JPH067193A - Monoclonal antibody - Google Patents

Abstract

PURPOSE:To provide a monoclonal antibody recognizing a specific site of a lipoprotein-associated coagulation inhibitor(LACI) having a specific amino acid sequence. CONSTITUTION:A polypeptide having an amino acid sequence expressed by the formula which is the first Kunitz site from N end of a lipoprotein-associated coagulation inhibitor(LACI) is synthesized and the polypeptide is bound to keyhole limpet hemocyanin(KLH) to prepare a synthetic peptide-hemocyanin complex. The complex is administered to a mouse together with an adjuvant to immunize the mouse and a spleen cell is collected after final immunization and fused with a mouse myeloma cell and the fused cell is cultured in a HAT medium to prepare a hybridoma, and an antibody producing clone is selected and the hybridoma is subjected to monocloning by a limiting dilution analysis. Then, the monocloned hybridoma is cultured to provide the objective monoclonal antibody capable of recognizing and binding the polypeptide of the formula.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a monoclonal antibody that binds to a specific site of a blood coagulation inhibitor. More specifically, tissue factor inhibitor (Tiss
ue Factor Inhibitor: TFI, Lipoprotein Associated Coagul
ation Inhibitor: LACI or Tissue Factor Pathway Inhibitor: TF
PI) to a monoclonal antibody that binds to a specific site.

[0002]

2. Description of the Related Art Extrinsic blood coagulation is initiated by the contact of blood with tissue thromboplastin (hereinafter referred to as tissue factor or TF). Tissue factor (TF) is
Membrane protein, especially in large amounts in the brain and lungs. Contact with TF causes Factor VII or its active form VIIa
The factor forms a complex with TF and then factor X is proteolytically activated to factor Xa.

Previous studies of TF-initiated regulation of extrinsic blood coagulation have revealed that incubation of TF with serum suppresses its in vitro activity. At least one of these factors is defined as tissue factor inhibitor (TFI) or lipoprotein-binding coagulation inhibitor (LACI).

According to Broze et al., Hep G2 cells (human
Has been shown to secrete an inhibitory component with similar properties to TFI present in serum (plasma) (Broze et al; Blood, 69, 150-155 (1
987)). About LACI, Nature, vol, 338,518-
520 (1989) describes the amino acid sequence and secondary structure.

Recent studies suggest that the function and physiological activity of this lipoprotein-binding coagulation inhibitor (hereinafter referred to as LACI) is important in relation to thrombosis and arteriosclerosis. ing.

Therefore, if the action mechanism of LACI can be elucidated and the structure-activity relationship of proteins can be elucidated, it is considered to have a very important meaning in the fields of basic medicine and clinical medicine.

[0007] Further, if the content, distribution and existing form of LACI in human blood can be analyzed, it may be possible to diagnose thrombotic diseases and development of arteriosclerosis.

On the other hand, a monoclonal antibody is specific to a single antigenic determinant and can stably produce an antibody having the same specificity. Therefore, analysis of the function and structure of an antigen protein or immunoassay ( EIA, RIA)
In recent years, it has become widely used. In particular, for functional analysis and molecular analysis of a protein having physiological activity, finding an antibody that recognizes a site involved in the function of the protein or a special structural site can be a powerful means.

[0009] Therefore, the present inventors have paid attention to three structural units of LACI called Kunitz domain, and have studied by using a partially synthetic peptide corresponding to each Kunitz domain as an antigen. A monoclonal antibody was found.

[0010]

DETAILED DESCRIPTION OF THE INVENTION According to the present invention, the amino acid sequence of the first Kunitz site (hereinafter, referred to as K1 site) from the N-terminal of LACI is: Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala Ile Met Lys Arg Phe Phe Phe. LACI, which is a monoclonal antibody that recognizes and binds to a polypeptide of Asn Ile Phe ... [I], is an inhibitor of the blood coagulation system,
It is a monoclonal antibody that can be detected with high sensitivity.

In the present specification, the amino acid sequence is abbreviated by the method adopted by the IUPAC-IUB Biochemistry Committee (CBN), and the following abbreviations are used, for example.

Ala L-alanine Arg L-arginine Asn L-asparagine Asp L-aspartic acid Cys L-cysteine Gln L-glutamine Glu L-glutamic acid Gly glycine His L-histidine Ile L-isoleucine Leu leu leu leucine Leu Leu Met L-methionine Phe L-phenylalanine Pro L-proline Ser L-serine Thr L-threonine Trp L-tryptophan Tyr L-tyrosine Val L-valine

LACI purified using the monoclonal antibody of the present invention has factor Xa inhibitory activity and TF inhibitory activity, as will be shown in the Examples below.

The present invention is also characterized in that a human LACI-containing mixture is brought into contact with an adsorbent in which the above-mentioned monoclonal antibody against human LACI is bound to an insoluble carrier to bind the human LACI to the adsorbent. A method for separating and purifying human LACI having high anticoagulant activity from a mixture containing human LACI.

Furthermore, when the monoclonal antibody of the present invention is used, human blood (plasma) can be obtained as will be shown in Examples below.
The LACI inside can be detected with high sensitivity.

According to the present invention, the monoclonal antibody against LACI is characterized in that, in the immunological assay reagent by the sandwich method, either the antibody bound to the insoluble carrier or the labeled antibody is the above monoclonal antibody. It is an immunoassay reagent for LACI using an antibody.

The hybridoma cell producing the monoclonal antibody of the present invention is the method of Kohler & Milstein [Kohler &Milstein; Nature: 256 , 495-497 (1975)].
Obtained by

That is, after immunizing a mouse with a partial synthetic peptide of LACI represented by the above amino acid sequence [I], the spleen cells of this mouse are fused with mouse myeloma cells, and the obtained hybridoma cells are placed in a microtiter plate. Antibodies that react with the immobilized peptide are systematically tested and selected.

The monoclonal antibody of the present invention is obtained from the product produced by such a novel hybridoma cell, and specifically acts on the K1 site of human LACI.

The monoclonal antibody of the present invention is human LA
Since it has a property of highly specifically binding to the K1 site of CI, it is very advantageous when human LACI is separated and purified while maintaining its anticoagulant activity. That is, one kind of the monoclonal antibody of the present invention is immobilized on an insoluble carrier, and blood or other human LACI-containing mixture,
Alternatively, human LACI is adsorbed and separated from the crude extract, the crude product, and the solution thereof, and the appropriate buffer (eg, 20
mM Tris-HCl, 0.5M NaCl, pH
After washing with 7.4), the solution can be replaced with a solution containing chaotropic ions or the like (for example, 3M NaSCN, pH 7.0) to elute human LACI.

A method for measuring the presence or absence of an antigen or the amount thereof using an antibody that binds to two different positions of an antigen is generally called a sandwich method.

The immunoassay reagent for human LACI of the present invention is an antibody that recognizes different antigenic determinant sites of LACI as two types of monoclonal antibodies to be used, and particularly recognizes any of the three Kunitz sites of LACI. Antibodies to be used. At that time, the antibody of the present invention is used for either one. According to the present invention, it is possible to measure human LACI in a solution state (for example, in blood) constantly and accurately with no difference in quality of reagents. Further, since the LACI is directly detected and measured, the measurement can be performed accurately and in a short time without being affected by other contaminants.

Therefore, according to the present invention, there have been provided reagents and kits capable of accurately and rapidly measuring human LACI, which have not been reported in the past.

Next, a specific method for producing the monoclonal antibody of the present invention will be described in detail.

A. Antigen The antigen used in the present invention is represented by the amino acid sequence represented by the following formula [I] Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala Ile Met Lys Arg Phe Phe Phe Asn Ile Phe ... [I]. In particular, the peptide is labeled with KLH (Keyhole Limpet Hemocyanin
Those bound to a carrier protein such as) are preferred.

B. Immunization of mice with the above antigens Female Balb / c mice can be used, but other strains (st
rain) mouse may be used. At that time, the immune plan,
And the concentration of the antigen should be chosen such that lymphocytes are formed which have received a sufficient amount of antigen stimulation. For example, mice are immunized with 50 μg of antigen 3 times in the abdominal cavity at intervals of 2 weeks,
A further 30 μg is administered intravenously. Spleen cells are removed for fusion several days after the final immunization.

C. Cell fusion Aseptically remove the spleen of the mouse immunized as described above,
From there, a single cell suspension is prepared. The spleen cells are fused with mouse myeloma cells from an appropriate line by using an appropriate fusion promoter. The preferred ratio of spleen cells to myeloma cells is in the range of about 20: 1 to about 2: 1. The use of 0.5-1.5 ml of fusion medium for about 10 ⁸ spleen cells is suitable.

Mouse myeloma cells used for cell fusion are well known, but in the present invention, P3-X63-Ag8-U is used.
1 cell (P3-U1) [Yelton, DF et al, Current.
Topics in Microbiology and Immunology, 81, 1 (1978)
Reference] is preferred.

As the preferred fusion promoter, for example, polyethylene glycol having an average molecular weight of 1000 to 4000 can be advantageously used, but other fusion promoters known in the art can also be used.

D. Selection of Fused Cells In a separate container (eg, a microtiter plate), a mixture of unfused spleen cells, unfused mouse myeloma cells and fused hybridoma cells in a selective medium that does not support unfused mouse myeloma cells. Dilute and incubate for a sufficient time (about 1 week) to kill unfused cells. The medium is drug resistant (eg 8-azaguanine resistant) and does not support unfused mouse myeloma cells (eg HA
T medium) is used. Unfused myeloma cells die in this selective medium. Since these unfused spleen cells are non-neoplastic cells, they die after a certain period of time (1 week). The cells fused to these have the tumorigenicity of the parental cells of myeloma and the properties of the parental splenocytes, and thus can survive in the selective medium.

E. Confirmation of resistance in each container After the hybridoma cells were thus detected, the culture supernatant was collected, and an enzyme-linked immunosorbent assay (Enzyme Linked Immuno-Sor) was performed on the antibody against the synthetic peptide of the formula [I].
Bent Assay) for screening.

F. Hybridoma that produces the desired antibody
Cloning of cells When a hybridoma cell producing an antibody of interest is cloned by an appropriate method (eg, limiting dilution method), the antibody is produced by two different methods. According to the first method, by culturing the hybridoma cells in an appropriate medium for a certain period of time, the monoclonal antibody produced by the hybridoma cells can be obtained from the culture supernatant.
According to the second method, the hybridoma cells can be injected into the abdominal cavity of a mouse having an isogenic or semiisogenic gene. The monoclonal antibody produced by the hybridoma cells can be obtained from the blood and ascites of the host animal after a certain period of time.

G. Human L from a mixture containing human LACI
Separation / Purification of ACI The monoclonal antibody against human LACI of the present invention is immobilized or bound to an insoluble carrier to obtain an adsorbent. The insoluble carrier used at that time may be any insoluble carrier generally used as a base material of a measurement reagent or a measurement kit using a monoclonal antibody. For example, sepharose, polyacrylamide, cellulose, dextran, maleic acid polymer or a mixture thereof is preferably used as the material. The form of these insoluble carriers can be various forms such as powder, granules, pellets, beads, and fibers. Also generally plasma,
Alternatively, it is advantageous to use a plate (well) having a large number of concave depressions used for measuring and separating the fractional components.

When the adsorbent is used and the mixture containing human LACI is brought into contact with the adsorbent, the monoclonal antibody fixed to the adsorbent binds to human LACI, and as a result, human LACI binds to the adsorbent. By doing so, it is possible to separate or remove human LACI.

Further, human LACI was bound to the adsorbent as in the previous period, and if possible, the remaining mixture was washed and removed,
Then, when human LACI bound to the adsorbent is contacted with or washed with an appropriate solution containing chaotropic ions (SCN ⁻ ), human LACI is released from the adsorbent, and human HACI having anticoagulant activity is isolated. LACI
Can be isolated.

H. Method for Measuring Human LACI Content The monoclonal antibody (first antibody) against human LACI of the present invention is immobilized on an appropriate insoluble carrier (for example, a plastic container) (hereinafter referred to as "immobilized antibody"). The surface of the insoluble carrier is then coated with a suitable substance (eg bovine serum albumin) to avoid non-specific binding between the insoluble carrier and the reagent or analyte sample to be measured.

The thus obtained insoluble carrier on which the first antibody has been immobilized is brought into contact with a sample for a fixed time and at a constant temperature to react.

During this period, the immobilized antibody and LAC in the specimen sample
I bind. Then, after washing with an appropriate washing solution, a solution of an antibody against LACI (second antibody) labeled with an appropriate labeling substance (eg enzyme) is contacted with LACI bound to the immobilized antibody of the insoluble carrier for a certain time and at a certain temperature. Second
React with antibody.

After washing this with an appropriate washing solution, the amount of the labeling substance labeled on the second antibody present on the insoluble carrier is measured (for example, if the labeling substance is an enzyme, a chromogenic synthetic substrate is used. Measure the absorbance). Thus, the LACI content in the sample can be calculated from the value.

Thus, using the monoclonal antibody of the present invention, human LA from a mixture containing human LACI
The removal of CI, the separation and purification of human LACI from the mixture, the measurement of the content of human LACI in the mixture, etc. are achieved by extremely simple operations.

Furthermore, the monoclonal antibody of the present invention is
Since it is a highly specific antibody that recognizes the first Kunitz site from the N-terminus of LACI, it can be used for immunological staining and imaging of tissues. For example, it is considered that the method can be applied to the accumulation of LACI in arteriosclerotic lesions and the diagnosis and identification of arteriosclerotic lesion sites in a living body.

Hereinafter, the present invention will be described in more detail with reference to Examples.

[0043]

Example 1 Preparation of synthetic peptide-hemocyanin (KLH) complex 0.3 mg of KLH (keyhole limpet hemocyanin) and synthetic peptide [amino acid sequence; Ala Phe Lys A]
la Asp Asp Gly Pro Cys Ly
s Ala Ile Met Lys Arg Phe
1 mg of Phe Phe Asn Ile Phe] was dissolved in 0.5 ml of distilled pure water. Add EDCI to this solution
(1-ethyl-3- (3-dimethylaminopropyl) carbod
30 mg of iimide-HCl) was added, and the mixture was allowed to react overnight at room temperature while protected from light. The reaction solution was thoroughly dialyzed against 2 liters of distilled water. Thus, a synthetic peptide-bonded KLH solution (about 0.
6 mg / ml concentration) was obtained. The solution was used as an antigen for immunization.

[0044]

Example 2 Immunization of Mouse Synthetic peptide-bonded KLH solution obtained in Example 1 (0.
(6 mg / ml) 0.5 ml and Complete Freund's Adjuvant (0.5 ml) were mixed using two syringes, and the mixture was intraperitoneally administered to two mice by 0.5 ml each. did.

Two weeks later, 0.5 ml of the above mixture was similarly administered to the abdominal cavity of the mouse. Then, 4 weeks later, 0.5 ml of a synthetic peptide-bound KLH solution (0.6 mg / ml) and Incomplete Freund's Ad
0.5 ml of juvant) was mixed, and 0.5 ml of this mixture was administered to the abdominal cavity of the mouse. Four weeks later, 50 μg / 100 μl of the synthetic peptide-bonded KLH solution was intravenously administered to the above two mice, and three days after that, the spleen was excised and cell fusion was performed as shown in Example 3 below.

[0046]

Example 3 Cell fusion and production of desired monoclonal antibody
Selection and acquisition of hybridoma cells Spleen cells of the excised mouse and myeloma cells (P3U1) of the syngeneic mouse were mixed at a ratio of about 6: 1, and cell fusion was performed using 50% polyethylene glycol 1540 as a fusion promoter. . The cells after fusion were RPMI 1 containing 10% bovine serum so that the cell concentration was 1 × 10 ⁶ cells / ml.
The cells were suspended in 640 medium and 100 μl was dispensed into each well of a 96-well microplate.

The hybridoma (fused cell) was cultured in a CO ₂ incubator (5% CO ₂ , 37 ° C.), and the medium was replaced with a medium containing hypoxanthine, aminopterin and thymidine (HAT medium), and the medium was transformed into HAT medium. Were screened for hybridomas consisting of spleen cells and myeloma cells.

The antibody in the culture supernatant of the hybridoma was detected by the ELISA method using a microtiter plate on which the above synthetic peptide was adsorbed. Cloning by the limiting dilution method was repeated twice for 8 wells among the antibody-producing positive wells, which had high hybridoma growth ability and strong binding to the synthetic peptide. EL
By the ISA method, four monoclones with high antibody-producing ability and strong antigen-binding ability were selected. 10 clones were obtained.
Suspended in 90% bovine serum solution containing% DMSO and stored in liquid nitrogen. The monoclonal antibody produced by each clone was purified by growing the clone in the abdominal cavity of Balb / c mice, and purifying it from the ascites using a protein A-Sepharose 4B column.

[0049]

Example 4 Determination of Class of Purified Monoclonal Antibody Subclasses were selected for IgG of each clone purified from mouse ascites by the Ouchterlony method.

[0050]

[Table 1]

[0051] The 1G1 system and the 2F2 system recognized different epitopes. Two
Purification of LACI with F2D9 and 2F2E2 yielded both active LACI.

[0052]

Example 5 Monochrome from human hepatocyte (Hep G2) serum-free culture supernatant
Purification of LACI by internal antibody column Monoclonal antibody 2F2D9 [Kd = described in Example 4
5 mg of 1 × 10 ⁻⁹ M] was coupled to bromocyan (CNBr) activated sepharose (Pharmacia Co., Ltd.) to prepare 2.5 ml of antibody sepharose column. Subsequently, serum-free culture supernatant of human hepatocytes (Hep G2) (ITES
-ERDF medium) (1 liter) was passed through the column at a flow rate of 40 ml / hour for contact. 0.5M NaCl,
200 ml of 20 mM Tris-HCl (pH 7.4) containing 0.05% Tween 20 and 10 U / ml aprotinin
After washing the column with 3M NaSCN (pH 7.0) 1
The protein adsorbed on the column was eluted with 5 ml. The total protein amount of the eluted fraction was about 50 μg.

[0053]

Example 6 LACI X purified by monoclonal antibody column
Effect on factor a activity 10 μl LACI purified by a monoclonal antibody column and prepared at various concentrations and factor Xa (bovine, 0.
15 μg / ml) 10 μl, and 50 mM Tris-H
Cl pH 7.8 (0.15M NaCl, 5 mg / ml B
SA (containing 5 mM CaCl ₂ ) 10 μl, and
The reaction was carried out at 37 ° C for 10 minutes. This was followed by 160 μl / well 50 mM Tris-HCl pH beforehand.
20 μl of the reaction solution was added to a 96-well microplate containing 7.8 (containing 0.15 M NaCl and 5 mg / ml BSA). Finally 2.5 mM Spectrozyme X
a (synthetic substrate MeO-CO-D-CHG-Gly-Ar
10 μl of g-pNA) was added to the well and ELISA A
Absorbance A ₄₀₅ nm was measured by NALYZER (Toyo Sokki Co., Ltd., ETY-96). The results are summarized in Fig. 1.

As a result, it was found that all the purified LACIs inhibited the activity of factor Xa in a concentration-dependent manner.

[0055]

Example 7 LACI T Purified by Monoclonal Antibody Column
Effect on F activity Rabbit brain thromboplastin (40 mg acetone powder, Sigma, 2 ml) 0.125 M containing 1 mg / ml BSA
A TF solution was prepared by diluting 1/20 with NaCl solution. 200 μl of TF solution and 20 μl of 25 mM CaCl ₂ solution were mixed and reacted at 37 ° C. for 2 minutes. 200 μl of this was added to 120 μl of human plasma supplemented with 20 μl of 20 μg / ml concentration of LACI solution, and prothrombin time (PT) was measured by a blood coagulation time measuring device (Sysmex
Company, CA-100). The results are summarized in Fig. 2.

As a result, LACI inhibits TF activity,
It was found to prolong blood coagulation time (PT).

From the results of the examinations of Examples 6 and 7 above, LACI purified by a monoclonal antibody was used.
Was confirmed to have factor Xa inhibitory activity and TF inhibitory activity.

[0058]

Example 8 LA in human blood by anti-LACI monoclonal antibody
Detection of CI Prior application P25614-0 by the present inventor (same as the applicant)
2A1H8 [Kd = 0.9 × 10 ⁻ , which is one of the monoclonal antibodies described in 1 (October 31, 1991 application), that is, one of the monoclonal antibodies that recognizes and binds to the third Kunitz site from the N-terminus of LACI. 20 μg of ⁹ M]
After adsorbing to the microtiter plate (96 well plate) at a concentration of 1 / ml, 1% BSA and 0.15M NaCl
The solid phase was blocked with 20 mM Tris-HCl (pH 7.4) containing

Cleaning solution (0.05% Tween 20, 0.
20 mM containing 5% BSA and 0.15 M NaCl
The wells were washed twice with Tris-HCl (pH 7.4).

Next, human plasma (derived from a healthy person) was diluted 5,50,200 times with a washing solution, and each was added to a well, and the monoclonal antibody adsorbed on the plate solid phase and 3
The reaction was carried out at 7 ° C for 2 hours. After washing 3 times with a washing solution, a 2F2E2 [Kd = 1 × 10 ⁻⁹ M] solution (400 ng / ml), which is one of the monoclonal antibodies of the present invention and labeled with peroxidase enzyme, was added and reacted at 37 ° C. for 2 hours. Let After washing three times with a washing solution, a substrate solution (ABTS) was added, and ELISA ANALYZER (Toyo Sokki Co., Ltd.,
ETY-96) absorbance at a wavelength of 415 nm A ₄₁₅
Was measured. LAC in human blood at each dilution ratio
The rate of detection of I is shown in FIG.

Using the monoclonal antibody of the present invention,
It is possible to detect LACI in human blood with high sensitivity.

[0062]

Comparative Example Monoclonal antibody 2A shown in Example 8
Similarly diluted human blood was made to react with the well in which 1H8 was immobilized. After washing, peroxidase enzyme-labeled monoclonal antibody (one type of antibody that recognizes and binds to the second Kunitz site [K2] from the N-terminal of LACI)
2G9F1 [Kd = 1 × 10 ⁻⁹ M] solution (400 ng / m
l) was added and the mixture was reacted at 37 ° C. for 2 hours. A washing solution and a substrate solution (ABTS) were added, and the absorbance was measured. FIG. 4 shows the proportion of LACI detected in human blood at each dilution ratio.
It was shown to. As a result, it was revealed that the detection sensitivity of LACI was low and the quantification was inferior as compared with Example 8.

For example, LAC in human blood 1/5 diluted solution
The ratio of detection of I (absorbance A ₄₁₅ nm) is 0.695 in FIG. 3, whereas it is 0.3 in FIG. 4 (comparative example).
It was 16, and the ratio was less than half.

Therefore, as the combination of antibodies used in the LACI measuring system, it is necessary to use the monoclonal antibody recognizing K1 of the present invention as the immobilized antibody or the enzyme-labeled antibody in view of the characteristics of the measuring system. it is conceivable that.

Monoclonal antibody 2 recognizing K2
G1F1 was obtained using a synthetic polypeptide having the following amino acid sequence Phe Leu Glu Glu Asp Pro Gly Ile Cys Arg Gly Tyr Ile Thr Arg Tyr Phe Tyr Asn Asn Gln, and Examples 1 to 4 were used. The 2G9F1 H chain was γ _2b and the L chain was κ.

[0066]

[Sequence list]

[0067] SEQ ID NO: 1 Length of sequence: 21 SEQ type: amino acid Topology: linear sequence type: peptide fragment type: intermediate portion fragment sequence: Ala Phe Lys Ala Asp Asp Gly Pro Cys 1 5 Lys Ala Ile Met Lys Arg Phe Phe Phe ^{10 15} Asn Ile Phe ²⁰

[0068] SEQ ID NO: 2 Length of sequence: 21 SEQ type: amino acid Topology: linear sequence type: peptide fragment type: intermediate portion fragment sequence: Phe Leu Glu Glu Asp Pro Gly Ile Cys 1 5 Arg Gly Tyr Ile Thr Arg Tyr Phe Tyr ^{10 15} Asn Asn Gln ²⁰

[Brief description of drawings]

FIG. 1 L purified by a monoclonal antibody column
It shows the effect of ACI on Factor Xa activity.

FIG. 2 L purified by a monoclonal antibody column
It shows the effect of ACI on TF activity.

FIG. 3: LAC in human blood by monoclonal antibody
It is shown with respect to the detection of I.

FIG. 4 shows detection of LACI in human blood in a comparative example.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication C07K 17/02 8517-4H G01N 33/53 D 8310-2J 33/537 8310-2J 33/577 B 9015-2J // C07K 7/10 ZNA 7537-4H C12N 15/06 (C12P 21/08 C12R 1:91) C07K 99:00

Claims (5)

[Claims] 1. A monoclonal antibody which recognizes and binds to a polypeptide of amino acid sequence Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala Ile Met Lys Arg Phe Phe Phe Asn Ile Phe. 2. A Lipoprotein Associated Coagulation Inhibitor:
A monoclonal antibody that recognizes and binds to the first Kunitz site from the N-terminus of LACI). 3. The monoclonal antibody according to claim 1, which recognizes and binds a protein substance having a tissue factor inhibitory activity and a blood coagulation factor Xa inhibitory activity. 4. A human LACI-containing mixture is brought into contact with an adsorbent in which the monoclonal antibody according to any one of the above 1, 2, and 3 is bound to an insoluble carrier, and the adsorbent is adsorbed with human LCI.
A method for separating human LACI from a mixture containing human LACI, which comprises binding ACI. 5. In the immunoassay reagent by the sandwich method, the antibody bound to the insoluble carrier and the labeled antibody are:
Immunization of LACI using a monoclonal antibody against LACI, characterized in that it is a monoclonal antibody that recognizes and binds to different Kunitz sites of LACI, and one of them is the monoclonal antibody of any of the above items 1, 2 and 3. Measurement reagents.