WO2012002345A1 - Procédé de dosage de pivka-ii, réactif de dosage de pivka-ii et anticorps - Google Patents

Procédé de dosage de pivka-ii, réactif de dosage de pivka-ii et anticorps Download PDF

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WO2012002345A1
WO2012002345A1 PCT/JP2011/064724 JP2011064724W WO2012002345A1 WO 2012002345 A1 WO2012002345 A1 WO 2012002345A1 JP 2011064724 W JP2011064724 W JP 2011064724W WO 2012002345 A1 WO2012002345 A1 WO 2012002345A1
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pivka
antibody
monoclonal antibody
vitamin
hybridoma
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Japanese (ja)
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吉康 狩野
卓味 大村
紀光 荒井
順 西村
宮崎　修
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エーディア株式会社
積水メディカル株式会社
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Publication of WO2012002345A1 publication Critical patent/WO2012002345A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/974—Thrombin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis

Definitions

  • the present invention relates to a method for measuring PIVKA-II, a reagent for measuring PIVKA-II, and an antibody, and in particular, determination of the degree of liver damage in liver cancer by measuring PIVKA-II caused by vitamin K deficiency in a specimen, and The present invention relates to a method for measuring PIVKA-II capable of increasing specificity, a reagent for measuring PIVKA-II, and a monoclonal antibody.
  • PIVKA-II Protein Induced by Vitamin K Absence or Antagonist-II
  • AFP ⁇ -fetoprotein
  • PIVKA-II is a precursor of prothrombin, one of vitamin K-dependent plasma proteins.
  • Prothrombin blood coagulation factor II
  • Fragment 1 consists of 156 amino acids including a Gla domain composed of 41 amino acids from the N-terminus.
  • PIVKA-II can be said to be a decarboxylated form of the ⁇ -carboxyglutamic acid residue of normal prothrombin, and is sometimes called abnormal prothrombin.
  • PIVKA-II can be said to be a decarboxylated form of the ⁇ -carboxyglutamic acid residue of normal prothrombin, and is sometimes called abnormal prothrombin.
  • PIVKA-II produced by hepatoma cells remain Glu residues
  • conventional PIVKA-II measuring reagents using a monoclonal antibody obtained from cell line MU-3 are normal. It is known that 10 to 9 of the 10 Gla residues of prothrombin show strong reactivity with PIVKA-II, which is a Glu residue (Non-patent Document 2).
  • PIVKA-II is measured as follows. First, after first reacting magnetic beads, glass beads, plastic plates, latex, etc. adsorbed with a PIVKA-II specific monoclonal antibody or polyclonal antibody with serum or plasma, BF washing is performed, followed by enzyme, After adding a polyclonal antibody or monoclonal antibody specific to human prothrombin labeled with a fluorescent substance, radioisotope, Ru complex, etc., followed by BF washing, the immune complex formed by the antigen-antibody reaction The absorbance or luminescence amount of the bound enzyme, fluorescent substance, radioisotope, or Ru complex is measured (Patent Document 2).
  • a measurement method is also known in which a monoclonal antibody that does not react with prothrombin and reacts only with PIVKA-II and a monoclonal antibody that reacts with prothrombin and also reacts with PIVKA-II are combined (Patent Document 3).
  • Patent Document 3 since the Gla residue has calcium binding ability, it is known that the three-dimensional structure changes in the presence of calcium, and a measurement method using an antibody that specifically binds to PIVKA-II only in the presence of calcium Is also known (Non-patent Document 3).
  • Liver cancer is different from malignant tumors of other organs, and liver cancer is complicated by chronic hepatitis or cirrhosis. Therefore, in treating patients with liver cancer, not only the tumor side factors but also the degree of liver damage is considered. Therefore, it is necessary to decide the treatment policy.
  • PIVKA-II and AFP are used as tumor marker tests, but they do not necessarily reflect the degree of liver damage and prognosis required for the determination of the treatment policy. Under these circumstances, blood tests that reflect the degree of liver damage and prognosis have been desired.
  • the degree of liver injury is an index for evaluating the liver reserve capacity described in the “Primary Liver Cancer Handling Regulations”.
  • serum bilirubin level, serum albumin level, ICG R 15 , and prothrombin activity level are classified into 3 degrees, the severity for each item is obtained, and the liver damage degree corresponding to 2 or more items is taken.
  • the Child-Pugh classification shows 1 to 3 points for encephalopathy, ascites, serum bilirubin level, serum albumin level, and prothrombin activity level, and indicates the degree of liver damage classified from the lowest of the total points to AC. It is an indicator.
  • PIVKA-II has been found to be elevated even in cases of vitamin K deficiency and vitamin K antagonist administration even when liver cancer is not present.
  • the sensitivity of the reagent is increased, false positives in alcoholic hepatitis and the like are occasionally observed, and there are reports of cases in which liver cancer is not found by diagnostic imaging despite being positive for PIVKA-II. Under these circumstances, development of a measurement method with higher specificity for liver cancer has been desired.
  • PIVKA-II includes PIVKA-II produced by liver cancer cells in the liver and PIVKA-II caused by vitamin K deficiency (hereinafter referred to as PIVKA-II other than PIVKA-II produced by liver cancer cells).
  • PIVKA-II caused by vitamin K deficiency
  • PIVKA-II caused by vitamin K deficiency but it is difficult to distinguish them with conventional measurement reagents, and false positives are sometimes seen. It was rare.
  • the present invention makes it possible to determine the degree of liver damage and predict the prognosis in liver cancer, and to detect liver cancer with high sensitivity, a method for measuring PIVKA-II, a reagent for measuring PIVKA-II, and a determination of the degree of liver damage
  • the object is to provide a method and a method for determining liver cancer.
  • the present inventors have conducted extensive studies and measured PIVKA-II by a two-antibody sandwich method using two types of antibodies that react with PIVKA-II caused by vitamin K deficiency. It was found that it was possible to judge the degree and predict the prognosis. It is also possible to distinguish alcoholic hepatitis or cirrhosis from liver cancer by comparing measured values obtained using this antibody with PIVKA-II measured values obtained by conventional methods.
  • the present invention has been completed by finding that it is possible to predict the prognosis of liver cancer and to detect liver cancer with high specificity.
  • the method for measuring PIVKA-II of the present invention comprises (a) a step of measuring PIVKA-II caused by vitamin K deficiency by an immunological measurement method using a two-antibody sandwich method and obtaining a measurement value A.
  • a method for measuring PIVKA-II comprising using an antibody specific for PIVKA-II caused by vitamin K deficiency as the antibody used in the two-antibody sandwich method of step (a).
  • the method for measuring PIVKA-II of the present invention comprises (b) a step of measuring PIVKA-II by an immunoassay utilizing a two-antibody sandwich method to obtain a measurement value B, and (c) the measurement value A and A step of comparing the measured value B, wherein the anti-PIVKA- reacts with PIVKA-II purified from a human hepatoma cell culture cell line as one of the antibodies used in the two-antibody sandwich method of the step (b). It is preferable to use an anti-human prothrombin antibody that does not contain an antibody that reacts with human thrombin as the other antibody used in the two-antibody sandwich method of step (b), using a II monoclonal antibody.
  • the antibody used in the two-antibody sandwich method in step (a) does not react with PIVKA-II purified from a human hepatoma cell culture cell line.
  • the antibody used in the two-antibody sandwich method in the step (a) does not react with human thrombin.
  • the antibody used in the two-antibody sandwich method in the step (a) is a monoclonal antibody.
  • None of the antibodies used in the two-antibody sandwich method of step (a) is i) reacting with prothrombin in the antigen solid phase ELISA in the presence of Ca 2+ ions, and ii) decarboxylation of prothrombin. It is preferable to be a combination of things that are more reactive than those obtained after 30 minutes of decarboxylation compared to those obtained after 6 hours.
  • the reagent for measuring PIVKA-II of the present invention can be used as an antibody for measuring PIVKA-II caused by vitamin K deficiency by an immunological assay using a two-antibody sandwich method. It is characterized by using an antibody specific for II.
  • any of the antibodies used in the two-antibody sandwich method is a reaction with i) prothrombin in an antigen solid-phase ELISA in the presence of Ca 2+ ions. And ii) a combination of those having a higher reactivity to 30 minutes of decarboxylation compared to 6 hours of prothrombin decarboxylation.
  • a first aspect of the monoclonal antibody of the present invention is characterized in that it is produced by a hybridoma specified by accession number FERM BP-11258.
  • a second aspect of the monoclonal antibody of the present invention is characterized in that it is produced by a hybridoma specified by accession number FERM BP-11259.
  • the first aspect of the hybridoma of the present invention is characterized by being identified by the accession number FERM BP-11258.
  • the second aspect of the hybridoma of the present invention is characterized by being identified by the accession number FERM BP-11259.
  • the antibody specific for PIVKA-II resulting from vitamin K deficiency is a monoclonal antibody produced by a hybridoma identified by accession number FERM BP-11258, and a trustee A monoclonal antibody produced by a hybridoma identified by the number FERM BP-11259 is preferred.
  • the method for determining the degree of liver damage according to the present invention is characterized in that the degree of liver damage is determined based on the value obtained by the measurement method of PIVKA-II according to the present invention.
  • liver cancer determination method of the present invention is characterized in that liver cancer is determined based on the value obtained by the PIVKA-II measurement method of the present invention.
  • the present invention it is possible to accurately determine the determination of the degree of liver damage and prognosis prediction in liver cancer, which has been difficult in blood tests, and to detect liver cancer with high sensitivity. Furthermore, according to the present invention, it is possible to distinguish hepatic cancer from alcoholic hepatitis or cirrhosis that does not develop liver cancer, and it is possible to predict the prognosis of liver cancer and to detect highly specific liver cancer. Is possible.
  • Example 4 It is the figure which showed the common site
  • the method for measuring PIVKA-II of the present invention comprises (a) measuring PIVKA-II caused by vitamin K deficiency by an immunological measurement method using a two-antibody sandwich method, and obtaining a measurement value A.
  • -II measurement method wherein an antibody specific for PIVKA-II caused by vitamin K deficiency is used as the antibody used in the two-antibody sandwich method of step (a).
  • PIVKA-II resulting from vitamin K deficiency measured in the step (a) may be referred to as NX-PVKA.
  • the antibodies used in the two-antibody sandwich method of step (a) are each an antibody that reacts with PIVKA-II caused by vitamin K deficiency produced using PIVKA-II caused by vitamin K deficiency as an antigen. It is manufactured by the following method.
  • a hybridoma was prepared by a known method using PIVKA-II purified from coumadin plasma of a non-hepatic cancer case (plasma of a patient administered with a vitamin K antagonist), that is, PIVKA-II resulting from vitamin K deficiency.
  • a hybridoma strain that reacts with PIVKA-II due to vitamin K deficiency is selected.
  • hybridoma that is highly reactive to PIVKA-II with a high ⁇ -carboxylation rate for 10 glutamic acid residues of prothrombin, and specifically, the decarboxylation time of normal prothrombin It is preferable to select hybridomas that exhibit high reactivity with PIVKA-II, which has a short PIVKA. For example, when comparing the reactivity to PIVKA-II prepared from prothrombin at 30 minutes and 6 hours with decarboxylation, the reactivity to PIVKA-II prepared at 30 minutes is the reactivity to PIVKA-II prepared at 6 hours. It is preferred to select a hybridoma that is greater than 1 when divided by. As the hybridoma, a hybridoma specified by the accession number FERM BP-11258 and a hybridoma specified by the accession number FERM BP-11259 are preferable.
  • a monoclonal antibody-producing hybridoma using the selected hybridoma and obtain a monoclonal antibody that reacts with PIVKA-II caused by vitamin K deficiency by a known method using the monoclonal antibody-producing hybridoma.
  • the immunoassay method using the two-antibody sandwich method in the step (a) is performed, for example, by the following method.
  • two types of monoclonal antibodies are prepared using PIVKA-II caused by vitamin K deficiency as an antigen, one of which is used in a solid phase and the other is used after being labeled.
  • the two types of antibodies are antibodies that do not cross-react with each other.
  • the method for immobilizing the antibody is not particularly limited, but for example, it is preferable to immobilize the antibody on a solid phase such as a magnetic bead or a microplate.
  • the method for labeling the antibody is not particularly limited, but labeling with a labeling substance such as Ru is preferable.
  • the measurement may be performed according to a normal procedure in an immunological measurement method using a two-antibody sandwich method.
  • an example using an electrochemiluminescence immunoassay was shown, but the present invention is not limited to this, for example, a known measurement such as a chemiluminescence method or a radioisotope method. The law can be widely used.
  • the degree of liver injury in liver cancer can be determined by obtaining the measurement value A of NX-PVKA measured in the step (a). Further, by comparing the measured value A with the measured value of PIVKA-II measured by a conventional method, it is possible to distinguish alcoholic hepatitis or cirrhosis that does not develop liver cancer from liver cancer.
  • the conventional method for measuring PIVKA-II that rises in liver cancer is not particularly limited. For example, (b) PIVKA-II is measured by an immunoassay using the two-antibody sandwich method, and the measured value B is calculated.
  • Examples of the other antibody used in the two-antibody sandwich method in step (b) include a method of using an anti-human prothrombin antibody that does not contain an antibody that reacts with human thrombin.
  • the means for comparing the measured value A and the measured value B is not particularly limited. For example, a method of calculating a ratio or a difference can be used. The ratio value can be obtained by dividing the measured value B by the measured value A. Is preferred.
  • Example 1 Preparation of Monoclonal Antibody (1) Preparation of Hybridoma PIVKA-II (1 mg / mL) purified from coumadin plasma (manufactured by UNIGLOBE RESEARCH CORPORATION) and Floyd's complete adjuvant (manufactured by GIBCO) are 1: 1. After mixing and emulsifying in 50 ⁇ g / 100 ⁇ mL (emulsion) subcutaneously in 8-week-old female BALB / C mice (Nihon Charles River Co., Ltd.) 4 times at intervals of 2 weeks, the spleen 3 days after the final immunization Extracted.
  • coumadin plasma manufactured by UNIGLOBE RESEARCH CORPORATION
  • GIBCO Floyd's complete adjuvant
  • Spleen cells obtained from the removed spleen and myeloma cells SP2 / O-Ag14 were mixed at a ratio of 10 to 1, and cell fusion was performed in the presence of 50% polyethylene glycol 1540 (Wako Pure Chemical Industries, Ltd.). I let you.
  • the fused cells were suspended as spleen cells in HAT medium so as to be 2.5 ⁇ 10 6 / mL, and 0.2 mL was dispensed into 96-well culture plates (CORNING).
  • the above PIVKA-II was immobilized on a microplate (manufactured by NUNC) at 0.1 ⁇ g / mL. After each culture supernatant was reacted with this, a peroxidase-labeled anti-mouse IgG goat antibody was reacted, and then a peroxidase substrate solution containing orthophenylenediamine (manufactured by Tokyo Chemical Industry Co., Ltd.) was added to cause color development. After stopping the color development by adding 5N sulfuric acid, a hybridoma strain that reacts with PIVKA-II was selected by measurement with a microplate reader (Abs. 492 nm). After this hybridoma was cloned by the limiting dilution method, IgG was purified from the culture supernatant using a protein A column (Pharmacia).
  • 0.1 mol / L EDTA ⁇ 2Na was added to the dialyzed solution so that the final concentration was 10 mmol / L, and the solution was allowed to stand at room temperature for 30 minutes. This solution was again dialyzed against ammonium bicarbonate solution at 4 ° C. for 2 hours. The dialyzed solution was dispensed into 6 heat-resistant vials with screw caps in equal amounts and lyophilized. The vial was then filled with nitrogen gas and heated at 110 ° C. for each time (0 minutes, 30 minutes, 1 hour, 2 hours, 6 hours, 23 hours). 1 mL of saline was added to each vial to dissolve the contents and stored frozen at ⁇ 80 ° C. until use.
  • PIVKA-II having a decarboxylation treatment time of 30 minutes and 6 hours, and a microplate on which prothrombin was immobilized at 0.1 ⁇ g / mL, calcium chloride at a concentration of 4 mmol / L.
  • the reactivity of the purified IgG of the selected hybridoma was measured under the coexisting conditions. Under these conditions, a hybridoma that does not react with prothrombin and shows high reactivity to PIVKA-II with a decarboxylation treatment time of 30 minutes compared to PIVKA-II with a decarboxylation treatment time of 6 hours was selected.
  • Hybridoma 11 Antibody-producing hybridomas 24211 and 24216 (hereinafter sometimes referred to as “hybridoma 11” and “hybridoma 16”, respectively) were obtained.
  • the obtained hybridomas 11 and 16 are deposited at the Patent Organism Depositary Center (National 1st, East 1-chome, Tsukuba City, Ibaraki Prefecture, Japan) (Received date: May 28, 2010). ), The accession number of the hybridoma 11 is FERM BP-11258, and the accession number of the hybridoma 16 is FERM BP-11259.
  • PIVKA-II was prepared by decarboxylating prothrombin in the same manner as described above except that the decarboxylation time was changed as shown in Table 1 and FIG.
  • Reactivity with PIVKA-II and prothrombin was measured by the same method as described above. The results are shown in Table 1 and FIG. In Table 1 and FIG.
  • P-11 monoclonal antibody and P-16 monoclonal antibody were prepared from hybridoma 11 and hybridoma 16 by the following method, respectively.
  • the hybridoma was intraperitoneally administered to a 12-week-old female BALB / C mouse that had been injected intraperitoneally with 0.5 mL of pristane two weeks ago in advance in an amount of 0.5 ⁇ 10 6 cells.
  • ascites was collected and centrifuged to obtain a supernatant.
  • the supernatant was mixed with an equal volume of buffer for adsorption (3 mol / L NaCl-1.5 mol / L Glycine-NaOH, pH 8.5) and then filtered.
  • the filtrate was passed through a protein A column (manufactured by Pharmacia) equilibrated with an adsorption buffer solution, and the antibody was adsorbed onto the column, followed by elution with a 0.1 mol / L citrate buffer solution (pH 3.0). Monoclonal antibody was purified.
  • Example 2 Using the P-16 monoclonal antibody and P-11 monoclonal antibody obtained in Example 1, PIVKA-II caused by vitamin K deficiency was measured by the following method.
  • (1) Preparation of P-16 monoclonal antibody solid phase magnetic beads Take 1 mL of magnetic beads (4.5 micron) 30 mg / mL into a test tube, trap with a magnet, discard the supernatant, and then add P- 1 mL of 16 monoclonal antibody 0.5 mg / mL (150 mM phosphate buffer, pH 7.8) was added and allowed to react at room temperature with stirring overnight.
  • the obtained Ru-labeled P-11 monoclonal antibody was diluted to 1 ⁇ g / mL with a diluted Ru solution at the time of use.
  • the composition of the diluted Ru solution is shown below. Composition of Ru dilution: 0.015 mol / L Hepes buffer (pH 7.8), 0.150 mol / L NaCl, 0.013 mol / L CaCl 2 , 0.1% surfactant, 0.1% NaN 3 , 0.1% benzamidine, 1 ⁇ g / mL, 5% rabbit serum (heated), 0.01% mouse IgG
  • Table 2 and FIG. 2 (a) show the results of calculating the average values of the measured values of Example 2 according to the Child-Pug classification.
  • ROC analysis was performed with the discriminating group as the death example and the control group as the survival example.
  • the obtained ROC curve is shown in FIG.
  • the area under the curve in the ROC analysis was 0.710.
  • AFP was measured using a Mutus Wako AFP-L3 measurement kit (manufactured by Wako Pure Chemical Industries, Ltd.) by a laser-induced fluorescence detection method (LAB method). The results are shown in Table 2, FIG. 2 (c) and FIG. The area under the curve in the ROC analysis was 0.651.
  • NX-PVKA of Example 2 reflected the Child-Pugh classification (classification for evaluating liver function). Further, as is clear from the results of FIG. 3, NX-PVKA of Example 2 reflected most prognosis prediction.
  • Example 3 Serum samples from 28 hepatocellular carcinoma patients, 20 cirrhosis patients and 9 chronic hepatitis patients were used as specimens, and the amounts of PIVKA-II and NX-PVKA were measured. A ratio value (NX-PVKA-R) was calculated by dividing the measured value of PIVKA-II by the measured value of NX-PVKA. The amount of NX-PVKA was measured in the same manner as in Example 2. PIVKA-II was measured using a Picolmi PIVKA-II measurement kit (manufactured by Eadia Co., Ltd.). The measured values are shown in Table 3.
  • FIG. 5 shows a graph of the ratio result obtained by dividing the measured value of PIVKA-II by the measured value of NX-PVKA.
  • HCC is hepatocellular carcinoma
  • CH chronic hepatitis
  • LC cirrhosis
  • B hepatitis B
  • C hepatitis C
  • NBNC non-B non-C hepatitis
  • NASH non Alcoholic liver disorder
  • AIH is autoimmune hepatitis
  • ALD is alcoholic liver disorder.
  • Example 4 The epitopes of the P-16 monoclonal antibody and P-11 monoclonal antibody obtained in Example 1 were examined by the following method.
  • PV002 ANTFLE ⁇ VRKGNL ⁇ R ⁇ PV003: ANTREEEVRKGNL ⁇ R ⁇ PV004: ANTREEEVRKGNLER ⁇ Amino acid sequence of PV002 Ala Asn Thr Phe Leu Glu Gla Val Arg Lys Gly Asn Leu Gla Arg Gla (SEQ ID NO: 1) Amino acid sequence of PV003 Ala Asn Thr Phe Leu Glu Glu Val Arg Lys Gly Asn Leu Gla Arg Gla (SEQ ID NO: 2) Amino acid sequence of PV004 Ala Asn Thr Phe Leu Glu Glu Val Arg Lys Gly Asn Leu Glu Arg Gla (SEQ ID NO: 3)
  • each well was dispensed with 25 ⁇ L / well of peptide solution of each concentration (20, 4, 0.8, 0.16, 0.032 ⁇ mol / L) diluted with BSA-PBST. Then, each monoclonal antibody (P-16, P-11) solution diluted with BSA-PBST to a concentration of 200 ng / mL was dispensed at 25 ⁇ L / well and allowed to stand at room temperature for 1 hour.
  • the P-16 monoclonal antibody showed equivalent reactivity to all three peptides.
  • the epitope of P-16 monoclonal antibody is PIVKA-II 1-6 residues (aa1-6 shown in FIG. 7), or the amino acid sequence of PIVKA-II, which is the common site of the three peptides. It was suggested that there are 8 to 13 residues (aa8-13 shown in FIG. 7) from the N-terminus of. On the other hand, it was found that the P-11 monoclonal antibody did not react with any peptide.
  • an XA-Y residue from the N-terminus of PIVKA-II is referred to as aaXY.
  • 0.1 mg / mL PIVKA-II, 0.1 mg / mL prothrombin, and 10 U / mL thrombin were each mixed 1: 1 with a mercaptoethanol-containing SDS treatment solution (manufactured by Cosmo Bio) and boiled for 10 minutes.
  • a sample was added to a polyacrylamide gel (Multigel II Mini 4/20) manufactured by Cosmo Bio Inc. in an amount of 5 ⁇ L / well and subjected to electrophoresis (SDS-PAGE) at 30 mA for 1 hour.
  • SDS-PAGE electrophoresis
  • the gel after electrophoresis was transferred to a PVDF membrane using a semi-drive lotter (manufactured by Cosmo Bio) (100 mA, 45 minutes).
  • the PVDF membrane was cut into lanes, immersed in BSA-PBST, and blocked overnight at 4 ° C. After washing once with PBST, each monoclonal antibody solution having a concentration of 5 ⁇ g / mL was brought into contact with the PVDF membrane and allowed to stand at room temperature for 1 hour. After washing 3 times with PBST, it was transferred to a container containing a solution of HRP-labeled goat anti-mouse IgG antibody (manufactured by DAKO) diluted 2000 times with BSA-PBST and gently shaken at room temperature for 1 hour.
  • HRP-labeled goat anti-mouse IgG antibody manufactured by DAKO
  • the conventional reagent antibody did not react with either fragment 1 or 2 derived from prothrombin.
  • the P-16 monoclonal antibody was highly reactive against fragment 1, while the P-11 monoclonal antibody was weak but reactive.
  • neither P-11 nor P-16 monoclonal antibody was reactive with fragment 2. This suggested that the epitopes of the P-11 and P-16 monoclonal antibodies are present within 156 residues (aa1-156) from the N-terminus of fragment 1, ie, prothrombin.
  • amino acid sequence of aa1-16 Ala Asn Thr Phe Leu Glu Glu Val Arg Lys Gly Asn Leu Glu Arg Glu (SEQ ID NO: 4) aa7-22 amino acid sequence Glu Val Arg Lys Gly Asn Leu Glu Arg Glu Cys Val Glu Glu Thr Cys (SEQ ID NO: 5) amino acid sequence of aa13-28 Leu Glu Arg Glu Cys Val Glu Glu Thr Cys Ser Tyr Glu Glu Ala Phe (SEQ ID NO: 6) amino acid sequence of aa19-34 Glu Glu Thr Cys Ser Tyr Glu Glu Ala Phe Glu Ala Leu Glu Ser Ser (SEQ ID NO: 7) aa25-40 amino acid sequence Glu Glu Ala Phe Glu Ala Leu Glu Ser Ser Thr Ala Thr Asp Val Phe (SEQ ID NO: 8) amino acid sequence of aa31-46 Leu Glu Ser Ser Thr Ala Thr
  • the epitope of P-16 monoclonal antibody is that of prothrombin fragment 1 based on the reactivity of the antibody to the peptides described in (1) and (4) above and the reactivity to PIVKA-II and prothrombin described in (2) above. It was found to be in the range of 5 residues from the N-terminus (aa1-5 represented by SEQ ID NO: 14). The amino acid sequence of aa1-5 Ala Asn Thr Phe Leu (SEQ ID NO: 14)
  • the epitope of the P-11 monoclonal antibody reacts with fragment 1 (aa1-156) in (3) above and does not react with each peptide contained in aa1-70 in (4) above, the prothrombin fragment 1 was considered to be in the range of aa60-156.
  • the PIVKA-II measuring method and measuring reagent of the present invention can be used for determination of liver damage degree and prognosis prediction, prognosis prediction of liver cancer, and detection of highly specific liver cancer.

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  • Genetics & Genomics (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un procédé de dosage de PIVKA-II, un réactif de dosage de PIVKA-II, un procédé destiné à déterminer la sévérité d'un trouble hépatique et un procédé d'identification du cancer du foie, chacun de ceux-ci étant caractérisé en ce qu'il n'emploie pas de réactif de dosage conventionnel qui mesure l'accroissement de PIVKA-II en présence d'un cancer du foie, en ce qu'il est capable de déterminer la sévérité ou de prédire le pronostic d'évolution d'un trouble hépatique en cancer du foie sur la base d'une valeur de mesure obtenue à l'aide d'un anticorps spécifique à la PIVKA-II accrue en présence d'une carence en vitamine K, et en ce qu'il est capable de détecter un cancer du foie avec une sensibilité élevée. Plus précisément, l'invention concerne un procédé de dosage de PIVKA-II, comportant une étape (a) consistant à mesurer la PIVKA-II induite par la carence en vitamine K par un procédé de mesure immunologique employant une technique à double sandwich d'anticorps pour obtenir une valeur de mesure (A), un anticorps spécifique à la PIVKA-II induite par la carence en vitamine K étant utilisé en tant qu'anticorps à employer dans la technique à double sandwich d'anticorps de l'étape (a).
PCT/JP2011/064724 2010-06-29 2011-06-28 Procédé de dosage de pivka-ii, réactif de dosage de pivka-ii et anticorps WO2012002345A1 (fr)

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ITMI20130513A1 (it) * 2013-04-05 2014-10-06 Euroclone S P A Metodo diagnostico per patologie autoimmuni del fegato
WO2019012019A1 (fr) 2017-07-13 2019-01-17 F. Hoffmann-La Roche Ag Nouvel agent de liaison et dosage pour pivka
CN114634576A (zh) * 2021-05-26 2022-06-17 中元汇吉生物技术股份有限公司 抗pivka-ii单克隆抗体及其应用
CN118005798A (zh) * 2024-04-08 2024-05-10 北京大学第三医院（北京大学第三临床医学院） 一种抗维生素k2抗体及其应用

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ITMI20130513A1 (it) * 2013-04-05 2014-10-06 Euroclone S P A Metodo diagnostico per patologie autoimmuni del fegato
WO2019012019A1 (fr) 2017-07-13 2019-01-17 F. Hoffmann-La Roche Ag Nouvel agent de liaison et dosage pour pivka
US11466098B2 (en) 2017-07-13 2022-10-11 Roche Diagnostics Operations, Inc. Binding agent and assay for PIVKA
CN114634576A (zh) * 2021-05-26 2022-06-17 中元汇吉生物技术股份有限公司 抗pivka-ii单克隆抗体及其应用
CN114634576B (zh) * 2021-05-26 2023-07-25 中元汇吉生物技术股份有限公司 抗pivka-ii单克隆抗体及其应用
CN118005798A (zh) * 2024-04-08 2024-05-10 北京大学第三医院（北京大学第三临床医学院） 一种抗维生素k2抗体及其应用

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