JPH06153981A - Immunological assay of laci, kit therefor and monoclonal antibody - Google Patents

Abstract

PURPOSE:To selectively determine free LACI in high sensitivity by immunologically determining LACI with two antibodies recognizing respective specific sites of LACI. CONSTITUTION:Free LACI in a human examination specimen is immunologically determined by a monoclonal antibody recognizing the polypeptide (K3) having the amino acid sequence of formula I and a monoclonal antibody recognizing the polypeptide (K1) having the amino acid sequence of formula II. One of the antibodies is immobilized on an insoluble carrier and the other antibody is labeled and used for the determination.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a free lipoprotein-binding coagulation system inhibitor ( L ipopro) in human test samples.
tein about A ssociated C oagulation I nhibitor) immunological assay of. More specifically, it relates to a method for highly sensitively measuring a free lipoprotein-binding coagulation inhibitor in a human test sample using two kinds of monoclonal antibodies, and a kit therefor.

The term "LACI" as used herein is intended to mean the lipoprotein-binding coagulation inhibitor.

LACI is an extrinsic coagulation inhibitor (Ex
trinsic Pathway Inhibitor (EPI) or Tissue Factor Pathway Inhibito
r: TFPI).

[0004]

Extrinsic blood coagulation is initiated by the contact of blood with tissue thromboplastin (hereinafter referred to as tissue factor (TF)). Tissue factor (TF) is a membrane protein with a molecular weight of 58,000, which is abundant especially in the brain and placenta. Upon contact with TF, factor VII or its active form factor VIIa forms a complex with TF, and then factor X is proteolytically activated to factor Xa, activating the coagulation system.

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).

The tissue factor inhibitory activity by LACI is T
It refers to the function of inhibiting the initiation of blood coagulation by the F-VIIa complex.

Broze et al. Have shown that Hep G2 cells (human hepatoma cell line) secrete an inhibitory component with properties similar to TFI present in serum (plasma) (Broze et al. Blood, 69, 15
0-155 (1987)). Also for LACI, Broze
et al by Nature, vol.338, 518-520 (1989) 1
The 76 amino acid sequence and its secondary structure are described.

According to the above-mentioned Nature, LACI has three Kunitz domains,
The first Kunitz domain from the N-terminus is TF-
It has been reported that it is a site that binds to VIIa, and that the second Kunitz domain is a site that binds to factor Xa, but the third Kunitz domain from the N-terminal has no function. It has been reported that not. And this Nature does not describe anything about the measurement and significance of measurement of free LACI in blood.

[0009]

Therefore, the present inventors have found that
Advances research on free LACI in blood and its measurement
I have A part of LACI in blood is LDL (Low Density).
ity Lipoprotein) and other lipoproteins
But some of it is present as free LACI,
The free LACI is deeply involved in the suppression of thrombus formation
It is considered to be one. Especially in the blood vessels such as pulmonary thrombus
In thrombotic disorders where thrombus is formed, α ₂PI
(Α₂-Plasmin Inhibitor) or fibrinogen
Are used as indicator substances to monitor the condition of the pathology.
It has been known for a long time that these substances are not suitable for
Accordingly, it has recently been reported that it does not change. Therefore above
Changes in the pathophysiology of thrombotic diseases such as
It was requested to find a new indicator that

On the other hand, the present inventors focused on the 3rd Kunitz domain from the N-terminal of LACI, synthesized a part of the polypeptide, and prepared a monoclonal antibody that specifically recognizes the polypeptide to produce LDL. (Human L
Further studies were conducted on the inhibition of the binding between DL) and LACI.

As a result, it was found that the third Kunitz domain of LACI may be one binding site of LDL (Low Density Lipoprotein). By utilizing the property of the above monoclonal antibody to inhibit the binding of LACI to LDL, this monoclonal antibody is used to selectively and highly sensitively detect free LACI in blood (LACI not bound to LDL). We were able to create a system that can measure It has been found that this measurement system is useful for diagnosing thrombotic diseases in which micro thrombus formation occurs in the organism.

[0012]

The present invention has been achieved on the basis of the above-mentioned findings, and provides a free lipoprotein-binding coagulation system inhibitor LACI in human test samples.
A method for immunologically measuring using a first antibody immobilized on an insoluble carrier and a labeled second antibody, comprising:
(I) Either the first antibody or the second antibody has the following amino acid sequence: Polypeptide recognizing the following amino acid sequence: Leu Thr Pro Ala Asp Arg Gly Leu Cys Arg Ala Asn Glu Asn Arg Phe Tyr Asn Ser K3. (Ii) the other antibody has the following amino acid sequence: A method for immunologically measuring free LACI, which is a monoclonal antibody recognizing K1) (hereinafter referred to as "K1-antibody").

Further, according to the present invention, there is provided a kit for immunologically measuring free LACI in a human test sample, which comprises (1) a first antibody immobilized on an insoluble carrier (2) a label A second antibody (3) a lysing agent (4) a detergent and (5) when the labeling substance is an enzyme, a combination of a substrate and a reaction terminator for measuring the enzyme activity, and (I) one of the first antibody and the second antibody is a K3-antibody,
i) An immunoassay kit for free LACI is provided, characterized in that the other antibody is a K1-antibody.

The present invention will be described in more detail below. In this specification, the amino acid sequence is IUPAC-I.
It is abbreviated by the method adopted by the UB 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 leucine Leu Leu Met L-methionine Phe L-phenylalanine Pro L-proline Ser L-serine Thr L-threonine Trp L-triprofan Tyr L-tyrosine Val L-valine

[I] Monoclonal antibody and preparation thereof: The present inventors synthesized polypeptides having the following three amino acid sequences and used them as antigens to prepare monoclonal antibodies. The specific method will be described below.

A. Antigens As the antigens, synthetic polypeptides (K ₁ , K ₂ and K ₃ ) having the following three amino acid sequences were used. Polypeptide _{K 1 Ala Phe Lys Ala Asp Asp} Gly Pro Cys Lys Ala Ile Met Lys Arg Phe Phe Phe Asn Ile Phe polypeptide _{K 2 Phe Leu Glu Glu Asp Pro} Gly Ile Cys Arg Gly Tyr Ile Thr Arg Tyr Phe Tyr Asn Asn Gln polypeptide _{K 3 Leu Thr Pro Ala Asp Arg} Gly Leu Cys Arg Ala Asn Glu Asn Arg Phe Tyr Tyr Asn Ser Val these polypeptides, carrier protein, for example KLH
(Keyhole Limpet Hemocyanin) or the like is preferably used as an antigen.

B. Immunization of mice with the above antigens Female Balb / c mice can be used, but mice of other strains may also be used. In doing so, the immunization regimen and antigen concentration should be chosen such that a sufficient amount of primed lymphocytes are formed. For example, a mouse is immunized with 50 μg of the antigen 3 times in the abdominal cavity at intervals of 2 weeks, and then 30 μg is intravenously administered. 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 (197
8) Reference] is preferable.

As a 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 cells have the tumorigenic properties of the parental cells of myeloma and the properties of the parental splenocytes, and thus can survive in the selective medium.

E. Confirmation of Antibodies in Each Container Thus, after the hybridoma cells were detected, the culture supernatant was collected, and the antibody against the synthetic peptide of the above formula [I] was assayed by the enzyme immunoassay (Enzyme Linked Immuno-Solution).
rbent Assay). Positive ones are tested for binding to LACI.

F. Hybridoma that produces the desired antibody
Cloning of cells After the hybridoma cells producing the antibody of interest have been cloned by a suitable method (eg limiting dilution), the antibody can be produced in two different ways. According to the first method, the hybridoma cells are cultured for a certain period of time in an appropriate medium, whereby 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.

[II] Immunological measurement A. Monoclonal Antibody In the measurement of free LACI in the human test sample of the present invention, a monoclonal antibody (K3-antibody) having a specific recognition site for the amino acid sequence (K3) and a specific recognition for the amino acid sequence (K1) are used. Monoclonal antibodies having sites (K1-antibody) are used in combination.

The monoclonal antibody (K2-antibody) having a specific recognition site for the amino acid sequence (K2) is K3.
-Either in combination with the antibody or K1-in combination with the antibody,
It is difficult to measure free LACI with high sensitivity.

The K3-antibody and K1-antibody used may be either whole antibodies, Fab or F
It may be any of the fragments such as (ab ′) ₂ .

Further, the first antibody immobilized on the insoluble carrier may be either K3-antibody or K1-antibody, but the first antibody is K3-antibody and the labeled antibody is K1.
The combination that is an antibody gives more favorable results.

B. Human test sample As the sample to be measured for free LACI in the present invention, various human body fluids that need to be tested for the presence of free LACI are used. Generally, it is serum, plasma, urine or the equivalent thereof, and plasma is particularly preferably used.

C. Measuring means The free LACI in the present invention can be measured by an immunological measuring method known per se (for example, a sandwich method) as long as the K3-antibody and the K1-antibody are used. The method may be a one-step method or a two-step method.

That is, either the K3-antibody or the K1-antibody is immobilized on a suitable insoluble carrier. 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 test sample to be measured. The thus obtained insoluble carrier on which the first antibody is immobilized is contact-reacted with a human test sample for a certain time and a certain temperature. Thus, free LACI binds to the first antibody. Then, after washing with a suitable washing agent, a solution of the second antibody labeled with a suitable labeling substance (eg, enzyme) is contacted with free LACI bound to the first antibody in the insoluble carrier for a certain time and a certain temperature. React. This is washed with a suitable detergent, and then the amount of the labeled substance of the second antibody (labeled antibody) bound on the insoluble carrier is measured.

The two-step method described above can also be carried out by a one-step method. That is, the first antibody immobilized on an insoluble carrier, the second antibody labeled and the human test sample are mixed at the same time, these are reacted at a constant temperature for a fixed time, and the amount of the labeling substance is measured. Can be done.

[III] Immunological Assay Kit The kit for assaying free LACI of the present invention comprises:
(I) A reagent in a usual sandwich method except that either one of the first antibody and the second antibody is a K3-antibody, and (ii) the other antibody is a K1-antibody. A kit is constructed based on the combination of.

That is, the immunological assay kit of the present invention comprises (1) a first antibody immobilized on an insoluble carrier, (2)
When the labeled second antibody, (3) lysing agent, (4) detergent and (5) labeling substance are enzymes, they contain a substrate for measuring enzyme activity and a reaction terminator.

Examples of the insoluble carrier include polymers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, cross-linked dextran and polysaccharides, other paper, glass, metal, agarose and combinations thereof. be able to.

The shape of the insoluble carrier may be various shapes such as tray shape, spherical shape, fibrous shape, rod shape, plate shape, container shape, cell and test tube.

As the labeling substance of the labeled antibody, it is advantageous to use an enzyme, a fluorescent substance, a luminescent substance, a radioactive substance or the like. Enzymes include peroxidase, alkaline phosphatase, β-D-galactosidase,
Fluorescein isothiocyanate and phycobiliprotein as fluorescent substances, isorucinol and lucigenin as luminescent substances, and radioactive substances as radioactive substances.
^{Although 125} I, ¹³¹ I, ¹⁴ C, ³ H and the like can be used, these are not limited to the exemplified ones, and other ones can be used as long as they can be used in the immunological assay method.

When the labeling substance is an enzyme, a substrate and, if necessary, a color former are used to measure its activity.

When peroxidase is used as the enzyme, H ₂ O ₂ is used as the substrate and _{2, 2} as the color former.
2'-azinodi- [3-ethylbenzthiazolinesulfonic acid] ammonium salt (ABTS), 5-aminosalicylic acid, o-phenylenediamine, 4-aminoantibilin, 3,3 ', 5,5'-tetramethylbenzidine, etc. ,
When alkaline phosphatase is used as the enzyme, o-nitrophenyl phosphate is used as the substrate and β-
When D-galactosidase is used, fluorescein-di- (β-D-galactopyranoside), 4-
Methylumbelliferyl-β-D-galactopyranoside and the like can be used.

In the immunoassay kit (3)
The lysing agent may be one that is usually used for immunological measurement, for example, phosphate buffer, Tris-HCl buffer,
A preferable example is one having an acetic acid buffer solution or the like and having a pH in the range of 6.0 to 8.0. Furthermore, as the detergent (4), those generally used for immunological measurement are used as they are. Examples thereof include physiological saline, phosphate buffer, Tris-HCl buffer, and mixed solutions thereof. These detergents may be further added with a nonionic surfactant such as Triton X100, Tween 20 or Brig35, and an ionic surfactant such as sodium dodecyl sulfate.

[0041]

The present invention will be described in more detail with reference to the following examples. In the examples, "%" is based on g / ml (for example, 0.01 g / ml corresponds to 1%).

[0042]

Example 1 Synthesis of synthetic polypeptide-hemocyanin (KLH) complex
0.3 mg of adult KLH (keyhole limpet hemocyanin) and 1 mg of each of the synthetic polypeptides (K3), (K1) or (K2) were dissolved in 0.5 ml of distilled pure water. EDCI (1-ethyl-3- (3-dimethylamin
30 mg of opropyl) carbodiimide-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, each synthetic peptide-bound KLH solution (concentration of about 0.6 mg / ml) was obtained. The solution was used as an antigen for immunization.

[0043]

Example 2 Immunization of Mouse Each antigen was immunized according to the following procedure.

The synthetic polypeptide-bound KLH solution (0.6 mg / ml) obtained in Example 1 (0.5 ml) and Complete Freund's Adjuvant.
Mix using 2 syringes connected with 5 ml, and the mixture is 0.5 ml in the abdominal cavity of 2 mice (female, Balb / c).
Each was administered. Two weeks later, 0.5 ml each of the above mixed solution 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 adjuvant (Incomplete Freund's
0.5 ml of Adjuvant) was mixed, and 0.5 ml of this mixture was administered intraperitoneally to the mouse. Four weeks later, 50 μg / 100 μl of a synthetic peptide-bonded KLH solution was intravenously administered to the above two mice, and three days later, the spleen was removed,
Hereinafter, cell fusion was performed as shown in Example 3.

[0045]

Example 3 Cell fusion and production of desired monoclonal antibody
Selection and Acquisition of Hybridoma Cells that Exclude Spleen cells of the excised mouse and myeloma cells (P3U1) of syngeneic mouse are mixed at a ratio of about 6: 1, and cell fusion is performed using 50% polyethylene glycol 1540 as a fusion promoter. It was 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.

Hybridomas (fused cells) were 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 cells were placed in HAT medium. Were screened for hybridomas consisting of spleen cells and myeloma cells. Then, it was conditioned in HT medium, and further conditioned with 10% FCS-RPMI 1640 medium.

The antibody in the culture supernatant of the hybridoma was detected by the ELISA method using a microtiter plate on which the above-mentioned synthetic peptide was adsorbed. The positive wells were further examined for binding to crudely purified LACI. From the antibody production positive wells, 8 wells having strong hybridoma growth ability, strong binding to the synthetic peptide and crude purified LACI were selected (first screening). Cloning by the limiting dilution method was repeated twice for the above wells. Clones having binding ability to the peptide and the crudely purified LACI were selected by the ELISA method (second screening).

Regarding the selected clones, the number of cells in the well and the mouse monoclonal antibody concentration were determined as E.
The levels of produced antibody concentration and the amount of antibody bound to the antigen (absorbance) measured by LISA were measured, and those showing high values for and were selected (third screening). After cell fusion and until the end of the third screening, K3 antibody took more than 1 month and other antibodies took less than 2 months. During this period, each hybridoma stably produced the antibody. From the high-performance clones subjected to the third screening, four representative monoclones having high antibody-producing ability and strong binding to synthetic peptides and crude purified LACI were selected. The resulting clone is 10% D
Suspended in 90% bovine serum solution containing MSO and stored in liquid nitrogen. The monoclonal antibody produced by each clone was used to grow the clone in the abdominal cavity of Balb / c mice,
The ascites was purified using a Protein A-Sepharose 4B column.

[0049]

[Table 1]

[0050]

Example 4 (1) Class of purified monoclonal antibody (K3-antibody)
Of Examples 1 to 3 above using a synthetic polypeptide (K3)
I of each clone purified from mouse ascites obtained by
A subclass was selected by the Ouchterlony method for gG. The results are shown in Table 2 below.

[0051]

[Table 2]

2A1C5 and 2A1H8 recognized different epitopes, and 3B1E7 and 3B1G3 competitively inhibited. The 2A1 system and the 3B1 system recognized different epitopes.

[0053]

Example 5 Determination of Class of Purified Monoclonal Antibody (K1-Antibody) Examples 1 to 3 above using synthetic polypeptide (K1)
Ig of each clone purified from mouse ascites fluid obtained from
A subclass of G was selected by the Ouchterlony method. The results are shown in Table 3 below.

[0054]

[Table 3]

The 1G1 system and the 2F2 system recognized different epitopes.

[0056]

Example 6 Determination of Class of Purified Monoclonal Antibody (K2-Antibody) Examples 1 to 3 above using synthetic polypeptide (K2)
Ig of each clone purified from mouse ascites fluid obtained from
A subclass of G was selected by the Ouchterlony method. The results are shown in Table 4 below.

[0057]

[Table 4]

2G9E9, 2G9F1 and 2G9G8
Recognized the same epitope. The 2G9 system and 2C1B1 recognized different epitopes.

[0059]

Example 7 Free LACI in human blood by monoclonal antibody
20 μg of 2A1C5 [Kd = 0.9 × 10 ⁻⁹ M], which is a kind of monoclonal antibody (K3-antibody) that recognizes and binds to the third Kunitz site from the N-terminal of the detected LACI
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

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

To prepare a calibration curve, LACI was set to 5,
Dilute with washing solution to a concentration of 10, 15, 25 ng / ml,
Calibration curve solutions were prepared and added to each well. Separately, using human plasma (healthy person or patient) with washing solution 1
Sample solutions were prepared by diluting to / 5 and each was added to another well. The calibration curve solution and the sample solution were reacted with the monoclonal antibody adsorbed on the plate solid phase at 37 ° C. for 2 hours. After washing, peroxidase enzyme-labeled 2F2D9 [Kd = 1 × 10 ⁻⁹ M] solution (400 ng) was added to the wells.
/ Ml) was added and reacted at 37 ° C. for 2 hours. Then, after washing three times with the washing solution, the substrate solution (ABTS) was added to
SA ANALYZER (Toyo Sokki Co., Ltd., ETY-9
In 6), the absorbance A ₄₁₅ at a wavelength of 415 nm was measured.

The calibration curve obtained is shown as a line in FIG.
The results for the sample liquid are shown in Table 5 below. Healthy person (N
= 5), LACI is present in blood at a concentration of 104 ng / ml, but in thrombotic diseases, it is about 1/3 to 1 in comparison with healthy subjects.
It was found that the blood level was reduced to about / 2.
Therefore, the detection of LACI concentration in blood is considered to be useful for diagnosing the presence or absence of the formation of minute thrombus in the living body.

[0063]

[Table 5]

[0064]

[Example 8] Monoclonal antibody 2 shown in Example 7
The well to which A1C5 was adsorbed was reacted with the LACI solution diluted in the same manner as in Example 7. After washing, peroxidase enzyme-labeled monoclonal antibody (LACI N
One type of antibody that recognizes and binds to K2 at the second Kunitz site from the end) 2G9F1 (FRI entry number FERMB
P-4027) [Kd = 1 × 10 ⁻⁹ M] solution (400 ng
/ Ml) was added and reacted at 37 ° C. for 2 hours. After washing, a substrate solution (ABTS) was added and the absorbance was measured. The result is shown as a line in FIG.

The combination of K3-antibody and K1-antibody is K3.
-Higher measurement sensitivity than the combination of antibody and K2-antibody,
It was proved to be excellent in quantification. K3-antibody and K
With the combination with the 2-antibody, the LACI concentration in human plasma could not be measured (quantified). K1 again
With the combination of —antibody and K2-antibody, the measurement sensitivity did not increase and LACI could not be detected.

[0066]

[Example 9] Distribution of LACI in human in vivo tissues Blood vessels of human (healthy human) placenta were formalin-fixed and paraffin-embedded to obtain tissue samples. The tissue was stained with K3-antibody (2A1C5) to examine the localization of LACI.

As a result, it was revealed that LACI is localized in the media (smooth muscle cells) in blood vessels. When the distribution of other tissues was examined in the same manner, it was revealed that free LACI was present in the myocardium and the glycogen field of hepatocytes.

[0068]

Example 10 Characteristics of various monoclonal antibodies Various monoclonal antibodies at various concentrations were added to 200 μl of human plasma and reacted at 37 ° C. for 30 minutes (this reaction causes LACI in human blood to bind to the antibody). Then, 100 μl of tissue factor (TF) each
(10 μg / ml concentration) was added and the blood coagulation time was changed to Blood Co
It was measured using an agulation Analyzer (manufactured by Sysmex). The result is shown in FIG.

As a result, the K1-antibody shortened the blood coagulation time in a concentration-dependent manner. That is, it was revealed that blocking the K1 site of LACI in blood with an antibody promotes blood coagulation. This effect was not observed with K2-antibody and K3-antibody.

[0070]

Example 11 Activity of Polypeptides K1, K2 and K3 Synthetic Polypeptides K1, K2 and K shown in Example 1
Various concentrations of 3 were added to 200 μl of human plasma each. Then, 100 μl of tissue factor (50 μg / ml concentration)
Blood Coagulation Analyses.
It measured using yzer (made by Sysmex). The result is shown in FIG.

As a result, it was revealed that the polypeptides K2 and K3 did not have the activity, but only the polypeptide K1 had the activity of inhibiting thrombus formation. From this, it is considered that the active site is contained in the amino acid sequence of polypeptide K1 (21 residues).

Furthermore, even if the K2-antibody was reacted with LACI in blood, the LA reported by Broze et al. (Nature) was reported.
The action of the second Kunitz domain from the N-terminus of CI remained.

[0073]

Example 12 Various monoclonal antibodies against the activity of polypeptide K1
Body effect Polypeptide K1 (40 nM) and various monoclonal antibodies were reacted at 37 ° C. for 30 minutes and then added to 200 μl of human plasma. Tissue factor (TF) 100 μl (50 μg / ml
Concentration) to adjust blood coagulation time to Blood Coagulation An
It was measured using an alyzer (manufactured by Sysmex). The result is shown in FIG.

As a result, the monoclonal antibody 2F2D9 that recognizes the K1 portion of LACI was K1 (at about 80 nM).
It was found that the peptide inhibits the thrombus formation inhibitory activity.

[0075]

INDUSTRIAL APPLICABILITY The present invention provides an immunoassay method capable of highly sensitively measuring free LACI in a human test sample and a kit therefor. By accurately measuring free LACI in a human specimen sample by distinguishing it from LACI bound to other proteins, it has been difficult to determine the pathological condition in which minute thrombus is formed in the living body. It is expected that the pathological condition of a patient with a thrombotic disease such as an early thrombotic condition can be properly diagnosed. Also provided are useful monoclonal antibodies for use therein.

The following are examples of embodiments of the present invention. 1. Free Lipoprotein Associated Coagulatio Inhibitor in Human Test Samples
Inhibitor: LACI) is immunologically measured using a first antibody immobilized on an insoluble carrier and a labeled second antibody, wherein (i) the first antibody or the second antibody One of the antibodies is a monoclonal antibody (K3) that recognizes a polypeptide (K3) “K3” or less, which is a polypeptide having the following amino acid sequence: Leu Thr Pro Ala Asp Arg Gly Leu Cys Arg Ala Asn Glu Asn Arg Phe Tyr Tyr Asn Ser Val (hereinafter “K3”) (Ii) The other antibody is the following amino acid sequence: Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala Ile Met Lys Arg Phe Phe Phe Asn Ile Phe Polypeptide (K1) which recognizes a polypeptide (K1). -Antibody " Immunoassay of free LACI which is a U).

2. The K3-antibody is a low-density lipoprotein (Lo
The method according to 1 above, which is a monoclonal antibody that inhibits the binding of LACI to w density lipo protein (hereinafter referred to as LDL).

3. The K3-antibody is hybridoma 2A
The method according to 1 above, which is a monoclonal antibody produced from 1C5 (FRI deposit number FERMBP-4017).

4. The method according to any one of 1 to 3 above, wherein the K1-antibody is a monoclonal antibody that inhibits the binding of LACI to the tissue factor-VIIa complex and suppresses the tissue factor inhibitory activity of LACI.

5. The K1-antibody is a hybridoma 2F
The method according to any one of 1 to 3 above, which is a monoclonal antibody produced from 2D9 (FRI deposit number FERMBP-4018).

6. The method according to any one of 1 to 5 above, wherein the first antibody is a K3-antibody and the second antibody is a K1-antibody.

7. A kit for immunologically measuring a free lipoprotein-binding coagulation system inhibitor (LACI) in a human test sample, comprising: (1) a first antibody immobilized on an insoluble carrier (2) labeling Second antibody (3) Lysis agent (4) Detergent and (5) When the labeling substance is an enzyme, it contains a combination of substrate and reaction terminator for measuring enzyme activity, and ( i) either the first antibody or the second antibody is a K3-antibody, (i
i) The immunoassay kit for free LACI, wherein the other antibody is a K1-antibody.

8. The K3-antibody is LA against LDL
The above 7 which is a monoclonal antibody that inhibits CI binding.
By kit.

9. The K3-antibody is hybridoma 2A
The kit according to 7 above which is a monoclonal antibody produced from 1C5 (FRI deposit number FERMBP-4017).

10. The K1-antibody is a tissue factor-VIIa
Inhibits the binding of LACI to the complex, and LACI
The kit according to any one of 7 to 9 above, which is a monoclonal antibody that suppresses the tissue factor inhibitory activity of.

11. The K1-antibody is hybridoma 2
The kit according to any one of 7 to 10 above, which is a monoclonal antibody produced from F2D9 (FRI deposit number FERMBP-4018).

12. The kit according to any of 7 to 11 above, wherein the first antibody is a K3-antibody and the second antibody is a K1-antibody.

13. A monoclonal antibody that recognizes a polypeptide (K3) of the following amino acid sequence: Leu Thr Pro Ala Asp Arg Gly Leu Cys Arg Ala Asn Glu Asn Arg Phe Tyr Tyr Asn Ser Val.

14. The monoclonal antibody according to the above 13, which inhibits the binding of LACI to LDL.

15. Hybridoma 2A1C5 (FRI
A monoclonal antibody according to the above 13, produced from deposit number FERM BP-4017).

16. A monoclonal antibody that recognizes the following amino acid sequence: Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala Ile Met Lys Arg Phe Phe Phe Asn Ile Phe polypeptide (K1).

17. L for tissue factor-VIIa complex
The monoclonal antibody according to the above 16, which inhibits ACI binding and suppresses tissue factor inhibitory activity of LACI.

18. Hybridoma 2F2D9 (FRI
A monoclonal antibody according to above 16, produced from deposit number FERM BP-4018).

[0094]

[Sequence list]

SEQ ID NO: 1 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence: Ala Phe Lys Ala Asp Asp Gly Pro Cys 15 Lys Ala Ile Met Lys Arg Phe Phe Phe 10 15 Asn Ile Phe 20

SEQ ID NO: 2 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence: Phe Leu Glu Glu Asp Pro Gly Ile Cys 15 Arg Gly Tyr Ile Thr Arg Tyr Phe Tyr 10 15 Asn Asn Gln 20

SEQ ID NO: 3 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence: Leu Thr Pro Ala Asp Arg Gly Leu Cys 15 Arg Ala Asn Glu Asn Arg Phe Tyr Tyr 10 15 Asn Ser Val 20

[Brief description of drawings]

FIG. 1 shows a calibration curve for LACI measurement using a combination of K3-antibody and K1-antibody according to the present invention. Furthermore, a combination of K3-antibody and K2-antibody is also shown as a comparative example.

FIG. 2 shows the relationship between antibody concentration and blood coagulation time when various antibodies are used.

FIG. 3 shows the results of examining the blood coagulation time when various synthetic polypeptides were added to plasma and further Tissue Factor was added.

FIG. 4 shows the results of examining the effect of addition of various monoclonal antibodies on the activity of synthetic polypeptide (K1).

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI Technical display location C12N 15/06 (C12P 21/08 C12R 1:91) C07K 99:00

Claims (4)

[Claims] 1. A free lipoprotein-associated coagulation inhibitor in a human test sample (Lipoprotein Associated C).
oagulation Inhibitor (hereinafter sometimes abbreviated as "LACI") is immunologically measured using a first antibody immobilized on an insoluble carrier and a labeled second antibody, i) Either the first antibody or the second antibody recognizes the following amino acid sequence: Polypeptide of Leu Thr Pro Ala Asp Arg Gly Leu Cys Arg Ala Asn Glu Asn Arg Phe Tyr Asn Ser Kv3 polypeptide. It is a monoclonal antibody (hereinafter referred to as "K3-antibody"), and (ii) the other antibody has the following amino acid sequence: ) Objects Immunoassay of free LACI which is a polyclonal antibody (hereinafter "K1-antibody" hereinafter). 2. A kit for immunologically measuring free LACI in a human test sample, comprising: (1) a first antibody immobilized on an insoluble carrier (2) a labeled second antibody When the antibody (3) the lysing agent (4) the detergent and (5) the labeling substance is an enzyme, a combination of a substrate and a reaction terminator for measuring the enzyme activity is included, and (i) its first Either the first antibody or the second antibody is a K3-antibody, and (i
i) The immunoassay kit for free LACI, wherein the other antibody is a K1-antibody. 3. A monoclonal antibody which recognizes a polypeptide (K3) having the following amino acid sequence: Leu Thr Pro Ala Asp Arg Gly Leu Cys Arg Ala Asn Glu Asn Arg Phe Tyr Tyr Asn Ser Val. 4. A monoclonal antibody which recognizes the polypeptide (K1) of the following amino acid sequence Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala Ile Met Lys Arg Phe Phe Phe Asn Ile Phe.