JPH0236353A - Immunoassay - Google Patents

Abstract

PURPOSE:To allow immunoassay with high sensitivity by incorporating a specific mol.wt. of casein decomposition product into an immune reaction soln. at the time of executing the immunoassay by utilizing an antigen-antibody reaction. CONSTITUTION:The casein of about 1,000-26,000mol.wt. is incorporated into the immune reaction liquid and the final concn. in the immune reaction soln. of the casein decomposition product is adjusted to 0.05-2.5wt.% at the time of making the immunoassay utilizing the antigen-antibody reaction. Cow's milk is often used as the casein of the casein decomposition product. A hydrolysis method, etc., by digestive enzyme such as chymotrypsin and papain are used as the method for decomposing the casein. The effect of suppressing serum interference to about 1/20 appears in case of using such casein decomposition product to the immunoassay system utilizing the antigen-antibody reaction. The exact measurement of the antigen quantity in the liquid to be inspected is possible.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Industrial field of application The present invention provides a method for immunologically measuring the amount of a substance using an antigen-antibody reaction. Eliminating serum interference (hereinafter referred to as serum interference) caused by ingredients,
The present invention relates to a novel immunoassay method for accurately measuring a substance to be measured in a test liquid.

(01 Conventional technology An immunoassay method that utilizes the specific reaction between an antigen and an antibody is
This method is widely used due to its high specificity and extremely high measurement sensitivity, and further development is expected with the discovery of monoclonal antibodies in 1975. Radioimmunoassay (RIA), in which the detection means of this immunoassay method is a radioactive substance (Yalow et al., Nature,
184. P1648-1649. 1959) is
Due to its specific selectivity and particularly high sensitivity measurement, it has been used to measure microscopic substances in biological samples such as serum and urine tissue fluid.

After that, enzyme immunoassay using enzymes as a detection means (
1+u+uno chemistry 8: 871
~874°1971) and methods using fluorescent substances have been developed, and in particular, EIA is based on the following: 1) Enzymes are more stable and economical during long-term storage than radioactive substances, and 2) The measurement method is simple. It has become rapidly popular in recent years because it has advantages such as versatility and measurement sensitivity equivalent to that of RIA.

Among EIA, it is a competitive method, the Sanderutsch method (enzyme immunoassay, 2nd edition, Igaku Shoin, 1982, pages 30-49).

The simultaneous sandwich method and the like are widely used, and in both methods, a group of substances to be measured (antigens) contained in a test liquid is measured based on a standard curve obtained using a standard substance of known concentration.

Now, when constructing these immunoassay methods, standard substances that have been purified by extraction or separation from body fluids are often used, whereas serum or plasma are often used as test samples. It will be done. The test liquid contains components other than the substance to be measured, and these substances affect the immune response during measurement, causing a phenomenon called serum interference. Therefore, differences in immune reactions occur between the standard substance and the test solution, and accurate measurement values are often not obtained.

Therefore, suppressing this serum interference as low as possible is an important technical point for completing an accurate immunoassay method. Therefore, various attempts have been made in the past.

The most widely used method is (1) to add an additive to the measurement buffer to suppress serum interference.

For example, JP-A-56-42142 discloses a method in which a gelatin decomposition product or a gelatin decomposition product and salts are added in coexistence;
JP-A-55-152458 describes a method of adding 1% of a nonionic or anionic surfactant, and a method of adding a hydrophobic protein and salts. In addition, there is a known example in which serum interference in a human growth hormone measurement system was suppressed by increasing the salt concentration of the buffer used during the immune reaction (Nashida S, et al., Cl1n-Chii-Acta, 19
83 Dec, 30. 135<3),
P 263-73).

Other methods include (1) suppressing serum interference by increasing the dilution ratio of the test solution and reducing the amount of test solution added to the measurement system.

(c) Problems to be solved by the invention However, many of the conventional methods described so far are empirical, and moreover, they are all limited to specific measurement systems and cannot eliminate serum interference over a wide range. There was a problem.

In the Zarani (2) method, there were often differences in quality between purchased lots of added substances, resulting in variations in the serum interference suppression effect. Particularly when animal serum is used, large variations are observed, and there is a problem that serum interference cannot be suppressed with good reproducibility.

Furthermore, when changing the salt concentration of a buffer solution, the concentration is often increased to have an inhibitory effect, but increasing the salt concentration simultaneously inhibits specific reactions, resulting in a decrease in measurement sensitivity. It was not the preferred method.

Next, method (2) of increasing the dilution ratio of the test liquid and adding it to the measurement system cannot be used when the measurement target in the test liquid is slightly crushed, as the measurement sensitivity decreases due to dilution. There were problems such as not being able to do so.

In view of this situation, the present inventors have conducted detailed studies and intensive research on substances and methods for suppressing serum interference. Serum interference can be suppressed by adding the degraded product so that the final concentration in the immune reaction solution falls within a predetermined range, eliminating the difference in immune reaction between the standard substance and the test solution and ensuring accurate measurement values. The present invention was achieved by discovering that this method can be obtained and that highly sensitive immunoassays can be performed.

That is, in the present invention, when performing an immunoassay using an antigen-antibody reaction, an immunoreaction solution containing a molecular weight of about 1000 to 26
An immunoassay method characterized by containing a casein decomposition product having a molecular weight of about 1,000 to 26,000, more specifically, when performing an immunoassay using an antigen-antibody reaction in a solution, a casein decomposition product with a molecular weight of about 1,000 to 26,000 is added to the immunoreaction solution. This immunoassay method is characterized in that the final concentration of the casein decomposition product in the immunoreaction solution is adjusted to 0.05 to 2.5% by weight.

Casein, the casein decomposition product of the present invention, is contained in large amounts in the milk of mammals, and is purified and used; however, a large amount of milk is used in the membrane.

Methods for decomposing casein include, for example, hydrolysis using digestive enzymes such as chymotrypsin, papain, trypsin, vancreatin, etc.; i! Alternatively, a hydrolysis method using an alkali, etc. can be used, and the molecular weight is about 100~
5oooo of casein decomposition products are obtained. Such digestive enzymes can be used alone or in combination of two or more, but as an example of using two or more, for example, casein, pig innards slices, and bank creatine powder are mixed and digested for about 10 days to degrade casein. You can also use the method of obtaining things (J
.

Mowared Mueller et al., J. Ba.
CteriOl 67P-271~277 (195
4) ).

When casein (molecular weight approximately 75,000 to 375,000) was used as it was, almost no effect of suppressing serum interference was observed, whereas among the above casein decomposition products, those with molecules 1 of approximately 1,000 to 26,000 particularly inhibited serum interference. The effect was excellent.

That is, the casein decomposition product of the present invention has a molecular weight of about 1000.
~26,000.

As such a casein decomposition product, a trypsin decomposition product of casein is particularly preferred, and among these, for example, NZ case (@uik).

5heHeld Cheg+1ca1).

Although the mechanism of expression of the serum interference suppressing effect of the present invention is not fully elucidated, the molecular members of the casein decomposition products are approximately 1000 to
It is estimated that adding 26,000 to the reaction solution during the immune reaction has the effect of keeping the environment during the immune reaction constant regardless of the presence or absence of serum or plasma.

In the present invention, the casein decomposition product is contained in the immunoreaction solution, and it is particularly preferable to adjust the final concentration in the immunoreaction solution to 0.05 to 2.5% by weight. The inhibitory effect on serum interference begins to appear at a casein decomposition product concentration of 0.03% by weight, but the inhibitory effect on serum interference is still insufficient at concentrations below 0.05% by weight, and at concentrations higher than 2.5% by weight. The casein decomposition product cannot be completely dissolved in the immunoreaction solution, and if left at room temperature, it will precipitate and become suspended, causing variations in the immune reaction. Therefore, the final concentration of the casein decomposition product should be adjusted to 0.05 to 2. A range of 5% by weight was determined. More preferably, it is in the range of 0.1 to 1.5% by weight.

For example, in the case of EIA, such a casein decomposition product is used as a component of a reagent kit for immunoassay, or as an insolubilized antibody or a labeled antibody.

It can also be included in any reagent kit consisting of an assay buffer, a standard substance, and the like. In the latter case, labeled antibodies or assay buffers are preferred among others;
In particular, assay buffers are preferred. For example, when performing an immunoassay using an antigen-antibody reaction in a solution, for example, the concentration of the casein decomposition product in the assay buffer is adjusted so that the final concentration in the immunoreaction solution becomes a predetermined concentration. . Or, for example, so-called dry EIA (Japanese Patent Publication No. 82-5
00121), the test liquid.

Any reagent kit consisting of a labeled antibody or the like can contain such a casein decomposition product, particularly preferably by adjusting the final concentration in the immunoreaction solution to a predetermined concentration.

The immunoassay method of the present invention uses polystyrene, polycarbonate, polyester, and polyolefin.

E: Reacting an immobilized antibody in which the antibody is insolubilized on a carrier such as beads, tubes, plates, latex, molded porous bodies, etc. made of cellulose acetate, nylon, glass fiber mineral fiber, etc., and a solution containing the analyte to be measured.
A system that uses LISA system or liposome to insolubilize the antibody, destroys the liposome through antigen-antibody reaction and the action of complement, and measures the antigen concentration by the leakage amount of the labeled substance contained in the liposome. It can also be applied to

(e) Effect of the invention The casein decomposition product of the present invention has a final concentration of 0.05 to 2.
．． When used in an immunoassay system that utilizes antigen-antibody reaction at a concentration of 5% by weight, the effect of suppressing serum interference to approximately 1/20 is exhibited, making it possible to accurately measure antigen m in the test liquid. It has become possible.

That is, when the casein degradation product according to the present invention is used by adding it to a buffer solution when measuring human placenta-derived acid glutathione s-h transferase (placenta-derived acid GST) present in serum, high specificity and high It is possible to provide an immunoassay method that allows easy measurement with sensitivity and provides accurate measurement values. In such cases, the anti-human placenta-derived acidic GST antibody may be either a polyclonal antibody or a monoclonal antibody. Such monoclonal antibodies can be obtained by the method described in Re-publication Publication No. 1988-62803377 filed by the present applicant.

The present invention will be specifically explained below using examples, but the present invention is not limited to the examples.

In the examples, % means weight %.

Example 1 N in human placenta-derived acidic GST measurement system
Suppressing effect of serum interference by Zcase (1) Preparation of polyclonal antibody-immobilized beads After thoroughly washing polystyrene beads (6 tuts in diameter), add 20 μg/L of rabbit anti-human placenta-derived acidic GSTS po-clonal antibody.
0.01 M phosphoric acid 0.15 M with a concentration of d
The samples were left in a NaCl pH 7,4 (PBS) solution at a temperature of 4°C for one day and night, then washed with PBS and placed in a PBS solution containing 1% bovine serum albumin (BSA) at a temperature of 4°C.
A post-coating process was carried out by leaving the beads day and night to obtain polyclonal antibody-immobilized beads.

(Anti-human placenta-derived acidic GST monoclonal antibody 6A manufactured by m-2 horseradish peroxidase-labeled monoclonal antibody (
1) obtained by the method described in Re-publication Publication No. 1988-803377). Add N to the PBS solution of OIIg/d.
-(m-maleimidobenzoic acid)-N-succinimide ester (MBS) 50μ of a 10μ/- dimethylformamide solution was added and allowed to react at a temperature of 25°C for 30 minutes. Next, gel filtration was performed with 0.1 M phosphate buffer (pl-16,0) using a column filled with Sephadex G-25 to separate the maleimidated monoclonal antibody and unreacted MBS.

Horseradish peroxidase (HRP)
1. N-succinimidyl-3-(2-pyridylthio)propionate (SP
An ethanol solution of DP) with a concentration of 110 l1/d was added and reacted at a temperature of 25° C. for 30 minutes. Next, using a column packed with Sephadex G-25, 0.1
Purified by gel filtration with M acetic acid mii solution (pH 4, 5),
The fraction containing pyridyl disulfidated HRP was collected and concentrated about 10 times in a collodion bag while cooling with water, and then mixed with 0.1 M containing 0.85% NaCl and 0.1 M dithiothreitol. Acetate buffer (pH
4,5) lad was added and stirred at a temperature of 25° C. for 30 minutes to introduce it into the HRP molecule and reduce the pyridyl disulfide group. After reduction, the mixture was subjected to gel filtration using a Sephadex G-25 column, mixed with thiolated HRP, concentrated to a protein concentration of 4/- using a collodion pack under water cooling, and left at a temperature of 4 DEG C. for one day and night.

Thereafter, gel filtration was performed using a column packed with Ultrogel Ac A44 (LKB) to obtain an HRP-labeled monoclonal antibody.

(3) Preparation of acidic GST-deficient serum derived from human placenta (
b) Preparation of anti-human placenta-derived acidic GST antibody-immobilized column Agarose gel activated with CNBr, such as CNBr-activated Sepharose 4 from Pharmacia.
An anti-human placenta-derived acidic GST polyclonal antibody was chemically bonded to B via an amino group. In that case, add 5 to 1019 antibody proteins to gel 11d in 0.1M carbonate 0.15M NaCl buffer (pH 8,5) 2at
The mixture was dissolved in E and fixed at a temperature of 4° C. for 16 hours with slow stirring. Next, the gel and antibody solution were separated using a glass filter, and the gel was washed with 0.1M acetic acid and 0.15M
NaCl buffer (DH4,0> and 0,1M carbonate.

o, unfixed antibodies were removed by three alternating washes with 15 M Na Cl buffer (pHa, s).

Then, CNBr activation 3 epharose 4 B
(7) The remaining active site was removed using 1M ethanolamine (DH8).
,0>Solution 5d was added and slowly stirred at room temperature for 2 hours to block. After blocking, it was transferred onto a glass filter again and washed in the same manner as the washing method after antibody fixation. Next, PBS used for affinity chromatography
Washed twice.

The gel thus prepared was packed into a column.

[0] Affinity chromatography anti-human
S immobilized with placenta-derived acidic GST polyclonal antibody
After equilibrating the epharose4 B column with PBS, healthy human serum was passed twice to remove acidic 0 from human placenta.
8-deletion-deficient serum was obtained.

(4) Serum interference suppression test in this assay method Immunoassay buffer (1.0% BSA, 0.1% skim milk PB)
S) of human placenta-derived acidic GST of 0, 12.5 nO/
d solution was prepared, and 100 μp of human placenta-derived acidic GST-deficient serum diluted 2 times with 100 μp of each buffer and HRP-labeled anti-human placenta-derived acidic GST
Monoclonal antibody 6A (republication publication 1988-803)
100 µm of the same buffer solution containing the above (obtained by the method described in No. 37T) was placed in a glass test tube to obtain a sample with serum. Similarly, acidic GS derived from human placenta
Same as above in place of T-deficient Xiangqing! l! A "no serum" sample was obtained using LLI fluid. Next, 100% of the same buffer solution containing NZ case prepared in 1 i so that the final concentration was 0 to 2%.
μΩ was added and mixed well. To this, one bead immobilized with a rabbit anti-human acidic GST polyclonal antibody from placenta burr was placed in each test tube and incubated at a temperature of 37° C. for 2 hours. Next, after removing the solution in the test tube by suction, the test tube was washed 3 times with 2 d of physiological saline, and then 3.3'
, 5.5' Tetramethylbenzidine salt M salt 0.02
Add 0.4 of 0.1M phosphoric acid-citrate M buffer (r)H4,0) containing %, H2022,5111M to each test tube and incubate for 30 minutes at a temperature of 37 °C, and then start the reaction. As a stop agent, 1Nlii! The enzymatic reaction was stopped by adding 1 d of l acid aqueous solution.

Next, the absorption intensity of this solution at 450 rv was measured using a spectrophotometer using purified water as a control, and the obtained results are shown in FIG.

From Figure 1, acidic GST derived from human placenta 0°12.5μ
g/IIdl, when the added amount of NZ case is 0%, “No serum J (O mark, ・ mark)
Although there is a difference in the absorbance between "with serum" and "with serum" (triangle marks, mu marks), the effect of suppressing serum interference started to appear when the amount of NZ case added was 0.03%. The extent of this difference is that when the absorbance (marked with O) without serum at 12.5 μg/d of human placenta-derived acidic GST is taken as 100%, the absorbance with the presence of serum (marked Δ) is NZCas.
When e is 0%, there is a 38% difference in absorbance due to serum interference, but when 0.1% NZ case is added, the difference becomes 1.5%. It was found that when 0.5% NZcase was further added, the difference was 3.3%, and accurate measured values could be obtained.

Example 2 Simultaneous sandwich enzyme immunoassay for human placenta-derived acidic GST 81 beads immobilized with rabbit anti-human placenta-derived acidic GSTS pork clonal antibodies and purified human placenta-derived acidic GST0. 6.25, 12.5.25.50ng/
jlIl! Containing 1% BSA, 0.5% NZc
ase.

0.1% skim milk, 200μ of PBS solution, and HR
1% BSA containing P-labeled anti-human placenta-derived acidic GST monoclonal antibody 6A, 0.5% N Zcase,
200 μA of 0.1% skim milk in PBS was added to each glass test tube and incubated at a temperature of 37° C. for 2 hours. Next, after removing the solution in the test tube by suction, it was washed three times with physiological saline 2IR1, and then
',5.5'-Tetramethylbenzidine hydrochloride 0.
0.1M phosphoric acid-citric acid containing 0.02%, 8202 2.51M! 1Ili solution (DH4,0) was added to 0.1Ili solution (DH4,0).
After adding 4 d into each test tube and incubating at a temperature of 37° C. for 30 minutes, 1 d of 1N sulfuric acid aqueous solution was added as a reaction terminator to stop the enzyme reaction. This solution was then measured using a spectrophotometer at 450 ni using purified water as a control.
By measuring the absorption intensity at and plotting it against the concentration of the standard substance, a calibration curve with good concentration dependence was obtained. The results are shown in Figure 2.

Example 31 Measurement of human placenta-derived acidic GST in bed specimens Serum from healthy individuals and patients with colorectal cancer was collected, and 50μ of each serum was collected.
Add 1% BSA to a glass test tube.

After diluting by adding 0.5% N Z CaSe, 0.1% skim milk, and 150μ of PBS solution, add one bead immobilized with rabbit anti-human/placenta-derived acidic GSTS pork clonal antibody and HRP-labeled anti-human.・1% BSA containing placenta-derived acidic GST monoclonal antibody 6A,
0.5% NZ case, 0.1% skim milk, and 200 uu of PBS solution were each added to a test tube and incubated at 37° C. for 2 hours.

Next, the test of Example 2! After washing, enzymatic reaction, and reaction termination were carried out in exactly the same manner as for preparing the sample, the absorption intensity at 450 nm was measured using a spectrophotometer using purified water as a control, and the concentration was determined from a calibration curve. As a result, the human placenta-derived acidic G5Ti1 degree in the serum of a healthy person was 12.8 nM tel, and the human placenta-derived acidic GSTII degree in the serum of a colon cancer patient was 51.0 n (J/d).

[Brief explanation of the drawing]

Figure 1 shows N in the human placenta-derived acidic GST measurement system.
z casel 1 degree and serum interference suppressing effect. In Figure 1, O1Δ marks are human placenta-derived acidic G S.
T 12.5 nM d Te blood m L/, l) Re'fi: Show L/, . The mu marks are human placenta-derived acidic GSTOng/li! Shows without serum and with serum. FIG. 2 shows a calibration curve for human placenta-derived acidic GST in the human-placenta-derived acidic GST measurement system. Patent applicant Teijin Ltd. NZ
αself(,,,sit force.%(%) Self-ST-ru paste (-1/,,l)

Claims (1)

[Claims] 1. When performing immunoassay using antigen-antibody reaction,
An immunoassay method characterized in that an immunoreaction solution contains a casein decomposition product having a molecular weight of about 1,000 to 26,000. 2. When performing an immunoassay using an antigen-antibody reaction in a solution, add a substance with a molecular weight of about 1000 to 2 to the immunoreaction solution.
6,000 of casein decomposition product, and the final concentration of the casein decomposition product in the immunoreaction solution was 0.05-2.
An immunoassay method characterized by adjusting the amount to 5% by weight. 3. The immunoassay method according to claim 2, wherein the casein decomposition product is a casein decomposition product using a digestive enzyme. 4. The immunoassay method according to claim 2, wherein the measurement target is human placenta-derived acid glutathione S-transferase.