JP2867325B2 - Anti-PIVKA-II antibody producing hybridoma and immunological assay method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a PIVKA-protein in a sample as a liver cancer marker or an indicator for diagnosis.
Measured II, in the development of rapid and accurate and convenient PIVKA- II measurements and methods aimed at to aid in medical diagnosis and treatment of, based on its.

[0002]

2. Description of the Related Art PIVKA- II is a precursor of prothrombin which is a blood coagulation factor II. In the presence of reduced vitamin K in hepatocytes, 10 glutamate residues (Glu) near the NH ₂ terminus are present. Is converted to γ-carboxyglutamic acid (Gla) by vitamin K-dependent carboxylase to form mature prothrombin. However, it is known that due to a vitamin K deficiency state or administration of a vitamin K antagonist such as warfarin, Glu is not converted to Gla and is released into the blood as it is as abnormal prothrombin. This abnormal prothrombin has Glu not converted to Gla,
It cannot have the ability to bind to Ca ions, and consequently does not have normal clotting activity and appears in plasma.
K A - II (or notation for both PIVKA-2) is called. 1984 Liebman et al., Since reported that in liver cancer patients PIVKA- II is detected at a high rate, PIVKA- II is, attention as an indicator substance for the different new liver cancer marker or diagnosed as α-feto-protein Have been. Up to now, PIVKA- II has been measured by two-dimensional immunoelectrophoresis using the presence or absence of binding to Ca salt, or after prothrombin is adsorbed with Ca or Ba salt, the antigenicity of abnormal prothrombin is measured. But their methods are specific, sensitive,
There was a problem in terms of operability. In particular, it has been difficult to more specifically and sensitively determine whether a part or most of Gla present in only 10 residues in the prothrombin remains as Glu.

[0003] As a method for solving such problems, the development of immunological assays using antibodies has been promoted. The measurement method using a polyclonal antibody specific to PIVKA- II has a low sensitivity, and there is a problem that a radioactive substance having a large problem in terms of environment and handling must be used. Under these circumstances, Motohara et al.
-It has been reported that a monoclonal antibody specifically recognizing II can be produced, and that it can be used for measurement by a more sensitive EIA. In such a method of measuring PIVKA- II using a monoclonal antibody, PI
An enzyme immunoassay method has been adopted in which both a monoclonal antibody specifically recognizing VKA- II and a polyclonal antibody against prothrombin are used, and one of them is used as an immobilized antibody and measured as an enzyme-labeled antibody. However, it has been pointed out that in this method, the operation is complicated and a long time is required for the measurement, or that the measurement sensitivity is still unsatisfactory. As a method for solving these problems, (1) human PIV
A first group of monoclonal antibodies that specifically react with KA- II but do not react with human prothrombin, (2)
Three types of monoclonal antibodies were prepared: a second group of monoclonal antibodies reactive to both human thrombin and human prothrombin, and (3) a third monoclonal antibody reactive to human prothrombin but not reactive to human thrombin. (A) combining the first monoclonal antibody group and the second or third monoclonal antibody group, or (B) combining the first monoclonal antibody group and the second monoclonal antibody group. Using a combination of the three with the third monoclonal antibody group,
A method aimed at quick, accurate and simple measurement of PIVKA- II has been proposed.

[0004]

Therefore, it is an object of the present invention to develop a method for measuring PIVKA- II which is excellent in simplicity and sensitivity by using a monoclonal antibody which can be easily obtained. In addition, there has not been any report on the identity of PIVKA- II derived from liver cancer patients and PIVKA- II derived from coagulation diseases or PIVKA- II derived from patients receiving drugs such as vitamin K antagonists, including the three-dimensional structure. For use as a liver cancer marker or an indicator for diagnosis, a monoclonal antibody that can more specifically recognize PIVKA- II derived from a liver cancer patient as an antigen to be measured is considered to be desirable. There is no report that a monoclonal antibody was produced using PIVKA- II derived directly from liver cancer.

[0005]

Means for Solving the Problems The present inventors have found that PIVKA has a feature in specificity and has an advantage in measurement sensitivity.
As a result of intensive studies to measure II , the liver cancer cell line PCL / PRF / 5 (Alexande
r) that PIVKA- II obtained from the culture supernatant was used as an antigen to produce a monoclonal antibody having specificity, and that a plurality of monoclonal antibodies thus obtained were used. The present inventors have found that excellent reactivity can be obtained by using a combination of monoclonal antibodies having different reaction specificities, and completed the present invention. The present invention relates to PIVKA- II in a sample.
Is characterized by using at least two kinds of monoclonal antibodies against PIVKA- II
A method for measuring IVKA- II is provided. As the two types of monoclonal antibodies against PIVKA- II used in the above method, for example, a reaction between (1) a monoclonal antibody that does not react with prothrombin but reacts only with PIVKA- II and (2) a reaction with prothrombin is also recognized. , PI
VKA- II also includes a combination of the monoclonal antibody of the above (1) with a monoclonal antibody having substantially the same reactivity. However, although VKA- II is identical in terms of other monoclonal antibodies to PIVKA- II , it substantially corresponds to other monoclonal antibodies. It can be a monoclonal antibody capable of specifically recognizing characteristics, for example, each kringle region other than the 10 amino acid residues near the NH ₂ end of the prothrombin structure.

According to the present invention, in addition to the above two or more kinds of monoclonal antibodies, (i) a group of monoclonal antibodies reactive to both human thrombin and human prothrombin; and (ii) a reaction to human prothrombin. ,
Any combination of a monoclonal antibody that does not react with human thrombin and (iii) a polyclonal antibody against human thrombin can be used in combination. Preferably, a monoclonal antibody is used as the partner of the combination. When used as a mixed antibody, a result was obtained in which the reactivity of prothrombin was unexpectedly completely suppressed. Furthermore, for example, when comparing the case of using only a single antibody and the case of mixing at least two antibodies, not only the effect on the measured value due to the difference of the solid-phase antibody is not recognized,
It was observed that by using an antibody having a recognition site close to that of the mixture, the reactivity was dramatically increased, and the sensitivity of the measurement system could be further improved. The use of a mixture of two kinds of antibodies not only increases the reactivity, but also leads to the result of exaggerating the specificity of one. It is difficult to explain the cause of this, but it is speculated that the affinity of the antibody may be increased from the change in the three-dimensional structure when adsorbed to the solid phase by mixing. The production of monoclonal antibodies against PIVKA- II, it is necessary to human PIVKA- II which may be used as immunogenic antigen. P as antigen
For preparation of IVKA- II , for example, the plasma of a person who has been administered a vitamin K antagonist such as warfarin is treated with an alkaline earth metal salt such as barium sulfate or barium carbonate, and prothrombin is adsorbed and removed, followed by ion exchange chromatography. After obtaining the crude product, it is further purified by affinity chromatography using a polyclonal antibody against the intersection of prothrombin and the C-terminal side of PIVKA- II . Such naturally derived PI
In addition to VKA- II , preferably cultured cells, such as PIV
PIVKA- II can be obtained using a KA- II producing cell line. PIVKA- II producing cell lines include P
Examples include cells producing IVKA- II and α-fetoprotein (AFP), and include, for example, cells derived from human liver cancer. For example, human liver cancer-derived cells PLC / PRL / 5
(Alexander) or the like can be suitably used. The PIVKA- II- producing cell line is cultured using a medium or culture method known in the art or a method substantially similar thereto, and preferably cultured in the presence of a vitamin K antagonist such as warfarin. PIVKA- II produced in Qing is subjected to ion exchange chromatography as described above, and prothrombin and PIV
Purified human PIVKA- II is obtained by affinity chromatography using a polyclonal antibody against the common portion of KA- II with the C-terminal side. In addition, recombinant PIVKA- II can be used. The PIVKA- II thus prepared may be further used as an immunogenic conjugate or the like, but it can be used as it is for immunizing animals by mixing it with an appropriate adjuvant.

[0007] As described above, antigens can be obtained from various raw materials, for example, cultured cells, cultured tissues, and other antigen-producing materials such as transformed cells by a conventionally known method, for example, salting out by ammonium sulfate precipitation, gel filtration by Sephadex, or the like. Method, for example, ion-exchange chromatography using a carrier having a diethylaminoethyl group or a carboxymethyl group or the like, for example, a hydrophobic chromatography method using a carrier having a hydrophobic group such as a butyl group, an octyl group, or a phenyl group, It can be obtained by purification by dye gel chromatography, electrophoresis, dialysis, ultrafiltration, affinity chromatography, high performance liquid chromatography, and the like. Preferably, it can be purified and separated by polyacrylamide electrophoresis or affinity chromatography in which an antibody such as a monoclonal antibody that specifically recognizes an antigen is immobilized. Furthermore, PIVKA- II selects a characteristic sequence region based on the amino acid sequence deduced from the fragmented or cloned and sequenced cDNA sequence,
The polypeptide fragment may be a polypeptide fragment obtained by designing and chemically synthesizing the polypeptide, and the fragment may be bound to various carrier proteins via a suitable condensing agent to form an immunogenic conjugate such as a hapten-protein. It can be used as a gate to design a monoclonal antibody that can recognize only a specific sequence using this. A cysteine residue or the like can be added to the designed polypeptide in advance so that the immunogenic conjugate can be easily prepared. Upon binding to carrier proteins, the carrier proteins can first be activated. For such activation, introduction of an activation bonding group may be mentioned. Activating linking groups include:
(1) Activated dithio group, such as 2-pyridyldithio group, etc. (2) Activated ester or activated carboxyl group, such as nitrophenyl ester group, pentafluorophenyl ester group, 1-benzotriazole ester group, N-succinimide ester And the like.
As carrier proteins, keyhole, limpet,
Hemocyanin (KLH), bovine serum albumin (BS
A), polypeptides such as ovalbumin, globulin, and polylysine, and bacterial cell components such as BCG.

The present invention provides at least two types of monoclonal antibodies that specifically bind to PIVKA- II . The monoclonal antibody according to the present invention can be obtained by immunizing an animal by a known method using the purified human PIVKA- II obtained by the present invention as an immunogen, and then obtained by a method known in the art or a method similar thereto. Milstein et al.'S method (Nature, 256: 495-4).
97, 1975). For example, the monoclonal antibody used in the present invention includes (1) immunization of an animal with an immunogenic antigen, (2) preparation and preparation of myeloma cells, (3) cell fusion between antibody-producing cells and myeloma cells, and (4) hybridoma. (Fusion cell) selection and monocloning, (5) production of monoclonal antibody,
(6) If necessary, it can be produced and obtained in steps such as culturing and ascites for obtaining a large amount. Immunization of animals with immunogenic antigens To immunize animals, for example, Shigeru Muramatsu, et al., Experimental Biology Course 14, Immunobiology, Maruzen Co., Ltd., Showa 60
Ed., Biochemical Society of Japan, ed. 5
New Chemistry Experiment Course 12, Molecular Immunology III, Antigen / Antibody /
Complement, Tokyo Kagaku Dojin, 1992, and the like. Adjuvants used with antigens include, for example, Freund's complete adjuvant, Ribi adjuvant, pertussis vaccine, BC
G, lipid A, liposome, aluminum hydroxide, silica and the like. Immunization is performed using an animal such as a mouse such as BALB / c. The dose of the antigen is, for example, about 1 to 400 μg for a mouse.
/ Injection, generally intraperitoneally or subcutaneously in the host animal,
Thereafter, the booster is boosted intraperitoneally, subcutaneously, intravenously or intramuscularly every 1 to 4 weeks, preferably every 1 to 2 weeks.
Repeat about 0 times. BAL as immunization mouse
In addition to B / c mice, F1 mice of BALB / c mice and other mice can also be used. If necessary, an antibody titer measurement system is prepared, and the antibody titer is measured to confirm the degree of animal immunity. On the other hand, according to the present invention, using recombinant PIVKA- II , a polyclonal antibody against PIVKA- II and its production are also possible. In such a case, mammals and birds can be used as animals to be used. For example, cattle, horses, goats, sheep,
Pigs, rabbits, mice, rats, guinea pigs, monkeys, dogs, cats, chickens and the like can be mentioned. The antibody may be an antiserum or a more purified one. For example, the isolation and purification can be performed in the same manner as the monoclonal antibody described below.

Prior to cell fusion, it is necessary to first prepare the myeloma cells to be used. The infinitely proliferative cell line (tumor cell line) used for cell fusion can be selected from cell lines that do not produce immunoglobulin, and is preferably P3-derived from mouse myeloma MOPC-21 cell line.
X63-Ag8-U1 (P3U1, Current topics in
Microbiol. And Immunol., 81, 1-7, 1978), for example, SP2 / 0-Ag14 (SP2, Nature,
276, 269-270, 1978), P3-X63-Ag8 (X6
3, Nature, 256, 495-497, 1975), P3-X63-
Ag8-653 (653, J. Immunol., 123, 1548-155).
0, 1979), PAI, PAI-0 and the like. The 8-azaguanine-resistant mouse myeloma cell line is a Dulbecco MEM medium (DMEM medium), RPMI-
To a cell medium generally known in the art such as 1640 medium, for example, an antibiotic such as penicillin, streptomycin, amikacin, fetal calf serum (FCS) and the like are added.
Further, 8-azaguanine (for example, 5-45 μg / ml)
The cells can be passaged in a normal medium 2 to 5 days before cell fusion to prepare a required number of cell lines. The cell strain used was prepared by completely thawing the cryopreserved strain at about 37 ° C., washing it three times or more with a normal medium such as RPMI-1640 medium, and then culturing it in a normal medium to prepare a required number of cell lines. There may be.

[0010] Cell fusion between an antibody-producing cell and a myeloma cell can be carried out as follows. An animal, such as a mouse, immunized according to the above immunization step, the spleen is removed 2 to 5 days after the final immunization, and a spleen cell suspension is obtained therefrom. In addition to spleen cells, lymph node cells from various parts of the body can be obtained and used for cell fusion. The thus obtained spleen cell suspension and the myeloma cell line prepared according to the above-mentioned steps are used, for example, in a minimal essential medium (MEM medium), DME
The medium is placed in a cell culture medium such as M medium or RPMI-1640 medium, and a cell fusion agent such as polyethylene glycol is added. Preferably, for example, 0.5 to 2 ml of 30 to 60% polyethylene glycol can be added, polyethylene glycol having a molecular weight of 1,000 to 8,000 can be used, and further, a molecular weight of 1,000 to 4,000 can be used.
A polyethylene glycol of 00 can be more preferably used. The concentration of polyethylene glycol in the fusion medium
For example, it is preferable to set it to 30 to 60%.
If necessary, fusion can be promoted by adding a small amount of, for example, dimethyl sulfoxide. Other various cell fusion agents known in the art can be used. Examples of such a cell fusion agent include inactivated Sendai virus (HVJ: Hemagglutinating v).
irs of Japan). The ratio of spleen cells (lymphocytes) to myeloma cell line used for the fusion may be, for example, 1: 1 to 20: 1, and more preferably 4: 1 to 7: 1. The fusion reaction was performed for 1 to 10 minutes and then RPMI-16
Add a cell medium such as 40 medium. The fusion reaction treatment can be performed plural times. After the fusion reaction, the cells are separated by centrifugation or the like, and then transferred to a selection medium.

The hybridomas (fused cells) thus obtained are selected and cloned. As a selection medium, for example, hypoxanthine,
MEM containing FCS containing aminopterin and thymidine
Medium, such as RPMI-1640 medium, so-called HAT
A medium is mentioned. The method of replacing the selective medium is generally such that the volume and the equivalent volume dispensed to the culture plate are added the next day, and then half of the HAT medium is replaced every 1 to 3 days. It can also be done with modifications. On the 8th to 16th days after the fusion, the medium can be exchanged every 1 to 4 days with a so-called HT medium excluding aminopterin. As a feeder, for example, mouse thymocytes can be used, which may be preferred. The culture supernatant of the culture well in which the proliferation of the hybridoma is extensively analyzed by a measurement system such as a radioimmunoassay (RIA), an enzyme immunoassay (ELISA), and a fluorescence immunoassay (FIA), or a fluorescence-induced cell separation device (FACS).
For example, screening or isolation is performed by using PIVKA- II or a fragment peptide thereof as an antigen, or by measuring a target antibody using a labeled anti-mouse antibody. For example, the screening method can be performed by the following three methods (EIA), and the target clone can be selected;
Check IgG producing clones using the anti-mouse IgG solid phase-culture supernatant-peroxidase (POD) labeled anti-mouse IgG system; PIVKA- II purified antigen solid phase-culture supernatant-POD labeled anti-mouse IgG system To check for PIVKA- II producing clones; and to check for prototonbin producing clones using the prototonbin antigen solid phase-culture supernatant-POD labeled anti-mouse IgG system. The hybridoma producing the antibody of interest is cloned. The cloning can be performed by picking up a colony in an agar medium or by limiting dilution. It can be more preferably performed by a limiting dilution method. Cloning is preferably performed multiple times.

The cloned hybridomas (fused cells) can be cultured and used for the production of monoclonal antibodies. The resulting hybridoma strain is F
The cells are cultured in an appropriate growth medium such as a CS-containing MEM medium or RPMI-1640 medium, and a desired monoclonal antibody can be obtained from the culture supernatant. In order to obtain a large amount of antibodies, ascites of the hybridoma can be mentioned. In this case, each hybridoma is transplanted and grown in the abdominal cavity of a histocompatible animal of the same type as the myeloma cell-derived animal, or, for example, each hybridoma is transplanted and grown in a nude mouse or the like and produced in the ascites of the animal. The obtained monoclonal antibody can be recovered and obtained. Animals were treated with pristane (2,6,6) prior to hybridoma transplantation.
Mineral oil such as (10,14-tetramethylpentadecane) can be administered intraperitoneally. After the treatment, hybridomas can be grown and ascites can be collected. Ascites fluid as it is or conventionally known methods such as salting out such as ammonium sulfate precipitation, gel filtration by Sephadex, etc., ion exchange chromatography, electrophoresis, dialysis, ultrafiltration, affinity chromatography And purified by high performance liquid chromatography or the like and used as a monoclonal antibody. Preferably, the ascites fluid containing the monoclonal antibody is fractionated after ammonium sulfate fractionation, such as DEAE-Sepharose,
Purification and separation can be performed by treating with an anion exchange gel and an affinity column such as a protein A column. Particularly preferred are affinity chromatography in which an antigen or antigen fragment (for example, a synthetic peptide, a recombinant antigen protein or peptide, a site specifically recognized by an antibody, etc.) is immobilized, and affinity chromatography in which protein A is immobilized. No. The antibody can preferably be a purified antibody that has been denatured. For example, when the effect of the denaturation treatment of the
In the case of an antibody, the reactivity of the antibody can be increased by about 10 times by denaturation treatment as compared with untreated treatment. Examples of the denaturing reagent include 6M guanidine hydrochloride.
As a factor for increasing the reactivity, it is considered that the denaturing effect of the Fc portion of IgG may increase the adsorption ability to the solid phase, and any treatment for such a purpose can be adopted. These antibodies are treated with enzymes such as trypsin, papain, and pepsin, and optionally reduced to obtain Fab, F
It may be used as an antibody fragment such as ab 'and F (ab') ₂ . As an antibody for providing a label, I
The specific binding site Fab ′ obtained by reducing after the gG fraction and further pepsin digestion can be used.

[0013] Labels include enzymes, enzyme substrates, enzyme inhibitors, prosthetic molecules, coenzymes, enzyme precursors, apoenzymes, fluorescent substances, dye substances, chemiluminescent compounds,
A luminescent substance, a coloring substance, a magnetic substance, a metal particle such as a gold colloid, and a radioactive substance can be given. Enzymes include dehydrogenases, reductases, oxidoreductases such as oxidases, and transfer enzymes that catalyze transfer of amino groups, carboxyl groups, methyl groups, acyl groups, phosphate groups, and the like, such as ester bonds, Examples include a hydrolase that hydrolyzes a glycosidic bond, an ether bond, a peptide bond, and the like, a lyase, an isomerase, and a ligase. Enzymes can be used for detection by using a plurality of enzymes in combination. For example, enzymatic cycling can be used. Representative radioisotope labeling isotopes include [ ³² P], [ ¹²⁵ I], [ ¹³¹ I],
[ ³ H], [ ¹⁴ C], [ ³⁵ S] and the like. Representative enzyme labels include peroxidase such as horseradish peroxidase, galactosidase such as Escherichia coli β-D-galactosidase, maleate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose oxidase, glucoamylase,
Examples include alkaline phosphatase such as acetylcholinesterase, catalase, bovine small intestine alkaline phosphatase, and Escherichia coli alkaline phosphatase.
When alkaline phosphatase is used, substrates such as umbelliferone derivatives such as 4-methylumbelliferyl phosphate, phosphorylated phenol derivatives such as nitrophenyl phosphate, enzymatic cycling systems using NADP, luciferin derivatives, and dioxetane derivatives are used. It can be measured by the fluorescence, luminescence, etc. generated by use. Luciferin and luciferase systems can also be used. When catalase is used, it reacts with hydrogen peroxide to generate oxygen, and the oxygen can be detected by an electrode or the like. The electrode may be a glass electrode, an ion electrode using a poorly soluble salt film, a liquid film electrode, a polymer film electrode, or the like. The enzyme label can be replaced with a biotin label and an enzyme-labeled avidin (streptavidin). A plurality of different types of labels may be used. In such a case, it may be possible to make multiple measurements continuously or discontinuously, and simultaneously or separately.

For labeling, a reaction between a thiol group and a maleimide group, a reaction between a pyridyl disulfide group and a thiol group,
The reaction can be carried out by utilizing the reaction between an amino group and an aldehyde group, and the method can be appropriately selected from known methods or methods which can be easily performed by those skilled in the art, or modified methods. In addition, a condensing agent that can be used for the preparation of the immunogenic complex, a condensing agent that can be used for binding to a carrier, and the like can be used.
Examples of the condensing agent include glutaraldehyde, hexamethylene diisocyanate, hexamethylene diisothiocyanate, N, N′-polymethylenebisiodoacetamide, N, N′-ethylenebismaleimide, ethylene glycol bissuccinimidyl succinate, bismuth Diazobenzidine, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, succinimidyl 3- (2
-Pyridyldithio) propionate (SPDP), N-
Succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), N-
Sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, N-succinimidyl (4-iodoacetyl) aminobenzoate, N-succinimidyl 4- (1-maleimidophenyl) butyrate, N- (ε- (Maleimidocaproyloxy) succinimide (EMCS), iminothiolane, S-acetylmercaptosuccinic anhydride, methyl-
3- (4′-dithiopyridyl) propionimidate,
Methyl-4-mercaptobutyrylimidate, methyl-
3-mercaptopropionimidate, N-succinimidyl-S-acetylmercaptoacetate and the like.

The detection and measurement in the present invention can be carried out by immunostaining, for example, tissue or cell staining, immunoassay, for example, competitive immunoassay or non-competitive immunoassay, using radioimmunoassay, ELISA, latex agglutination, etc. And the measurement can be performed with or without BF separation. Preferably, a latex agglutination method or an enzyme immunoassay is used, and a sandwich-type assay is further preferable. For example, in a sandwich-type assay, PIVKA-
One of the antibodies to II is detectably labeled. Another antibody that can recognize the same antigen is immobilized on a solid phase. Incubation is performed to react the sample with the labeled antibody and the solid-phased antibody sequentially as necessary. Here, the unbound antibody is separated, and the labeled substance is measured. The amount of label measured is proportional to the amount of antigen, ie, PIVKA- II . This assay is referred to as a simultaneous sandwich-type assay, a forward sandwich-type assay, a reverse sandwich-type assay, or the like, depending on the order of addition of the insolubilized antibody or the labeled antibody. For example, washing, stirring, shaking, filtration, pre-extraction of antigen, etc. are appropriately adopted in these measurement steps under specific circumstances. Other measurement conditions such as the concentration of a specific reagent, buffer, etc., temperature, or incubation time can be changed according to factors such as the concentration of the antigen in the sample and the properties of the sample. Those skilled in the art can appropriately select the optimum conditions effective for each measurement and perform the measurement using ordinary experimental methods.

For example, an enzyme immunoassay using a sandwich type assay according to the present invention can be carried out as follows. The measurement system is a solid phase, an antibody against at least two types of PIVKA- II as immobilized monoclonal antibody components (for example, (i) a monoclonal antibody that does not react with prothrombin and reacts only with PIVKA- II and (ii) a prothrombin And PIVKA- II
A standard antigen or a sample for measurement, an enzyme-labeled antibody (second antibody), and a substrate. As the solid phase, those described below can be used. Immobilization of the monoclonal antibody component can be carried out, for example, by placing the monoclonal antibody component dissolved in a buffer in a solid phase, for example, a polystyrene well, and leaving it at about 4 ° C. overnight to coat the solid phase surface. The method described below can be used as appropriate. The uncoated solid phase surface can be treated with bovine serum albumin or the like to suppress non-specific reactions. As a standard antigen, PIVKA- II- containing plasma with a known concentration can be used. Normally, the standard antigen may be diluted with a buffer containing bovine serum albumin or the like, placed in the above-mentioned coated well, reacted, and then unreacted substances may be removed by washing. Next, as the enzyme-labeled antibody (second antibody), P
An OD-labeled anti-human prothrombin antibody can be suitably used. This anti-human prothrombin antibody is obtained by using the PI
It is obtained by immunizing an animal using human prothrombin as an antigen in the same manner as the antibody against VKA- II . Examples of animals used include mammals and birds, and examples include cows, horses, goats, sheep, pigs, rabbits, mice, rats, guinea pigs, monkeys, dogs, cats, chickens, and the like. For example, Shigeru Muramatsu,
Other editions, Experimental Biology Course 14, Immunobiology, Maruzen Co., Ltd., 1985, edited by The Biochemical Society of Japan, Seminal Chemistry Laboratory Course 5, Immunobiochemical Research Methods, Tokyo Chemical Dojin, 1986, edited by The Biochemical Society of Japan , Shinsei Chemistry Laboratory 12, Molecular Immunology II
I, antigen / antibody / complement, Tokyo Chemical Dojin, 1992, etc. The antibody may be an antiserum or a more purified one,
For example, the isolation and purification can be performed in the same manner as in the above monoclonal antibody. An anti-human prothrombin monoclonal antibody can also be obtained using human prothrombin as an antigen in the same manner as the above-mentioned antibody against PIVKA- II .

Many carriers on which an antigen or an antibody can be immobilized are known, and in the present invention, any of them can be appropriately selected and used. As the carrier, various carriers used for an antigen-antibody reaction and the like are known, and in the present invention, of course, any of these known carriers can be used. Particularly preferably used are, for example, glass, such as activated glass, porous glass, silica gel, silica-
Inorganic materials such as alumina, alumina, magnetized iron, magnetized alloys, polyethylene, polypropylene, polyvinyl chloride,
Crosslinking such as polyvinylidene fluoride, polyvinyl acetate, polymethacrylate, polystyrene, styrene-butadiene copolymer, polyacrylamide, cross-linked polyacrylamide, styrene-methacrylate copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer Natural or denatured cellulose such as albumin, collagen, gelatin, dextran, agarose, cross-linked agarose, cellulose, microcrystalline cellulose, carboxymethyl cellulose, cellulose acetate, polyamide such as cross-linked dextran, nylon, and organic polymers such as polyurethane and polyepoxy resin Substances, and those obtained by emulsion polymerization thereof, such as latex particles (eg, polystyrene latex particles, styrene- Such Tajien copolymer latex particles), cells, red blood cells, etc., can be mentioned those which had been introduced a functional group with a silane coupling agent if necessary. Further, filter paper, beads, inner walls of test containers, for example, test tubes, titer plates, titer wells, cells made of synthetic materials such as glass cells, synthetic resin cells, glass rods, rods made of synthetic materials, and thicker ends. Alternatively, the surface of a solid material (object) such as a thinned rod, a rod having a round or flat projection at its end, or a thin plate-shaped rod may be used.

An antibody can be bound to these carriers, and preferably, a monoclonal antibody that specifically binds to PIVKA- II obtained in the present invention can be bound thereto. The binding between the carrier and those involved in the antigen-antibody reaction is performed by a physical method such as adsorption, a condensing agent, or a chemical method using an activated material, or a mutual method. It can be performed by a method utilizing a chemical binding reaction or the like. In the present invention, 4-hydroxyphenylacetic acid, 1,2-phenylenediamine, tetramethylbenzidine, and the like, together with horseradish peroxidase (POD), umbelliferyl galactoside, nitrophenylgalactoside, and the like are used to form a signal such as a substrate. It can be used together with an enzyme reagent such as D-galactosidase and glucose-6-phosphate dehydrogenase, and includes quinol compounds such as hydroquinone, hydroxybenzoquinone and hydroxyanthraquinone;
Those which can form thiol compounds such as lipoic acid and glutathione, phenol derivatives, ferrocene derivatives and the like by the action of enzymes and the like can be used. Examples of the fluorescent substance or the chemiluminescent compound include fluorescein isothiocyanate, for example, rhodamine derivatives such as rhodamine B isothiocyanate and tetramethylrhodamine isothiocyanate, dansyl chloride, dansyl fluoride,
Examples include fluorescamine, phycobiliprotein, acridinium salts, luminols such as lumiferin, luciferase, and equolin, imidazole, oxalate, rare earth chelate compounds, coumarin derivatives, and the like.

According to the measuring method of the present invention, a labeled antibody reagent such as a monoclonal antibody in which a substance to be measured is labeled with an enzyme and the like and an antibody bound to a carrier can be sequentially reacted, or simultaneously. You can also. The order in which the reagents are added depends on the type of carrier system chosen. When sensitized plastic beads or the like are used, a labeled antibody reagent such as a monoclonal antibody labeled with an enzyme or the like is first put together with an analyte sample containing a substance to be measured in an appropriate test tube, and then, The measurement can be performed by adding beads such as sensitized plastic. The measurement can be performed in an appropriate buffer system so as to maintain the optimum pH, for example, about pH 4 to 9. Particularly suitable buffers include, for example, acetate buffers, citrate buffers, phosphate buffers, Tris buffers,
Examples include a triethanolamine buffer, a borate buffer, a glycine buffer, a carbonate buffer, and a Tris-HCl buffer. Buffering agents can be used by mixing them at an arbitrary ratio. Preferably, the antibody-antigen reaction is performed at a temperature between about 0 ° C and 60 ° C.

An antibody reagent such as a monoclonal antibody labeled with an enzyme or the like, and an antibody reagent bound to a carrier;
In addition, the incubation of the substance to be measured
The reaction can be performed until equilibrium is reached, but at a much earlier point in time when the equilibrium of the antibody-antigen reaction is achieved, the solid phase and the liquid phase can be separated and the reaction can be stopped after a limited incubation treatment. The extent of the presence of a label, such as an enzyme, in either the phase or the solid phase can be measured. The measurement operation can be performed using an automated measurement device, and a luminescence detector, a photo detector, or the like is used to detect a display signal generated by the conversion of a substrate by the action of an enzyme and to perform measurement. You can also. In the antibody-antigen reaction, the reagent used, the substance to be measured, and further stabilize the label such as an enzyme,
Appropriate measures can be taken to stabilize the antibody-antigen reaction itself. In addition, proteins, stabilizers, surfactants, chelating agents, etc. should be added to the incubation solution to remove non-specific reactions and reduce inhibitory effects, or to activate measurement reactions. Can be added. As the chelating agent, ethylenediaminetetraacetic acid salt (EDTA) is more preferable. It may be subjected to a blocking treatment to prevent a non-specific binding reaction commonly used in the art or known to those skilled in the art, for example, normal serum proteins such as mammals,
Albumin, skim milk, fermented milk, collagen,
It can be treated with gelatin or the like. These methods can be used without any particular limitation as long as the purpose is to prevent a non-specific binding reaction. As the sample to be measured by the measurement method of the present invention, any form of solution or colloid solution, a non-fluid sample and the like can be used, preferably a biological sample, such as blood, plasma, synovial fluid, cerebrospinal fluid, Saliva, amniotic fluid, urine, other body fluids, cell cultures, tissue cultures, tissue homogenates, biopsy samples, tissues, cells and the like. For example, blood, plasma, hepatocyte culture, liver tissue culture,
Examples include liver tissue, liver tissue homogenate, and liver biopsy sample.

According to the present invention, there is provided a reagent used directly in the above-mentioned measuring method. Such a reagent can be a reagent set in a form suitable for performing a measurement using at least two types of monoclonal antibodies against PIVKA- II . Representative reagents included in this reagent set include:
A solid phase on which at least two monoclonal antibodies against PIVKA- II are immobilized, such as (i) a monoclonal antibody that does not react with prothrombin and reacts only with PIVKA- II , and (ii) reacts with prothrombin; And a plastic well coated with both monoclonal antibodies that also react with PIVKA- II . In addition, the reagent set includes a standard antigen, an enzyme-labeled antibody (second antibody) such as a labeled anti-prothrombin antibody, a substrate, a diluent, a lysis solution, a reaction terminator, a buffer, and the like. Good. These components may optionally be combined for convenient measurement.

[0022]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples, and various embodiments will be readily apparent to those skilled in the art. It should be understood that they also fall within the scope of the invention if they use features of the invention. Example 1 (Preparation and method of reagent) 1) Purification of PIVKA- II PIVKA- II was purified from the culture supernatant using human liver cancer-derived cells PLC / PRL / 5 (Alexander), which is a strain producing PIVKA- II and AFP. Get II . Culture conditions, the Alexander cultured in the presence of fetal calf serum (FCS), confluent conditions was changed to FCS the Free at (confl u ent) conditions, a vitamin K antagonist Waferin (0.08mg / ml) Was administered to promote the production of PIVKA- II . P in culture supernatant
The IVKA- II concentration was 4.2 Au / ml and the AFP concentration was 200 ng / ml. The addition of warfarin increased the amount of production, yielded a plateau at a concentration of 20 μg / ml or more and a culture period of 6 to 8 days. For the purpose of completely inhibiting the metabolism of prothrombin precursor to prothrombin, the concentration of warfarin was set to 80 μg / ml. The culture supernatant obtained as described above was collected and purified as follows. First,
Crude purification was performed by ion exchange chromatography using DE-52 or CM-52 Cellulose. With respect to the pH of the equilibration buffer (3.0 to 7.0), conditions are set such that PIVKA- II and contaminant proteins can be separated from each other in a gradient elution condition of 0.1 to 0.3 M NaCl. For purification. It was confirmed that PIVKA- II and prothrombin dissociated favorably in a buffer solution of pH 5.0. In addition, prothrombin and PIV
Purification was performed by affinity chromatography using a polyclonal antibody against the common portion on the C-terminal side of KA- II . The affinity column is CN-Br activated Sepharose 4B ( trade name: Pharma ci)
Using a ), 18.5 mg of ligand was coupled per 1 g of swollen gel by a conventional method, and used as a carrier for affinity chromatography. The column equilibration buffer was 0.2 M PB, 0.2 M NaCl p.
At H6.5, elution was with 5M guanidine hydrochloride. PIVKA- II obtained by the above purification method was obtained as a substantially single band by 5% acrylamide electrophoresis.

2) Preparation and purification of anti-PIVKA- II monoclonal antibody PIVKA- II (0.06 mg /
0.3ml) a complete Freund's adjuvant of the same capacity (Complete Fre un d Adjuvan
t) with BALB / C mice (female, 6 weeks) subcutaneously, and after 2 weeks, the same amount of antigen as in the beginning was added to incomplete Freund's adjuvant (incomplete Freund).
be administered with un d Adju v ant). further,
After booster immunization with the same amount of antigen (two weeks later), two weeks later, 0.03 mg of the antigen was further administered as an abdominal empty Booster, and five days later, spleen cells were collected to perform cell fusion. 1.8 × 10 ⁸ spleen cells and myeloma cells (PA
I) 6 × 10 ⁷ were fused using 50% polyethylene glycol. For fusion, both cells suspended in serum-free RPMI medium were centrifuged at 1500 rpm, and the supernatant was removed with an aspirator.
Add gently and stir gently for another minute. After adding serum-free RPMI medium slowly, 1500r for 5 minutes
centrifugation at pm, the supernatant is removed with an aspirator, and HAT
Medium (serum free RPMI medium containing hypoxanthine (10
0 μM), aminopterin (0.4 μM) and thymidine (16 μM) were added, and the cell concentration was adjusted to 5 × 10 ⁵ cells / well in a 96-well plate (5.5 plates). Incubate for 10 days. 2, 3, 5,
On day 8, half of the medium (about 0.1 ml) was replaced with fresh HAT medium, and on day 10, half of the medium was replaced with fresh HT medium (HAT without aminopterin). Screening was performed on all wells where hybridoma growth was visually observed. The screening method is
A clone was selected by the following three methods (EIA).

Checking method for IgG-producing clones / A
anti mouse IgG solid phase-culture supernatant-POD
Anti mouse IgG PIVKA- II Producing clone check method / PIV
KA- II purified antigen solid phase-culture supernatant-POD Anti
mouse IgG Prothro m bin-producing clones of the check method / Prothro m bin antigen solid phase - the culture supernatant -POD
Anti mouse IgG In each screen, a 96-well polystyrene plate was coated with each antigen and then washed with PBS for washing (0.
Washing was carried out using 0.05% Tween 20 to remove unadsorbed peptides. In addition, one uncoated part of each well
Blocked with% BSA. 0.1 ml of the supernatant of the well in which the growth of the hybridoma was confirmed was added to each well, and the well was allowed to stand at room temperature for about 1 hour. Horseradish peroxidase (POD) -labeled goat anti-mouse immunoglobulin (Cappel Lab.) Was added as a secondary antibody, and the mixture was allowed to stand at room temperature for about 1 hour. Next, the substrate hydrogen peroxide and ο
-Phenylenediamine was added, and the degree of color development was measured at 492 nm using a microplate absorbance meter (MRP-A4, Tosoh). Of these three methods, P
IVKA- II and will react, but began to select clones from that of the Prothro m bin do not react (law). Then, cloning was performed three times by the limiting dilution method, and two types of anti-PIVKA- II antibody-producing strains (2G4, 1C9) were established. The monochrome produced by this cell line
The null antibody is called 2G4, 1C9. The above PIVKA
- II antibody-producing hybridoma cell line 2G4 is entrusted to the Agency of Industrial Science and Technical Research Laboratories number FERM P-1
Deposited as 5070. PIVKA-
The II antibody-producing hybridoma cell line 1C9 was obtained from the National Institute of Bioscience and Biotechnology at the Accession No. FERM P-15.
No. 069. The immunoglobulin class of each antibody belongs to IgG, and its subclass is G1.
Met. These are mouse monoclona
l antibody isotyping Kit
(Amersham). Cryopreservation of the established high <br/> Buridoma is, 10% FCS containing RPMI16
The cells grown in a 40 medium and grown logarithmically in 4 × 10 ⁶ to
Store frozen at a number of 10 ⁷ cells / m. As a cryopreservation solution, RPMI-16 containing 20% FCS and 10% DMSO
Forty media were used. In order to purify antibodies from the obtained hybridomas, 2G4 and 1C9 cells were first cultured and increased, and 3 × 10 ⁶ to 1 × 10 ⁷ cells / ml were added to pristane one week beforehand under FCS Free conditions. Intraperitoneally administered mice (BALB / c system, ♀, 6 weeks old). About 7 to 10 days later, ascites is collected and 40%
Salting out was performed twice with ammonium sulfate, and the precipitate was stored frozen. Purification from ascites was performed using Protein A cellofine.
Chromatography was performed. subc
This antibody having a G1 of lass is a buffer solution of pH 8.5 (50
mM Tris - HCl, 150 mM NaCl) and pH 5.0 (100 mM Citrate , 15 mM).
Elution is performed under mild conditions (0 mM NaCl) to obtain a purified antibody. After dialysis against 20 mM PBS, 0.01% Na
N3 is added and used.

3) Preparation of peroxidase-labeled human prothrombin IgG Rabbits were immunized with human prothrombin from DAKO, and the obtained serum was subjected to Q-Sepharose chromatogram.
ogrphy (50 mM Tris - HCl, pH
8.0) 0-0.2 M NaCl Gradie
Elute the IgG fraction with nt. The obtained IgG was converted to human prothrombin affinity chromato.
Anti-human prothrombin rabbit I using graphy
The specific antibody of gG was purified. The affinity column is coupled with 1.4 mg prothrombin / g gel using Formyl Cellulofine. The equilibration buffer was 0.1 M PB (pH 7.0), and the elution buffer was 0.1 citrate buffer. The ratio of the obtained specific antibody was 3 to 5% of the total IgG. Anti-human prothrombin IgG obtained by the above operation
2.5mg plus horseradish peroxidase (HRP)
2.0 mg was labeled by a maleimide method. 2mg /
0.3 ml of HRP (0.1 mol / l, pH 7.0)
Was added with 0.3 mg / 30 μl of N- (ε-maleimidocaproyloxy) succinimide (EMCS) at 30 ° C.
And displace the product buffer. On the other hand, 2.5 mg / 0.25 ml of IgG (0.1 mol /
l, phosphate buffer, pH 7.5)
Add 2 mg / 2 μl, stir at room temperature for 30 minutes, then add 0.1 μl / l EDTA / 2Na 20 μl, 1 m
50 μl of ol / l Tris-HCl (pH 7.0) and 50 μl of 1 mol / l hydroxylamine hydrochloride are added, and the mixture is stirred at 30 ° C. for 5 minutes to replace the buffer of the product. The two were mixed in equal amounts, left at 4 ° C. overnight, and then subjected to gel filtration to obtain a labeled product. HRP / Ig of the obtained label
G (molar ratio) was in the range of 1.5 to 3.0. Also w
The IgG concentration per cell was 1.4 to 3.0 ng. The dilution used in the reaction system is about 1 to 20,000 times. In addition, a mouse was immunized with human prothrombin from DAKO and subjected to cell fusion to obtain an anti-human prothrombin monoclonal antibody (the immunization method and the fusion method were PIVKA-
II ). Peroxidase labeling is performed in the same manner as in 3) above to obtain a labeled antibody. Approximately the same efficacy was observed in the results of measurement of the samples. The use of monoclonal antibodies has the advantage that the step of purification to specific antibodies can be omitted. Similarly, a labeled antibody was prepared for the 1C9 antibody.

Example 2 (ELISA Method and Measurement Conditions) 1) Selection of Immunoplate In selecting an ELISA plate, it is necessary to select a plate having a high solid phase adsorption property of an antibody and a low nonspecific reaction. The selection of the immunoplate was examined based on the standard.
The solid phase buffer contains 10 mM Tris-HCl 0.1
Using M NaCl (pH 7.0), 0.1 ml of 2G4 antibody adjusted to 10 μg / ml is added, and the solid phase is immobilized at 4 ° C. overnight. Wash solution (10mPBS, 0.05% Tween
After washing 3 times with -20), 0.2% of 10% skim milk
l for 2 hours at room temperature.
g). After washing, PIVKA- II standard product (St)
0.1 ml is added and the reaction is carried out at room temperature for 2 hours. Peroxidase labeled anti-human prothrombin IgG (0.1M
Tris - HCl, 0.075M NaCl, 0.2%
Tween-20, 5% NRS, 50% Block A
(ce, pH 7.0), react at room temperature for 1 hr, add 0.1 ml of substrate solution, and after 30 min, stop the reaction with 2N sulfuric acid. Colorimetry is measured with a 450/650 nm filter. As a result, a plate having high antibody solid-phase adsorption and low nonspecific reaction could be selected. Examples of such a plate include a plate manufactured by Labosystem, a plate manufactured by Corning, and a plate manufactured by Nunc.
axsorb plates and the like. 2) Selection of reaction buffer In the ELISA, if the sample serum is directly reacted, the reaction to the solid phase antibody may be inhibited due to the effect of a certain substance in the serum component, and even in the PIVKA- II system, This phenomenon is observed. One way to reduce this is to dilute the sample. This dilution effect also suppresses non-specific reactions, but must not affect the sensitivity of the measurement system. Therefore, conditions such as a dilution buffer and its components were examined. As a basic buffer, 25 mM buffer (Buffer) /
0.075M NaCl / 0.1% BSA / 0.01%
Tested using Tween-20. As a result, it was determined that it was appropriate to use a Tris-HCl buffer (pH 7.0 and its vicinity) as the buffer. It was confirmed that it can be selected from the viewpoint of reactivity and stability. It was also confirmed that conditions having high reactivity, little inhibition by serum components, and an effect of suppressing nonspecific reactions could be selected. 3) Examination of reaction time The sandwich method comprises a first reaction (reaction between the primary antibody and the sample), a second reaction (reaction between the antigen reacted with the primary antibody and the labeled secondary antibody), and a third reaction (labeling). Reaction between a secondary antibody and a substrate). Which step of the reaction time had a large effect on the measurement result was compared between the case where the first reaction was at room temperature and the case where the first reaction was at 4 ° C. The reaction conditions are shown below. Reaction time Reaction temperature (room temperature) Reaction temperature (4 ° C.) First reaction 1 hr 2 hr 2 hr ON ON ON ON ON Second reaction 1 hr 1 hr 1 hr 1 hr 1 hr 2 hr 3 hr 4 hr Third reaction 0.5 hr 0.5 hr 1 hr 0.5 hr 1 hr 1 hr 1 hr 1 hr Examination As a result, it was judged that the first reaction was suitable for 2 hours, the second reaction was also 2 hours, and the third reaction was 1 hour. 4) Solid phase concentration and solid phase time The antibody solid phase concentration was set under the condition that the reactivity reached a plateau. The immobilization of the 2G4 antibody alone was compared with a denatured antibody or a non-denatured antibody. Further, it was confirmed whether the solid phase was immobilized at the same concentration even when the 2G4 antibody and the 1C9 antibody were mixed. Antibody solid phase concentration is 10 μg / ml
Was determined to be sufficient, but 20 μg / ml was determined to be appropriate in order to reduce non-specific reactions. In the case of solid-phase immobilization, sufficient results were obtained by treatment at 4 ° C. for 1 day in the case of the mixed antibody, but it was recognized that treatment with 4G at 1 ° C. for 1 day was not sufficient with the 2G4 antibody alone in some cases. . 5) Preparation of enzyme substrate solution 0.17 g of tetramethylbenzidine (TMB) was added to DM
After dissolving in 9.9 ml of SO, the first reagent to which 0.8 ml of a saturated EDTA-2Na solution was added, and 0.1 M sodium acetate, 10 mM EDTA-2Na and 10% dimethylformamide were adjusted to pH 5.0 with 1 M citric acid. Make up to 1 liter (second reagent). Add the first reagent to the second reagent and leave it at room temperature overnight before use. In use, the color of the substrate solution is colorless. 2N sulfuric acid was adjusted and used as a reaction stopping solution.

Example 3 (Monoclonal antibody (2G
Confirmation of characteristics of 4 / 1C9) 1) Characteristics of monoclonal antibody 2G4 (characteristics of denatured antibody) Purification of 2G4 antibody is performed by a method using Protein G. This method uses glycine / HCl (p
H2.2) A method using a buffer solution, which is recognized to have denaturation to the antibody. Next, Protein A
And elution conditions were changed to citrate buffer (p
H5.0), the purity of the purified IgG was further increased. However, the purified antibodies (Protein G and Pro
teinA) was used as a solid phase and used in an ELISA system. As a result, the reactivity of the purified antibody with Protein A was low. Therefore, it was examined whether or not the reactivity of the antibody purified by Protein A was improved when the antibody was denatured. The study was performed using the 2G4 antibody.
The method is the following two methods. (Treatment 1) 0.5 ml of antibody was reacted with 0.5 ml of 0.5 M glycine HCl (pH 2.2) solution for 30 minutes at room temperature, and then 0.5 ml of neutralization buffer was added to obtain a treated antibody. Was solid-phased and used for the reaction. (Treatment 2) 0.1 ml of the antibody and 0.1 ml (× 2) /0.4 (× 5) /0.9 (× 10) of a 6M-guanidine HCl solution were reacted at room temperature for 60 minutes and then dialyzed ( 20 mM P
BS), and the solid phase was used for the reaction. Table 1 shows the results.

[0028]

[Table 1]

As a result of examining the denaturing effect of the purified antibody,
It was found that the reactivity of the G4 antibody can be increased about 10 times by the denaturation treatment as compared with the untreated G4 antibody. When compared at a solid phase concentration of 25 μg / ml, the effect of the denaturing reagent 6M guanidine hydrochloride was the highest. It is suggested that the reason for the increase in the reactivity is that the denaturing effect of the Fc portion of IgG may increase the adsorption ability to the solid phase.

2) Two types of antibodies (2G
Specificity when immobilized by mixing 4, 1C9) The ELISA system using the 1G9 antibody as a solid phase also reacts with normal prothrombin, but on the other hand, results have been obtained in detecting end-stage liver cancer. . Since the characteristic that the 2G4 antibody hardly reacts with normal prothrombin and that it reacts with PIVKA- II has been obtained, the case where both were mixed and used was examined. By the way, when both are mixed and used, it is expected that the reactivity with normal prothrombin will surface and the specificity of PIVKA- II will decrease, but it was also examined whether or not such a result would be obtained. The mixing method is to add 2G4 antibody or 1C9 antibody to each of 40 μg / m
The solid phase was adjusted at the following mixing ratio. (2G4 antibody / 1C9 antibody) = (5/0), (4 /
1), (2/1), (1/1), (1/2), (1 /
4), (0/5) The method of ELISA after immobilization is the same as the method described so far. The reactivity with PIVKA- II or prothrombin is examined in an ELISA system using, as a solid phase, the 2G4 and 1C9 antibodies purified individually in the above (2) or a mixture of both. Microplate (Labosystem Breakable, F
8) Using 10 mM Tris - HC in the solid phase buffer
l, 0.1 M NaCl (pH 7.0)
g / ml 2G4 or 1C9 antibody at 0.1
Add the solid and immobilize overnight at 4 ° C. Washing solution (10 mM
After washing three times with PBS, 0.05% Tween-20),
(0.2 ml of 10% skim milk) is added and left at room temperature for 2 hours to perform Blocking. After washing three more times, PIV
KA- II Standard (Warfarin-administered patient plasma: commercial product) or Prothrombin Standard
(Commercially available product) is added in an amount of 0.1 ml, and reacted at room temperature for 2 hours. After washing, the peroxidase-labeled anti-human prothrombin IgG (0.1 M T) obtained in 3) of Example 1 above was used.
ris-HCl, 0.1 M NaCl, 0.2% Tw
een-20.5% NRS, 50% Block Ac
e) was added, and the mixture was reacted at room temperature for 1 hr.
After 1hr added _{MB) 0.1ml 2N-H 2 SO} 4
Add 0.05 ml to stop the reaction, and perform colorimetry with a 450/650 nm filter. The results are shown in Table 2, Table 3, Table 4, Table 5, and Table 6.

[0031]

[Table 2]

In each case, the antibody was immobilized by changing the mixing ratio of the antibody at a certain concentration, and the results of ELISA showed that the reactivity when mixed was about 10 times higher than that of the solid phase containing only 2G4 antibody. I have. Almost no reactivity was observed with the 1C9 antibody alone. There is not much difference in reactivity depending on the mixing ratio. Furthermore, in order to examine the reactivity of the 1C9 antibody with prothrombin, first, the reactivity with prothrombin when a mixed antibody was prepared was examined in a buffer system and then in a serum system. The results are shown in Tables 3, 4, 5, and 6.

[0033]

[Table 3]

[0034]

[Table 4] In the buffer-added system, the reactivity with prothrombin increases as the ratio of the 1C9 antibody increases. Therefore, a system to which serum was added was also examined.

[0035]

[Table 5]

[0036]

[Table 6]

When used as a mixed antibody, a result was obtained in which the reactivity of prothrombin was completely suppressed by the addition of serum. Further, in order to confirm the validity of this method, a sample was measured, and comparative data were summarized for a case where only the 2G4 antibody was used. The conditions of the solid phase antibody used were a system using only the 2G4 antibody and a 2G4 / 1C9 (1/1) mixed antibody system. There was no effect on the measured values due to the difference in the solid phase antibody. It was observed that by using an antibody having a recognition site close to that of the mixture, the reactivity was dramatically increased, and the sensitivity of the measurement system could be further improved. The use of a mixture of two kinds of antibodies not only increases the reactivity, but also leads to the result of exaggerating the specificity of one. Although it is difficult to explain the cause of this, the change in the three-dimensional structure when adsorbed on the solid phase by mixing indicates that a
It is speculated that the fifinity may increase.

[0038] 3) specificity for 2G4 antibody PIVKA- II, confirmed by inhibition tests for 2G4 antibody PIVKA- II antigen (8/4/2/1 / 0.5 / 0Au
/ Ml) and 2G4 antibody (40/20/10/5 / 2.5)
/ 0 μg / ml), immediately add to the 2G4 immobilized plate, and react overnight (4 ° C.). The following is a normal EI
Performed according to A. That is, 100 µg / ml of a POD-labeled anti-prothrombin antibody was added, and after reacting at 4 ° C for 1 hour, 200 µg / ml of the substrate was added, followed by treatment at room temperature for 30 minutes, and 50 µg / ml of a reaction stop solution was added. From these results, the specificity of the 2G4 antibody for PIVKA- II and the test for inhibition of the 2G4 antibody in the PIVKA- II antigen were used to determine the affinity of the present antibody.

Example 4 (Non-Specific Reaction in ELISA System) 1) Non-Specific Reaction Using Autoantibody (RAPA) Specimen It is the presence or absence of a non-specific reaction in the ELISA system. There are various possible causes for this. There is a reaction of the labeled antibody to non-specific adsorption to the solid phase plate, or a non-specific reaction to autoantibodies. These can be considerably improved or not at all possible by selecting a buffer component. PIV of the present invention
Non-specific reactions in the KA- II ELISA system were examined. The measurement was performed using a sample of RAPA, which is a test item for autoantibodies. The detected sample was measured again using a commercially available kit and confirmed. The method is as follows: 0.1 ml of a buffer solution and 0.1 ml of a sample are added to a solid-phased plate (dried and stored in a pack), and the mixture is reacted at 4 ° C. overnight. Subsequent operations were performed as usual. As a result, there was an example in which a sample with a high RAPA measurement value was positive for PIVKA- II, but the same result was obtained with a commercially available kit, and there was no correlation with the measurement result of the autoantibody.

2) Suppression of Nonspecific Reaction A PIVKA- II negative sample was left at 56 ° C. for 30 minutes to be immobilized, and this was used as an artificially prepared nonspecific sample to be used as an index of the suppression effect. The method was carried out as usual, and the inhibitory effect of the buffer component on the nonspecific reaction in the measurement system was examined. Whether mouse IgG or rabbit serum is added to the buffer; the basic buffer is 0.01 M Tris - HCl / 0.07
5M NaCl / 0.01% Tween-20,
The study was conducted under two conditions: a condition of adding to the buffer of the primary reaction system and a condition of adding to the buffer for dilution of the labeled antibody of the secondary reaction system. Presence or absence of addition Primary reaction Reaction buffer (+) (+) (-) Secondary reaction Labeled antibody dilution buffer (+) (-) (+) As a result of the measurement, addition of rabbit serum to the primary reaction exceeded a certain level. Then, a result showing a remarkably nonspecific reaction was obtained, but it was found that the addition of rabbit serum to the secondary reaction was suppressed even in a large excess. On the other hand, when the addition of mouse IgG became more than a certain amount in the secondary reaction, the whole reaction itself was suppressed, and it was determined that the addition to the secondary reaction system was more effective.

Under the conditions selected in, NaCl and Tw
een-20 was added, and the addition concentration was examined. 0.01% mIgG, 0.1% mIgG in basic buffer,
5.0% NRS was added, and the following concentration of NaCl or Tw was added.
Add een-20. NaCl (M) 0.075 0.15 0.3 0.6 Tween-20 (%) 0.01 0.2 0.5 1.0 As a result, when NaCl was 0.75 M or more, the reactivity was reduced to almost し, and the non-specific reaction was also proportional thereto. When Tween-20 becomes 0.2% or more, mouse I
In the system to which gG was added, the reactivity tended to decrease, but the non-specific reaction did not change much. From these results,
As an additive to the basic buffer to suppress non-specific reactions, 0.01% mIgG or rabbit serum
It is considered appropriate to add 75 M NaCl and 0.2% Tween-20.

Example 5 (Measurement of Sample) 1) A standard curve was prepared using a 10-fold dilution of a PIVKA- II standard, warfarin-administered patient plasma, which was diluted to 8 Au / ml, and doubled. As the monoclonal antibody-immobilized plate, a 2G4 / 1C9 mixed antibody was used, and an HRP-labeled rabbit polyclonal antibody was used as a labeled anti-prothrombin antibody. 0.2 ml of the mixed antibody of 0.2 μg / ml was added to an immunoplate (Lab System Breakable Plate) (0.2 ml / well), incubated at 4 ° C. overnight, washed three times, and then washed with 50% Block Ace. The solution was treated with 0.25 ml and
Incubate for hours. Then 2% after 3 washes
0.25 ml of a secondary coating solution composed of saccharose and 1% BSA is added, left at room temperature for 2 hours, decanted for 30 minutes, and dried at room temperature overnight to obtain a solid-phased plate. The measurement was performed by adding 0.1 ml of the measurement buffer and 0.1 ml of the sample to the solid-phased plate, incubating overnight at 4 ° C., washing 4 times, adding 0.2 ml of HRP-labeled anti-prothrombin antibody, Incubate for 2 hours, wash 5 times, and wash with substrate solution (TM
B) Add 0.2 ml, incubate for 1 hour at room temperature, add 0.05 ml of reaction stop solution and add colorimetric (5
40/650 nm). Similarly, ascites from liver cancer was measured.

[0043]

[Table 7]

[0044]

[Table 8]

Similarly, for 127 clinical cases including hepatocellular carcinoma, P was determined using the measurement system using the mixed antibody of the present invention.
IVKA- II was measured. α-fetoprotein (AF
P) and other liver disease related items, GOT, GPT,
The results for total bilirubin and albumin are summarized in Tables 9 to 15.

[0046]

[Table 9]

[0047]

[Table 10]

[0048]

[Table 11]

[0049]

[Table 12]

[0050]

[Table 13]

[0051]

[Table 14]

[0052]

[Table 15]

As a result, it has been found that a sufficient measurement result can be obtained in the measurement system using the mixed antibody of the present invention.

EFFECT OF THE INVENTION PIVKA- II produced by liver cancer-derived cells
Excellent sensitivity can be achieved by using a mixture of at least two of the monoclonal antibodies recognizing PIVKA- II obtained by using as a immunogen. In particular, in a measurement system in which this mixed antibody is immobilized, PIVKA- II can be specifically measured even if an anti-PIVKA- II antibody that recognizes prothrombin is used unexpectedly. Thus PIVKA
- II can specifically and simply measure, it is possible to apply to such diagnosis as liver cancer marker.

[Brief description of the drawings]

FIG. 1 shows the relationship between the mixing ratio of a 2G4 / 1C9 antibody mixture and the reactivity in PIVKA- II measurement.

FIG. 2 shows the relationship between the mixing ratio of the 2G4 / 1C9 antibody mixture and the reactivity in PIVKA- II measurement.

FIG. 3 shows the relationship between the mixing ratio of a 2G4 / 1C9 antibody mixture and the reactivity with prothrombin.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-5-284994 (JP, A) JP-A-7-20127 (JP, A) JP-A-60-60557 (JP, A) 249108 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) G01N 33/53 G01N 33/577

Claims (6)

(57) [Claims] (1) sandwiching PIVKA- II in a sample
Upon using the type assay measuring immunologically, at least a monoclonal antibody against the following two PIVKA- II: (i) a and PIVKA-II does not react with prothrombin
Only reacted with monoclonal antibodies (ii) prothrombin to react and PIVKA-II with anti
A method for measuring PIVKA- II , wherein a corresponding monoclonal antibody is blended and used as an immobilized antibody . 2. Sandwiching PIVKA- II in a sample
Using a type assay Upon immunologically measured, and does not react with (a) (i) Prothrombin PIVKA-
Both monoclonal antibodies reactive II only with reaction to and PIVKA- II monoclonal antibody reactive with (ii) prothrombin is immobilized to a solid support, the the immobilized carrier of the mixed antibody, analyte (B) then, if necessary, washing the carrier , and (iii) a contact reaction with a labeled anti-prothrombin antibody. (C) Then, after optionally washing the carrier, a signal generated by the label is obtained. A method for measuring PIVKA- II , wherein the measurement is performed as an index of PIVKA- II by detecting. 3. Sandwiching PIVKA-II in a sample
In performing immunological measurement using a type assay, (a) (i) Not reacting with prothrombin and PIVKA-
Reacts only with II and hybridizes with FERM P-15070
Monoclonal antibody 2G4 produced by Doma
(Ii) reacts with prothrombin and is also PIVKA-II
React, FER To the hybridoma of MP-15069
Monoclonal antibody 1C9 produced from solid phase carrier
The immobilized carrier of the mixed antibody immobilized on
Contact reaction with (b) Next, after washing the carrier if necessary, (iii) the labeled anti-pro
Contact reaction with thrombin antibody, (c) Next, the carrier is washed, if necessary, and then generated by a label.
By detecting the signal, it becomes the index of PIVKA-II
Of measuring PIVKA-II characterized by performing measurement by using
Law. 4. Monocloner reacting with PIVKA-II
Le antibody is a monoclonal antibody that recognizes more produced human PIVKA- II hybridoma obtained by fusion of tumor cells that do not produce spleen cells and immunoglobulin immunized with human hepatoma cells derived PIVKA- II
The method according to any one of claims 1 to 3, wherein
Law. 5. A method for sandwiching PIVKA-II in a specimen.
Used for immunological measurement using type assay
At least the following two types of immobilized antibodies for the measurement.
Monoclonal antibodies against PIVKA-II: (i) not reactive with prothrombin and with PIVKA-II
Only reacted with monoclonal antibodies (ii) prothrombin to react and PIVKA-II with anti
It is characterized by containing an antibody immobilized by blending the corresponding monoclonal antibody
PIVKA-II measurement reagent. 6. The method according to claim 6, wherein the immobilized antibody does not react with (i) prothrombin and has PIVKA-II.
Only reacts and hybridizes FERM P-15070
Reacts with the monoclonal antibody 2G4 produced by MA and (ii) prothrombin and also with PIVKA-II
In response to the FERM P-15069 hybridoma
Mixed antibody with monoclonal antibody 1C9
It is immobilized on a solid support, and as a labeled antibody for the measurement, an anti-prothrombin antibody
The measurement according to claim 5, wherein the body is contained.
reagent.