JP5856956B2 - Blood test method - Google Patents

Description

  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 resulting from 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) is widely measured in clinical laboratories as a marker for diagnosing liver cancer along with AFP (α-fetoprotein).
PIVKA-II is a precursor of prothrombin, which is one of vitamin K-dependent plasma proteins. Prothrombin (blood coagulation factor II) is a protein having a molecular weight of 71,600 and is composed of three regions of fragments 1 and 2 and thrombin, and its amino acid sequence has also been clarified (Non-patent Document 1). Fragment 1 consists of 156 amino acids including a Gla domain composed of 41 amino acids from the N-terminus. Those in which 10 γ-carboxyglutamic acid (Gla) residues in this Gla domain are normally synthesized are normal prothrombin, those that are not normally synthesized, and all or part of them remain as glutamic acid (Glu) residues Is PIVKA-II. Therefore, PIVKA-II can be said to be a decarboxylated form of the γ-carboxyglutamic acid residue of normal prothrombin, and is sometimes called abnormal prothrombin. Depending on how many of the 10 glutamic acid residues are subjected to γ-carboxylation, several types of PIVKA-II exist in a mixed state (Non-patent Document 2).

  Most PIVKA-II produced by hepatoma cells remain Glu residues, and the conventional PIVKA-II measuring reagent using a monoclonal antibody (Patent Document 1) obtained from the cell line MU-3 is 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).

Generally, PIVKA-II is measured as follows. First, magnetic beads, glass beads, plastic plates, latex, etc. adsorbed with a PIVKA-II specific monoclonal or polyclonal antibody are first reacted with serum or plasma, followed by BF washing, 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).
In addition, since the Gla residue has a 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 a 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”. Ascites, 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. Or it can also be evaluated using the Child-Pugh classification. The Child-Pugh classification shows the degree of liver damage that classifies from 1 to 3 points for the encephalopathy, ascites, serum bilirubin level, serum albumin level, and prothrombin activity level, and classifies from the lowest of the total points to AC. It is an indicator.

  PIVKA-II has also been found to be elevated in vitamin K deficient and vitamin K antagonist administered cases even when liver cancer is not present. As the sensitivity of the reagent is increased, false positives in alcoholic hepatitis and the like are occasionally observed, and there are cases where liver cancer is not found by image diagnosis even though PIVKA-II is positive. Under these circumstances, development of a measurement method with higher specificity for liver cancer has been desired.

Japanese Patent Publication 5-43357 JP-A-5-249108 JP 9-43237 A

Biochemistry. 1987 Sep 22; 26 (19): 6165-77 Biochem Biophys Acta. 2002 Apr 24; 1586 (3): 287-98 2002 J Immunoassay. 1995 May; 16 (2): 213-29.

As described above, 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). Is called “PIVKA-II caused by vitamin K deficiency”), but it is difficult to distinguish them with conventional measurement reagents, and false positives are sometimes seen. Therefore, the development of a measurement method with fewer false positives is desired. It was rare.
The present invention makes it possible to determine the degree of liver injury 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 method for determining the degree of liver injury The object is to provide a method and a method for determining liver cancer.

  The present inventors have conducted intensive 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, thereby causing liver damage in liver cancer. It was found that it was possible to judge the degree and predict the prognosis. Moreover, it is possible to distinguish alcoholic hepatitis or cirrhosis from liver cancer by comparing the measured value obtained using this antibody with the measured value of PIVKA-II performed by a conventional method. 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.

  That is, 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. 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, and anti-PIVKA-reactive 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 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.

  It is preferable that the antibody used in the two-antibody sandwich method in the step (a) does not react with PIVKA-II purified from a human hepatoma cell culture cell line.

  It is preferable that the antibody used in the two-antibody sandwich method in the step (a) does not react with human thrombin.

  It is preferable that 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 measurement reagent for PIVKA-II of the present invention can be used as an antibody when PIVKA-II caused by vitamin K deficiency is measured by an immunological assay using a two-antibody sandwich method. It is characterized by using an antibody specific for II.

In the measurement reagent for PIVKA-II caused by vitamin K deficiency according to the present invention, any of the antibodies used in the two-antibody sandwich method is reacted 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 by being produced by a hybridoma specified by accession number FERM BP-11258.

  A second aspect of the monoclonal antibody of the present invention is characterized by being 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 accession number FERM BP-11258.

  The second aspect of the hybridoma of the present invention is characterized by being identified by accession number FERM BP-11259.

  In the method and reagent for measuring PIVKA-II of the present invention, the antibody specific to PIVKA-II resulting from vitamin K deficiency is a monoclonal antibody produced by a hybridoma identified by the accession number FERM BP-11258 and the accession It is preferably a monoclonal antibody produced by a hybridoma identified by the number FERM BP-11259.

  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 method for measuring PIVKA-II according to the present invention.

  The method for determining liver cancer of the present invention is characterized by determining liver cancer based on the value obtained by the method for measuring PIVKA-II of the present invention.

  According to the present invention, it is possible to accurately determine the determination of the degree of liver damage and prediction of prognosis in liver cancer, which has heretofore been difficult with 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.

It is the graph which showed the relationship between the reactivity of an antibody, and the decarboxylation time of prothrombin. It is the graph which showed the Child-Pugh classification | category average value of the measured value of Example 2 and Comparative Examples 1-3, (a) is the result of Example 2, (b) is the result of Comparative Example 1, (c). Shows the results of Comparative Example 2, and (d) shows the results of Comparative Example 3, respectively. It is the graph which showed the result of the ROC analysis of Example 2 and Comparative Examples 1-3. It is a graph which shows the measurement result of PIVKA-II of Example 3. 10 is a graph showing a ratio value of Example 3. It is a graph which shows the reactivity of the antibody with respect to the Gla residue containing peptide of Example 4 (1). It is the figure which showed the common site | part of the peptide of Example 4 (1). It is a figure which shows the reactivity with respect to the modified | denatured prothrombin and modified | denatured PIVKA-II of Example 4 (2). It is a figure which shows the reactivity with respect to the fragment 1 and the fragment 2 of Example 4 (3).

  DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiments of the present invention will be described below with reference to the accompanying drawings. However, the illustrated examples are illustrative only, and various modifications can be made without departing from the technical idea of the present invention. .

  The method for measuring PIVKA-II of the present invention comprises the step of (a) measuring PIVKA-II caused by vitamin K deficiency by an immunological assay utilizing 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). In the present specification, 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-hepatoma case (plasma of a vitamin K antagonist administered patient), that is, PIVKA-II resulting from vitamin K deficiency as an immunogen. A hybridoma strain that reacts with PIVKA-II due to vitamin K deficiency is selected. When selecting the hybridoma strain, it is preferable to select a hybridoma strain that does not react with PIVKA-II or human thrombin purified from a human hepatoma cell culture cell line. In particular, it is preferable to select a hybridoma that is highly reactive to PIVKA-II with a high γ-carboxylation rate for 10 glutamic acid residues of prothrombin, and specifically, a decarboxylation time of normal prothrombin It is preferable to select a hybridoma that shows a high reactivity with PIVKA-II, which is short. For example, when comparing the reactivity to PIVKA-II prepared from prothrombin at 30 minutes and 6 hours of 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.

  It is preferable to produce 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.
According to the above method, two kinds 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.

  PIVKA-II caused by vitamin K deficiency in the sample is measured using the solid-phased antibody and the labeled antibody. The measurement may be performed according to a normal procedure in an immunological measurement method using a two-antibody sandwich method. In the examples to be described later, 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.

In the present invention, 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). Moreover, by comparing the measured value A with the measured value of PIVKA-II measured by a conventional method, alcoholic hepatitis or cirrhosis that does not develop liver cancer can be distinguished 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 immunological measurement method using a two-antibody sandwich method, and the measured value B is calculated. An anti-PIVKA-II monoclonal antibody that 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 step (b), 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.

  The present invention will be described more specifically with reference to the following examples. However, these examples do not limit the scope of the present invention.

Example 1 Preparation of Monoclonal Antibody (1) Preparation of Hybridoma 1: 1 PIVKA-II (1 mg / mL) purified from coumadin plasma (UNIGLOBE RESEARCH CORPORATION) and Floyd's complete adjuvant (GIBCO) After mixing and emulsifying in 50 μg / 100 μmL (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. Spleen cells obtained from the removed spleen and myeloma cells SP2 / O-Ag14 are mixed at a ratio of 10 to 1, and cell fusion is performed in the presence of 50% polyethylene glycol 1540 (manufactured by 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). This was cultured in a 5% CO ₂ incubator at 37 ° C., and after approximately 2 weeks, the culture supernatant of the well in which the hybridoma had grown was evaluated according to the ELISA method shown below, and the antibody reacting with PIVKA-II Were selected.

  Specifically, first, the PIVKA-II was immobilized on a microplate (manufactured by NUNC) at 0.1 μg / 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 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).

  In order to investigate the specificity of the obtained monoclonal antibody, the method of Bajah et al. (S. Paul Bajah, Paul A. Price, and William A. Russell, Decarboxylation of γ-Carboxyglutamic Acid Residues in Human Prothrombin) , Journal of Biological Chemistry 257 (7): 3726, 1982), various PIVKA-II having different decarboxylation times were prepared. Specifically, 0.78 mL of ammonium bicarbonate solution (0.1 mol / L, pH 8.0) was added to 0.22 mL of the 4.6 mg / mL prothrombin solution, and this was further added to the ammonium bicarbonate solution at 4 ° C. Dialyzed overnight. 0.1 mol / L EDTA · 2Na was added to the solution after dialysis 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.

  Of the various PIVKA-II, PIVKA-II having a decarboxylation time of 30 minutes and 6 hours, and a microplate on which 0.1 μg / mL of solid thrombin was immobilized, 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. 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 were deposited at the Patent Organism Depositary Center of the National Institute of Advanced Industrial Science and Technology (1st, 1st East, 1-chome, Tsukuba City, Ibaraki, Japan) (Reception date: May 28, 2010) ), The accession number of hybridoma 11 is FERM BP-11258, and the accession number of hybridoma 16 is FERM BP-11259.

  Prothrombin was decarboxylated in the same manner as described above except that the decarboxylation time was changed as shown in Table 1 and FIG. 1, and five types of PIVKA-II were prepared. The P-11 monoclonal antibody prepared from the hybridoma 11, the P-16 monoclonal antibody prepared from the hybridoma 16, and the anti-PIVKA-II monoclonal antibody of the conventional reagent (Picolmi PIVKA-II (manufactured by Adia Co., Ltd.)). Reactivity with PIVKA-II and prothrombin (decarboxylation time 0 minutes) was measured by the same method as described above. The results are shown in Table 1 and FIG. In Table 1 and FIG. 1, (1) is a P-11 monoclonal antibody, (2) is a P-16 monoclonal antibody, and (3) is a conventional anti-PIVKA-II monoclonal antibody (MU-3). The P-11 monoclonal antibody and the P-16 monoclonal antibody were prepared by the method described later.

(2) Preparation of monoclonal antibody P-11 monoclonal antibody and P-16 monoclonal antibody were prepared from the hybridoma 11 and the hybridoma 16, respectively, by the following method.
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. About 14 days later, ascites was collected and centrifuged to obtain a supernatant. The supernatant was mixed with an equal amount 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 in 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. After washing the magnetic beads, 2 mL of 1% BSA / phosphate buffer was added and blocked with stirring overnight at room temperature to prepare P-16 monoclonal antibody solid phase magnetic beads. At the time of use, it was diluted with a bead diluent to a magnetic bead amount of 1 mg / mL. The composition of the bead dilution is shown below.
Composition of bead diluent: 0.05 mol / L Tris buffer (pH 7.5), 0.150 mol / L NaCl, 0.01% Tween 20, 0.1% NaN ₃ , 10% rabbit serum (heated), 0. 1% mouse serum (clarified)

(2) Preparation of Ru-labeled P-11 monoclonal antibody To 1 mL of the prepared P-11 monoclonal antibody 1 mg / mL, 68 μL of succinimide group-modified ruthenium tri-dipyridyl Ru complex compound was added and reacted at room temperature for 30 minutes with stirring. After the reaction, 50 μL of 2 mol / L glycine was added to stop the reaction, and the reaction was further continued with stirring at room temperature for 10 minutes. Finally, the sample was passed through Sephadex G-25 (equilibrated with 10 mmol / L phosphate buffer), and Ru-bound protein fractions were collected to prepare Ru-labeled P-11 monoclonal antibody. The obtained Ru-labeled P-11 monoclonal antibody was used after diluting to 1 μg / mL with Ru diluted solution. 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 μg / mL, 5% rabbit serum (heated), 0.01% mouse IgG

(3) Measurement of PIVKA-II caused by vitamin K deficiency Vitamin K deficiency in a sample by electrochemiluminescence immunoassay using the prepared P-16 monoclonal antibody solid phase magnetic beads and Ru-labeled P-11 monoclonal antibody The measured value A of PIVKA-II (NX-PVKA) resulting from the above was determined.
The amount of NX-PVKA was measured according to the following method using serum samples of 63 patients with hepatocellular carcinoma who had not been operated on and had a known prognosis after 5 years.

  To 50 μL of each specimen, 25 μL of P-16 monoclonal antibody solid phase magnetic beads and 150 μL of Ru-labeled antibody solution containing 1 μg / mL Ru-labeled P-11 monoclonal antibody were added and reacted at 30 ° C. for 9 minutes. After the reaction, the magnetic beads were washed three times with a Picormi BF washing solution (manufactured by Adia Co., Ltd.) while trapping with a magnet. Add 300 μL of Picolmi Luminescent Electrolyte containing 0.1 mol / L Tripropylamine (manufactured by Adia Co., Ltd.), send it to the electrode surface, and measure the amount of light emitted from Ru bound to the magnetic beads. )), And the amount of NX-PVKA in the specimen was determined.

  Table 2 and FIG. 2 (a) show the results of calculating the average values of the measured values of Example 2 for each of the Child-Pug classifications. Regarding the measured values, 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.

(Comparative Example 1)
PIVKA-II was measured using a Picolmi PIVKA-II measurement kit (manufactured by Eadia Co., Ltd.). The results are shown in Table 2, FIG. 2 (b) and FIG. The area under the curve in the ROC analysis was 0.653.

(Comparative Example 2)
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.

(Comparative Example 3)
AFP-L3 (fractional ratio based on lectin reactivity of AFP) was measured by the same method as in Comparative Example 2. The results are shown in Table 2, FIG. 2 (d) and FIG. The area under the curve in the ROC analysis was 0.665.

  As is clear from the results shown in Table 2 and FIG. 2, only NX-PVKA of Example 2 reflected the Child-Pugh classification (classification for evaluating liver function). Moreover, as is clear from the results of FIG. 3, NX-PVKA of Example 2 reflected the prognosis prediction most.

(Example 3)
Serum samples of 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) obtained by dividing the PIVKA-II measured value by the NX-PVKA measured value was calculated. 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 Adia Corporation). The measured values are shown in Table 3.

A graph showing the measured values of PIVKA-II is shown in FIG. FIG. 5 shows a graph of the ratio value obtained by dividing the PIVKA-II measurement value by the NX-PVKA measurement value. In Table 3, FIG. 4 and FIG. 5, HCC is hepatocellular carcinoma, CH is chronic hepatitis, LC is cirrhosis, B is hepatitis B, C is hepatitis C, NBNC is non-B non-C hepatitis, NASH is non Alcoholic liver disorder, AIH is autoimmune hepatitis, and ALD is alcoholic liver disorder.
As is clear from the results shown in Table 3, FIG. 4, and FIG. 5, the ratio value (NX-PVKA-R in FIG. 5) was obtained by dividing the PIVKA-II measurement value obtained by the conventional measurement method by the NX-PVKA measurement value. ), It was possible to distinguish samples that were false positives in the conventional measurement method, and showed high specificity for liver cancer.

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.

(1) Reactivity to Gla residue-containing peptides Three peptides represented by the following SEQ ID NOs: 1 to 3 (PV002, including Gla residues (denoted as γ) considered to exist at the N-terminal 16 residues of PIVKA-II PV003 and PV00 (4) were synthesized.
PV002: ANTFLEγVRKGNLγRγ
PV003: ANTREEEVRKGNLγRγ
PV004: ANTREEEVRKGNLERγ
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)

In order to examine the reactivity to these three peptides, competitive ELISA was performed by the following method.
PIVKA-II with a decarboxylation treatment time of 1 hour was diluted with PBS to a concentration of 0.1 μg / mL, then dispensed into a 96-well plate for ELISA, 50 μL / well, and allowed to stand at 4 ° C. overnight. Each well was washed three times with PBS containing 0.05% Tween 20 (hereinafter referred to as “PBST”) (400 μL / well), and PBST containing 1% BSA (hereinafter referred to as “BSA-PBST”) was divided into 100 μL / well. Then, blocking was performed by allowing to stand at room temperature for 1 hour. After washing 3 times with PBST, each well was dispensed with 25 μL / well of peptide solutions of each concentration (20, 4, 0.8, 0.16, 0.032 μmol / 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 μL / well and allowed to stand at room temperature for 1 hour. After washing 3 times with PBST, a solution of HRP-labeled goat anti-mouse IgG antibody (manufactured by DAKO) diluted 3000 times with BSA-PBST was dispensed at 50 μL / well and allowed to stand at room temperature for 1 hour. After washing three times with PBST, a substrate solution containing orthophenylenediamine (manufactured by Tokyo Chemical Industry Co., Ltd.) was dispensed at 50 μL / well and allowed to stand at room temperature for 10 minutes. To this, 1.5 N sulfuric acid was dispensed at 50 μL / well to stop the reaction, and then the absorbance at 492 nm of the microplate reader was measured. The result is shown in FIG.

  First, the P-16 monoclonal antibody showed equivalent reactivity to all three peptides. From this, the epitope of P-16 monoclonal antibody is 1-6 residues (aa1-6 shown in FIG. 7) at the N-terminal of PIVKA-II which is a common site of three kinds of peptides, or the amino acid sequence of PIVKA-II 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. In the present specification, the X to Y residues from the N-terminus of PIVKA-II are referred to as aaX-Y.

(2) Reactivity to denatured PIVKA-II, denatured prothrombin, and denatured thrombin The reactivity of each monoclonal antibody to denatured PIVKA-II, prothrombin, and thrombin was examined by Western blotting. Specifically, reactivity to PIVKA-II having a decarboxylation treatment time of 2 hours, 0 minute prothrombin, and commercially available purified thrombin (manufactured by Benesis) was examined by the following method. 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. 5 μL / well of each sample was added to a polyacrylamide gel (Multigel II Mini 4/20) manufactured by Cosmo Bio, and electrophoresis (SDS-PAGE) was performed at 30 mA for 1 hour. 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 μg / 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 an HRP-labeled goat anti-mouse IgG antibody solution (manufactured by DAKO) diluted 2000 times with BSA-PBST, and gently shaken at room temperature for 1 hour. Each PVDF membrane was washed 3 times with PBST and once with PBS, then immersed in a substrate solution containing diaminobenzidine (manufactured by Dojindo Laboratories), reacted for 3 minutes, then transferred to purified water to stop the reaction. . The result of the obtained stained image is shown in FIG.

  First, the antibody of the conventional reagent reacted only to PIVKA-II and not to prothrombin, whereas both the P-11 and P-16 monoclonal antibodies reacted equally to PIVKA-II and prothrombin. On the other hand, none of the antibodies reacted with thrombin. This suggests that the P-11 and P-16 monoclonal antibodies recognize the common site of the primary amino acid sequences of PIVKA-II and prothrombin, that is, the site not containing the Gla residue.

(3) Reactivity to fragment 1 and fragment 2 The reactivity of each monoclonal antibody to fragments 1 and 2 of prothrombin was examined by Western blotting. Fragments 1 and 2 (manufactured by Enzyme Research Laboratories) were each adjusted to a concentration of 0.05 mg / mL with PBS, mixed one-on-one with a mercaptoethanol-containing SDS treatment solution (manufactured by Cosmo Bio) for 10 minutes. Boiled.
Using the prepared fragments 1 and 2, the reactivity of each monoclonal antibody to fragments 1 and 2 was examined in the same manner as the Western blot method of (2) above. The result of the obtained stained image is shown in FIG.

  The conventional reagent antibody (MU-3) did not react with either fragment 1 or 2 derived from prothrombin. The P-16 monoclonal antibody was highly reactive against the fragment 1, while the P-11 monoclonal antibody was weak but reactive. On the other hand, 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 exist within 156 residues (aa1-156) from the N-terminus of fragment 1, ie prothrombin.

(4) Reactivity to a Gla residue-free peptide From the N-terminal of prothrombin to the 70th position and not containing a Gla residue (PIVKA-II in which all 10 Gla residues remain Gul residues) Ten partial peptides represented by 13 were synthesized.
The 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)
amino acid sequence of aa7-22
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)
Aa19-34 amino acid sequence
Glu Glu Thr Cys Ser Tyr Glu Glu Ala Phe Glu Ala Leu Glu Ser Ser (SEQ ID NO: 7)
amino acid sequence of aa25-40
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 Asp Val Phe Trp Ala Lys Tyr Thr Ala (SEQ ID NO: 9)
amino acid sequence of aa37-52
Thr Asp Val Phe Trp Ala Lys Tyr Thr Ala Cys Glu Thr Ala Arg Thr (SEQ ID NO: 10)
Aa43-58 amino acid sequence
Lys Tyr Thr Ala Cys Glu Thr Ala Arg Thr Pro Arg Asp Lys Leu Ala (SEQ ID NO: 11)
amino acid sequence of aa49-64
Thr Ala Arg Thr Pro Arg Asp Lys Leu Ala Ala Cys Leu Glu Gly Asn (SEQ ID NO: 12)
Aa55-70 amino acid sequence
Asp Lys Leu Ala Ala Cys Leu Glu Gly Asn Cys Ala Glu Gly Leu Gly (SEQ ID NO: 13)

  The reactivity of each monoclonal antibody and each peptide was examined by competitive ELISA similar to (1) above. The results are shown in Table 4. First, the P-16 monoclonal antibody showed reactivity to the aa1-16 peptide, but not to aa7-22 and other peptides. On the other hand, it was found that the P-11 monoclonal antibody did not react with any peptide.

The above results are summarized as follows.
The epitope of P-16 monoclonal antibody is determined from the reactivity of the antibody to the peptides described in (1) and (4) above and the reactivity of 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).
amino acid sequence of aa1-5
Ala Asn Thr Phe Leu (SEQ ID NO: 14)

  Moreover, since the epitope of 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 It was thought that it exists in the range of 1 aa60-156.

  The PIVKA-II measurement method and measurement reagent of the present invention can be used for determination of the degree of liver injury and prediction of prognosis, prediction of liver cancer prognosis, and detection of highly specific liver cancer.

Claims (5)

(A) a step of measuring PIVKA-II caused by vitamin K deficiency by an immunoassay using a two-antibody sandwich method and obtaining a measurement value A;
(B) measuring PIVKA-II by an immunoassay using a two-antibody sandwich method to obtain a measurement value B; and (c) comparing the measurement value A and the measurement value B.
A blood test method for determining the degree of liver damage in liver cancer based on the value obtained by the measurement method of PIVKA-II including
As an antibody used in the two-antibody sandwich method of the step (a), an antibody specific to PIVKA-II caused by vitamin K deficiency does not react with PIVKA-II purified from a human hepatoma cell culture cell line. use,
Using one anti-PIVKA-II monoclonal antibody that 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 step (b),
Characterized by using the anti-human prothrombin antibody that does not contain antibodies that react with human thrombin as the other antibody used in the double antibody sandwich method of the step (b), method.   The method according to claim 1, wherein the antibody used in the two-antibody sandwich method in the step (a) is a monoclonal antibody.   The method according to claim 1 or 2, wherein the epitope of the antibody used in the two-antibody sandwich method of step (a) is present in 156 residues from the N-terminus of prothrombin.   An antibody specific for PIVKA-II resulting from the vitamin K deficiency is produced by a monoclonal antibody produced by the hybridoma identified by accession number FERM BP-11258 and a hybridoma identified by accession number FERM BP-11259 The method according to any one of claims 1 to 3, wherein the method is a monoclonal antibody. (A) a step of measuring PIVKA-II caused by vitamin K deficiency by an immunoassay using a two-antibody sandwich method and obtaining a measurement value A;
(B) measuring PIVKA-II by an immunoassay using a two-antibody sandwich method to obtain a measurement value B; and (c) comparing the measurement value A and the measurement value B.
A blood test method for determining liver cancer based on a value obtained by a measurement method of PIVKA-II including
As an antibody used in the two-antibody sandwich method of the step (a), an antibody specific to PIVKA-II caused by vitamin K deficiency does not react with PIVKA-II purified from a human hepatoma cell culture cell line. use,
Using one anti-PIVKA-II monoclonal antibody that 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 step (b),
Characterized by using the anti-human prothrombin antibody that does not contain antibodies that react with human thrombin as the other antibody used in the double antibody sandwich method of the step (b), method.