JP3121166B2 - Anti-PIVKA-II monoclonal antibody - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention uses PIVKA-II produced by a cultured human hepatocyte cell line as an antigen to prepare a monoclonal antibody that reacts with the antigen and to use the antibody in biological samples in biological samples.
The present invention relates to a measurement reagent for measuring PIVKA-II.

[0002]

2. Description of the Related Art PIVKA-II is a precursor of prothrombin which is one of vitamin K-dependent plasma proteins.
-Incomplete degree of carboxylation. Those with a complete degree of carboxylation are called normal prothrombin. Therefore, PIVKA-II is normal prothrombin γ
-It can be said that it is a decarboxylated form of carboxyglutamic acid residue, and is sometimes called Abnormal prothrombin other than the name PIVKA-II. Depending on whether some of the 10 glutamic acid residues undergo γ-carboxylation, several types of PIVKA-
II exists 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, unless otherwise specified,
It refers to the mixed state of several types of PIVKA-II. 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-
Incomplete carboxylation results in the appearance of PIVKA-II in the blood, and its measurement as a marker of vitamin K deficiency or suppression is clinically important.
About PIVKA-II Protein induced by vitamin K absence-I
I is an abbreviation for I, 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 PIVKA-II is also found in α-fetoprotein-negative hepatocellular carcinoma patients, which has conventionally been a good marker for hepatocellular carcinoma. Has a high clinical utility equivalent to that of α-fetoprotein. For reference about the relationship between PIVKA-II and vitamin K and hepatocellular carcinoma 1)
To 3) are listed. 1) Motohara K. Kuroki Y. Kan H. Endo F. Mtsuda I.,
Detection of vitaminK deficiency by use of an enz
yme-linked immunosorbent assay for circulating ab
normal prothrombin. Pediatric Research., 19, 354-7,
1985. 2) Okuda H. Obata H. Nakanishi T. Furukawa R. Hashi
moto E., Production ofabnormal prothrombin (des-γ
-carboxyprothrombin) by hepatocellular carcinoma., J
ournal Hepatology, 4,357-63,1987. 3) Hattori N. Ohmizo R. Unoura M. Tanaka N. Kobayas
hi K., Abnormal prothrombin measurements in hepatoc
ellular carcionama., Journal of Tumor markeroncolo
gy, 3, 207-16, 1988.

[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-dependen
t carboxylation deficiency in liver disease.The N
ew England Journal of Medicine., 305, 242-8, 1981) or PIVKA-II remaining after absorption of normal prothrombin
Method for measuring thrombin activity of (Soulier J. et al.,
A new method toassay des-γ-carboxyprothrombin., G
astroenterology, 91: 1258-62, 1986), etc., but in any case, in the case of clinical examinations that handle a large number of clinical specimens due to complicated preparation of materials and complicated measurement systems. Not practical. For these methods,
Specific PIVK using anti-PIVKA-II monoclonal antibody
The A-II measurement method (Japanese Patent Application Laid-Open No. 60-60557) is very simple and has the characteristic that a large number of samples can be measured accurately. Currently, the only measurement reagent using this method is
It is widely used as a diagnostic agent for PIVKA-II measurement.

[0005] The anti-PIVKA-II monoclonal antibody disclosed in Japanese Patent Application Laid-Open No. 60557/1985 is obtained from the plasma of a patient administered a vitamin K antagonist.
PIVKA-II was purified and used as an immunizing antigen to immunize mice to prepare a monoclonal antibody against PIVKA-II.

[0006] The specific method for measuring PIVKA-II using the anti-PIVKA-II monoclonal antibody disclosed in Japanese Patent Application Laid-Open No. 60557/1985 is very simple and has the feature that a large number of samples can be accurately measured. Currently, a measuring reagent using this method is widely used as the only diagnostic agent for measuring PIVKA-II, and is used for diagnosis and follow-up of hepatocellular carcinoma.

[0007]

Problems to be Solved by the Invention Japanese Patent Application Laid-Open No. 60-60557
Specific PIVKA- using PIVKA-II monoclonal antibody
According to the II measurement method, the positive rate of hepatocellular carcinoma was 52.9%. (Shin Hattori, Clinical and Research, Vol. 65, No. 3, pp. 249-258, 1988
Year) The present inventors have prepared an antibody having characteristics different from those of the monoclonal antibody used in Japanese Patent Application Laid-Open No. 60557/1985 and constructed a measuring reagent to increase the positive rate in hepatocellular carcinoma. Considering the purpose, the present invention has been completed.

[0008]

DISCLOSURE OF THE INVENTION The present invention provides a method for culturing human hepatocytes instead of purifying PIVKA-II, which is an immunizing antigen for preparing a monoclonal antibody, from the blood of a patient to which a vitamin K antagonist has been administered as in the prior art. To obtain from a cell line. It has been reported that this human hepatocyte culture produces PIVKA-II (Hiroaki Okuda, Hepatobiliary pancreas Vol. 14, No. 5, May 1987)
759-766 a month)

[0009] PIVKA-II was purified from a cultured human hepatocyte cell line and administered to mice as an immunogen. The cells were fused with mouse spleen cells and mouse myeloma cells in the usual manner, and did not react with prothrombin. To establish a hybrid macrone that produces a monoclonal antibody that specifically reacts only with -II. In addition, ascites was obtained by intraperitoneally administering the established hybridoma clones to mice, and IgG was collected from the ascites.
Is purified. PIVKA-II in a biological sample is measured by a two-antibody immunoassay using the obtained IgG as a first antibody and an anti-prothrombin antibody as a second antibody.

Hereinafter, the present invention will be described in detail. Purification of PIVKA-II, an immunizing antigen for preparation of monoclonal antibodies, was performed using ammonium sulfate salting-out of the culture supernatant of a PIVKA-II producing human liver cultured cell line,
DEAE-Sephacel, Heparin-Sepharose, Blue-Sepharos
e, obtained by gel filtration of Ultrogel AcA44 and prothrombin affinity-column.

[0011] Anti-PIVKA-II monoclonal antibody-producing hybridoma cells are prepared by immunizing a mouse with purified PIVKA-II, collecting spleen cells from the immunized mouse, and using the method of Koehler G. et al.
stein C., Deviation of specific antibody-producting
culture and tumor lines by cell fusion., Eur.
Cell fusion and cloning with the myeloma cell line P3U1 were performed by Immunol., 6,511-9, 1976) to establish an anti-PIVKA-II antibody-producing hybridoma cell line that does not react with prothrombin but reacts only with the immunizing antigen PIVKA-II.

[0012] After administering an anti-PIVKA-II antibody-producing hybridoma cell line to a mouse to obtain ascites, the ascites is treated with Protein A.
Purify the IgG.

[0013] The PIVKA-II assay according to the present invention is based on a two-antibody sandwich method based on enzyme immunoassay, radioimmunoassay or other assay, and uses an anti-PIVKA-II monoclonal antibody as the first antibody as the antibody. Then, an antibody against a common antigen of PIVKA-II and prothrombin (referred to as an anti-prothrombin antibody) is used as the second antibody to measure PIVKA-II in the biological sample.

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 that 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. It is a set of combinations of 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 when the set includes an anti-human prothrombin antibody and an enzyme. It is free to provide both 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 lysis 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 of 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, the substrate is added and incubated, and the amount of degradation of the substrate is measured using a spectrophotometer. . As will be shown in the examples described later, the measurement reagent of the present invention enables PIVKA-II measurement at a sensitivity 30 times higher than the conventional measurement sensitivity for the first time.

[0015]

Industrial Applicability The present monoclonal antibody and the use thereof
By measuring with the PIVKA-II measurement reagent, the conventional PIVKA-II
PIVKA-II antigen, which could not be measured with the II measurement reagent, can be measured with 30 times the measurement sensitivity.

[0016]

The effects of the present invention will be described more specifically with reference to the following examples. Example 1 Cell culture of human hepatocyte culture and purification of PIVKA-II Human hepatocyte culture was prepared by using warfarin potassium 10ug / ml.
And then collect the supernatant. For the purification of PIVKA-II in the culture supernatant, an equal amount of 4M saturated ammonium sulfate was added to 40 liters of the culture supernatant, and the mixture was stirred for 2 hours and centrifuged. The precipitate
It was dissolved in 0.1 M phosphate buffer and dialyzed. 0M-0.6M NaCl fractionation is performed in DEAE-Sephacel, and the PIVKA-II fraction is collected, dialyzed, and passed through Heparin-Sepharose and Blue-Sepharose. Ul
PIVKA-II was prepared by gel filtration on trogel AcA44 and prothrombin affinity-column. Its final recovery was 10%.

Example 2 Establishment of anti-PIVKA-II monoclonal antibody-producing hybridoma cell line [0017] Purified PIVKA-II was immunized 5 times at 12.5 ug / mouse into a mouse, and its spleen cells were collected, followed by the method of Koehler G. et al. (Koehler
G. Milstein C. Deviation of specific antibody pro
ducting culture and tumor lines by cell fusion., E
ur. J. Immunol., 6, 511-9, 1976), cell fusion with myeloma cell line P3U1, 3 cloning by limiting dilution method, PI which does not react with prothrombin and does not react with prothrombin
An anti-PIVKA-II monoclonal antibody-producing hybridoma P19B7 that reacts only with VKA-II was established as a cell line.

Example 3 Measurement Method A method for immobilizing an anti-PIVKA-II monoclonal antibody on a multiplet for enzyme immunoassay is described in JP-A-60-6.
This is carried out according to the usual method described in 0557. The anti thus obtained
Inject 100 ul of the test sample per well into the solid phase of PIVKA-II monoclonal antibody coat, and incubate at 4 ° C overnight. 0.05M Tris-HCl buffer pH7.5 (0.05% Tween20)
After washing 3 times with 100 μl, add 100 μl of enzyme-labeled antibody labeled with enzyme to
To 0.05M Tris-HCl buffer pH 7.5 (0.05% Tween2
After washing 3 times with 0), add 100 ul of ABTS solution, stand still for 60 minutes, stop the reaction by adding 100 ul of 2 mM sodium azide, and measure the absorbance at 405 nm using a spectrophotometer. The standard curve measured by this method is shown in FIG. Healthy people 8
When the measurement of the example was 0.002 AU / ml or less, the detection limit of this measurement reagent was 0.002 AU / ml (FIG. 2).

FIG.

FIG. 2

Example 4 Characteristics of Obtained Monoclonal Antibody The monoclonal antibody was immobilized on a microcup, and the characteristics of the monoclonal antibody were compared using PIVKA-II positive plasma according to the method described in Example 3. The results are shown in FIG. The monoclonal antibody obtained by the present invention is disclosed in
Activity is 10 compared to the monoclonal antibody described in No. 60-605575
Twice as high.

FIG. 3

Example 5 Measurement of PIVKA-II in Serum of Liver Disease Patients Serum obtained from 214 liver disease patients was obtained according to Japanese Patent Laid-Open No. 60605605/1985.
The PIVKA-II measurement values were compared between the PIVKA-II measurement reagent using the monoclonal antibody described in No. 1 and this measurement reagent. The result is shown in FIG. The monoclonal antibody according to the present invention is a PIVKA-protein that could not be measured with a conventional PIVKA-II measurement reagent.
II could be measured, and the positive rate increased by 28.3% (Table 1). Also, there was no correlation with r2 = 0.252 with the conventional PIVKA-II measurement reagent (FIG. 4).

FIG. 4

[Table 1]

As described above, the human hepatocyte culture cell line produces
An anti-PIVKA-II monoclonal antibody was successfully prepared using PIVKA-II. Also, the reactivity is about 30 times higher than the anti-PIVKA-II monoclonal antibody of JP-A-60-605575,
PIVKA-II, which was not measured with the PIVKA-II measurement reagent, became measurable with this antibody.

[Brief description of the drawings]

FIG. 1 is a standard curve of a measuring reagent using a monoclonal antibody prepared according to the present invention.

FIG. 2 Distribution diagram of measurement of PIVKA-II in liver disease patient sample

FIG. 3 Activity comparison between monoclonal antibodies

[FIG. 4] Correlation of PIVKA-II measurement values between a measurement reagent using a monoclonal antibody described in JP-A-60-605575 and a PIVKA-II measurement reagent according to the present invention

 ────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-60-60557 (JP, A) Hiroaki Okuda et al. “Liver” Vol. 28, No. 2, page 270 CANCER RESEARCH 49, 6493-6497, 1989

Claims (2)

(57) [Claims] 1. An anti-PIVKA-II reagent which is an immunochemical measurement reagent for PIVKA-II in a biological sample and which is prepared using PIVKA-II purified from a human liver cancer cell culture cell line as an antigen.
PIVK characterized by using a monoclonal antibody
A-II measurement reagent. 2. The cultured human hepatoma cell line is PLC / PRF / 5, huH-
The P of claim 1, which is 1, huH-2, HepG2 or Hep3B.
IVKA-II measurement reagent.