JP2000346841A - Pivka-ii measurement method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To make correctly measurable the concentration of an abnormal prothrombin (PIVKA-II) without being effected by various interfering substances existing in a specimen by using a buffer liquid containing lactoprotein as the buffer. SOLUTION: A buffer is used for reaction between a specimen and a primary antibody solid phase and/or for reaction with a marker secondary antibody, and preferably the buffer used for the reaction between the specimen and the primary antibody solid phase contains lactoprotein. 0.1 wt.% or more, especially 0.1-10 wt.%, and preferably 1-5 wt.% of the lactoprotein should be added into the buffer in terms of attraction prevention effect of interfering substance. Measurement is carried out by performing reaction through addition of lactoprotein contained buffer to the primary antibody solid phase beads and the specimen, then performing reaction with the secondary antibody, and measuring a signal of the marker secondary antibody connected with the primary antibody solid phase beads. By taking out the beads from a reaction vessel and then performing the measurement, an accurate measurement can be made. Even if an interfering substance is attracted to the reaction vessel, there is no influence on the measurement since only the primary antibody solid phase is measured.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for measuring abnormal prothrombin (PIVKA-II), and more particularly, to measuring the concentration of PIVKA-II in a biological sample and the effect of an interfering substance present in the sample. It relates to a method of accurately measuring without receiving.

[0002]

2. Description of the Related Art Prothrombin, a blood coagulation factor, is produced in normal hepatocytes. However, in an anemia state in which vitamin K is deficient, normal prothrombin is not produced, and some of the glutamate contained in the molecule is not produced. Abnormal prothrombin (PI) with a structural abnormality in which carboxylation of residues is incomplete
VKA-II) was known to be produced. This PIVKA-
II is a protein in which a part of normal prothrombin is abnormal, and has a structure very similar to normal prothrombin. For this reason, PIVKA-
The measurement became possible by using an antibody that recognizes II.

[0003] In 1984, Liebman et al. Reported that the concentration of PIVKA-II released into the blood of patients suffering from hepatocellular carcinoma was higher than that of normal subjects by radioimmunoassay using antibodies. He found the possibility of diagnosing hepatocellular carcinoma.

Further, a sandwich measurement method of PIVKA-II was developed utilizing the principle of a sandwich measurement method of sandwiching a substance to be measured using two kinds of antibodies which were widely applied at that time (Japanese Patent Laid-Open No. 60557/1985). issue). This method is called PIVKA-
An antibody specific for II (referred to as primary antibody or primary antibody) is immobilized on a reaction vessel, PIVKA-II is bound thereto, and another antibody (secondary antibody or secondary antibody) labeled with an enzyme or the like is used. In this method, the amount of the labeled secondary antibody increases in accordance with the amount of PIVKA-II, and the concentration of PIVKA-II can be measured by detecting the signal. PIVKA-II
Is a modification of the N-terminal structure of prothrombin (specific structure), but this sandwich method uses PIVKA-
In the antibody sandwich method that recognizes only II, there is a special circumstance that measurement cannot be performed well because both antibodies try to react with a narrow specific structure. For this reason, PIVKA-II
Immobilize an antibody that recognizes the specific structure of
The other secondary antibody used was an antibody that recognized the common region between PIVKA-II and normal prothrombin. At this time, the labeled secondary antibody has a property of binding to not only PIVKA-II but also normal prothrombin and its degraded product, thrombin and fragments, and at the time of measurement, these normal prothrombin and its degraded product, thrombin and In some cases, fragments were unexpectedly nonspecifically adsorbed to the reaction vessel, in which case PIVKA-II alone could not be measured correctly due to the binding of the labeled secondary antibody to these substances. That is, when normal prothrombin and its decomposed products, thrombin and fragments, are non-specifically adsorbed to the reaction vessel and the labeled secondary antibody binds to these substances, the labeled product reacted with PIVKA-II is labeled. In order to detect the signal without distinguishing it from the secondary antibody, PIVKA-
It gives false results, such as measuring II.

On the other hand, Japanese Patent No. 2702616 describes a method for measuring PIVKA-II using an anti-human prothrombin antibody that does not contain an antibody that reacts with human thrombin as a secondary antibody. This method focuses only on thrombin among the various interfering substances as described above. Even if the thrombin in the sample binds to the reaction container, if there is no antibody that reacts with the labeled secondary antibody, thrombin adsorption is not performed. PIVKA-II in a sample can be measured correctly without being affected.

[0006]

However, this method has a problem that an absorption operation using a thrombin affinity gel is required in order to remove an antibody that reacts with thrombin from the secondary antibody. However, above all, it is presumed that this method can avoid the effect of thrombin, but it cannot avoid the effect of adsorbing interference substances such as normal prothrombin other than thrombin and fragments of its degradation products. In particular, interfering substances include not only thrombin but also various related substances, and their concentrations in these samples are all higher than the concentration of PIVKA-II. There was a problem how to avoid it. That is, even in the study of the present inventor, when not only thrombin but also prothrombin or a fragment thereof was unexpectedly adsorbed to the reaction vessel in the measurement of PIVKA-II, the effect was larger than that when thrombin was adsorbed. , PIVKA-II could not be measured correctly. At this time, the method of using a secondary antibody that does not react with the adsorbed substance, that is, the method of using a labeled secondary antibody that does not react with the adsorbed thrombin as in Japanese Patent No. 2702616, has the problem of adsorbing prothrombin and the like. Not resolved. That is, the labeled secondary antibody is PIVKA-II
If the method of removing the antibody that reacts with prothrombin is adopted for the adsorption of prothrombin to the reaction vessel, the PIVKA- This is because there is a contradiction that it cannot respond to II.

Accordingly, an object of the present invention is to provide an accurate method for measuring PIVKA-II, which excludes the influence of various interfering substances present in a sample.

[0008]

The present inventors have conducted various studies to prevent the interference of the interfering substance into the reaction vessel, instead of removing the antibody against the interfering substance. The present inventors have found that surfactants, serum-derived proteins and inorganic salts cannot provide a sufficient effect, and that the addition of milk protein to a buffer solution is effective in preventing adsorption of interfering substances, thus completing the present invention.

That is, the present invention relates to PIVK in a biological sample.
An object of the present invention is to provide a method for measuring PIVKA-II, which comprises using a buffer containing milk protein as a buffer in an immunoassay using the A-II two-antibody sandwich method.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The measuring method of the present invention is a two-antibody sandwich method, but a measuring method in which a primary antibody is a solid phase and a secondary antibody is a labeled antibody is preferable. Therefore, the buffer in the present invention is a buffer used for the reaction between the sample and the primary antibody solid phase and / or a buffer used for the reaction of the labeled secondary antibody, but is used for the reaction between the sample and the primary antibody solid phase. Preferably, the buffer contains milk protein. Among milk proteins, skim milk is particularly preferred because it is easily available.
The milk protein is preferably added to the buffer solution in an amount of 0.1% by weight or more, particularly 0.1 to 10% by weight, and more preferably 1 to 5% by weight from the viewpoint of the effect of preventing the adsorption of interference substances.

Further, it is preferable to add inorganic salts such as sodium chloride, and animal serum proteins such as bovine serum albumin and mouse gamma globulin to the buffer. The concentration of the inorganic salts is preferably 0.1 to 0.5M, particularly preferably 0.2 to 0.5M. The concentration of animal serum protein is 1 to 10% by weight, especially 3 to 7%.
% By weight is preferred.

The basic composition of the buffer is preferably a Tris-HCl buffer or the like.

A particularly preferred buffer composition is 5.0% bovine serum albumin, 50 mg / L normal mouse gamma globulin, 0.3 M sodium chloride, 0.01 M disodium ethylenediaminetetraacetate, 0.05% sodium azide and 0.05M Tris-HCl buffer (pH 8.0) containing 1.0% skim milk.

The measurement method of the present invention may be in accordance with the usual two-antibody sandwich method except that the above-mentioned buffer is used. As the primary antibody, it is preferable to use an anti-PIVKA-II antibody, particularly an anti-PIVKA-II monoclonal antibody that recognizes a structurally abnormal site of PIVKA-II. The secondary antibody may be any of an anti-prothrombin antibody and an anti-PIVKA-II antibody, and these may include an antibody that reacts with thrombin. Further, the secondary antibody may be a polyclonal antibody or a monoclonal antibody.

The primary antibody may be immobilized on a plate or beads, but beads are preferred. The label of the secondary antibody may be any of radioisotope such as ¹²⁵ I, a luminescent substance, a fluorescent substance, and an enzyme.
A luminescent substance or a fluorescent substance is particularly preferred in that the amount of the secondary antibody can be reduced.

In the measurement, the case where beads are used as the primary antibody solid phase is shown as an example. First, the primary protein-immobilized beads and the sample are allowed to react by adding the above-mentioned milk protein-containing buffer, and then labeled. The reaction is performed by reacting with the secondary antibody and measuring the signal of the labeled secondary antibody bound to the primary antibody solid-phase beads. At the time of this signal measurement, a more accurate measurement can be performed by removing the immunoreaction product to be measured, that is, the primary antibody solid phase (beads) from the reaction container and measuring it.
That is, by moving the primary antibody solid phase from the reaction vessel, even if an interfering substance is adsorbed on the reaction vessel, only the primary antibody solid phase is measured at the time of measurement, so this effect can be completely avoided.

[0017]

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

Production Example 1 (Preparation of Primary Antibody Solid Phase) Anti-PIVKA-II monoclonal antibody solution (5 μg / bead)
Then, plastic beads (6.4 mm ± 0.2 mm) are immersed overnight to bind the monoclonal antibody on the bead surface. After washing with purified water, the beads were washed with 0.3% Tween.
Soak for 30 minutes in the 20 solution. After washing again with purified water,
After immersing the beads in a 0.5% bovine serum albumin (BSA) solution for 3 hours or more, the beads were dried to prepare beads having immobilized monoclonal antibodies.

Production Example 2 (Preparation of labeled secondary antibody) About 37 MBq of Na was added to 84 μg of an anti-PIVKA-II monoclonal antibody (containing 3% of an antibody that reacts with thrombin).
¹²⁵ I (New England Nuclear) and 10 μg of chloramine T (Kishida Chemical) were added and reacted for 60 seconds, and the reaction was stopped by adding 10 μg of sodium metabisulfite (Pure Chemical). The reaction solution was purified with an ion exchange resin (Amberlite IRA-400T), diluted with a buffer solution for a secondary antibody to about 19 kBq / mL, and iodinated PIVKA-II monoclonal antibody ( ¹²⁵ I).
A solution (containing 3% of the antibody that reacts with thrombin) was prepared.

Production Example 3 An antibody reacting with human thrombin was purified from an anti-human prothrombin antibody (DAKO) using a human prothrombin affinity column and a human thrombin affinity column, and labeled with ¹²⁵ I in the same manner as in Production Example 2. A solution of human iodinated human thrombin antibody ( ¹²⁵ I) was prepared.

COMPARATIVE EXAMPLE 1 Human normal prothrombin was heated under vacuum, and decarboxylated prothrombin (abnormal prothrombin, PIVKA-I
I) was prepared and diluted with normal horse serum to an appropriate concentration to prepare a standard PIVKA-II solution. Dispense 100 μL of the standard PIVKA-II solution (decarboxylation) or the sample into a polystyrene tube, and then use phosphate buffer (1
100 μL of 0 mM, pH 7.4, 0.2% BSA, 0.05% sodium azide) and PIVKA-II antibody beads 1
Add each to each tube and shake for 2 hours at room temperature. After removing the reaction solution by suction, it is washed twice with 2 mL of physiological saline. Next, the iodinated PIVKA-II monoclonal antibody ( ¹²⁵ I) 20 diluted with the above phosphate buffer was used.
Add 0 μL to the tube and shake again at room temperature for 2 hours. After removing the reaction solution by suction, the beads are washed twice with 2 mL of physiological saline, and the amount of radioactivity in the tube containing the beads is measured using a gamma counter. From the standard curve drawn from the count of each standard PIVKA-II concentration, PIVKA-II in the sample
Find the concentration.

Example 1 A 50 mM Tris-HCl buffer (pH 8.0, 5%) was used as a buffer reagent instead of the phosphate buffer in Comparative Example 1.
BSA, 50 mg / L normal mouse gamma globulin, 0.3 M
Sodium chloride, 0.01 M disodium ethylenediaminetetraacetate, containing 0.05% sodium azide), and further contained 0.05% Tween 20 (surfactant) and 0.05% NP-40 (surfactant). Agent) or 1% skim milk. In addition, iodide PIVKA-II
10 mM buffer for monoclonal antibody ( ¹²⁵ I)
Phosphate buffer (pH 6.4, 1% BSA, 1% horse serum, 0.9% sodium chloride, 0.05% Tween2
(Containing 0, 0.05% sodium azide) in the same manner as in Comparative Example 1. Table 1 and FIG. 1 show the results of the measurement using the serum of a healthy adult as described above.
Since the PIVKA-II measurement value of a healthy person is less than 40 mAU / mL, the PIVKA-II measurement value is less than 40 mAU / mL when correct measurement can be performed without being affected by the interfering substance in the sample. At this time, when affected by the interfering substance in the sample, the measured value of PIVKA-II becomes an abnormally high value of 40 mAU / mL or more.

[0023]

[Table 1]

As is clear from Table 1, when the anti-adsorption agent is not added (Comparative Example 1), there are many cases where abnormally high values are obtained despite healthy subjects. On the other hand, Tween2
When 0 or NP-40 is added, the effect is improved to some extent, but the effect of the addition is not sufficient. On the other hand, when skim milk was added, the PIVKA-II measurement value of all healthy subjects was less than 40 mAU / mL, indicating that the measurement can be performed correctly.

Example 2 100 μL of a standard PIVKA-II solution (decarboxylation) or a sample was dispensed into each well of a reaction tray.
Add μL and one PIVKA-II antibody bead to each well and shake for 2 hours at room temperature. After removing the reaction solution by suction,
Wash twice with 2 mL of saline. Next, iodide PIVK
Add 200 μL of A-II monoclonal antibody ( ¹²⁵ I) to each well and shake again for 2 hours at room temperature. After aspiration of the reaction solution, the beads are washed twice with 2 mL of physiological saline, and the beads are transferred from the reaction tray to a counting tube. The amount of radioactivity in the tube containing the beads is measured with a gamma counter, and the PIVKA-II concentration in the sample is determined from the standard curve drawn from the count of each standard PIVKA-II concentration. FIG. 2 shows the results of the measurement using the serum and plasma of a healthy person as described above. FIG. 2 shows that when the antibody beads were transferred from the reaction vessel to another tube at the time of counting the labeled secondary antibody, more accurate PIVKA-II concentration measurement could be performed.

Example 3 The anti-PIVKA-II monoclonal antibody used as a secondary antibody in Examples 1 and 2 contained 3% of an antibody that reacts with thrombin, and was further obtained in Production Example 3. Those to which human thrombin antibody was added (10% or 30%) were used as secondary antibodies. Except using these secondary antibodies, the PIVKA-II concentration in the serum of a healthy individual was measured in the same manner as in Example 2.
As a result, as shown in Table 2, it was found that by adding the protein to the buffer solution, the PIVKA-II concentration in the serum could be accurately measured even when the secondary antibody contained a large amount of the antibody that reacts with thrombin. .

[0027]

[Table 2]

[0028]

According to the present invention, PIVK contained in a specimen
Since the adsorption of the interfering substance which influences the A-II measurement value to the reaction vessel can be almost prevented, the PIVKA-II can be measured accurately. Further, according to the method of the present invention, PIVKA-II in serum can be accurately measured even when an antibody containing an antibody that reacts with thrombin is used as the secondary antibody.

[Brief description of the drawings]

FIG. 1 is a graph showing the effect of adding milk protein to a buffer on the measured value of PIVKA-II concentration in serum.

FIG. 2 is a diagram showing the effect of adding milk protein in PIVKA-II concentration measurement by an antibody solid phase transfer method.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (reference) // C07K 16/36 C07K 16/36 F term (reference) 4B063 QA01 QA18 QQ03 QQ36 QR48 QR51 QR56 QR83 QS03 QS20 QS33 QS35 QS36 QX07 4H045 DA65 DA76 DA89 FA82

Claims (3)

[Claims] 1. An immunological assay using a two-antibody sandwich method for PIVKA-II in a biological sample, characterized in that a buffer containing milk protein is used as a buffer. Measurement method. 2. An immunological assay using a two-antibody sandwich method for PIVKA-II in a biological sample, wherein the immunological reaction product to be measured is removed from the reaction container and measured. The described measurement method. 3. The milk protein is skim milk.
Or the measuring method according to 2.