JPH0943237A - Hybridoma born from anti-pika-2 antibody and immunological measuring method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To quickly, accurately and simply measure PIVKA-2 in a specimen by using at least two kinds of monoclonal antibodies for PIVKA-2. SOLUTION: A monoclonal antibody which does not react with prothrombin and reacts only with PIVKA-2, and another nomoclonal antibody which reacts with both of them are combined and used to obtain excellent responsibility. In the production of these antibodies, an animal is immunized by making an immune source of human PIVKA-2, and cells born from the obtained antibody is cell-fused with myeloma cells, and detained antigen-forming cell is fused with myeloma cell. For instance, spleen cell suspension of an immunized mouse and the myeloma cell stocks are set in cultivation ground, and a cell fusion agent is added thereto. After fusion reaction processing, centrifuged cells are removed to another cultivation ground for selection, aimedhybridoma (cell fusion) is selected and cloned. The cloned hybridoma is cultivated in a suitable cultivation ground for breeding and a desired monoclonal antibody can be obtained from the supernatant of the cultivation ground.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to PIVKA-in a sample as a liver cancer marker or an indicator substance for diagnosis.
The purpose of the present invention is to develop a method aiming to be useful for rapid, accurate, and convenient PIVKA-2 measurement and medical diagnosis and treatment based on the measurement.

[0002]

PIVKA-2 is a precursor of prothrombin which is a blood coagulation factor II. In the presence of reduced vitamin K in hepatocytes, 10 glutamic acid residues (Glu) near NH ₂ terminus are present. Is converted into γ-carboxyglutamic acid (Gla) by vitamin K-dependent carboxylase, resulting in mature prothrombin. However, it is known that due to vitamin K deficiency or the administration of vitamin K antagonists such as warfarin, this Glu is not converted into Gla and is released into the blood as it is as an abnormal prothrombin. This abnormal prothrombin is because Glu is not converted to Gla,
It is unable to bind to Ca ions and, as a result, does not have normal coagulation activity and appears in plasma. Generally, such abnormal prothrombin is detected by PIV.
I call it K-2. In 1984, Liebman et al.
Since it was reported that PIVKA-2 was detected at a high rate in liver cancer patients, PIVKA-2 has been attracting attention as a new liver cancer marker different from α-fetoprotein or an indicator substance for diagnosis. Until now PIVKA
-2 was measured by a two-dimensional immunoelectrophoresis method using the presence or absence of the binding property with Ca salt, or after the prothrombin was adsorbed with Ca or Ba salt, the antigenicity of abnormal prothrombin was measured. However, those methods have problems in terms of specificity, sensitivity, operability and the like. In particular, it was difficult to more specifically and sensitively discriminate whether or not a part or most of Gla existing in only 10 residues in prothrombin remained as Glu.

As a method for solving these problems, development of an immunological assay method using an antibody has been promoted. The assay method using a polyclonal antibody specific to PIVKA-2 has a problem that the sensitivity is low, and a radioactive substance that has 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 that specifically recognizes -2 can be produced, and that it can be used for measurement by a more sensitive EIA. In the method for measuring PIVKA-2 using such a monoclonal antibody, PI
An enzyme immunoassay method has been adopted in which both a monoclonal antibody that specifically recognizes VKA-2 and a polyclonal antibody against prothrombin are used, and one of them is used as an immobilized antibody and the enzyme labeled antibody is measured. However, it has been pointed out that this method requires complicated operations and requires a long time for measurement, or that the measurement sensitivity still remains unsatisfactory. Furthermore, as a method for solving these problems, (1) human PIV
A first group of monoclonal antibodies that specifically react with KA-II, but not with human prothrombin, (2)
Three types of monoclonal antibodies were prepared: a second group of monoclonal antibodies reactive with both human thrombin and human prothrombin, and (3) a third monoclonal antibody reactive with human prothrombin but not human thrombin. Or (A) a combination of the first monoclonal antibody group and the second or third monoclonal antibody group, or (B) the first monoclonal antibody group and the second monoclonal antibody group. Using a combination of the above-mentioned third group of monoclonal antibodies,
A method aiming at rapid, accurate, and convenient PIVKA-2 measurement has been proposed.

[0004]

Therefore, it has been an object to develop a method for measuring PIVKA-2, which is superior in terms of simplicity and sensitivity, by using a monoclonal antibody that can be easily obtained. In addition, there is no confirmed report on the identity of PIVKA-2 derived from a liver cancer patient and PIVKA-2 derived from a coagulation disease-derived patient or a drug-administered patient such as a vitamin K antagonist, including a three-dimensional structure. In order to use it as a liver cancer marker or an indicator substance for diagnosis, a monoclonal antibody capable of more specifically recognizing PIVKA-2 derived from a liver cancer patient as an antigen to be measured is considered desirable, but until now, There is no report that a monoclonal antibody was produced using PIVKA-2 directly derived from liver cancer.

[0005]

DISCLOSURE OF THE INVENTION The inventors of the present invention have a feature of more specificity and an advantage in terms of measurement sensitivity.
-2 as a result of intensive studies to measure -2, hepatoma cell line PCL / PRF / 5 (Alexande
It was found that by using PIVKA-2 obtained from the culture supernatant of r) as an antigen, a monoclonal antibody having a specificity can be prepared, and further, a plurality of monoclonal antibodies thus obtained can be used. Therefore, they 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-2 in a sample.
P for measuring at least two PIVKA-2 monoclonal antibodies against PIVKA-2
A method for measuring IVKA-2 is provided. As the two types of PIVKA-2 monoclonal antibodies used in the above method, for example, (1) a monoclonal antibody that does not react with prothrombin but only PIVKA-2 and (2) a reaction with prothrombin are also recognized. , PI
Examples of the combination of VKA-2 and the monoclonal antibody having substantially the same reactivity as the monoclonal antibody of (1) above are also mentioned, but other monoclonal antibodies against PIVKA-2 are the same, but substantially other It can be a monoclonal antibody that can specifically recognize each kringle region in a region other than the 10 amino acid residues near the NH ₂ terminus of the prothrombin structure.

Further, according to the present invention, in addition to the above-mentioned two or more monoclonal antibodies, (i) a group of monoclonal antibodies showing reactivity with both human thrombin and human prothrombin, and (ii) showing a reaction with human prothrombin. ,
Both a monoclonal antibody that does not react with human thrombin and (iii) a polyclonal antibody against human thrombin can be used in combination, but it is preferable to use a monoclonal antibody as a partner of the combination. When used as a mixed antibody, the reactivity of prothrombin was unexpectedly completely suppressed. Furthermore, when comparing, for example, the case of using only a single antibody and the case of mixing at least two kinds of antibodies, not only the effect on the measured value due to the difference in solid phase antibody is not observed,
It has been observed that the use of a mixture of antibodies having similar recognition sites dramatically improves the reactivity and can further improve the sensitivity of the measurement system. By using two kinds of antibodies in combination, not only the reactivity is increased, but also the result that the specificity of one is maximized is derived. It is difficult to explain the factors that cause this, but it is speculated that the affinity of the antibody may increase from the change in the three-dimensional structure upon adsorption to the solid phase by mixing. The production of monoclonal antibodies against PIVKA-2 requires human PIVKA-2, which can be used as an immunogenic antigen. P as an antigen
For the preparation of IVKA-2, for example, 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 after adsorbing and removing prothrombin, it is subjected to 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-2. These naturally derived PIs
In addition to VKA-2, preferably cultured cells such as PIV
PIVKA-2 can be obtained using the KA-2 producing cell line. PIVKA-2 producing cell lines include P
Examples thereof include cells producing IVKA-2 and α-fetoprotein (AFP), and examples include cells derived from human liver cancer. For example, human hepatoma-derived cells PLC / PRL / 5
(Alexander) or the like can be preferably used. The PIVKA-2 producing cell line is cultured using a medium or a culture method known in the art or a method substantially similar thereto, preferably in the presence of a vitamin K antagonist such as warfarin, and then cultured. The PIVKA-2 produced in Qing was subjected to the above-mentioned ion exchange chromatography treatment, and prothrombin and PIV
Purified human PIVKA-2 is obtained by affinity chromatography treatment using a polyclonal antibody against the common portion with the C-terminal side of KA-2. Also, recombinant PIVKA-2 can be used. The PIVKA-2 thus prepared may be further used as an immunogenic conjugate or the like, but it can be used as it is in admixture with a suitable adjuvant to immunize an animal.

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

The present invention provides at least two monoclonal antibodies that specifically bind to PIVKA-2. The monoclonal antibody according to the present invention can be obtained by immunizing an animal with the purified human PIVKA-2 obtained according to the present invention as an immunogen by a known method, and then by a method known in the art or a method similar thereto. Milstein et al. (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, (4) hybridoma Selection of (fused cells) and monocloning, (5) production of monoclonal antibody,
(6) If necessary, it can be prepared and obtained by a process such as culturing and ascites for large-scale acquisition. Immunization of Animals with Immunogenic Antigens To immunize animals, for example, Shigeru Muramatsu, et al., Experimental Biology Course 14, Immunobiology, Maruzen Co., Ltd., 1985, Japan Biochemical Society, ed. 5. Immunobiochemistry research method, Tokyo Kagaku Dojin, 1986, edited by The Biochemical Society of Japan, New Chemistry Experimental Course 12, Molecular Immunology III, Antigen / Antibody / Complement, Tokyo Kagaku Dojin, 1992, etc. It can be carried out according to. Examples of the adjuvant used together with the antigen include Freund's complete adjuvant, Ribi adjuvant, pertussis vaccine, BCG,
Examples include lipid A, liposome, aluminum hydroxide, silica and the like. Immunization is performed using animals such as mice such as BALB / c. The dose of the antigen is, for example, about 1 to 400 μg / animal for a mouse, and it is generally injected intraperitoneally or subcutaneously into a host animal, and thereafter every 1 to 4 weeks, preferably every 1 to 2 weeks. , Subcutaneous, intravenous or intramuscular booster 2-10
Repeat about once. BALB as mouse for immunization
In addition to the / c mouse, an F1 mouse of BALB / c mouse and other mouse can also be used. If necessary, an antibody titer measuring system can be prepared and the antibody titer can be measured to confirm the degree of animal immunity. On the other hand, according to the present invention, recombinant PIVKA-2 can be used to produce a polyclonal antibody against PIVKA-2 and its production. In such a case, as the animal used, mammals and birds can be used, and for example, cow, horse, goat, sheep,
Pigs, rabbits, mice, rats, guinea pigs, monkeys, dogs, cats, chickens and the like can be mentioned. The antibody may be an antiserum or may be a more purified antibody, and its isolation and purification can be carried out in the same manner as the monoclonal antibody described below.

Before cell fusion, it is necessary to first prepare the myeloma cells to be used. The infinitely proliferative strain (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, a cell line other than SP2 / 0-Ag14 (SP2, Nature,
276, 269 to 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 can be used. The 8-azaguanine-resistant mouse myeloma cell line is a Dulbecco MEM medium (DMEM medium), RPMI-
To a cell culture medium generally known in the art such as 1640 medium, antibiotics such as penicillin, streptomycin, amikacin, fetal calf serum (FCS), etc. are added,
8-azaguanine (eg 5-45 μg / ml)
However, the required number of cell lines can be prepared by subculturing in a normal medium 2 to 5 days before cell fusion. 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.

Cell fusion between antibody-producing cells and myeloma cells can be carried out as follows. An animal, for example, a mouse immunized according to the above immunization step, has its spleen removed 2 to 5 days after the final immunization to obtain a splenocyte suspension. In addition to splenocytes, lymph node cells in various parts of the body can be obtained and used for cell fusion. The spleen cell suspension thus obtained and the myeloma cell line prepared according to the above-mentioned steps are used, for example, in a minimum essential medium (MEM medium), DME.
It 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 4,0.
A polyethylene glycol of 00 can be used more preferably. The concentration of polyethylene glycol in the fusion medium is
For example, it is preferably set to 30 to 60%.
If necessary, a small amount of dimethyl sulfoxide or the like can be added to promote fusion. As the cell fusion agent, various other ones known in the art can be used. Examples of such a cell fusion agent include inactivated Sendai virus (HVJ: Hemagglutinating v
irus of Japan) and the like. The ratio of spleen cells (lymphocytes): myeloma cell line used for fusion may be, for example, 1: 1 to 20: 1, and more preferably 4: 1 to 7: 1. The fusion reaction is performed for 1-10 minutes, then RPMI-16
Add cell culture medium, such as 40 medium. The fusion reaction treatment can be performed multiple 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 further cloned. Examples of the selection medium include hypoxanthine,
FCS-containing MEM containing aminopterin and thymidine
Medium, medium such as RPMI-1640 medium, so-called HAT
The medium may be mentioned. The selection medium can be replaced generally by adding the volume dispensed to the culture plate and the equivalent volume on the next day, and then replacing the HAT medium by half every 1 to 3 days. You can also make changes to. On the 8th to 16th days after the fusion, the so-called HT medium without aminopterin can be replaced every 1 to 4 days. Mouse thymocytes, for example, can also be used as a feeder, which may be preferred. The culture supernatant of the culture well in which the proliferation of the hybridoma is prominent is measured, for example, by a radioimmunoassay (RIA), an enzyme-immunoassay (ELISA), a fluorescence-immunoassay (FIA), or a fluorescence-induced cell separator (FACS).
For example, PIVKA-2 or a fragment peptide thereof is used as an antigen, or the target antibody is measured using a labeled anti-mouse antibody, etc. to perform screening or separation. For example, the screening method can be performed by the following 3 methods (EIA), and the target clone can be selected;
The IgG producing clones are checked using the anti-mouse IgG solid phase-culture supernatant-peroxidase (POD) -labeled anti-mouse IgG system; the PIVKA-2 purified antigen solid phase-culture supernatant-POD-labeled anti-mouse IgG system is used. The PIVKA-2 producing clones are used to check; and the prototonbin antigen solid phase-culture supernatant-POD labeled anti-mouse IgG system is used to check the prototonbin producing clones. The hybridoma producing the target antibody is cloned. The cloning can be performed by picking up a colony in an agar medium or by the limiting dilution method. The limiting dilution method can be used more preferably. Cloning is preferably performed multiple times.

The hybridoma (fused cell) thus cloned can be cultured and used for the production of a monoclonal antibody. The resulting hybridoma strain is F
The desired monoclonal antibody can be obtained from the culture medium by culturing in a suitable growth medium such as CS-containing MEM medium and RPMI-1640 medium. In order to obtain a large amount of antibody, ascites of the hybridoma may be mentioned. In this case, each hybridoma is transplanted into the abdominal cavity of a histocompatibility animal of the same strain as the myeloma cell-derived animal and proliferated, or each hybridoma is transplanted into, for example, a nude mouse or the like, proliferated, and produced in the ascites of the animal. The obtained monoclonal antibody can be recovered and obtained. The animals were treated with pristane (2, 6,
Mineral oil such as 10,14-tetramethylpentadecane) can be intraperitoneally administered, and after the treatment, hybridomas can be grown and ascites can be collected. The ascites fluid may be used as it is, or may be a conventionally known method, for example, salting out such as ammonium sulfate precipitation, gel filtration using Sephadex, ion exchange chromatography, electrophoresis, dialysis, ultrafiltration, affinity chromatography. It can be purified by high performance liquid chromatography or the like and used as a monoclonal antibody. Preferably, the ascites fluid containing the monoclonal antibody, after ammonium sulfate fractionation, such as DEAE-Sepharose,
It can be purified and separated by treatment with an anion exchange gel and an affinity column such as a protein A column. Particularly preferably, an affinity chromatography in which an antigen or an antigen fragment (for example, a synthetic peptide, a recombinant antigenic protein or peptide, a site specifically recognized by an antibody, etc.) is immobilized, an affinity chromatography in which protein A is immobilized, etc. Can be mentioned. The antibody may preferably be a purified antibody which has been subjected to denaturation treatment. For example, the result of examining the denaturation treatment effect of the purified antibody shows that 2G4
With respect to the antibody, the reactivity of the antibody can be increased by about 10 times by denaturing treatment as compared with untreated. Examples of the denaturing reagent include 6M guanidine hydrochloric acid.
As a factor for increasing the reactivity, it is considered that the denaturing effect of the Fc portion of IgG may enhance the adsorptivity to the solid phase, and treatment for such purpose can be adopted. Fab, F obtained by treating these antibodies with an enzyme such as trypsin, papain, pepsin, etc., and optionally reducing them.
It may be used as an antibody fragment such as ab ′ or F (ab ′) ₂ . As the antibody that gives the labeled substance, I
It is possible to use the gG fraction, and further the specific binding part Fab ′ obtained by reduction after digestion with pepsin.

As the label, an enzyme, an enzyme substrate, an enzyme inhibitor, a prosthetic group, a coenzyme, an enzyme precursor, an apoenzyme, a fluorescent substance, a pigment substance, a chemiluminescent compound,
Examples thereof include a luminescent substance, a coloring substance, a magnetic substance, metal particles such as gold colloid, and a radioactive substance. Examples of the enzyme include dehydrogenase, reductase, oxidoreductase such as oxidase, for example, amino group, carboxyl group, methyl group, acyl group, transferase that catalyzes transfer of phosphate group, for example, ester bond, Examples thereof include hydrolases that hydrolyze glycoside bonds, ether bonds, peptide bonds and the like, lyases, isomerases and ligases. Enzymes can also be used for detection by using multiple enzymes in combination. For example, enzymatic cycling can be utilized. Typical radioactive isotope labeling isotopes include [ ³² P], [ ¹²⁵ I], [ ¹³¹ I],
[ ³ H], [ ¹⁴ C], [ ³⁵ S] and the like can be mentioned. As typical enzyme labels, peroxidase such as horseradish peroxidase, galactosidase such as Escherichia coli β-D-galactosidase, maleate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose oxidase, glucoamylase,
Examples thereof include acetylcholinesterase, catalase, calf intestinal alkaline phosphatase, and alkaline phosphatase such as 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. The luciferin and luciferase system 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 type electrode, a polymer film electrode, or the like. The enzyme label can be replaced with a biotin label and an enzyme-labeled avidin (streptavidin). The label can also use a plurality of different types of labels. 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 a reaction between an amino group and an aldehyde group, and the method can be appropriately selected from known methods, methods that can be easily performed by those skilled in the art, and methods in which they are modified. Further, a condensing agent that can be used for preparing 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′-ethylene bismaleimide, ethylene glycol bissuccinimidyl succinate, and bis. 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 / measurement in the present invention can be carried out by immunostaining, for example, tissue or cell staining, immunoassay, for example, competitive immunoassay or noncompetitive immunoassay, and radioimmunoassay, ELISA, latex agglutination method, etc. are used. The measurement can be performed with or without BF separation. A latex agglutination method and an enzyme immunoassay method are preferable, and a sandwich type assay is more preferable. For example, in a sandwich type assay, PIVKA-
One of the antibodies to 2 is detectably labeled. Another antibody capable of recognizing the same antigen is immobilized on the solid phase. Incubation treatment is performed in order to sequentially react the sample with the labeled antibody and the immobilized antibody, if necessary, and after separating the unbound antibody, the labeled substance is measured. The amount of label measured is proportional to the amount of antigen, PIVKA-2. This assay is referred to as a simultaneous sandwich type assay, a forward sandwich type assay, or a reverse sandwich type assay, 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 or buffer solution, temperature or incubation treatment 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 while using a normal experimental method.

For example, the enzyme immunoassay method using the sandwich type assay method according to the present invention can be carried out as follows. The assay system is a solid phase, at least two types of antibodies against PIVKA-2 as solid phase immobilized monoclonal antibody components (for example, (i) a monoclonal antibody that does not react with prothrombin and (ii) prothrombin) Reacts with and PIVKA-2
(Reacting monoclonal antibody), a standard antigen or a sample sample for measurement, an enzyme-labeled antibody (second antibody) and a substrate. As the solid phase, those described below can be used. The immobilization of the monoclonal antibody component can be carried out by, for example, placing the monoclonal antibody component dissolved in a buffer solution in a solid phase, for example, a well made of polystyrene, and leaving it at about 4 ° C. overnight to coat the surface of the solid phase. The methods described below can be used as appropriate. The uncoated solid surface or the like can be treated with bovine serum albumin or the like to suppress nonspecific reactions. As the standard antigen, PIVKA-2-containing plasma having a known concentration can be used. Usually, the standard antigen may be diluted with a buffer solution containing bovine serum albumin or the like, placed in the coated well, reacted, and then unreacted substances are washed off. Next, as the enzyme-labeled antibody (second antibody), P
An OD-labeled anti-human prothrombin antibody can be preferably used. This anti-human prothrombin antibody is the above-mentioned PI
It can be obtained by immunizing an animal with human prothrombin as an antigen in the same manner as an antibody against VKA-2. The animals used include mammals and birds, and examples thereof include cattle, horses, goats, sheep, pigs, rabbits, mice, rats, guinea pigs, monkeys, dogs, cats, chickens, and the like. For example, Shigeru Muramatsu,
Others, Experimental Biology Course 14, Immunobiology, Maruzen Co., Ltd., 1985, Japan Biochemical Society Edition, Sequel Biochemistry Laboratory Course 5, Immunobiochemistry Research Method, Tokyo Kagaku Dojin, 1986, Japan Biochemical Society Edition , Shinsei Chemistry Laboratory 12, Molecular Immunology II
I, antigen / antibody / complement, Tokyo Kagaku Dojin, 1992 and the like. The antibody may be antiserum or may be more purified,
For example, the isolation and purification can be performed in the same manner as the above monoclonal antibody. The anti-human prothrombin monoclonal antibody can also be obtained by using human prothrombin as an antigen in the same manner as the above-mentioned antibody against PIVKA-2.

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 antigen-antibody reaction and the like are known, and of course in the present invention, it can be selected from these known carriers and 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,
Polyvinylidene fluoride, polyvinyl acetate, polymethacrylate, polystyrene, styrene-butadiene copolymer, polyacrylamide, crosslinked polyacrylamide, styrene-methacrylate copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer, etc. Albumin, collagen, gelatin, dextran, agarose, cross-linked agarose, cellulose, microcrystalline cellulose, carboxymethyl cellulose, natural or modified cellulose such as cellulose acetate, cross-linked dextran, polyamide such as nylon, polyurethane, polyepoxy resin and other organic polymers Substances, and those obtained by emulsion polymerization of them, such as latex particles (for example, 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. Furthermore, filter paper, beads, inner wall of test container, for example, test tube, titer plate, titer well, glass cell, cell made of synthetic material such as synthetic resin cell, glass rod, rod made of synthetic material, thickening the end, Alternatively, the surface of a solid substance (object) such as a thin rod, a rod having round protrusions or flat protrusions at its end, or a thin rod may be used.

An antibody can be bound to these carriers, preferably a monoclonal antibody which specifically binds to PIVKA-2 obtained in the present invention. The binding between the carrier and those involved in these antigen-antibody reactions may be carried out by a physical method such as adsorption, a chemical method using a condensing agent or the like, or an activated method, or the mutual method. It can be performed by a method utilizing a chemical bonding reaction. In the present invention, 4-hydroxyphenylacetic acid, 1,2-phenylenediamine, tetramethylbenzidine, etc. together with horseradish peroxidase (POD), and umbelliferylgalactoside, nitrophenylgalactoside, etc., are used in the formation of signals such as substrates. It can be used with enzyme reagents such as D-galactosidase, glucose-6-phosphate dehydrogenase, and quinol compounds such as hydroquinone, hydroxybenzoquinone, and hydroxyanthraquinone,
Those capable of forming thiol compounds such as lipoic acid and glutathione, phenol derivatives, ferrocene derivatives and the like by the action of enzymes can be used. Examples of the fluorescent substance or 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 salt, luminol such as lumiferin, luciferase, and equalin, imidazole, oxalate ester, rare earth chelate compound, and coumarin derivative.

According to the assay method of the present invention, a labeled antibody reagent such as a monoclonal antibody labeled with a substance to be assayed by an enzyme and the antibody bound to the carrier can be reacted successively or simultaneously. You can also The order of adding the reagents 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 carried out in an appropriate buffer system so as to maintain the optimum pH, for example, about pH 4-9. Particularly suitable buffers include, for example, acetate buffers, citrate buffers, phosphate buffers, Tris buffers,
Examples thereof include triethanolamine buffer, borate buffer, glycine buffer, carbonate buffer, Tris-hydrochloric acid buffer and the like. The buffering agents can be used as a mixture with each other in an arbitrary ratio. The antibody-antigen reaction is preferably carried out at a temperature between about 0 ° C and 60 ° C.

An antibody reagent such as a monoclonal antibody labeled with an enzyme and an antibody reagent bound to a carrier,
In addition, the incubation of the substance to be measured
It can be carried out until equilibrium is reached, but at a point much earlier than the equilibrium of the antibody-antigen reaction is achieved, the solid and liquid phases can be separated to stop the reaction after a limited incubation treatment, The extent of the presence of labels such as enzymes in either the phase or solid phase can be measured. The measurement operation can be performed using an automated measurement device, and the measurement is performed by detecting a display signal generated by the substrate being converted by the action of an enzyme using a luminescence detector, a photo detector, or the like. 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, reduce inhibitory effects, or activate measurement reactions. It can also 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, milk fermented substances, collagen,
It can be treated with gelatin or the like. As long as the purpose is to prevent non-specific binding reaction, those methods can be used without particular limitation. The sample to be measured by the measuring method of the present invention may be a solution or colloidal solution of any form, a non-fluid sample or the like, but preferably a biological sample such as blood, plasma, joint fluid, cerebrospinal fluid, Examples thereof include saliva, amniotic fluid, urine, other body fluids, cell culture medium, tissue culture medium, tissue homogenate, biopsy sample, tissue and cells. For example, blood, plasma, hepatocyte culture medium, liver tissue culture medium,
Examples thereof include liver tissue, liver tissue homogenate, and liver biopsy sample.

According to the present invention, there is provided a reagent which is directly used in the above measuring method. Such reagents can be a reagent set in a form suitable for measurement using at least two types of monoclonal antibodies against PIVKA-2. Typical reagents included in this reagent set are:
A solid phase on which at least two monoclonal antibodies against PIVKA-2 are immobilized, such as (i) a monoclonal antibody that does not react with prothrombin and (ii) a prothrombin that reacts only with PIVKA-2 and One characterized by including a plastic well coated with both monoclonal antibodies that also react with PIVKA-2. The reagent set also contains a standard antigen, an enzyme-labeled antibody (second antibody) such as a labeled anti-prothrombin antibody, and a substrate, a diluent, a lysate, a reaction terminator, and a buffer. 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, however, that they are also within the scope of the invention if they use the features of the invention. Example 1 (Preparation of reagent and method) 1) Purification of PIVKA-2 Using human liver cancer-derived cells PLC / PRL / 5 (Alexander), which is a producer strain of PIVKA-2 and AFP, from the culture supernatant, PIVKA- Get 2. The culture conditions were as follows: Alexander was cultured in the presence of fetal calf serum (FCS), and was changed to FCS Free under confluent conditions, and vitamin K antagonist Waferin (0.08 mg / ml) was administered. And promoted the production of PIVKA-2. PI in culture supernatant
VKA-2 concentration is 4.2 Au / ml, AFP concentration is 2
It was 00 ng / ml. The production amount increased by adding warfarin, and the result was that plateau was obtained under the condition that the concentration was 20 μg / ml or more and the culture period was 6 to 8 days. The concentration of warfarin was set to 80 μg / ml for the purpose of completely inhibiting the metabolism of prothrombin precursor to prothrombin. The culture supernatants obtained as described above were collected and purified as follows. First, D
Crude purification was performed by ion exchange chromatography using E-52 or CM-52 Cellulose. Under the elution conditions of 0.1-0.3 M NaCl gradient with respect to the pH of the equilibration buffer (3.0 to 7.0), set the conditions that enable separation of PIVKA-2 and contaminant proteins. And purified. It was confirmed that PIVKA-2 and prothrombin were favorably dissociated in the pH 5.0 buffer solution. In addition, prothrombin and PIV
Purification was performed by affinity chromatography using a polyclonal antibody against the common part on the C-terminal side of KA-2. As the affinity column, CN-Br activated Sepharose 4B (Pharmasis) was used, and 18.5 mg of the ligand per 1 g of the swollen gel was coupled by a usual method, which was used as a carrier for affinity chromatography. The equilibration buffer of the column was 0.2M PB, 0.2M NaCl pH 6.5,
For elution, 5M guanidine hydrochloride was used. PIVKA-2 obtained by the above purification method was obtained as a substantially single band by 5% acrylamide electrophoresis.

2) Preparation and purification of anti-PIVKA-2 monoclonal antibody PIVKA-2 purified in the above 1) (0.06 mg /
0.3 ml) of the same volume of Complete Freund's Adjuvant (Complete Freud Adjuva
nt) and subcutaneously administered to BALB / C mice (female, 6 week), and 2 weeks later, the same amount of the antigen as the initial amount was added to incomplete Freund's adjuvant (incomplete Fr).
euad Adjuvant). After boosting with the same amount of antigen (2 weeks later), 2
Weekly, 0.03 mg of antigen was added to the abdominal cavity Booster
And 5 days later, spleen cells were collected and cell fusion was performed. 1.8 × 10 ⁸ spleen cells and myeloma cells (P
AI) 6 × 10 ⁷ cells were fused with 50% polyethylene glycol. For fusion, both cells suspended in serum-free RPMI medium were centrifuged at 1500 rpm, the supernatant was removed with an aspirator, and then polyethylene glycol solution 1
Add ml gently and stir gently for 1 minute. Serum-free RPMI medium is slowly added and then 1500 minutes for 5 minutes
Centrifuge at rpm, remove supernatant with aspirator, and remove HA
T medium (serum-free RPMI medium with hypoxanthine (1
00 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,
On the 5th and 8th days, half of the culture medium (about 0.1 ml) was replaced with fresh HA.
Replace with T medium and half of the medium is replaced with fresh HT on day 10.
The medium was replaced with HAT medium containing no aminopterin.
Screening was performed for all wells in which the growth of hybridomas was visually observed. As the screening method, the following 3 methods (EIA) were performed to select clones.

Method for checking IgG-producing clone / A
anti mouse IgG solid phase-culture supernatant-POD
Check method of Anti mouse IgG PIVKA-2 producing clone / PIV
KA-2 purified antigen solid phase-culture supernatant-POD Anti
mouse IgG Prothronbin-producing clone check method / Prothronbin antigen solid phase-culture supernatant-POD
For each anti mouse IgG screen, a 96-well polystyrene plate was coated with each antigen and then washed with PBS (0.
The adsorbed peptide was removed by washing with (containing 05% Tween 20). Further, add 1 to the uncoated part of each well.
Blocked with% BSA. To each well was added 0.1 ml of the supernatant of the well in which the growth of hybridoma was confirmed, and the mixture 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. Then the substrate hydrogen peroxide and ο
-Phenylenediamine was added, and the degree of color development was measured at an absorbance of 492 nm using an absorptiometer for microplate (MRP-A4, Tosoh Corporation). Of these three methods, P
Clones that react with IVKA-2 but not (method) with Prothrombin were selected. Then, cloning was performed three times by the limiting dilution method to establish two types of anti-PIVKA-2 antibody-producing strains (2G4, 1C9). Hereinafter, the monoclonal antibody produced by this cell line is referred to as 2G4, 1C9. Above PIVKA-
The 2 antibody-producing hybridoma cell line 2G4 was placed under the consignment number FERM P-15 at the Institute of Biotechnology, Institute of Biotechnology, Industrial Technology Institute.
It has been deposited and stored as 070. Also PIVKA-2
The antibody-producing hybridoma cell line 1C9 was placed under the consignment number FERM P-150 at the Institute of Biotechnology, Institute of Biotechnology, Industrial Technology Institute.
It has been deposited and stored as No. 69. The immunoglobulin class of each antibody belonged to IgG, and its subclass was G1. These are mouse monoclonal
antibody isotyping Kit (A
Confirmation was carried out by Mersham Co.). The established hybridomas are cryopreserved by culturing in 10% FCS-containing RPMI1640 medium to logarithmically proliferate cells at 4 × 10 ⁶ to 10 ^7.
Store frozen at the number of cells / m. As a cryopreservation liquid, RPMI-1640 medium containing 20% FCS and 10% DMSO was used. In order to purify the antibody from the obtained hybridoma, first, 2G4 and 1C9cells were cultured to increase the number, and 3 × 10 ⁶ to 1 × were obtained under the condition of FCS Free.
10 ⁷ cells / ml is intraperitoneally administered to a mouse (BALB / c strain, ♀, 6 weeks old) to which pristane was intraperitoneally administered 1 week before. After about 7 to 10 days, ascites was collected, salted out twice with 40% ammonium sulfate, and the precipitate was frozen and stored. For purification from ascites, Protein A cellofine ch
It was performed by romatography. subclas
This antibody in which s is G1 is a buffer solution of pH 8.5 (50 mM
TrisHCl, 150 mM NaCl)
H5.0 (100 mM Cltric, 150 mM N
aCl) is eluted under mild conditions to obtain a purified antibody.
After dialysis with 20 mM PBS, 0.01% NaN3 is added for use.

3) Preparation of Peroxidase-Labeled Human Prothrombin IgG Rabbits were immunized with human prothrombin from DAKO, and the resulting sera were subjected to Q-Sepharose chromatography.
ography (50 mM TrisHCl, pH 8.
0) over 0-0.2M NaCl Gradient
Elute the IgG fraction with. The obtained IgG was used as human prothrombin affinity chromatogr.
Anti-human prothrombin / rabbit IgG using aphy
The specific antibody was purified. Formyl cellulofine is used for the affinity column, and 1.4m per 1g of gel
Couple g of prothrombin. The equilibration buffer was 0.1 M PB (pH 7.0), and the elution buffer was 0.1 citrate buffer. The ratio of the specific antibody obtained was 3 to 5% of total IgG. Anti-human prothrombin IgG2.5m obtained by the above operation
g and horseradish peroxidase (HRP) 2.0 mg
Was labeled by the maleimide method. 2 mg / 0.3 ml
HRP (0.1 mol / l, pH 7.0) with N- (ε
-Maleimidocaproyloxy) succinimide (EM
CS) (0.3 mg / 30 μl) is added, and the mixture is stirred at 30 ° C. for 60 minutes to replace the product buffer solution. 2.5m on the other hand
0.12 mg / 2 of AMSA in g / 0.25 ml IgG (0.1 mol / l, phosphate buffer, pH 7.5)
Add μl, stir at room temperature for 30 minutes, then 0.1mo
20 μl of 1 / l EDTA / 2Na, 50 μl of 1 mol / l Tris-HCl (pH 7.0) and 1 mol
/ L hydroxylamine hydrochloric acid 50μl was added, 30 ℃
Stir for 5 minutes at room temperature to displace the product buffer. Both were mixed in equal amounts and left at 4 ° C. overnight, and then gel filtered to obtain a labeled product. The HRP / IgG (molar ratio) of the obtained labeled product was in the range of 1.5 to 3.0. The IgG concentration per well was 1.4 to 3.0 ng. The dilution factor used in the reaction system is about 1 to 20,000 times. Besides, DA
Mice were immunized with human prothrombin from KO and cell fusion was performed to obtain an anti-human prothrombin monoclonal antibody (immunization method and fusion method conform to PIVKA-2). Peroxidase labeling is performed in the same manner as in 3) above to obtain a labeled antibody. As a result of measuring the samples, almost the same effectiveness was observed. The use of monoclonal antibodies has the advantage of eliminating the step of purification to specific antibodies. 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 the antibody and a low nonspecific reaction. The selection of immunoplates was examined based on the criteria.
For the solid phase buffer, 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 solid phase immobilization is performed at 4 ° C. overnight. Wash solution (10mPBS, 0.05% Tween
After washing 3 times with -20), add 0.2m of 10% skim milk.
1 at room temperature for 2 hr blocking (Blockin
g). After cleaning, PIVKA-2 standard product (St)
0.1 ml is added, and it is made to react 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) was added to the reaction solution at room temperature for 1 hr, 0.1 ml of the substrate solution was added, and after 30 minutes, the reaction was stopped with 2N sulfuric acid. Colorimetry is measured with a 450/650 nm filter. As a result, it was possible to select a plate having a high solid phase adsorption property of the antibody and a low nonspecific reaction. Examples of such a plate include a plate made by Labosystem, a plate made by Corning, and an M made by Nunc.
Ax sorp plate etc. are mentioned. 2) Selection of reaction buffer In ELISA, when the sample serum is directly reacted, the reaction to the solid phase antibody may be inhibited due to the influence of a certain substance in the serum component, and even in the PIVKA-2 system. This phenomenon is observed. One way to reduce this is to dilute the sample. This diluting effect also results in suppressing non-specific reactions, but it should not affect the sensitivity of the measurement system. Therefore, the conditions such as the dilution buffer and its components were examined. As a basic buffer, 25 mM buffer (Buffer) /
0.075M NaCl / 0.1% BSA / 0.01%
Tested with Tween-20. As a result, it was judged appropriate to use a Tris-HCl buffer solution (pH 7.0 and its vicinity) as the buffer solution. It was confirmed that it can be selected from the viewpoint of reactivity and stability. It was also confirmed that it is possible to select a condition that has high reactivity, little inhibition by serum components, and has an effect of suppressing non-specific reaction. 3) Examination of reaction time The sandwich method consists of the first reaction (reaction of the primary antibody and the sample), the second reaction (reaction of the antigen reacted with the primary antibody and the labeled secondary antibody), and the third reaction (labeled). Reaction of the secondary antibody with the substrate). The reaction time of which step has a large influence on the measurement result was compared between when the first reaction was at room temperature and when it was 4 ° C. The reaction conditions are shown below. Reaction time Reaction temperature (room temperature) Reaction temperature (4 ° C) 1st reaction 1hr 2hr 2hr ON ON ON ON ON 2nd reaction 1hr 1hr 1hr 1hr 1hr 2hr 3hr 4hr 3rd reaction 0.5hr 0.5hr 1hr 0.5hr 1hr 1hr 1hr 1hr Study As a result, it was determined that the first reaction was suitable for 2 hours, the second reaction for 2 hours, and the third reaction for 1 hour. 4) Solid-Phase Concentration and Solid-Phase Time The antibody solid-phase concentration was set so that the reactivity reached a plateau. Immobilization of 2G4 antibody alone was compared with denatured antibody or undenatured antibody. Moreover, it was confirmed whether the solid phase was immobilized at a similar concentration 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 addition, solid-phase immobilization gave sufficient results by treatment at 4 ° C for 1 day in the case of mixed antibodies, but it was confirmed that treatment with 2G4 antibody alone at 4 ° C for 1 day may not be sufficient. . 5) Preparation of enzyme substrate solution 0.17 g of tetramethylbenzidine (TMB) was added to DM
After dissolving in 9.9 ml of SO, 0.8 ml of a saturated EDTA-2Na solution was added, 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 (2nd reagent). The first reagent is added to the second reagent, and the mixture is left at room temperature overnight before use. At the time of use, the color of the substrate liquid is colorless. 2N sulfuric acid was prepared and used as a reaction stop solution.

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

[0028]

[Table 1]

As a result of examining the denaturing effect of the purified antibody, 2
It was found that the denaturation treatment of the G4 antibody can increase the reactivity of the antibody by about 10 times 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 denaturing effect of the Fc portion of IgG may increase the adsorptivity to the solid phase as a factor for increasing the reactivity.

2) Two types of antibodies (2G
4,1C9) specificity when mixed and solid-phased The ELISA system using the 1G9 antibody as the solid-phase also reacts with normal prothrombin, but on the other hand, it has been found that end-stage liver cancer is detected. . The 2G4 antibody hardly reacts with normal prothrombin, and the characteristic that it reacts with PIVKA-2 has been obtained. Therefore, the case of using both as a mixture was examined. By the way, it is predicted that when both are mixed and used, the reactivity with normal prothrombin is surfaced and the specificity of PIVKA-2 is lowered, but it was also examined whether such a result could be obtained. The mixing method was 40 μg / m 2G4 antibody or 1C9 antibody each.
It was adjusted to 1 and immobilized 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 to PIVKA-2 or prothrombin is examined in an ELISA system using the 2G4 and 1C9 antibodies purified in (2) above alone or as a mixture of both antibodies. Microplate (Labosystem Breakable, F
8) and 10 mM TrisHC in solid phase buffer
1, 0.1 M NaCl (pH 7.0), 50 μ
0.1 g of 2G4 or 1C9 antibody adjusted to g / ml
Add ml and solidify overnight at 4 ° C. Wash solution (10 mM
After washing 3 times with PBS, 0.05% Tween-20),
10% skim milk (0.2 ml) is added and left at room temperature for 2 hours for Blocking. After washing three more times, PIV
KA-2 Standard (Warfarin administration patient plasma: commercial product) or Prothrombin Standard
(Commercially available product) (0.1 ml) is added, and the mixture is reacted at room temperature for 2 hours. After washing, the peroxidase-labeled anti-human prothrombin IgG (0.1M 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
0.1 ml of e) was added and reacted for 1 hr at room temperature, then the substrate solution (T
After 1hr added _{MB) 0.1ml 2N-H 2 SO} 4
The reaction is stopped by adding 0.05 ml, and colorimetry is performed 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]

The results of ELISA in the case of immobilizing the antibodies of fixed concentrations at different mixing ratios showed that the reactivity when mixed was increased by about 10 times as compared with the solid phase of 2G4 antibody alone. There is. 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 in the case of using a mixed antibody was examined with a buffer system and then with a serum system. The results are shown in Table 3, Table 4, Table 5 and Table 6.

[0033]

[Table 3]

[0034]

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

[0035]

[Table 5]

[0036]

[Table 6]

When used as a mixed antibody, the result showed that the reactivity of prothrombin was completely suppressed by the addition of serum. Furthermore, in order to confirm the validity of this method, a sample was measured and comparison data was summarized with the case of using only 2G4 antibody. The conditions of the solid phase antibody used were 2G4 antibody only system and 2G4 / 1C9 (1/1) mixed antibody system. No effect on the measured values due to the difference in solid phase antibody was observed. It has been observed that the use of a mixture of antibodies having similar recognition sites dramatically improves the reactivity and can further improve the sensitivity of the measurement system. By using two kinds of antibodies in combination, not only the reactivity is increased, but also the result that the specificity of one is maximized is derived. Although it is difficult to explain the factors that cause such a change, the a
It is speculated that finity may increase.

3) The specificity of the 2G4 antibody for PIVKA-2 was confirmed by an inhibition test against the 2G4 antibody PIVKA-2 antigen (8/4/2/1 / 0.5 / 0Au)
/ Ml) and 2G4 antibody (40/20/10/5 / 2.5)
/ 0 μg / ml), and immediately added to the 2G4 immobilized plate, and reacted overnight (4 ° C.). The following is a normal EI
It carried out according to A. That is, 100 μg / ml of POD-labeled anti-prothrombin antibody was added, 200 μg / ml of substrate was added after reaction for 1 hour at 4 ° C., treatment was performed for 30 minutes at room temperature, and 50 μg / ml of reaction stop solution was added. From this result, the affinity of this antibody was determined from the specificity of the 2G4 antibody for PIVKA-2 and the inhibition test of the 2G4 antibody on the PIVKA-2 antigen.

Example 4 (Non-Specific Reaction in ELISA System) 1) Non-Specific Reaction Using Autoantibody (RAPA) Specimen What matters in the ELISA system is the presence or absence of non-specific reaction. 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 an autoantibody. And, these can be improved considerably or not at all by selecting the buffer solution component. PIV of the present invention
The non-specific reaction in the KA-2 ELISA system was examined. The measurement was performed using a RAPA sample, which is a test item for autoantibodies. Regarding the detected sample, it was decided to perform measurement again using a commercial kit to confirm. In the method, 0.1 ml of the buffer solution and 0.1 ml of the sample are added to a solid-phased plate (after drying and stored in a pack), and the mixture is reacted at 4 ° C. overnight. Subsequent operations were performed as usual. As a result, there were cases where PIVKA-2 positives were found in samples with high RAPA measurement values, but this was the same with commercial kits, and no correlation with the autoantibody measurement results was observed.

2) Suppression of non-specific reaction The PIVKA-2 negative sample was left to inactivate at 56 ° C for 30 minutes, and this was used as an artificially prepared non-specific sample to be used as an index of the suppression effect. The method was carried out as usual, and the inhibitory effect of the non-specific reaction of the buffer component in the measurement system was examined. Whether or not mouse IgG or rabbit serum was added to the buffer; the basic buffer was 0.01 M Tris HCl /
0.075M NaCl / 0.01% Tween-2
It was set to 0, and two series of examinations were performed, that is, the condition of adding to the buffer solution of the primary reaction system and the condition of adding to the labeled antibody dilution buffer 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, the addition of rabbit serum to the primary reaction was above a certain level. In that case, a result showing a remarkable 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 exceeded a certain level in the secondary reaction, the whole reaction itself was suppressed, and it was judged 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% mlgG, 0.1% mlgG in basic buffer,
5.0% NRS is added, and the following concentration NaCl or Tw
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, the reactivity of NaCl decreased to about 1/2 at 0.75 M or more, and the non-specific reaction was proportional to it. 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 nonspecific reaction did not change so much. From these results,
As an additive to the basic buffer for suppressing non-specific reaction, 0.01% mlgG or rabbit serum was added with 0.0
It is considered appropriate to add 75M NaCl and 0.2% Tween-20.

Example 5 (Measurement of Specimen) 1) A standard curve was prepared using a PIVKA-2 standard product, which was obtained by diluting 10 times the plasma of a patient with warfarin administration to 8 Au / ml. As the monoclonal antibody-immobilized plate, 2G4 / 1C9 mixed antibody was used, and HRP-labeled rabbit polyclonal antibody was used as a labeled anti-prothrombin antibody. 0.2 μg / ml mixed antibody (0.2 ml) was added to the immunoplate (labo system breakable plate) (0.2 ml / well) and incubated at 4 ° C. overnight, and washed 3 times, then 50% Block Ace The solution is treated with 0.25 ml of liquid and allowed to stand at room temperature
Incubate for an hour. 2% after washing 3 times
A secondary coating solution of 0.25 ml consisting of sucrose and 1% BSA is added, the mixture is left at room temperature for 2 hours, decanted for 30 minutes, and then 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 sample to the solid-phased plate, incubating overnight at 4 ° C., washing 4 times, and adding 0.2 ml of HRP-labeled anti-prothrombin antibody at room temperature. After incubating for 2 hours and washing 5 times, the substrate solution (TM
B) 0.2 ml was added, the mixture was incubated at room temperature for 1 hour, 0.05 ml of the reaction stop solution was added, and the colorimetric (5
40/650 nm). Similarly, the ascites derived from liver cancer was also measured.

[0043]

[Table 7]

[0044]

[Table 8]

Similarly, for 127 clinical cases including hepatocellular carcinoma, P using the assay system using the mixed antibody of the present invention.
IVKA-2 was measured. α Fetoprotein (AF
P) and other liver disease-related items, GOT, GPT,
The results of total bilirubin and albumin are also 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 of the above, it was found that sufficient measurement results can be obtained with the measurement system using the mixed antibody of the present invention.

EFFECT OF THE INVENTION PIVKA-2 produced by liver cancer-derived cells
Excellent sensitivity can be achieved by mixing and using at least two of the monoclonal antibodies that recognize PIVKA-2 obtained by using as a immunogen. In particular, in a measurement system in which this mixed antibody is immobilized, unexpectedly, PIVKA-2 can be specifically measured even if an anti-PIVKA-2 antibody that recognizes prothrombin is used. In this way PIVKA
-2 can be specifically and easily measured and can be applied to diagnosis and the like as a liver cancer marker.

[Brief description of drawings]

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

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

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

Claims (6)

[Claims] 1. A method for measuring PIVKA-2, which comprises using at least two kinds of monoclonal antibodies against PIVKA-2 in measuring PIVKA-2 in a sample. 2. The monoclonal antibodies used are at least two of the following: (i) not reacting with prothrombin and PIVKA-2
A method according to claim 1, which is a monoclonal antibody that reacts only with (ii) a monoclonal antibody that reacts with prothrombin and also with PIVKA-2. 3. When measuring PIVKA-2 in a sample, (i) it does not react with prothrombin and PIVK
A monoclonal antibody that reacts only with A-2, such as 2G
4 antibody reacts with (ii) prothrombin and PIVK
A monoclonal antibody that also reacts with A-2, eg 1C9
The antibody is immobilized on a solid-phase carrier, the solid-phased carrier of the mixed antibody is contact-reacted with the sample, and then the carrier is optionally washed and then contacted with the labeled anti-prothrombin antibody,
Next, a method for measuring PIVKA-2, which comprises measuring the signal generated by labeling after washing the carrier, if necessary, as an index of PIVKA-2. 4. A monoclonal antibody that recognizes human PIVKA-2 produced by a hybridoma obtained by fusing spleen cells immunized with human liver cancer cell-derived PIVKA-2 and tumor cells that do not produce immunoglobulin. 5. The monoclonal antibody comprises at least two of the following: (i) not reacting with prothrombin and PIVKA-2
A monoclonal antibody that reacts only with (ii) a monoclonal antibody that reacts with prothrombin and also with PIVKA-2. 6. The monoclonal antibody is 2G4 and / or
Or a 1C9 antibody.
The monoclonal antibody as described.