JP2702616B2 - PIVKA-II measurement reagent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method and a reagent for measuring PIVKA-II. For more information, see PIVK
The present invention relates to a measurement method and a measurement reagent for measuring A-II by an immunoassay using a two-antibody sandwich method.

[0002]

2. Description of the Related Art PIVKA-II is a precursor of prothrombin, one of vitamin K-dependent plasma proteins, and has an incomplete degree of γ-carboxylation of ten glutamic acid residues in the amino terminal region. Say something. Those with a complete degree of carboxylation are called normal prothrombin. Therefore, it can be said that PIVKA-II is a decarboxylated form of γ-carboxyglutamic acid residue of normal prothrombin.
In addition to the name VKA-II, abnormal prothrombin (Ab
It is sometimes called normal prothrombin. Depending on how many of the 10 glutamic acid residues undergo γ-carboxylation, several types of PIVKA-II
Exist in a mixed state. Since the present invention is mainly aimed at measuring PIVKA-II in a biological sample, PIVKA-II in the present invention refers to a mixed state of several kinds of PIVKA-II unless otherwise specified.
Those with a complete degree of carboxylation for 10 glutamic acid residues are called normal prothrombin.

[0003] Regarding the clinical usefulness of PIVKA-II measurement, in the case of vitamin K deficiency or suppression, the γ-carboxylation is incomplete and as a result, P
Since IVKA-II appears in the blood, its measurement as a marker for vitamin K deficiency or suppression is clinically important. About PIVKA-II
induced by vitaminK absen
This is an abbreviation for ce-II, which is named based on the above physiological viewpoint. Recently, it has been found that PIVKA-II appears in the blood with hepatocellular carcinoma, and PIVK-II is also found in hepatocellular carcinoma patients who are negative for α-fetoprotein, which is conventionally a good marker for hepatocellular carcinoma.
A-II may appear at a high concentration, so that α-II
Clinical utility equivalent to fetoprotein has been recognized. The following references 1) to 3) are listed for reference regarding the relationship between PIVKA-II, vitamin K and hepatocellular carcinoma. 1) Motohara K. Kuroki Y. Kan
H. Endo F. Matsuda I. Detection of vitamin K de
ficiencyby use of an enzyme
me-linked immunosorbent a
ssay for circulating abno
rmal prothrombin. Pediatric Research. 1985; 1
9: 354-7 2) Okuda H .; Obata H .; Nakanis
hi T. Furukawa R. Hashimoto
E. FIG. Production of abnormal pr
othrombin (des-γ-carboxy p
rothrombin) by hepatocelllu
lar carcinoma. Journal Hepatology. 1987;
4: 357-63 3) Hattori N.R. Ohmizo R. Unou
ra M. TanakaN. Kobayashi K. et al. Abnormal prothrombin meas
announcements in hepatocellular
carcionoma Journal of Tu
mor marker oncology. 1988;
3: 207-16

[0004] As a method for measuring PIVKA-II, a competitive radioimmunoassay using a polyclonal anti-PIVKA-II antibody (Blanchard R. et.
al. Acquired vitamin K-dep
end carboxylation defi
science in river disease. T
he New England Journal of
Medicine. 1981; 305: 242-
8) Alternatively, a method of measuring the thrombin activity of PIVKA-II remaining after absorption of normal prothrombin (So
ulier J. et al. A new method
to assay des-γ-carboxypr
othobin. Gastroenterolog
y. 1986; 91: 1258-62), but all of these methods are practical in a clinical laboratory where a large number of clinical specimens are handled due to complicated material preparation and complicated measurement systems. is not. In contrast to these methods, a specific method for measuring PIVKA-II using an anti-PIVKA-II monoclonal antibody (Japanese Patent Application Laid-Open No. 60-6055)
No. 7) is very simple and has the characteristic that a large number of samples can be accurately measured. Currently, a measurement reagent using this method is widely used as the only diagnostic reagent for PIVKA-II measurement.

[0005] Measurement of PIVKA-II is performed together with measurement of α-fetoprotein for the diagnosis and follow-up of hepatocellular carcinoma. The measurement of α-fetoprotein can be used in serum or plasma as a sample. The method of measuring PIVKA-II based on Japanese Patent Application Laid-Open No. 60-60557 has a serious drawback in that accurate measurement cannot be performed with serum and plasma must be used as a sample (Shin Hattori, Research, Vol. 65, No. 3, page 257, left column states, "Some serum samples showed abnormally high values, indicating that stable quantitative values could not be obtained." ). In addition, even if plasma samples are frequently frozen and thawed during the storage period, or if they are stored at high temperatures, the blood coagulation reaction progresses and the condition becomes close to that of serum. May be. This means that two types of blood, serum and plasma, must be collected from the patient who needs diagnosis, and the burden on the patient, as well as the reliable measurement of PIVKA-II in the serum sample as well as in the measurement side. The development of a reagent that can be used is desired. Further, although accurate PIVKA-II measurement is possible in a plasma sample, the reason why a stable measurement value cannot be obtained in a serum sample is unknown, and no solution has been found yet.

[0006]

SUMMARY OF THE INVENTION In view of the above circumstances, the present inventors have developed PIVKA-plasma samples, not only plasma samples, but also blood coagulation reactions, especially serum samples.
The purpose is to develop a reagent that can measure II.

[0007]

Means for Solving the Problems The present inventors have proposed PIVKA.
In the immunoassay using the two-antibody sandwich method for -II, when an anti-human prothrombin antibody to be used as the second antibody is prepared by immunizing an animal with human prothrombin by a usual method, even if the human prothrombin affinity is Even when the antibody was purified using a column (JP-A-60-60557), it was found that an antibody cross-reacting with human thrombin appeared in the anti-human prothrombin antibody. Furthermore, as a result of earnestly studying the properties of the antibody, it has been found for the first time that an antibody that cross-reacts with human thrombin has a bad effect on the PIVKA-II measurement system of serum samples. That is, it has been found for the first time that PIVKA-II can be accurately measured not only in a plasma sample but also in a serum sample if an anti-human prothrombin antibody used as a second antibody in a PIVKA-II immunoassay system does not react with human thrombin. The present invention has been completed.

That is, in the present invention, an anti-PIVKA-II monoclonal antibody is used as a primary antibody in a PIVKA-II measuring reagent based on a two-antibody sandwich method in an oxygen immunoassay, a radioimmunoassay, or another assay. The second antibody used is an antibody against a common antigen of PIVKA-II and prothrombin (called an anti-prothrombin antibody). At this time, the antibody used as the second antibody is thrombin regardless of whether it is a monoclonal antibody or a polyclonal antibody. By using an antibody that does not cross-react with serum, serum samples as well as plasma samples
An object of the present invention is to provide a completed reagent capable of accurately measuring IVKA-II. Since the reagent thus produced does not react with thrombin, PIVKA-II can be measured without being affected by thrombin present in a large amount in serum. On the other hand, when an anti-prothrombin antibody that cross-reacts with thrombin is used, the measurement value is increased by reacting with thrombin in addition to PIVKA-II in serum, and accurate measurement is impossible.

Hereinafter, the present invention will be described in detail. The anti-prothrombin antibody which does not react with thrombin according to the present invention is produced, for example, as follows. First, from fresh human plasma, Shapiro et al. (Shapiro S. et al.
The purification of human
prothrombin. Thromb. Diat
h. Haemorph. , 1966; 16: 469-9.
Purified human prothrombin is obtained by the method of 0). Next, a rabbit is immunized with this human prothrombin, and blood is collected to obtain an antiserum. Ammonium sulfate is added to the antiserum for salting out, dialyzed, and ion-exchanged with DE-52 Cellulose. This is called human prothrombin affinity
Apply to a column and elute with 4M guanidine hydrochloride to obtain anti-human prothrombin rabbit IgG antibody. After dialysis to remove guanidine hydrochloride, the fraction is passed through a thrombin affinity column, and the flow-through fraction is collected to obtain an anti-prothrombin antibody that does not react with thrombin. In addition to the above polyclonal antibody, a mouse was immunized with purified human prothrombin, and its spleen cells were collected. (Koehler G. Milstein C. Dev)
iation of specificibod
y-producting culture and
tumor lines by cell fusio
n. Eur. J. Immunol. 1976; 6:51.
A cell established as an anti-prothrombin antibody-producing cell line that undergoes cell fusion with myeloma cell line P3U1 according to 1-9), performs cloning three times by limiting dilution, and reacts with PIVKA-II and normal prothrombin without reacting with thrombin May be used as an anti-prothrombin antibody that does not react with thrombin.

The anti-PIVKA-II monoclonal antibody according to the present invention is disclosed in, for example, a published patent (JP-A-60-6055).
As described in 7), it is manufactured as follows.
First, BaSO ₄ and BaC were obtained from plasma of warfarin recipients.
O ₃ treatment to adsorb and remove human prothrombin,
Performed ion exchange with E-52 Cellulose, and finally adsorbed to an affinity column using an anti-prothrombin antibody that reacts with PIVKA-II and normal prothrombin, and eluted with 4M guanidine hydrochloride.
Dialyze and concentrate to obtain purified PIVKA-II. Next, a mouse was immunized with the purified PIVKA-II, and its spleen cells were collected. Cell fusion with the myeloma cell line P3U1 by the method described above, cloning is performed three times by the limiting dilution method, and cells established as an antibody-producing cell line that reacts only with PIVKA-II without reacting with normal prothrombin are secreted. Monoclonal antibodies can be used as anti-PIVKA-II monoclonal antibodies.

Next, a measurement method using the two-antibody sandwich method in the present invention is carried out, for example, as follows. Here, the case of an enzyme immunoassay is shown, but it goes without saying that the present invention can also be used in a radioimmunoassay or other methods. The specific embodiment of the measuring reagent of the present invention is as follows. That is, the measurement reagent of the present invention comprises an anti-human prothrombin antibody which does not contain an antibody that reacts with human thrombin as an essential component, and comprises a monoclonal anti-PIVKA-II antibody (alone or immobilized), a standard antigen, an enzyme and a substrate. This is a set obtained by combining those arbitrarily selected from a group. Here, when the solid phase is included in the set, the solid phase is provided in a state coated with the monoclonal anti-PIVKA-II antibody, or the standard anti-human prothrombin antibody and the enzyme are included in the set. If
Both may be provided in a conjugated state, and these are also included in the embodiment of the measurement reagent of the present invention. In addition, an antigen diluent, a reaction diluent, a substrate lysing solution, a reaction stop solution, and the like that are suitable for the benefit of performing the measurement may be freely attached to the set, and these are not limitations on the present invention. . In the measurement, a standard antigen or a test biological sample (blood, plasma or serum) is added to an anti-PIVKA-II monoclonal antibody-coated solid phase and incubated. After washing the solid phase, an enzyme-labeled anti-prothrombin antibody (which does not react with thrombin) is added and incubated again, washed, and finally, a substrate is added and incubated.
The amount of decomposition of the substrate is measured using a spectrophotometer. As shown in the examples described later, the measurement reagent of the present invention enables PIVKA-II measurement in a serum sample for the first time.

[0012]

The present invention enables the measurement of PIVKA-II in serum, which has been impossible to measure accurately in the past, and the measurement of PIVKA-II is effective for the diagnosis and follow-up of hepatocellular carcinoma by the measurement of α-fetoprotein. Can be measured with the same serum as above, eliminating the burden on the patient of separately collecting serum and plasma.

[0013]

The effects of the present invention will be described more specifically with reference to the following examples. Embodiment 1 FIG. Preparation of anti-prothrombin antibody that does not react with thrombin To 1 liter of fresh human plasma, 80 ml of 1M BaCl ₂ is added, and the mixture is stirred for 1 hour, and then the precipitate is collected by centrifugation. After adding 200 ml of 0.2 M EDTA and stirring for 2 hours, a 25-65% saturated fraction is collected with ammonium sulfate. After dialysis against 0.1 M phosphate buffer, DEAE-S
Perform 0M to 0.6M NaCl fractionation on ephacel. The prothrombin fraction was collected, dialyzed, and purified by Hapar.
in-Sepharose and Blue-Sepha
Pass through rose. Gel filtration was performed using Ultragel AcA44 to obtain purified normal human prothrombin. Its final recovery was 30-40%. A rabbit was immunized with the obtained normal human prothrombin, 35% ammonium sulfate was added to the antiserum to obtain a precipitate, which was then dialyzed, applied to a DEAE-Cellulose column, and subjected to ion exchange chromatography with a 0.017 M phosphate buffer. To obtain the IgG fraction. The purified normal human prothrombin is bound to Sepharose activated with BrCN to form an affinity column. The purified IgG fraction is passed through an affinity column, and the bound antibody is eluted with 4M guanidine hydrochloride to obtain an anti-human prothrombin rabbit IgG antibody. After dialysis to remove guanidine hydrochloride, the solution is passed through a thrombin affinity column to collect a flow-through fraction. The antibody thus purified is used as an anti-prothrombin antibody that does not react with thrombin.

Embodiment 2 FIG. Preparation of enzyme-labeled antibody 5mg purified anti-prothrombin antibody that does not react with thrombin
Was dialyzed against 0.1M acetate buffer (pH 4.2).
0.2 mg of pepsin was added and incubated at 37 ° C. for 24 hours. After adjusting the pH to 7.0, Ultragel
0.1M acetate buffer pH 7.0 over an AcA44 column.
And gel filtration is performed to obtain F (ab ') ₂ . F (a
b ′) After dialysis of ₂ against 0.1 M phosphate buffer pH 6.0,
Add 50 μl of 0.1 M mercaptoethylamine and add
Incubated at BR> ° C for 90 minutes. Seph equilibrated with 0.1 M phosphate buffer pH 6.0 (5 mM, EDTA)
dialyzed through an adex G25 column and Fab
-SH was obtained. On the other hand, 2 mg of horseradish peroxidase (abbreviated as HRP) was used as an enzyme in a 0.1 M phosphate buffer pH
After dissolving in 7.0, 0.7 mg of N-succinimidyl m-maleimidobenzoate (dissolved in N, N-dimethylformamide) was added and incubated at 30 ° C. for 60 minutes. Sep equilibrated with 0.1 M phosphate buffer pH 6.0
The solution was dialyzed through a hadex G25 column to obtain maleimidated HRP. Fab-SH and maleimidated H
Mix with RP and incubate overnight at 4 ° C.
Ultrogel equilibrated with 1 M phosphate buffer pH 6.5
Gel filtration was performed on an AcA44 column to obtain an enzyme-labeled antibody.

Embodiment 3 FIG. Measuring method PIVKA-II monoclonal naral body is disclosed in
It is prepared according to a conventional method described in JP-A-557-557. The method of immobilizing this antibody on a multiplate for enzyme immunoassay is carried out by a conventional method described in the publication. Inject 100 µl of the test sample per well into the solid phase coated with the anti-PIVKA-II monoclonal antibody, and incubate at 4 ° C overnight. 0.05M Tris monohydrochloride buffer pH7.
After washing three times with 5 (0.05% Tween 20), 100 μl of enzyme-labeled antibody is added and incubated at 4 ° C. for 1 hour. 0.05M Tris-HCl buffer pH 7.5 (0.0
After washing three times with 5% Tween 20), the ABTS solution 10
After adding 0 μl and leaving the mixture to stand for 60 minutes, the reaction is stopped by adding 100 μl of 2 mM sodium azide, and the absorbance at a wavelength of 405 nm is measured by a spectrophotometer.

Embodiment 4 FIG. Measurement of Plasma and Serum Samples of PIVKA-II Negative Healthy Person Plasma samples and serum samples were simultaneously collected from 40 PIVKA-II negative healthy persons, and PIVKA-II was measured for each of them according to the method described in Example 3. The results are shown in FIG. 1 and FIG. FIG. 1 shows a plasma sample, a method using an anti-human prothrombin antibody containing an antibody reactive with human thrombin (A) and a method of the present invention using an anti-human prothrombin antibody not containing an antibody reactive with human thrombin ( The result of B) is shown. Since the detection limit of this measurement reagent is 0.0625 AU / ml, methods A and B
In any case, it is shown that the plasma sample of the healthy subject is below the detection limit. That is, the healthy person is PIVK
A-II is not detected and indicates an accurate value that no person has a high measured value. On the other hand, as shown in FIG.
0.0625-0.13AU /
In many cases, it shows a high measured value distributed to ml, and a stable value cannot be obtained. As described above, the method B of the present invention enables measurement of not only a plasma sample but also a serum sample, and the method A does not allow accurate measurement of a serum sample as has been described so far.

Embodiment 5 FIG. Measurement of serum and plasma samples of PIVKA-II-positive hepatocellular carcinoma patients Serum samples and plasma samples were simultaneously collected from 25 PIVKA-II-positive hepatocellular carcinoma patients, and each was measured according to the measurement method. 3 and 4 show the results of plotting the correlation between the serum sample and the plasma sample collected at the same time.
Shown in FIG. 3 is a result of plotting the correlation between a serum sample and a plasma sample by a reagent using an anti-prothrombin antibody not containing an antibody that reacts with human thrombin. The correlation coefficient is 0.989 and the slope is 0.981 (FIG. 3).
The solid line is a straight line having a slope of 1, and the curve plotting the serum sample and the plasma sample is indicated by a dotted line), and accurate measurement was possible even with the serum sample. On the other hand, FIG. 4 shows the result of using an anti-prothrombin antibody containing an antibody that reacts with human thrombin. The correlation coefficient was as good as 0.991, but the slope was 0.949. Quantitative value in PIVKA-II increases the non-specific reaction of thrombin.
Is measured simultaneously with the specific reaction of
The solid line is a straight line having a slope of 1, and the curve plotting the serum sample and the plasma sample is indicated by a dotted line).

As described above, when an anti-prothrombin antibody that reacts with thrombin is used, measurement using a serum sample is impossible. Similarly, serum specimens can be used.

[Brief description of the drawings]

FIG. 1 shows a method using an anti-human prothrombin antibody containing an antibody that reacts with human thrombin as a second antibody (A)
PIVKA by a method (B) using an anti-human prothrombin antibody not containing an antibody that reacts with human thrombin
-Measured value of PIVKA-II in plasma of 40 II-negative healthy persons.

FIG. 2 shows a method using an anti-human prothrombin antibody containing an antibody that reacts with human thrombin as a second antibody (A)
PIVKA by a method (B) using an anti-human prothrombin antibody not containing an antibody that reacts with human thrombin
-Measured value of PIVKA-II in serum of 40 II-negative healthy persons.

FIG. 3 shows a correlation between a plasma sample and a serum sample of a PIVKA-II positive patient by a method using an anti-prothrombin antibody containing no antithrombin antibody.

FIG. 4 shows a correlation between a plasma sample and a serum sample of a PIVKA-II positive patient by a method using an anti-prothrombin antibody including an anti-thrombin antibody.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location A61K 39/44 A61K 39/44

Claims (5)

(57) [Claims] 1. An anti-human prothrombin antibody that does not contain an antibody that reacts with human thrombin as a second antibody in measuring PIVKA-II in a biological sample by an immunoassay using a two-antibody sandwich method. A reagent for measuring PIVKA-II, characterized by using: 2. An anti-human prothrombin antibody which does not react with human thrombin is an antibody produced by removing an anti-human thrombin antibody from an antibody obtained by immunizing a non-human animal with human prothrombin. Item 7. The reagent for measuring PIVKA-II according to Item 1. 3. The reagent for measuring PIVKA-II according to claim 1, wherein the anti-human prothrombin antibody that does not react with human thrombin is a mouse monoclonal antibody. 4. The reagent for measuring PIVKA-II according to claim 1, wherein the biological sample is plasma or serum. 5. An anti-human prothrombin antibody that does not contain an antibody that reacts with human thrombin as a second antibody in measuring PIVKA-II in a biological sample by an immunoassay using a two-antibody sandwich method. A method for measuring PIVKA-II, comprising using: