JP2001192400A - Anti-lcat antibody and method for measuring lcat - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an anti-LCAT antibody having high bond specificity to human-derived LCAT and to provide a method for measuring a human-derived LCAT in a specimen by using the antibody simply in a high yield. SOLUTION: This anti-LCAT antibody is reacted with a human-derived LCAT and recognizes any one sequence of sequence number 1 to 5 in the specification or its fragment. This hybridoma produces the monoclonal antibody. This method for producing the polyclonal antibody uses a peptide of sequence number 1 to 5. This reagent for measuring LCAT contains the anti-LCAT antibody, its fragment or its marker. This method for measuring LCAT uses the reagent. This method for separating/purifying a human-derived LCAT is carried out by affinity chromatography using an immobilized anti-LCAT antibody obtained by immobilizing the anti-LCAT antibody or its fragment to an insoluble carrier or an immobilized fragment.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an anti-LCAT antibody reactive to human lecithin cholesterol acyltransferase (LCAT), a hybridoma producing an anti-LCAT monoclonal antibody, a method for producing an anti-LCAT polyclonal antibody, and an LCAT measuring reagent. , And LCAT
It relates to a method of purification.

[0002]

2. Description of the Related Art Cholesterol in free form, long chain fatty acid form and ester form is present in all tissues and plasma of living organisms. The former plays an important role in the composition of cell membranes. It is physiologically inert and has a strong storage form. Cholesterol in the living body is derived from uptake from the small intestine due to food intake or biosynthesis in each tissue, particularly in the liver. Most of the cholesterol is derived from biosynthesis in tissues such as the liver. Free cholesterol biosynthesized in the liver is taken up by very low-density lipoprotein (VLDL), and in the blood, by the action of lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL), medium-density lipoprotein (ID)
After L), it is metabolized to low density lipoprotein (LDL).
LDL is taken up into peripheral cells via the LDL receptor and free cholesterol is supplied to the cells. Contrary to such a flow from the liver to peripheral cells, there is a flow of cholesterol from peripheral cells to the liver called a reverse cholesterol transfer system. That is, excess free cholesterol supplied from the liver to peripheral cells is extracted by high-density lipoprotein (HDL) in the blood, and cholesterol ester is acted upon by the action of lecithin / cholesterol acyltransferase (hereinafter abbreviated as “LCAT”). Converted to blood HDL
Stored inside. Cholesterol esters stored in HDL are transferred to blood VLDL, IDL and LDL by the action of cholesterol ester transfer protein (CETP). VLDL, IDL and LDL that have received cholesterol esters
Under the action of the LDL receptor, cholesterol is indirectly transferred to the liver.

The reverse cholesterol transfer system has recently attracted attention as a mechanism for preventing cholesterol from accumulating in peripheral cells and preventing arteriosclerosis. In fact, regarding HDL which plays an important role in this reverse cholesterol transfer system,
Numerous epidemiological studies have shown that the reduction of blood HDL cholesterol is one of the risk factors for coronary artery disease, and HDL is widely recognized as a lipoprotein with anti-atherosclerotic effects. became. Further elucidating the importance of blood HDL and the relationship between LCAT and various diseases such as hyperlipidemia, hypoHDL cholesterol, hypercholesterolemia, hypolipidemia, arteriosclerosis, diabetes and nephrotic syndrome This has been a long-standing challenge. Also,
Since LCAT is a glycoprotein with a short half-life synthesized in the liver, it is attracting attention as an extremely organ-specific diagnostic marker that reflects liver function and nutritional status.

[0004] In order to elucidate the relationship between LCAT and various diseases as described above, methods for quantifying LCAT in body fluids such as plasma of healthy individuals and patients suffering from various diseases as described above include: Mainly, two types of methods having different principles are used. Regarding the method of quantifying LCAT by activity, for example,
Stock (KT Stokke) et al. [Journal of Clinical Laboratory Investigation (Jour)
nal of Clinical Laboratory Investigation), Chapter 27
Vol.21, 1971], Nagasaki Toshihide et al. [Clinical Chemistry, No.4]
Vol. 306, 1976], Hiroshige Itakura et al. [Records of the Japanese Society of Clinical Metabolism, Vol. 4, 136, 1987], and JA Glomset et al. [Biochimica et Biochemica et Biophysica et al. Biophysica Acta), 8th
9, p. 266, 1964]. However, in these quantification methods based on activity measurement, there is a case where one of the measurement methods is normal and the other measurement method shows an abnormal value depending on the disease state. In other words, the difference between the test values depending on the measurement method,
This is due to the fact that what is observed differs depending on the substrate used. Further, it has a drawback in terms of operational complexity such as the use of radioisotopes.

[0005] On the other hand, a method for quantifying LCAT in body fluids such as plasma of immunologically normal persons and patients suffering from the above various diseases using antibodies has been studied.
Radioimmunoassay (RIA) using AT polyclonal antibody or enzyme immunoassay (EIA, ELIS
Attempts have been made to develop a quantification method using an immunoassay such as A), and to develop an anti-LCAT antibody for use in such a quantification method.

[0006] Regarding the quantification method using an anti-LCAT polyclonal antibody, see, for example, JJ Alberts et al. [Journal of Clinical Investigation, No. 67].
Pp. 141-148, 1981] report a quantification method by an RIA method using an anti-LCAT polyclonal antibody and labeled LCAT. However, this quantification method required a complicated operation of purifying LCAT from plasma and labeling it with isotopes. Furthermore, since LCAT purified from plasma is pretreated to immunize goats to obtain anti-LCAT polyclonal antibodies, there is a difference between the measurements, which is not practical. For the production of an anti-LCAT polyclonal antibody, see, for example, Lima (VL. Lima) et al. [Brazilian Journal of Medical and Biological Research (Brazilian Journal of M.
edical & Biological Research), Vol. 29, No. 8,
957-968, 1996], and KG. Varma et al. [Biochimica et Biophysica acta (Biochi.
mica et Biophysica Acta), Vol. 486, No. 2, No. 378
384 pages, 1977] has produced a polyclonal antibody from rabbits or goats, but none of them has yet established a quantitative system for LCAT.

[0007] There has been no report on a quantification method using an anti-LCAT monoclonal antibody. For the production of anti-LCAT monoclonal antibodies, see, for example, A. Khalil
Et al. [BioChemica E Biophysica Acta (Bioc
himica et Biophysica Acta), Volume 878, Issue 1, Issue 12
7-130, 1986]. But this anti-LCAT
Monoclonal antibodies (B _10), not only LCAT, are those would also recognize phospholipase A ₂ closely related, were those difficult to say that the specific antibodies.

[0008]

As described above, anti-LCAT monoclonal antibodies and polyclonal antibodies that clearly recognize a specific site of LCAT have not been known so far, and LCAT in blood can be easily obtained. There have been no reports of successful quantification.

Accordingly, the present invention provides an anti-LCAT antibody having high binding specificity to human-derived LCAT and a method for simply and highly sensitively measuring human-derived LCAT in a sample using the same. Aim.

[0010]

Under such circumstances, the present inventors have produced various anti-LCAT antibodies specifically recognizing human-derived LCAT and a peptide which is a part of its amino acid sequence, and determined the reactivity thereof. As a result of the examination, purified human-derived LC
An anti-LCAT antibody having extremely high antigen specificity for AT and any one of SEQ ID NOs: 1 to 5 was successfully obtained, and furthermore, according to an immunoassay using the antibody, LCAT in human body fluid was intact. The antibody can be measured easily and with high sensitivity.
The present invention was found to be extremely useful for the separation / purification of DNA and that SEQ ID NOS: 1 to 5 have excellent antigenicity.

That is, the present invention provides an anti-LCAT antibody or a fragment thereof that reacts with human-derived LCAT and recognizes any one of SEQ ID NOS: 1 to 5.

The present invention also provides a hybridoma producing such a monoclonal antibody.

The present invention also provides a method for producing such a polyclonal antibody by using the peptide of SEQ ID NOS: 1 to 5 or a conjugate thereof with a carrier as an antigen.

The present invention also provides an LCAT measuring reagent containing the above-mentioned anti-LCAT antibody or its fragment or a labeled product thereof, and a method for measuring LCAT using the same.

Further, the present invention provides an immobilized anti-LCAT obtained by immobilizing the above anti-LCAT antibody or a fragment thereof on an insoluble carrier.
It is intended to provide a method for separating or purifying human-derived LCAT by affinity chromatography using an antibody or an immobilized fragment.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION Anti-LCAT Antibody The anti-LCAT antibody of the present invention is characterized by having reactivity with human LCAT. As the anti-LCAT antibody of the present invention,
Both monoclonal antibodies and polyclonal antibodies are included. Particularly preferred specific examples include monoclonal antibodies 36487 produced by hybridoma 36487 deposited as FERM P-17610 and recognizing SEQ ID NO: 1, and FERM P-
Monoclonal antibody 3644 produced by hybridoma 36442 deposited as 17609 and recognizing SEQ ID NO: 2
2.Hybridoma 36454 deposited as FERM P-17608
A monoclonal antibody 36454 recognizing SEQ ID NO: 3, a hybridoma 36405 recognizing SEQ ID NO: 4 produced by hybridoma 36405 deposited as FERM P-17611, and a hybridoma 36486 deposited as FERM P-17607. Produced, SEQ ID NO: 5
And a monoclonal antibody 36486 that recognizes

The monoclonal antibody of the present invention is described in "Experimental Medicine (separate volume) Cell Engineering Handbook" (edited by Toshio Kuroki et al., Published by Yodosha, 66-74, 1992), "Introduction to Monoclonal Antibody Experimental Procedures" [ Tamoe Ando et al., Published by Kodansha, 1991
For example, the antibody can be produced from a hybridoma (fused cell) produced by so-called cell fusion according to a general method for producing a monoclonal antibody as described in J. Am. Such a hybridoma is obtained by immunizing a mammal with an antigen to obtain an antibody-producing cell, preparing a hybridoma from the antibody-producing cell and a myeloma cell line (myeloma cell) having no autoantibody-producing ability, and cloning the hybridoma. And a clone producing a monoclonal antibody showing specific affinity for the antigen used for immunization.

Specifically, an intact, intact, biologically active, purified human LCAT is used as an antigen, or any of SEQ ID NOS: 1 to 5 which are part of the amino acid sequence of the human LCAT. A peptide consisting of a sequence or a conjugate of the peptide and a carrier as an antigen, and using the antigen as a mammal (including transgenic animals such as human antibody-producing mice that have been genetically engineered to produce human antibodies); Immunization is preferably carried out by injecting one or several times into mice, rats, hamsters, guinea pigs, rabbits, etc., more preferably subcutaneously, intramuscularly, intravenously, in a footpad or intraperitoneally of mice, rats or hamsters. Apply. Usually, immunization is performed about 1 to 4 times about every 1 to 14 days after the initial immunization, and antibody-producing cells are obtained from the immunized mammal about 1 to 5 days after the final immunization.

Hybridomas can be created, for example, by the method of Koehler and Milstein [Nature,
Vol. 256, pp. 495-497, 1975] and a modification method based thereon. That is, the spleen, lymph node obtained from the mammal immunized as described above,
Bone marrow, tonsils and the like, preferably antibody-producing cells contained in the spleen, preferably a mouse, rat, guinea pig, hamster, rabbit, mammals such as humans, more preferably mouse, rat or human autoantibody-derived ability of Hybridomas are produced by cell fusion with non-myeloma cells. Examples of myeloma cells include mouse-derived P3 / X63-AG8.653 (653), P3 / NS1 / 1-Ag4-1 (N
S-1), P3 / X63-Ag8.U1 (P3U1), SP2 / O-Ag14 (Sp2 / O, S
p2), PA1, FO, BW5147, etc., 210RCY3-Ag.2.
3.Human-derived U-266AR1, GM1500-6TG-A1-2, UC729-
6, CEM-AGR, D1R11, CEM-T15, etc. can be used.

Screening of hybridomas producing monoclonal antibodies is carried out by culturing the hybridomas, for example, in a microtiter plate, and examining the reactivity of the culture supernatant in the wells in which proliferation has been observed with the antigen used for immunization of mammals. For example, it can be measured by an enzyme immunoassay such as RIA and ELISA. here,
When purified human-derived LCAT is used as an antigen in immunization, antibodies to human LCAT can be efficiently produced by evaluating reactivity with the sequences of SEQ ID NOs: 1 to 5 in addition to purified human-derived LCAT Hybridomas can be selected.

The production of the monoclonal antibody by the hybridoma can be carried out by culturing the hybridoma in vitro or in vivo and isolating it from the obtained culture supernatant or mammalian ascites. In vitro culture can be carried out using a known nutrient medium used for the production of monoclonal antibodies, or any nutrient medium derived and prepared from a known basal medium. As the basic medium, for example, a low calcium medium such as Ham'F12 medium, MCDB153 medium, low calcium EME medium; MCDB104 medium, ME
M medium, D-MEM medium, RPMI1640 medium, ASF104 medium, high calcium medium such as RD medium and the like, the basic medium,
Depending on the purpose, for example, serum, hormones, cytokines and / or various inorganic or organic substances can be contained. Culturing in vivo can be carried out with mouse, rat, guinea pig, hamster, rabbit and the like, preferably mouse or rat, and more preferably mouse ascites. Monoclonal antibody isolation and purification is performed by using the above-mentioned culture supernatant or ascites fluid with saturated ammonium sulfate,
Euglobulin precipitation method, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE, DE52, etc.),
It can be carried out by subjecting it to affinity column chromatography such as an anti-immunoglobulin column and a protein A column.

The polyclonal antibody of the present invention can be prepared according to a general method described in the literature similar to that described for the preparation of the monoclonal antibody. That is, mammals such as mice, rats, guinea pigs, rabbits, goats, pigs, cows, etc., preferably rats, guinea pigs, rabbits or goats, the peptide of any of SEQ ID NOs: 1 to 5, or a mixture thereof, or a carrier thereof. The conjugate is immunized in the same manner as described in the method for producing a monoclonal antibody, and serum containing an antibody reactive with human-derived LCAT produced thereby is isolated in the same manner as in the case of the monoclonal antibody described above. Can be prepared by purification. The production of a polyclonal antibody using a purified protein is a well-known method, but there is a problem that if there is a very small amount of antigen-containing contaminants, an antibody against them will be produced together. In addition, when a purified protein is to be obtained from human serum, there are many problems, such as ethical problems, problems of worker health due to infectious substances in the raw materials, and problems of product lot differences. On the other hand, the method using the sequences of SEQ ID NOs: 1 to 5 of the present invention has no problems derived from the raw materials, and can ignore the difference between production lots.
The sequences of (5) to (5) can be obtained from any facilities having protein expression and peptide synthesis facilities, and can be easily obtained by a contractor who performs custom synthesis. It has many advantages and is very useful.

In the production of a monoclonal antibody or a polyclonal antibody, when a peptide represented by any of SEQ ID NOs: 1 to 5 or a conjugate thereof with a carrier is used as the mammalian immunogen, General methods as described in similar literature, for example
It can be synthesized according to the tBOC method, Fmoc method, MAP (Multiple Antigen Peptide) method and the like. As an expression method, a gene arranged to express each amino acid sequence is inserted into an expression vector, and a host cell is transformed with the expression vector.
It can be produced by culturing the transformed cells. As host cells, any of prokaryotic cells (eg, E. coli) and eukaryotic cells (eg, CHO cells) can be used. An amino acid such as cysteine, lysine, or arginine can be added to the N- or C-terminal of the peptide represented by any of SEQ ID NOs: 1 to 5 in order to effectively bind to the carrier. For example, when cysteine is added to the N or C terminus, the amino group or carboxy group of cysteine is made free and the other end is blocked. Alternatively, the synthesis may be performed with the amino terminal free, the carboxy terminal converted to an amide, and a bisimide ester may be used for bonding to the carrier. Examples of the peptide carrier include mammalian proteins such as albumin and globulin; proteins such as keyhole limpet hemocyanin; microorganisms such as inactivated Mycobacterium tuberculosis; polyamino acids such as polylysine and polyasparagine; polysaccharides; Substances can be used.

The anti-LCAT antibody of the present invention obtained as described above can be treated with an antibody fragment, ie, Fab 'or F (ab'), by a conventional method such as treatment with a protease such as pepsin or papain. ₂ ), in the measurement and purification of human-derived LCAT described below, the anti-LCAT of the present invention
Not only antibodies but also such antibody fragments can be used.

Measurement of human-derived LCAT It is possible to easily detect or quantify human-derived LCAT in a test sample by using the above-mentioned anti-LCAT antibody or a fragment thereof or a label thereof. The test sample is not particularly limited, and examples thereof include a body fluid sample such as plasma, a culture supernatant, a centrifugal supernatant, and the like. Here, the anti-LCAT antibody or a fragment thereof used includes those labeled with another substance.

The substance used for the labeling is not particularly limited as long as it can be generally used for a measurement method utilizing an immunological reaction. For example, insoluble carriers such as glass, polystyrene latex, beads, magnetic carriers, and plastic plates; Enzymes such as peroxidase, alkaline phosphatase and β-D-galactosidase; fluorescent substances such as fluorescein isothiocyanate; ³ H, ¹⁴ C, ¹²⁵ I, ¹³¹
Radioisotopes such as I; substances having mutual affinity such as biotin and avidin; proteins such as albumin and globumin; water-soluble carriers such as polysaccharides.

In the present invention, any of these labeling substances can be used, but in view of high detection sensitivity or quantitative sensitivity and convenience of operation, peroxidase and biotin are preferable.

The method of measuring LCAT according to the present invention includes:
There is no particular limitation as long as the antigen and the antibody of the present invention are used.
The method can be applied to any of ISA, EIA, immunoturbidimetry, immunoprecipitation, EMIT, immunochromatography, etc. In addition, the present invention can be applied to a method as described in "Enzyme immunoassay" (3rd edition, edited by Eiji Ishikawa et al., Published by Medical Shoin, 1987). In the case of ELISA, which is a typical immunoassay, it is preferable to immobilize the monoclonal antibody 36486 of the present invention on a plastic plate and bind peroxidase to 36487 or a fragment thereof.

Separation / Purification of LCAT A human-derived LCAT antibody or a fragment thereof is immobilized on an insoluble carrier by affinity chromatography using an immobilized anti-LCAT antibody or an immobilized fragment. LCAT can be separated or purified.

Examples of the insoluble carrier used for the separation or purification of such human LCAT include (1) a filter made of a water-insoluble substance represented by plastics such as polystyrene resin, polycarbonate resin, silicone resin and nylon resin and glass. , A membrane, etc., (2) a cellulose-based carrier, an agarose-based carrier, a polyacrylamide-based carrier, a dextran-based carrier, a polystyrene-based carrier, a polyvinyl alcohol-based carrier, a polyamino acid-based carrier, a porous silica-based carrier, etc. it can.

As such an affinity chromatography, for example, the (A) filter, membrane or the like of the above (1) is used as an insoluble carrier, and the anti-LC of the present invention is added thereto.
After immobilizing the AT antibody or its antibody fragment, a method of separating human-derived LCAT contained in the sample by contacting the sample with the insoluble carrier, (B) the cellulose-based carrier of the above (2) as the insoluble carrier The anti-LCAT antibody of the present invention or an antibody fragment thereof is immobilized thereto by a conventional method (physical adsorption, polymerization by crosslinking, sealing in a matrix, non-covalent bonding, etc.), and the insoluble carrier is By filling a column made of glass, plastic, stainless steel, or the like, and eluting the sample (for example, a body fluid sample such as plasma, a culture supernatant, or a centrifuge) into the column (for example, a columnar column). A method for separating or purifying human-derived LCAT contained in the sample,
The method (B) is preferred.

Specific examples of the insoluble carrier (2) used in the affinity chromatography (B) are not particularly limited as long as the anti-LCAT antibody of the present invention or the antibody fragment thereof can be immobilized thereon. A commercial product, Sepharose 2 from Pharmacia
B, Sepharose 4B, Sepharose 6B, CNBr-Activated Seph
arose 4B, AH-Sepharose 4B, CH-Sepharose 4B, Activa
ted CH-Sepharose 4B, Epoxy-activated Sepharose 6
B, Activated thiol-Sepharose 4B, Sephadex, CM-Seph
adex, ECH-Sepharose 4B, EAH-Sepharose 4B, NHS-acti
Bio-GelA, Cellex, Cellex AE, manufactured by Bio-Rad, such as vated Sepharose, Thiopropyl Sepharose 6B,
Cellex-CM, Cellex PAB, Bio-Gel P, Hydrozide Bio-Ge
l P, Aminoethyl Bio-Gle P, Bio-Gel CM, Affi-Gel 1
0, Affi-Gel 15, Affi-Prep 10, Affi-Gel Hz, Affi-Pr
ep Hz, Affi-Gel 102, CMBio-Gel A, Affi-Gel hepari
n, Affi-Gel 501, Affi-Gel 601 etc., chroma gel A, chroma gel P, Enzafix PH manufactured by Wako Pure Chemical Industries, Ltd.
Z, Enzafix P-SH or Enzafix PA
AE-Cellurose, CM-Celluro made by Selva (Serva)
se, PAB Cellurose and the like.

[0033]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

Reference Example 1 Measurement of LCAT activity In each of the following examples, LCAT activity was measured according to the method described in JP-A-59-116.
According to the method described in Japanese Patent No. 577, etc., the activity was measured as shown below according to the package insert using an in vitro diagnostic drug Anasorb LCAT (manufactured by Daiichi Kagaku) for lecithin-cholesterol-acyltransferase measurement. . Dispense LCAT stop solution drop by drop into small test tubes (T0), (T1), (St) and (B1). Take a 200 μl sample in a stoppered test tube, add 500 μl of the substrate solution, mix immediately, add 200 μl of this mixture to a small test tube (T0) to stop the LCAT reaction, and in a 37 ° C constant temperature bath for 40 minutes. Heat. The remaining mixture is stoppered and heated in a thermostat at 37 ° C. for 40 minutes to obtain a reaction solution. The reaction solution is mixed well, and 200 μl is dispensed into a small test tube (T1) to stop the LCAT reaction. Dispense 200 µl of the standard solution into a small test tube (St) and 200 µl of purified water into the small test tube (B1). Small test tubes (T0), (T1),
Add 3000 μl of the enzyme solution to (St) and (B1), mix well, and mix
Heat at ℃ for 10 minutes. After returning to room temperature, the absorbance is measured at a wavelength of 545 nm using purified water as a control within 60 minutes. From the measured values, LCAT activity is determined according to the following formula. With this, LCAT
The activity is defined as “1U = ΔF-CHOnmol / ml · hr (37 ° C.)”.

LCAT activity (U) = {E (T0) -E (T1)} / {E (St) -E
(B1)} × 500 ^{* 1} × 3.5 ^{* 2} × 60/40 ^{* 3} [where E (T0), E (T1), E (St) and E (B1) indicate the absorbance of each small test tube. . * 1: Free cholesterol (F-CHO) concentration in the standard solution (5
* 2: Dilution ratio * 3: Reaction time (40 minutes)]

Reference Example 2 Preparation of purified human-derived LCAT According to the method described in JP-A-61-257580, human-derived LCAT
CAT was purified. The following operations were all performed at 4 ° C. or on an ice bath according to a conventional method.

(1) Purification by Butyl Toyopearl Column Chromatography Peripheral blood collected from a plurality of healthy persons is subjected to centrifugation according to a conventional method to remove hemocytes, and human plasma is obtained at −80 ° C. until use. saved. 880 ml of this human plasma was dissolved in a thermostat at 37 ° C, and centrifuged (5000 xg, 30 minutes,
(4 ° C.), and an equal volume of 20 mM Tris buffer (pH 7.4) containing 1 M sodium chloride was added to the supernatant. 400 ml of butyl toyopearl gel (BUTYL-TOYOPEARL 650M, manufactured by Tosoh Corporation) equilibrated with 20 mM Tris buffer (pH 7.4) containing 0.5 M sodium chloride was added, and the mixture was slowly stirred in an ice bath for 2 hours. The butyl toyopearl gel to which the hydrophobic component of plasma was bound was filtered by suction using a Buchner funnel. The butyl toyopearl gel was suspended in 20 mM Tris buffer (pH 7.4) containing 0.5 M sodium chloride, stirred slowly in an ice bath for 2 hours, and then filtered by suction using a Buchner funnel and washed. After suspending again, suction filtration, and washing, butyl toyopearl gel was added to 0.
20 mM Tris buffer (pH 7.4) containing 5 M sodium chloride
And filled in a glass column tube (10 × 40 cm, manufactured by Bio-Rad). After washing with 500 ml of 20 mM Tris buffer (pH 7.4) containing 0.5 M sodium chloride, elution was carried out with 5% aqueous ethanol (20 ml / tube, 50 ml).
Book). The elution pattern was observed by measuring the absorbance at a wavelength of 280 nm and the LCAT activity, and an active fraction containing human-derived LCAT was collected.

(2) Purification by Hydroxyapatite Column Chromatography The active fraction collected in (1) was diluted by adding an equal amount of purified water, and this was diluted with a glass column tube (10 × 40 cm, manufactured by Bio-Rad). Hydroxyapatite column (Hydroxylapatite, manufactured by Bio-Rad Co.)
Was added. After washing with 500 ml of purified water, the mixture was eluted with a concentration gradient of 0 to 25 mM phosphate buffer (pH 7.4) (10 ml / tube, 120 tubes). The elution pattern was measured with the absorbance at 280 nm.
Observation was performed by measuring the LCAT activity, and an active fraction containing human-derived LCAT was collected.

(3) Purification by Anion Exchange Column Chromatography An equal amount of a 40 mM phosphate buffer (p
H7.4), packed in a glass column tube (2 × 40 cm, manufactured by Bio-Rad) and equilibrated with 20 mM phosphate buffer (pH 7.4) (DEAE TOYOPEARL 650M, manufactured by Tosoh Corporation) Was added. 200 mM of 20 mM phosphate buffer (pH 7.4)
After washing with ml, the mixture was eluted with a 20 mM phosphate buffer (pH 7.4) containing 0 to 220 mM sodium chloride (5 ml / tube, 100 tubes). The elution pattern was observed by measuring the absorbance at a wavelength of 280 nm and the LCAT activity, and an active fraction containing human-derived LCAT was collected to obtain purified human-derived LCAT.

(4) Characteristic analysis of purified human-derived LCAT The protein amount of the purified human-derived LCAT obtained in (3) above was determined by the Bradford method (Protein Assay kit, manufactured by Bio-Rad).
When the amount of LCAT activity was determined by the method of Reference Example 1, the enzyme activity (specific activity) was 6680 U (unit) / mg.
Met. Further, the molecular weight of the purified human-derived LCAT was analyzed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) according to a conventional method. This result is shown in FIG.
Shown in It was confirmed that the purified human-derived LCAT obtained in this Reference Example was composed of one species having a molecular weight of about 65,000. Human origin
The purification of LCAT has been attempted by many researchers so far, all of which have a molecular weight of about 65,000 [CH Chen et al., Biochimica et Biophysica Acta, Vol. 834. , 18th
8, 1985]. From comparison with these values, it was confirmed that the human-derived LCAT obtained in the present Reference Example had extremely high purity.

Example 1 Preparation of Antibody Human LCAT Monoclonal Antibody An anti-human LCAT monoclonal antibody was prepared according to a general method.

(1) Selection of immunizing antigen and antigen for screening In order to prepare an anti-human LCAT monoclonal antibody,
The following method (a) or (b) was used. (a): immunizing a mammal with purified human-derived LCAT, fusing spleen cells and myeloma cells of the immunized animal, and obtaining the purified human-derived LCAT or the following
Hybridoma cells that specifically react with the peptide selectively synthesized in step (2) were selected. (b): synthesizing a peptide constituting a part of the amino acid sequence of human-derived LCAT, immunizing a mammal with this peptide or a conjugate thereof with a carrier thereof, and fusing spleen cells of the immunized animal with myeloma cells Then, from the obtained hybridoma cells, hybridoma cells that specifically reacted with the purified human-derived LCAT or the peptide selectively synthesized in the following step (2) were selected.

(2) Method for Designing and Synthesizing Peptides Using the basic properties of the proteins described below, a sequence of a synthetic peptide having a clear hydrophilic region and a clear primary structure between animal species was selected. The protein structure of human-derived LCAT was analyzed in order to use a highly hydrophilic site in the amino acid sequence, which is generally considered to be an externally exposed site as a protein structure, in screening for an immunogen or a hybridoma cell. Dedicated software for analysis of nucleotide and amino acid sequences
DNASIS-Mac v.3.6 (Hitachi Software) was used. The Hop and Wood method (Hopp & Woods method, Proceedings National Academy of Sciences (Pr.)
oc. Natl. Acad. Sci. USA), Vol. 78, No. 3824-3828.
1981). In addition, when immunizing a certain animal species with a heterologous antigen, the amino acid sequence presented as the antigen is considered to be important to immunize a region where the species difference of the animal is clearly recognized in the amino acids of the antigen. Was done. FIG. 2 shows the calculation results of hydrophobicity calculated from the amino acid sequence of human-derived LCAT. Although it is strongly hydrophobic as a whole, there are hydrophilic regions at the center and C-terminal. In addition, as a result of a homology search between the LCAT amino acid sequences of the animals whose gene sequences have been determined (Multiple alignment), the amino acid sequence of LCAT has almost no difference in animal species overall, and only a slight species difference at the C-terminus is observed. there were. Estimation of the above-mentioned hydrophobic site and homology evaluation of the amino acid sequence of LCAT of different origin animals, furthermore, the overall structural characteristics of individual amino acids, comprehensively judge the binding to the carrier, and use it as an antigen or produce an antibody The sequences used for hybridoma selection were determined. From the above viewpoint, the peptides synthesized and used in this example are peptides consisting of the amino acid sequence at positions 27 to 48 in the amino acid sequence of human-derived LCAT represented by SEQ ID NO: 6 (hereinafter referred to as “peptide 1”), A peptide consisting of the 74th amino acid sequence (hereinafter referred to as “peptide 2”), 159-1
A peptide consisting of the 79th amino acid sequence (SEQ ID NO: 1;
A peptide comprising the amino acid sequence at positions 258 to 273 (SEQ ID NO: 2; hereinafter referred to as “peptide 4”); a peptide comprising the amino acid sequence at positions 273 to 294 (SEQ ID NO: 3; hereinafter referred to as “peptide 5”) "), 35
A peptide consisting of the amino acid sequence at positions 2 to 376 (SEQ ID NO: 4; hereinafter referred to as “peptide 6”), a peptide consisting of the amino acid sequence at positions 384 to 407 (hereinafter referred to as “peptide 7”) and the amino acid sequence at positions 415 to 440 (SEQ ID NO: 5; hereinafter, referred to as “peptide 8”). The synthesis of these peptides is tBOC (tert-butoxycarbonyl)
And Fmoc (9-fluorenylmethoxycarbonyl) method.

(3) Method of binding synthetic peptide to carrier (i) When bovine thyroglobulin is used as carrier: 0.5 ml of synthetic peptide (2 mg / ml distilled water) and 0.5 ml of bovine thyroglobulin (manufactured by Sigma) are mixed and mixed with 1M acetic acid. Add ammonium to bring the pH to 7.0. Add 0.54 ml of 0.02 M glutaraldehyde, stir at room temperature for 5 hours, and add
Dialyze for 1 day against 100 volumes of distilled water. The dialyzed product was sonicated for 10 minutes with an ultrasonic washing machine and subjected to an immunogen or enzyme immunoassay.

(Ii) When using keyhole limpet hemocyanin (KLH) as a carrier 10 mg of KLH for carrier (Pierce) is dissolved in 1 mL of distilled water, and 2 mg of Sulfo-MSCC (Pierce) is dissolved in 1 mL of distilled water. It was dissolved in 1 mL of distilled water, 100 µl of the solution was mixed with 200 µl of the KLH solution, and the mixture was stirred at room temperature for 1 hour. It was added to a PD-10 column (Pharmacia Biotech) equilibrated with a 0.1 M phosphate buffer (pH 7.4). Elution was performed with a 0.1 M phosphate buffer (pH 7.4), and the elution pattern was measured by absorbance at a wavelength of 280 nm.
After collecting maleimide-activated KLH according to the elution pattern,
Add 0.5 ml of synthetic peptide (4 mg / ml distilled water) and add 2 ml at room temperature.
The mixture was stirred for 100 hours, dialyzed against 100 volumes of PBS, and subjected to immunoassay or enzyme immunoassay.

(4) Preparation of monoclonal antibody (i) Preparation of spleen cells of immunized animal Purified human-derived LC prepared from BALB / c mouse in Reference Example 2
The cells were immunized with AT or the conjugate of the peptide prepared in (3) above and a carrier, and spleen cells were prepared from the immunized mice. The immunization was carried out by subcutaneously, intravenously or intraperitoneally in BALB / c mice of 8 to 10 weeks old using the purified human-derived LCAT prepared in Reference Example 2 or the conjugate of the peptide prepared in (3) above and a carrier. A suitable adjuvant [for example, Freund's adjuvant or Libya adjuvant system (Ribi Adjuvant Syste
m, manufactured by RIBI IMMUNOCHEM RESEARC, sold by Funakoshi))
Were immunized for the first time (day 0).
On days 14, 28 and 42 from the first immunization, booster immunization was performed by injecting subcutaneously or intraperitoneally the purified human-derived LCAT or the conjugate of the peptide and the carrier, and further producing the monoclonal antibody described below Similarly, two days before and the day before the preparation of the hybridoma, the final immunization was performed in the same manner, and spleen cells were prepared from the mouse and used for cell fusion.

(Ii) Preparation of mouse myeloma cells 8-azaguanine-resistant mouse bone marrow cells P3-U1 were cultured in a normal medium (R
The cells were cultured (37 ° C., CO ₂ , 5% aeration) in PMI-1640 supplemented with 1.5 mM glutamine and 13% fetal calf serum, and after 4 days, 2 × 10 ⁷ or more cells were obtained.

(Iii) Preparation of hybridomas 1 × 10 ⁸ immunized mouse spleen cells and 2 × 10 ⁷ mouse myeloma cells thoroughly washed with RPMI-1640 (manufactured by Nissui Pharmaceutical Co., Ltd.) were mixed and mixed at 1500 rpm for 5 minutes. Centrifuged. After loosening the mixed cell group of splenocytes and P3-U1 obtained as a precipitate,
While stirring, 1 ml of a 50% polyethylene glycol and 10% DMSO solution was added, and 2 minutes later, RPMI-1640 was gradually added so that the total volume became 50 ml. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded, the cells were loosened gently, and HAT medium (hypoxanthine 10 ^-1 M, thymidine 1.5
30 ml of a medium containing × 10 ⁻⁵ M and aminopterin (4 × 10 ⁻⁷ M) was added, and the cells were gently suspended with a 5 ml dissolution pipette and cultured at 37 ° C. for 2 hours in a 5% CO ₂ incubator. After centrifugation at 1500 rpm for 5 minutes, the supernatant was discarded, the cells were loosened loosely, suspended in HAT medium, dispensed into a 96-well culture plate at 200 μl / well, and incubated at 37 ° C. in a 5% CO ₂ incubator at 37 ° C. Cultured for ~ 14 days.

(5) Screening of hybridomas Screening of hybridomas producing anti-human-derived LCAT monoclonal antibodies was carried out using an enzyme-linked immunosorbent assay in which an antigen was immobilized. The purified human-derived LCAT prepared in Reference Example 2 was prepared at a concentration of 1 μg / ml in a 20 mM phosphate buffer (pH 7.4) containing 150 mM sodium chloride, and then dispensed at 50 μl / well into a 96-well EIA plate. The mixture was allowed to stand at room temperature for 2 hours to immobilize the antigen on the plate. 20 mM phosphate buffer containing 0.05% Tween 20 and 150 mM sodium chloride (pH 7.4, T
-PBS) was dispensed at 350 μl / well and left at room temperature for 1 hour to block protein-binding residues on the bottom surface. 96 hole EIA
After the plate was washed twice with T-PBS, 50 μl of T-PBS was added per well to the plate, and 100 μl of the hybridoma culture supernatant was transferred to a 96-well EIA plate in a clean bench as a primary antibody. Left overnight or at room temperature for 2 hours. After washing the 96-well EIA plate twice with T-PBS, a second antibody containing 50 parts of a goat anti-mouse immunoglobulin-peroxidase conjugate (manufactured by TAGO, Cosmo Bio) was used.
The 00-fold dilution was dispensed at 50 μl / well and left at room temperature for 1 hour. After washing the 96-well EIA plate twice with T-PBS, OPD
Substrate solution [30% o-phenylenediamine dihydrochloride dissolved in 20 ml of citrate / phosphate buffer (pH 5.2)
Was added, and the reaction was stopped by dispensing 50 μl / well of 1N sulfuric acid solution. Absorbance measured by plate reader at main wavelength 492n
m, measured at an auxiliary wavelength of 620 nm. When purified human LCAT is used as the immunogen according to the above method, the purified human LCAT
Select hybridoma cells that specifically react with LCAT and the peptide selectively synthesized in (2), and (2) as an immunogen
In the case where a peptide selectively synthesized in step 1 or a conjugate thereof with a carrier was used, purified human-derived LCAT and hybridoma cells that specifically reacted with the synthetic peptide were selected. For holes in which hybridomas producing anti-human LCAT monoclonal antibodies were observed, cleaning was repeated 2 to 4 times by the limiting dilution method, and those in which antibody production was stably recognized were identified as anti-human-derived LCAT monoclonal antibody-producing hybridoma strains. Selected as As a result, a hybridoma 36487 producing a monoclonal antibody 36487 recognizing SEQ ID NO: 1; a hybridoma 36442 producing a monoclonal antibody 36442 recognizing SEQ ID NO: 2; a hybridoma 36454 producing a monoclonal antibody 36454 recognizing SEQ ID NO: 3; Five kinds of hybridomas, a hybridoma 36405 producing a monoclonal antibody 36405 recognizing SEQ ID NO: 4 and a hybridoma 36486 producing a monoclonal antibody 36486 recognizing SEQ ID NO: 5, were selected.
These hybridomas were deposited on October 14, 1999 with the Ministry of International Trade and Industry, Ministry of Industry and Technology,
RM P-17610, FERM P-17609, FERM P-17608, FERM P-176
11, and deposited as FERM P-17607.

(6) Large-scale preparation of monoclonal antibody Pristane treatment (2,6,10,14-tetramethylpentadecane
0.5 ml / animal was intraperitoneally administered and bred for 1-2 weeks)
0 week-old mice (BALB / c) are injected intraperitoneally with 1 × 10 ⁶ cells / animal of each of the hybridoma strains obtained above. 10-21
One day later, the hybridoma line became ascites cancer. After 10 to 21 days, ascites (4 to 10 ml / animal) was collected from the mice with accumulated ascites and centrifuged to remove solids. After the supernatant was salted out with 50% ammonium sulfate, it was dialyzed overnight against a 20 mM phosphate buffer (pH 7.4) containing 150 mM sodium chloride to obtain a crude monoclonal antibody.

(7) Determination of isotype Mouse monoclonal antibody isotype determination kit (ZY
Using MED (manufactured by Cosmo Bio Co., Ltd.), the procedure was performed according to the experimental operation protocol attached to the kit, and the isotype of each of the monoclonal antibodies 36487, 36442, 36454, 36405 and 36486 was determined. 36442, 36454 and 36486 are I
It was confirmed that gG ₁ and 36405 were IgG _2a and 36487 was IgG _2b .

(8) Purification of monoclonal antibody The monoclonal antibodies 36487, 36442, and 3645 obtained in (6)
An equal amount of MAPSII binding buffer (manufactured by Bio-Rad) was added to each of the crudely purified monoclonal antibodies 4, 36405 and 36486, and the mixture was filtered with a filter (manufactured by Millipore) to remove white precipitate. The obtained filtrate was added to a HiTrap Protein A affinity column (Pharmacia Biotech).
After washing with MAPSII binding buffer (Bio-Rad),
It was eluted with MAPSII elution buffer (Bio-Rad) (3 ml / tube, 10 tubes). Elution pattern at wavelength 280nm
The fraction containing the antibody was collected according to the elution pattern.

Example 2 Purification of human-derived LCAT by affinity chromatography The purified human-derived LCAT prepared in (2) of Reference Example 2 was subjected to affinity chromatography using the purified monoclonal antibodies 36487 and 36486 prepared in Example 1. It was further purified with high purity, and its properties were analyzed.

(1) Preparation of Immobilized Monoclonal Antibody (Adsorbent for Packing Column) Using CNBr-activated Sepharose 4B (Pharmacia Biotech), the operation was performed according to the attached protocol. That is, CNBr-activated Sepharose 4B is treated with 1 mM hydrochloric acid.
After swelling for 20 minutes, the plate was washed with 1 mM hydrochloric acid on a Buchner funnel with a glass filter having G3 grade pores. The gel is suspended in a buffer (hereinafter, referred to as a binding buffer) containing 0.5 M sodium chloride and 0.1 M sodium hydrogen carbonate (pH 8.3), and an antibody dialyzed against the binding buffer overnight is added thereto. The mixture was mixed to bind the antibody and the gel. 1000
The mixture was centrifuged at rpm for 5 minutes to remove the supernatant, suspended in 0.1 M Tris buffer (pH 8.0), and allowed to stand at room temperature for 2 hours. After filtration through a glass filter, the plate is washed alternately with a binding buffer and 0.1 M acetate buffer (pH 4.0) containing 0.5 M sodium chloride on the glass filter. It is suspended in a 20 mM phosphate buffer (pH 7.4) containing an appropriate amount of 150 mM sodium chloride and packed into a column.

(2) Purification by Affinity Chromatography A solution containing purified human-derived LCAT prepared in (2) of Reference Example 2 was mixed with a 20 mM phosphate buffer (p.m.) containing 150 mM sodium chloride.
H7.4, hereinafter referred to as PBS. ) Was dialyzed overnight, and then added to the affinity column prepared in (1). The column was washed with PBS and eluted with PBS containing 3M NaSCN. The pattern of the eluted fraction was measured by the absorbance at a wavelength of 280 nm.
The result is shown in FIG. According to the elution pattern, fractions containing human-derived LCAT were collected and dialyzed against PBS to prepare extremely high-purity purified human-derived LCAT.

(3) Characteristic analysis of purified human-derived LCAT Purification of each chromatography step was carried out by SDS-PA
Analyzed by GE. The result is shown in FIG. It was confirmed that the purified human-derived LCAT obtained in this example was composed of one species having a molecular weight of about 65,000. After purification by butyl toyopearl column chromatography and hydroxyapatite column chromatography, by using affinity chromatography purification, it was shown that human-derived LCAT can be purified more easily and with good reproducibility compared to Reference Example 2. In addition, the human origin obtained in this example
LCAT was confirmed to be of extremely high purity.

Test Example 1 Confirmation of Antigen Recognition Site of Anti-Human LCAT Monoclonal Antibody The enzyme immunoassay in which the synthetic peptide synthesized after theoretical selection and synthesized in Example 1 (2) was immobilized was used in Example 1. Prepared purified monoclonal antibodies 36487, 36442, 36454, 36405
And 36486 were confirmed. Example 1
1 mg / ml of peptides 1 to 8 synthesized after theoretical selection in (2)
Dissolved in high purity distilled water at a concentration of After preparing each of the dissolved peptides at a concentration of 10 μg / ml in 20 mM phosphate buffer (pH 7.4: hereinafter referred to as PBS) containing 150 mM sodium chloride,
50 μl / well was dispensed into a well EIA plate and left at room temperature for 2 hours to immobilize the antigen on the plate. After washing twice with BPS containing 0.05% Tween 20 (hereinafter referred to as T-PBS), 350 μl of blocking reagent (Block Ace, manufactured by Dainippon Pharmaceutical Co., Ltd.) was used.
Each well was left at room temperature for 1 hour to block protein-binding residues on the bottom surface. After washing twice with T-PBS, each of the purified monoclonal antibodies 36487, 36442, 36454, 36405 and 36486 prepared in Example 1 was prepared at a concentration of 10 μg / ml in T-PBS as a primary antibody. Dispense per hole and leave at room temperature for 2 hours. After washing twice with T-PBS, the second
As an antibody, a goat anti-mouse immunoglobulin-peroxidase conjugate (TAGO, sold by Cosmo Bio)
The 5000-fold diluted solution was dispensed at 50 μl / well and left at room temperature for 1 hour. After washing twice with T-PBS, an OPD substrate solution (a solution of 60 mg of o-phenylenediamine diamine dihydrochloride in 20 ml of a citrate / phosphate buffer (pH 5.2)) was added with 20 μl of 30% hydrogen peroxide. Is added and the color is developed, and then the reaction is stopped by dispensing a 1N sulfuric acid solution at 50 μl / well. The absorbance is measured with a plate reader at a main wavelength of 492 nm and a sub wavelength of 620 nm. Table 1 shows the results.

[0058]

[Table 1]

Monoclonal antibody 36487 established by immunization with purified human LCAT recognizes the amino acid sequence represented by SEQ ID NO: 1 (peptide 3). Monoclonal antibody 36442 established by immunization with purified human LCAT has the following sequence: Monoclonal antibody 36454, which recognizes the amino acid sequence of SEQ ID NO: 2 (peptide 4) and immunizes with the carrier conjugate of peptide 5 of SEQ ID NO: 3, establishes the amino acid sequence of SEQ ID NO: 3 (peptide 5) The monoclonal antibody 36405 established by immunization with the carrier-bound substance of peptide 6 represented by SEQ ID NO: 4 recognizes the amino acid sequence represented by SEQ ID NO: 4 (peptide 6), and
It was confirmed that the monoclonal antibody 36486 established by immunizing with AT recognized the amino acid sequence represented by SEQ ID NO: 5 (peptide 8). Thus, the monoclonal antibody of the present invention has extremely high specificity.

Example 3 Quantification of human-derived LCAT by sandwich ELISA (1) Preparation of immobilized monoclonal antibody 20 mM phosphate buffer containing 150 mM sodium chloride (pH 7.
4, hereinafter referred to as PBS), prepared at a concentration of 10 μg / ml of the purified monoclonal antibody 36486 prepared in Example 1, and dispensed into a 96-well EIA plate in an amount of 50 μl / well. Immobilized. PBS containing 0.05% Tween20
(Hereinafter referred to as T-PBS) twice, containing 1% BSA
350 μl / well of T-PBS was dispensed and left at room temperature for 1 hour to block protein-binding residues on the bottom surface. 1% BSA and 1
T-PBS containing 0% sucrose was dispensed at 350 μl / well, left at room temperature for 1 hour, air-dried, and washed twice with T-PBS.

(2) Preparation of anti-LCAT monoclonal antibody F (ab ') fragment The purified monoclonal antibody 36486 prepared in Example 1 was
Dialysis was performed against 0.1 M sodium acetate buffer (pH 4.5). To this solution was added pepsin (manufactured by Sigma) in an amount of 1 mg of IgG.
Was added, and reacted at 37 ° C. for about 24 hours.
1/10 volume of Tris buffer (pH 8.0) was added. FPLC
A chromatographic system (Pharmacia Biotech) was attached to a gel filtration column TSK gel G3000SW (Tosoh) using an automated chromatography with a 0.1 M phosphate buffer containing 1 mM magnesium chloride and 0.2 M sodium chloride ( pH 6.5), and F (ab ') was recovered. F (a
b ') ₂ in 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA
Dialyzed against. After dialysis, a 0.1 M phosphate buffer (pH 6.0) containing 0.1 M 2-mercaptoethylamine and 5 mM EDTA.
0) was added at 1/10 volume and reacted at 37 ° C. for 90 minutes. Next, this was added to a 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA.
Add to a column of Sephadex G-25 gel (1.5 × 30 cm, manufactured by Pharmacia Vitec) equilibrated in
b ') Fractions were collected.

(3) Peroxidase Labeling of Anti-LCAT Monoclonal Antibody Fragment N-Succinylmidyl-6-maleimidohexanate (DOJIN
A solution of 15 mg in 150 μl of N, N-dimethylformamide was added to 1.5 ml of 0.1 M phosphate buffer (pH 7.0).
It was added to a solution in which 0 mg of peroxidase (hereinafter referred to as HRP) was dissolved and reacted at room temperature for 45 minutes. Then, the reaction solution was
The mixture was added to a column of Sephadex G-25 gel (1.5 × 30 cm; manufactured by Pharmacia Vitec) equilibrated with M phosphate buffer (pH 6.0) to recover a maleimide / HRP fraction. Next, the F (ab ') fraction obtained in (2) and maleimide / HRP
The fractions were mixed and reacted at 4 ° C. for 3 days. After the reaction, 0.1
M containing 2-mercaptoethylamine and 5 mM EDTA.
One tenth volume of 1 M phosphate buffer (pH 6.0) was added. FP
Automated chromatography with a gel filtration column TSD gel G3000SW (manufactured by Tosoh Corporation) attached to an LC chromatography system (manufactured by Pharmacia Biotech) was performed using 0.1 M phosphate buffer (pH 6.5) containing 0.2 M sodium chloride. After equilibration, the HRP-labeled F (ab ') was recovered. BSA and chlorhexidine glucuronate were added at 0.1% and 0.0028%, respectively, aliquoted and stored at -80 ° C.

(4) Establishment of quantification method by sandwich ELISA Human-derived L by sandwich ELISA established in the present invention
The CAT quantification method is as follows. The immobilized monoclonal antibody 36486 (hereinafter referred to as immobilized microplate) prepared in the above (1) is used. To each well of the solid-phased microplate, 50 μl of a measurement sample (purified human-derived LCAT standard substance or human serum prepared in Example 2, (2)) diluted with T-PBS containing 0.2% BSA was added, and the mixture was cooled to room temperature. For 2 hours. After washing the immobilized microplate twice with T-PBS, the peroxidase-labeled anti-LCAT monoclonal antibody prepared in the above (3) diluted with T-PBS containing 0.2% BSA was dispensed in 50 μl / well portions, and the room temperature was kept at room temperature. For 1 hour. After washing the immobilized microplate twice with T-PBS,
PD substrate solution [30-ml o-phenylenediamine dihydrochloride dissolved in 20 ml of citrate / phosphate buffer (pH 5.2)
A solution containing 20 μl of 20% hydrogen peroxide] was dispensed at 50 μl / well, and after the color was developed, 1N sulfuric acid solution was dispensed at 50 μl / well to stop the reaction. Absorbance measured by plate reader
The measurement was performed at 492 nm and an auxiliary wavelength of 620 nm, and the amount of LCAT during the measurement was determined from a calibration curve prepared from a purified human-derived LCAT standard.

(5) Preparation of calibration curve Using the purified human-derived LCAT prepared in Example 2 (2) as a standard substance, a calibration curve was prepared using the sandwich ELISA established in the above (4). FIG. 5 shows the results. A good calibration curve was obtained in the concentration range of 0.1 to 10 μg / ml (correlation coefficient: R ² =
0.984).

(6) Comparison with the conventional quantification method In order to confirm the utility of the LCAT protein quantification method of the present invention and the LCAT protein quantification method of the present invention (Reference Example 1) using the conventional LCAT activity value measurement method as an index, Was tested. Thirty human serum samples, which are considered to have no particularly significant lipoprotein abnormality, were measured using the LCAT activity measurement method and the LCAT protein quantification method, and the correlation between the two measurement methods was examined. The results are shown in FIG. The correlation coefficient (r) between the conventional activity assay and the protein assay of the present invention showed a correlation of 0.773.

Test Example 2 Usefulness in Lipoprotein Metabolism Study (1) Distribution of LCAT in Human Serum Lipoprotein Fraction After lipoprotein was separated by gel filtration chromatography, the distribution of LCAT was examined. Automated chromatography with a gel filtration column Superose 6HR 10/30 (Pharmacia Biotech) attached to an FPLC chromatography system (Pharmacia Biotech) was performed using a 20 mM phosphate buffer (pH 7) containing 0.15 M sodium chloride. After equilibration in .4), the serum of a healthy individual was added and separated into VLDL, LDL and HDL lipoprotein fractions, and the LCAT protein content of each fraction was measured according to the method of Example 3. FIG. 7 shows the results. LCAT
Was almost distributed in the HDL fraction as conventionally pointed out in the literature. In this method, it is not necessary to perform a pretreatment with a surfactant and / or heating to avoid protein-protein and / or protein-lipoprotein interactions,
The denaturation of proteins in the sample can be avoided as much as possible, which is highly useful for studying lipid metabolism.

(2) Reactivity to serum of each animal Various animals are currently used as model systems for elucidating the etiology and pathology of dyslipidemia. It was examined whether the method of quantifying human-derived LCAT by sandwich ELISA shown in the present invention is applicable to these uses. The amount of LCAT protein was measured according to the method of Example 3 using rabbit serum as an animal. FIG. 8 shows the results. As shown in the figure,
It became clear that LCAT could be detected.

Example 4 Preparation of anti-LCAT polyclonal antibody An anti-LCAT polyclonal antibody was prepared according to a general method.

(1) Preparation of anti-LCAT antiserum Rabbits (Japanese white, female) were immunized with the conjugate of the peptide prepared in (2) of Example 1 with a carrier, and antiserum was obtained from the immunized rabbits. Prepare. Rabbit (Japanese white, female)
Subcutaneously, intracutaneously, intravenously or intraperitoneally of Example 1.
Synthetic peptide 1, 5, 6, 7 or 8 prepared in (2)
Immunization was carried out for the first time (day 0) by injecting the conjugate of the compound with the carrier together with an appropriate adjuvant (for example, Freund's adjuvant or Libya adjuvant system (manufactured by RIBI IMMUNOCHEM RESEARC, sold by Funakoshi)). Day 14, Day 28, 42 after first immunization
On days 56, 70, and 70, the conjugate of the peptide and the carrier was boosted by subcutaneous, intradermal, intravenous, or intraperitoneal injection of the conjugate, and two days before and one day before the antiserum preparation. Similarly, final immunization was performed, and blood was collected from the carotid artery or heart to prepare a large amount of antiserum.

(2) Purification of polyclonal antibody The antiserum was salted out with 50% ammonium sulfate and then dialyzed overnight against a 20 mM phosphate buffer (pH 7.4) containing 150 mM sodium chloride to obtain a crudely purified polyclonal antibody. . An equal amount of MAPSII binding buffer (manufactured by Bio-Rad) was added to the roughly purified polyclonal antibody, and the mixture was filtered with a filter (manufactured by Millipore) to remove white precipitate. The obtained filtrate was added to a HiTrap ProteinA affinity column (Pharmacia Biotech). After washing with MAPSII binding buffer (manufactured by Bio-Rad), elution was carried out with MAPSII elution buffer (manufactured by Bio-Rad) (3 ml / tube, 10 tubes). Elution pattern with wavelength
It was measured by absorbance at 280 nm, and the fraction containing the antibody was collected according to the elution pattern.

(3) Examination of Antibody Titer of Polyclonal Antibody The antibody titer of the polyclonal antibody was analyzed using an enzyme immunoassay in which an antigen was immobilized. After preparing the purified human-derived LCAT prepared in Reference Example 2 at a concentration of 1 μg / ml in a 20 mM phosphate buffer (pH 7.4) containing 150 mM sodium chloride, 50 μl / well was dispensed into a 96-well EIA plate, After leaving at room temperature for 2 hours, the antigen was immobilized on the plate. 0.05% Tween 20 and 150 mM
A 20 mM phosphate buffer solution (pH 7.4; hereinafter, referred to as T-PBS) containing 350 mM of sodium chloride was dispensed at 350 μl / well and allowed to stand at room temperature for 1 hour to block protein-binding residues on the bottom surface. 96 holes
After washing the EIA plate twice with T-PBS, T-PBS was added to the plate at 50 μl per well, and as a primary antibody,
96-well EIA of hybridoma culture supernatant in a clean bench
Transfer to a plate 100 μl and leave at 4 ° C. overnight or at room temperature for 2 hours. After washing the 96-well EIA plate twice with T-PBS,
As a second antibody, a 5000-fold diluted solution of a goat anti-mouse immunoglobulin-peroxidase conjugate (manufactured by TAGO and sold by Cosmo Bio Inc.) was dispensed in 50 μl / well portions, and the resulting solution was incubated at room temperature.
Left for hours. After washing the 96-well EIA plate twice with T-PBS, an OPD substrate solution [30% o-phenylenediamine dihydrochloride in a solution of 60 mg of o-phenylenediamine dihydrochloride in 20 ml of citrate / phosphate buffer (pH 5.2) was added. A solution containing 20 μl of hydrogen oxide] was dispensed at 50 μl / well, and after the color was developed, 1N sulfuric acid solution was dispensed at 50 μl / well to stop the reaction. The absorbance is measured with a plate reader at a main wavelength of 492 nm and a sub wavelength of 620 nm. FIG. 9 shows the results. Synthetic peptides 1, 5 synthesized in (2) of Example 1
When an antibody was prepared using a conjugate of 6, 7, or 8 and a carrier as an immunogen, peptide 5 (SEQ ID NO: 3), peptide 6
When (SEQ ID NO: 4) and peptide 8 (SEQ ID NO: 5) were used as antigens, a polyclonal antibody recognizing purified human-derived LCAT was obtained. Furthermore, these polyclonal antibodies are polyclonal antibodies that recognize a specific amino acid sequence of human LCAT, respectively, and have extremely high specificity. In addition, LCAT antigens (including recombinantly expressed and synthetic peptides) containing the sequences shown in SEQ ID NOs: 3 to 5 have been shown to be extremely useful as artificial antigens for producing anti-human LCAT antibodies. Was.

[0072] The cross-reactivity to phospholipase A ₂ anti-human-derived LCAT monoclonal antibody crossreactivity present invention to the test example 3 Phospholipase A ₂ family, was investigated using immobilized enzyme immunoassay method an antigen. Purified human-derived LCAT prepared in Reference Example 2, phospholipase A ₂ (bovine pancreas-derived,
Sigma), phospholipase A ₂ (derived from porcine pancreas, Sigma), and phospholipase A ₂ (derived from honey bee venom, manufactured by Sigma) were each converted to 20 mM containing 150 mM sodium chloride.
After adjusting to a concentration of 1 μg / ml in M phosphate buffer (pH 7.4), 96
50 μl / well was dispensed into a well EIA plate and left at room temperature for 2 hours to immobilize the antigen on the plate. After washing twice with PBS containing 0.05% Tween 20 (hereinafter referred to as T-PBS), 350 μl of blocking reagent (Block Ace, manufactured by Dainippon Pharmaceutical Co., Ltd.) was used.
Each well was left at room temperature for 1 hour to block protein-binding residues on the bottom surface. After washing twice with T-PBS, each of the purified monoclonal antibodies 36487, 36442, 36454, 36405 and 36486 prepared in Example 1 was prepared in T-PBS at a concentration of 10 μg / ml as a primary antibody. The mixture was dispensed into each hole and left at room temperature for 2 hours. At the same time, mouse myeloma IgG ₁ (manufactured by ZYMED) was prepared at a concentration of 10 μg / ml as a control, dispensed at 50 μl / well, and left at room temperature for 2 hours. T-PBS
After washing twice, a goat anti-mouse immunoglobulin-peroxidase conjugate (manufactured by TAGO,
A 5000-fold diluted solution of Cosmo Bio Co., Ltd.) was dispensed at 50 μl / well and left at room temperature for 1 hour. After washing twice with T-PBS, an OPD substrate solution [a solution of 60 mg of o-phenylenediamine dihydrochloride dissolved in 20 ml of citrate / phosphate buffer (pH 5.2) and 20 μl of 30% hydrogen peroxide were added. The solution was dispensed in 50 μl portions, and after color development, a 1N sulfuric acid solution was dispensed in 50 μl / well portions to stop the reaction. The absorbance was measured at a main wavelength of 492 nm and a sub wavelength of 620 nm using a plate reader. The result is shown in FIG. Anti-human-derived LCAT monoclonal antibodies of the present invention, which reacts to the purification LCAT, the phospholipase A ₂ family not reactive. From these results, each of the monoclonal antibodies of the present invention clearly showed an antigen recognition site different from the reported anti-LCAT monoclonal antibody, and proved to be specific monoclonal antibodies having no cross-reactivity.

[0073]

The anti-LCAT antibody of the present invention has high specificity for human LCAT, and can be easily quantified by applying it to an immunoassay.
High purity LCAT sample can be obtained by utilizing it for purification. Furthermore, because of its high binding specificity, the anti-LCAT antibody of the present invention is also useful as an extremely organ-specific diagnostic marker that reflects liver function and nutritional status.

The anti-LCAT monoclonal antibody of the present invention can be obtained by utilizing a peptide having the sequence of SEQ ID NOS: 1 to 5 for selection of an antibody-producing hybridoma,
An AT polyclonal antibody can be efficiently and conveniently produced by using the peptide as an immunogen.

[0075]

[Sequence List] SEQUENCE LISTING <110> Daiichi Pure Chemicals Co., Ltd. <120> Anti-LCAT Antibody <130> P00061201 <160> 6 <210> 1 <211> 21 <212> PRI <213> Artificial Sequence < 220> <223> Designed peptide based on hydrophilicity of human LCAT <400> 1 Arg Asp Glu Thr Val Arg Ala Ala Pro Tyr Asp Trp Arg Leu Glu Pro 1 5 10 15 Gly Gln Gln Glu Glu 20 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Designed peptide based on hydrophilicity of human LCAT <400> 2 Met Ser Ser Ile Lys Leu Lys Glu Glu Gln Arg Ile Thr Thr Thr Ser 1 5 10 15 <210 > 3 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Designed peptide based on hydrophilicity of human LCAT <400> 3 Ser Pro Trp Met Phe Pro Ser Arg Met Ala Trp Pro Glu Asp His Val 1 5 10 15 Phe Ile Ser Thr Pro Ser 20 <210> 4 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Designed peptide based on hydrophilicity of human LCAT <400> 4 Asp His Gly Phe Pro Tyr Thr Asp Pro Val Gly Val Leu Tyr Glu Asp 1 5 10 15 Gly Asp Asp Thr Val Al a Thr Arg Ser 20 25 <210> 5 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Designed peptide based on hydrophilicity of human LCAT <400> 5 Asn Ala Ile Leu Leu Gly Ala Tyr Arg Gln Gly Pro Pro Ala Ser Pro 1 5 10 15 Thr Ala Ser Pro Glu Pro Pro Pro Pro Glu 20 25 <210> 6 <211> 440 <212> PRT <213> Homo sapiens <400> 6 Met Gly Pro Pro Gly Ser Pro Trp Gln Trp Val Thr Leu Leu Leu Gly 1 5 10 15 Leu Leu Leu Pro Pro Ala Ala Pro Phe Trp Leu Leu Asn Val Leu Phe 20 25 30 Pro Pro His Thr Thr Pro Lys Ala Glu Leu Ser Asn His Thr Arg Pro 35 40 45 Val Ile Leu Val Pro Gly Cys Leu Gly Asn Gln Leu Glu Ala Lys Leu 50 55 60 Asp Lys Pro Asp Val Val Asn Trp Met Cys Tyr Arg Lys Thr Glu Asp 65 70 75 80 Phe Phe Thr Ile Trp Leu Asp Leu Asn Met Phe Leu Pro Leu Gly Val 85 90 95 Asp Cys Trp Ile Asp Asn Thr Arg Val Val Tyr Asn Arg Ser Ser Gly 100 105 110 Leu Val Ser Asn Ala Pro Gly Val Gln Ile Arg Val Pro Gly Phe Gly 115 120 125 Lys Thr Tyr Ser Val Glu Tyr Leu Asp Ser Ser Lys Leu Ala Gly Tyr 130 135 140 Leu Hi s Thr Leu Val Gln Asn Leu Val Asn Asn Gly Tyr Val Arg Asp 145 150 155 160 Glu Thr Val Valg Ala Ala Pro Tyr Asp Trp Arg Leu Glu Pro Gly Gln 165 170 175 Gln Glu Glu Tyr Tyr Arg Lys Leu Ala Gly Leu Val Glu Glu Met His 180 185 190 Ala Ala Tyr Gly Lys Pro Val Phe Leu Ile Gly His Ser Leu Gly Cys 195 200 205 Leu His Leu Leu Tyr Phe Leu Leu Arg Gln Pro Gln Ala Trp Lys Asp 210 215 220 Arg Phe Ile Asp Gly Phe Ile Ser Leu Gly Ala Pro Trp Gly Gly Ser 225 230 235 240 Ile Lys Pro Met Leu Val Leu Ala Ser Gly Asp Asn Gln Gly Ile Pro 245 250 255 Ile Met Ser Ser Ile Lys Leu Lys Glu Glu Gln Arg Ile Thr Thr Thr Thr 260 265 270 Ser Pro Trp Met Phe Pro Ser Arg Met Ala Trp Pro Glu Asp His Val 275 280 285 Phe Ile Ser Thr Pro Ser Phe Asn Tyr Thr Gly Arg Asp Phe Gln Arg 290 295 300 Phe Phe Ala Asp Leu His Phe Glu Glu Gly Trp Tyr Met Trp Leu Gln 305 310 315 320 Ser Arg Asp Leu Leu Ala Gly Leu Pro Ala Pro Gly Val Glu Val Tyr 325 330 335 Cys Leu Tyr Gly Val Gly Leu Pro Thr Pro Arg Thr Tyr Ile Tyr Asp 340 345 350 His Gl y Phe Pro Tyr Thr Asp Pro Val Gly Val Leu Tyr Glu Asp Gly 355 360 365 Asp Asp Thr Val Ala Thr Arg Ser Thr Glu Leu Cys Gly Leu Trp Gln 370 375 380 Gly Arg Gln Pro Gln Pro Val His Leu Leu Pro Leu His Gly Ile Gln 385 390 395 400 400 His Leu Asn Met Val Phe Ser Asn Leu Thr Leu Glu His Ile Asn Ala 405 410 415 Ile Leu Leu Gly Ala Tyr Arg Gln Gly Pro Pro Ala Ser Pro Thr Ala 420 425 430 Ser Pro Glu Pro Pro Pro Pro Glu 435 440

[Brief description of the drawings]

FIG. 1 is an electrophoretogram showing the degree of purification of human-derived LCAT purified by anion exchange column chromatography.

FIG. 2 is a view showing a calculation result of hydrophobicity calculated from an amino acid sequence of human-derived LCAT.

FIG. 3 is a view showing a chromatogram of human-derived LCAT by affinity chromatography using monoclonal antibodies 36486 and 36487.

FIG. 4 is an electrophoretogram showing the degree of purification of human-derived LCAT purified by affinity chromatography using monoclonal antibodies 36486 and 36487.

FIG. 5 is a diagram showing a calibration curve of a method for quantifying human-derived LCAT by sandwich ELISA using monoclonal antibodies 36486 and 36487.

FIG. 6 is a diagram showing a correlation between the LCAT protein quantification method of the present invention and a conventional LCAT activity quantification method (Reference Example 1).

FIG. 7 shows the distribution of LCAT in the lipoprotein fraction in human serum.

FIG. 8 shows the reactivity of the monoclonal antibody of the present invention with rabbit serum.

FIG. 9 is a view showing the antibody titer of the anti-LCAT polyclonal antibody of the present invention.

FIG. 10 shows purified LC of the anti-LCAT monoclonal antibody of the present invention.
FIG. ₂ is a view showing the reactivity to AT and various phospholipase A2.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/50 G01N 33/577 B 33/573 33/92 B 33/577 C12R 1:91) 33/92 (C12P 21/08 // (C12N 5/10 C12R 1:91) C12R 1:91) C12N 5/00 B (C12P 21/08 15/00 ZNAC C12R 1:91) C12R 1:91) F term (reference 2G045 BB14 BB41 BB51 DA20 DA62 DA69 FA29 FB03 FB06 FB07 GC10 4B024 AA11 BA44 BA61 DA02 GA03 HA01 4B064 AG27 CA10 CA20 CC24 DA13 4B065 AA92X AB05 AC14 BA08 CA25 CA46 4H045 AA11 BA17 BA18 CA40 DA76

Claims (14)

[Claims] 1. Reacting with human-derived LCAT, and
An anti-LCAT antibody or a fragment thereof which recognizes any one of the above-mentioned sequences. 2. The anti-LCAT antibody according to claim 1, which is a monoclonal antibody. 3. An antibody that recognizes SEQ ID NO: 1 is FERM P-176
The antibody produced by hybridoma 36487 deposited as No. 10 and recognizing SEQ ID NO: 2 was produced by hybridoma 36442 deposited as FERM P-17609, and the antibody recognizing SEQ ID NO: 3 was deposited as FERM P-17608 An antibody produced by 36454 and recognizing SEQ ID NO: 4 was produced by hybridoma 36405 deposited as FERM P-17611, and an antibody recognizing SEQ ID NO: 5 was produced by F
3. The anti-LCAT antibody according to claim 2, which is produced by hybridoma 36486 deposited as ERM P-17607. 4. The hybridoma 36487 deposited as FERM P-17610. 5. A hybridoma 36442 deposited as FERM P-17609. 6. The hybridoma 36454 deposited as FERM P-17608. 7. A hybridoma 36405 deposited as FERM P-17611. 8. The hybridoma 36486 deposited as FERM P-17607. 9. The anti-LCAT antibody according to claim 1, which is a polyclonal antibody. 10. The peptide obtained by using the peptide of SEQ ID NOS: 1 to 5 or its conjugate with a carrier as an antigen.
The anti-LCAT antibody described. 11. The anti-LCAT according to claim 9, wherein the peptide of SEQ ID NO: 1 to 5 or a conjugate thereof with a carrier is used as an antigen.
A method for producing an antibody. 12. An LCAT measuring reagent comprising the anti-LCAT antibody or fragment thereof according to any one of claims 1, 2, 3, 9 and 10, or a label thereof. 13. A method for measuring LCAT using the anti-LCAT antibody or a fragment thereof or a label thereof according to any one of claims 1, 2, 3, 9 and 10. 14. An affinity chromatograph using an immobilized anti-LCAT antibody or an immobilized fragment obtained by immobilizing the anti-LCAT antibody or a fragment thereof according to any one of claims 1, 2, 3, 9 and 10 on an insoluble carrier. A method for separating or purifying human-derived LCAT by chromatography.