTP protein portion having each NH ₂ end (recombinant human PLTP1-
165) and partially recombinant human PL having COOH termini
The TP protein portion (recombinant human PLTP167-476) was purified. In addition, the purified NH ₂ terminus and COOH
When the partially recombinant human PLTP having a terminal was confirmed by SDS-PAGE using 12.5% polyacrylamide gel, the molecular weight of the partially recombinant human PLTP having an NH ₂ terminal and a COOH terminal was about 22.0%.
00 and about 38,000 (FIG. 9).

Epitope Analysis of Monoclonal Antibody A partially recombinant human PLTP protein portion having each NH ₂ terminus (recombinant human PLTP) purified by the above method
1-165) and a partially recombinant human PLTP protein portion having a COOH terminus (recombinant human PLTP167-47).
6) was subjected to SDS-PAGE electrophoresis using a 12.5% polyacrylamide gel at a concentration of 100 ng / lane.

Next, the gel was transferred to a PVDF membrane, and the transferred membrane was blocked with a blocking reagent [PBS containing 5% skim milk].
(PH 7.4)] at 4 ° C. overnight.
Further, the transfer membrane was washed with PBS containing 0.1% Tween 20.
(PH 7.4) 5 times, and 0.1% Tween2
0, each purified monoclonal antibody (# 113, # 11) adjusted to a concentration of 1 μg / mL with PBS (pH 7.4).
4, # 110G) was reacted at room temperature for 2 hours.

After the reaction, the membrane was washed with 0.1% Tween20.
Wash 5 times with PBS (pH 7.4) containing 0.1% T
A secondary antibody (HRP-labeled mouse IgG antibody: manufactured by Zymet) diluted 4000-fold with PBS containing 20 (pH 7.4) was reacted at room temperature for 1 hour. After the reaction,
The transfer membrane was washed with PBS containing 0.1% Tween 20 (pH
In 7.4), the plate was washed 5 times, and reacted with a chemilumine reagent (manufactured by NEN Life Science Products) for 1 minute.

Next, the chemiluminescence reagent was removed from the film, an X-ray film was exposed to the film, the film was developed, and a band appeared on the film was detected. As a result, each purified monoclonal antibody (# 113, # 114, # 110G)
Among them, # 113 and # 114 are partially recombinant human PLTP proteins having COOH terminals (recombinant human PLTP1
67-476), a band corresponding to a molecular weight of about 38,000 was observed,

# 110G is a partially recombinant human PLTP protein having an NH ₂ terminus (recombinant human PLTP1-16).
In contrast to 5), a band corresponding to a molecular weight of about 22,000 was observed (FIG. 10). From these results, the purified monoclonal antibodies # 113 and # 114 were
167-476 of the mature amino acid sequence of
# 110G is 1- of the mature amino acid sequence of human PLTP.
It was shown to recognize 165 regions.

[Human PL by sandwich ELISA]
Establishment of TP Quantitative Method] Preparation of Immobilized Monoclonal Antibodies Each of the above purified monoclonal antibodies (# 113, # 11
4, # 110G) in phosphate buffer (10 ng / well to 1 μg / well, pH 7.4), and each of these monoclonal antibody solutions (100 μL) was subjected to ELISA
The solution was added to each well of a 96-well microplate for A (manufactured by Nunc), and incubated at 4 ° C. for 24 hours to adsorb each monoclonal antibody to the microplate. Each well was then washed four times with 200 μL of phosphate buffer. This washing solution is discarded, and a blocking reagent [200 μL of a phosphate buffer containing 1% Block Ace powder (manufactured by Dainippon Pharma Co., Ltd.)] is added to each well, and the mixture is incubated at room temperature for 2 hours. Blocked. Then, each well was filled with 0.1% Tw
The plate was washed four times with a phosphate buffer containing een20 (200 μL). Thus, the desired immobilized monoclonal antibody was prepared.

Preparation of Labeled Monoclonal Antibodies Each of the above purified monoclonal antibodies (# 113, # 11
4, # 110G: 1 mL of 1 mg / mL) was added to 0.1 M Na
Dialysis against HCO ₃ (pH 8.2-8.3) solution (4 ° C., 2
4 hours). Next, NHS-biotin (1 μg / mL, 60 μL, manufactured by Pierce) was added, and the mixture was stirred vigorously and incubated at room temperature for 4 hours. Subsequently, the mixture was dialyzed (4 ° C., 24 hours) with a phosphate buffer to prepare a desired biotin-labeled monoclonal antibody (hereinafter referred to as a labeled monoclonal antibody) for each monoclonal antibody.

The reactivity of the labeled monoclonal antibody thus prepared for purified recombinant human PLTP was measured using the immobilized purified recombinant human PLTP microplate described above.
It was examined similarly. As a result, each labeled monoclonal antibody was shown to react with immobilized and purified recombinant human PLTP (100 ng) at a concentration of 0.3 to 0.6 μg / mL (FIGS. 11 and 12). .

Confirmation of quantitative method by sandwich ELISA
Stand each immobilized monoclonal antibody (# 113, # 11
4, # 110G: immobilized at a concentration of 500 ng / well to 0.5 μg / well. Hereinafter, it is also referred to as “immobilized microplate”. ) In 0.1% Tween 2
Washed 5 times with 0-containing phosphate buffer. In each well of each immobilized microplate, a measurement sample diluted with a phosphate buffer containing 0.1% Tween 20 [plasma of a healthy person,
100 μL of a medium containing human recombinant PLTP) and incubated at room temperature for 2 hours.

After the incubation, the immobilized microplate was washed 5 times with a phosphate buffer containing 0.1% Tween 20, and each well was diluted with a phosphate buffer containing 0.1% Tween 20. (1 μg / mL at 100 μL) and incubated at room temperature for 2 hours.

Next, the immobilized microplate was placed in 0.
After washing 5 times with a phosphate buffer containing 1% Tween 20,
200% in phosphate buffer containing 0.1% Tween 20
HRP-labeled streptavidin (1 mg /
(mL: manufactured by Vector Laboratories) was added to each well, and incubated at room temperature for 30 minutes.

After the incubation, the immobilized microplate was washed with phosphate buffer containing 0.1% Tween 20 for 5 hours.
After washing twice, 100 μL of a 0.25 mg / mL OPD (manufactured by Sigma) solution containing 0.015% hydrogen peroxide was added to the immobilized microplate, followed by incubation at room temperature for 30 minutes. Further, the immobilized microplate was placed in 2N sulfuric acid (50 μl).
L) was added to each well to stop the reaction.

Then, the absorbance at a wavelength of 492 nm was measured with a microplate reader (manufactured by Labsystem). In such a measurement of PLTP in a measurement sample by sandwich ELISA, as shown in Examples, PLTP in a healthy human plasma or a medium containing recombinant human PLTP was used.
Was measured in a concentration-dependent manner, indicating that it is sufficiently possible to quantify the amount of PLTP in the measurement sample by the sandwich ELISA of the present invention (No. 13).
(FIG. 14, FIG. 14).

As a result, among the combinations of the labeled monoclonal antibody and the immobilized monoclonal antibody, in particular, the immobilized monoclonal antibody # 114 and the labeled monoclonal antibody # 113, or the immobilized monoclonal antibody # 1
It is preferable to measure the amount of PLTP in the measurement sample using any combination of 10G and labeled monoclonal antibody # 113.

The optimal concentration of immobilized monoclonal antibody and labeled monoclonal antibody for measuring PLTP in such a measurement sample is such that the concentration of immobilized monoclonal antibody is 0.5 μg / well and that of labeled monoclonal antibody Was shown by the above example to be 0.1 μg / well.

[0134]

Industrial Applicability According to the present invention, a monoclonal antibody against human phospholipid transfer protein (PLTP), which plays an important role in lipid metabolism in humans, a hybridoma used in the production process thereof, and lipid metabolism such as arteriosclerosis Provided are a method for measuring a human phospholipid transfer protein using a monoclonal antibody against the human phospholipid transfer protein, which can be an important indicator of a disease closely related to abnormality, and a measurement kit for performing the measurement method.

[0135]

[Sequence list]

SEQ ID NO: 1 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CAGCTCCACC GCTGAGCCCG CTC 23

SEQ ID NO: 2 Sequence length: 25 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence ACAGCTGCCA GCTTGGGGAT TGAGG 25

SEQ ID NO: 3 Sequence length: 37 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GGCAGGCGCA CATATGGAGT TCCCAGGCTG CAAGATC 37

SEQ ID NO: 4 Sequence length: 32 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GCAGGCGCGC ATGCAGAGTT CCCAGGCTGC AA 32

SEQ ID NO: 5 Sequence length: 36 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GACAGGGCAA AGCTTCTGGT TGAGGAGGAA GCGCAT 36

[Brief description of the drawings]

FIG. 1 is an electrophoretic image showing the molecular weight of PLTP purified from human plasma.

FIG. 2 is a construction diagram of a CHO cell expression vector pEF321-3.1.1.

FIG. 3 is an electrophoretic image showing the molecular weight of recombinant human PLTP purified by a Ni-NTA agarose column.

FIG. 4. Each purified monoclonal antibody # 113, # 114
4 is an electrophoretic image showing the reactivity of # 110G and # 110G with purified human PLTP.

FIG. 5. Each purified monoclonal antibody # 113, # 114
4 is an electrophoretic image showing the reactivity of # 110G and # 110G to PLTP in human plasma.

FIG. 6 shows purified monoclonal antibodies # 113 and # 114.
5 is an electrophoretic image showing the reactivity of # 110G and # 110G with PLTP in a human HDL fraction.

FIG. 7 shows purified monoclonal antibodies # 113 and # 114.
5 is an electrophoretic image showing the reactivity of # 110G and # 110G with PLTP in a mouse HDL fraction.

FIG. 8 shows purified monoclonal antibodies # 113 and # 114.
FIG. 3 is a drawing showing the reactivity of # 110G and # 110G to immobilized recombinant human PLTP.

FIG. 9 is an electrophoretic image showing the molecular weight of each partially modified PLTP having a purified NH ₂ end and a COOH end.

FIG. 10. Each purified monoclonal antibody # 113, # 11
4 is an electrophoretic image showing the results of epitope analysis for # 4 and # 110G.

FIG. 11: Biotin-labeled monoclonal antibody # 113
Fig. 3 is a drawing showing the reactivity of immobilized recombinant human PLTP.

FIG. 12 shows the reactivity of biotin-labeled monoclonal antibodies # 114 and # 110G to immobilized recombinant human PLTP.

FIG. 13 is a drawing showing the reactivity of a combination of labeled monoclonal antibody # 113 and immobilized monoclonal antibody # 110G in a sandwich ELISA system.

FIG. 14 is a drawing showing the reactivity of a combination of labeled monoclonal antibody # 113 and immobilized monoclonal antibody # 114 in a sandwich ELISA system.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI G01N 33/53 C12N 15/00 C (72) Inventor Takeo Exit 1361 Matoba, Kawagoe-shi, Saitama 1 B.M. EL Research Institute

Claims (18)

[Claims] 1. A monoclonal antibody against human phospholipid transfer protein. 2. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is a monoclonal antibody having the following properties: an antigen-antibody reaction against human PLTP; an antigen-antibody reaction against at least mouse PLTP. Is not recognized. 3. The mature amino acid sequence of human PLTP, 167.
3. The monoclonal antibody according to claim 1, which recognizes the region of -476. 4. The mature amino acid sequence of human PLTP 1-1.
3. The monoclonal antibody according to claim 1, which recognizes 65 regions. 5. The method according to claim 5, wherein the monoclonal antibody is a mouse hybridoma.
PLTP113 (deposit number: FERM P-16605), mo
use hybridoma PLTP114 (Deposit number: FERMP-16
606) and mouse hybridoma PLTP110G (deposit number: F
The monoclonal antibody according to claim 1 or 2, which is a monoclonal antibody produced from any hybridoma selected from the group consisting of ERM P-16607). 6. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is labeled with a labeling substance that produces a detectable signal by reacting the labeling substance alone or the labeling substance with another substance. The monoclonal antibody according to any one of the preceding claims. 7. The monoclonal antibody according to claim 6, wherein the labeling substance is any labeling substance selected from the group consisting of enzymes, fluorescent substances, chemiluminescent substances, biotin, avidin and radioisotopes. 8. An immobilized monoclonal antibody, wherein the monoclonal antibody according to any one of claims 1 to 7 is immobilized on an insoluble carrier. 9. The insoluble carrier is any one of the insoluble carriers selected from the group consisting of plates, test tubes, tubes, beads, balls, filters, membranes and insoluble carriers used in affinity chromatography. The immobilized monoclonal antibody according to the above. 10. The immobilized monoclonal antibody according to claim 8, wherein the insoluble carrier is any insoluble carrier selected from the group consisting of a filter, a membrane and an insoluble carrier used in affinity chromatography. 11. A hybridoma derived from an immunized animal, wherein the animal has been immunized with a human phospholipid transfer protein. 12. The hybridoma according to claim 11, wherein the human phospholipid transfer protein is a recombinant human phospholipid transfer protein. 13. The hybridoma is mouse hybridoma PL.
TP113 (Deposit No .: FERM P-16605), mous
e hybridoma PLTP114 (Deposit number: FERMP-166
06) and mouse hybridoma PLTP110G (deposit number: FE)
The hybridoma according to claim 11 or 12, which is any hybridoma selected from the group consisting of RMP-16607). 14. A method according to claim 1, wherein one or more of the monoclonal antibodies according to any one of claims 1 to 10 is used in an antigen-antibody reaction with a human phospholipid transfer protein in the sample. A method for measuring human phospholipid transfer protein, which measures lipid transfer protein. 15. A method for measuring a human phospholipid transfer protein, wherein the method for measuring a human phospholipid transfer protein according to claim 14 is an enzyme immunoassay. 16. A kit for measuring human phospholipid transfer protein, comprising one or more of the monoclonal antibodies according to any one of claims 1 to 10. 17. The kit for measuring a human phospholipid transfer protein according to claim 16, comprising the immobilized monoclonal antibody according to any one of claims 8 to 10. 18. The kit for measuring human phospholipid transfer protein according to claim 17, wherein the measurement principle for measuring human phospholipid transfer protein in the kit is an enzyme immunoassay.