JPH1123574A - Method combining enzyme with physiological active substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an enzyme labeling method increasing the number of labeled enzymes without raising background blank. SOLUTION: In this method, an enzyme is oxidizingly processed, the hydroxyl group of sugar chain in the enzyme is converted to an aldehyde group, this aldehyde group is reacted with an isolated amino group in a physiological active substance to generate a Shiff base, next, the Shiff base is processed by a reducing agent to generate second class amine, and the enzyme is bonded with the physiological active substance. In this case, buffer concentration at reacting the aldehyde group with the amino group is set to be over 50 mM and pH of the buffer solution is set to be over 10.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for binding an enzyme to a physiologically active substance, and more particularly, to an enzyme-labeled antigen or antibody used in a method for immunologically measuring a physiologically active substance such as a biological component. (Label).

[0002]

2. Description of the Related Art As one method of measuring a trace amount of a physiologically active substance present in a living body, for example, an enzyme immunoassay (EIA) or a radioimmunoassay (RIA) is known.
Immunoassays, etc., and these methods occupy an important position in clinical tests. In particular, since EIA has high specificity and can be measured with high sensitivity without using a radioactive substance, it has recently begun to spread rapidly. Recently, an EIA using a porous membrane as a solid phase has been developed, which enables measurement in the same time as a biochemical test.

[0003] An immunoassay is to react an antigen or an antibody to be measured in a sample with an antibody or an antigen having a specific affinity for the substance, and measure the degree of the reaction. A method for detecting an antigen or an antibody. EIA refers to an enzyme activity of a complex formed by an antigen-antibody reaction or an enzyme activity of an unreacted label using an enzyme-labeled antigen or antibody (label). Is a method of detecting an antigen or an enzyme in a sample by measuring the

In EIA, in order to measure an antigen or an antibody with high sensitivity, an enzyme-labeled antibody or an antigen (label) has high specificity and affinity for the antigen or antibody to be measured, and It is necessary that the enzyme activity is also high. In particular, enzyme activity is the most directly involved requirement for high-sensitivity measurement, and in order to obtain high enzyme activity, the amount of enzyme to be bound per antibody or antigen (labeled substance) to which the enzyme is bound is determined. It is desirable that the number is large. This is particularly required in a short-time EIA using a porous membrane as a solid phase.

[0005] Such an antigen or antibody (labeled substance) 1
Conventionally, methods for increasing the number of labeled enzymes per molecule include the glutaraldehyde method and the periodate method (J. Histochemistry and Cytochemistry 22,1084,197
4) etc. are known. In these methods, in the preparation of aggregated antibody-enzyme conjugates, the random cross-linking remains largely inaccessible for antigen-binding purposes because the antibody or antibody fragment may be buried deep within the aggregated complex. The law. It is difficult to prepare these amorphous polymer complexes with good reproducibility.
Immunoassays utilizing aggregated antibody-enzyme conjugates by the random cross-linking method have the disadvantage that the background blank is always very high due to non-specific binding, and the ultimate sensitivity that can be achieved with the immunometric assay is significantly reduced. (See JP-A-61-73067)
Publication, Ishikawa et al., Ann. Clim. Biochem., 19, 379
, (1982)].

[0006] Separately, a labeled product (JP-A-61-73067) labeled with a pre-polymerized enzyme is also known. However, it is difficult to control the method of binding many of these enzymes to the target substance during preparation, and it has been difficult to establish a method of always labeling the same number of enzymes. Furthermore, when the number of labeling enzymes is too large, there is a disadvantage that non-specific binding is large and measurement sensitivity cannot be obtained.

[0007]

According to the periodic acid method, in which an enzyme is bound to a physiologically active substance, a large number of enzymes can be bound to an object to be labeled, and the binding ratio can be increased by increasing the periodate concentration. It is known to produce high-molecular-weight labels (J. Histochemistry and Cytochemistr
y 22,1084,1974). However, there is a disadvantage that the background blank tends to be high. Background is
In the measurement system, it refers to a measured value when the concentration of the substance to be measured is 0.

[0008]

Means for Solving the Problems The present inventors have conducted intensive studies on a method for increasing the number of labeling enzymes without increasing the background blank, and as a result, the sugar chains of the enzymes are oxidized with periodic acid. During the dialysis, the temperature and concentration of the periodate treatment were kept constant, and in particular, in order to bind the enzyme having oxidized the sugar chain to the physiologically active substance, to remove the treated low-molecular substance and adjust the pH, It has been found that by selecting the concentration of the dialysate and the pH of the buffer solution, the labeling efficiency of the enzyme is significantly improved, and the present invention has been achieved.

[0009] That is, the present invention oxidizes the enzyme,
The hydroxyl group of the sugar chain in the enzyme is converted to an aldehyde group, and the aldehyde group is reacted with a free amino group of a physiologically active substance to generate a Schiff base. A method for generating a secondary amine and binding the enzyme to a physiologically active substance, wherein the concentration of the buffer during the reaction between the aldehyde group and the amino group is 50 mM or more;
Further, the present invention is a method for binding an enzyme to a physiologically active substance, wherein the pH of the buffer is 10 or more.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, the enzymes used include peroxidases containing sugar chains (peroxidase derived from horseradish, hereinafter sometimes abbreviated as HRP).
Examples include glucose oxidase, but are not limited to these.

In the present invention, examples of the physiologically active substance used include various polyclonal antibodies, monoclonal antibodies or fragments thereof, and antigens thereof. Particularly preferred is a monoclonal antibody.
Each of the antibody and the antigen is selected depending on the target to be detected. For example, CEA, AFP, C
K-MB, HBs-Ag, HBs-Ab, human IgG,
IgE is exemplified.

In the present invention, an enzyme is oxidized to convert a hydroxyl group of a sugar chain in the enzyme into an aldehyde group. Periodic acid is used as an oxidizing agent. These concentrations are between 0.04 and 0.16M. As a buffer, a sodium bicarbonate solution (pH 8.1) is usually used.

In the present invention, the concentration of the buffer during the reaction between the aldehyde group and the amino group is 50 mM or more, and
It is necessary that the pH of the buffer is 10 or more. When the buffer concentration is less than 50 mM, the binding ratio between the enzyme and the physiologically active substance is unstable, and there is a disadvantage that a conjugate having a constant ratio cannot be always obtained. When the pH is less than 10,
The bonding ratio is not much different from the conventional one. Furthermore, lack of either concentration or pH does not result in a preferred enzyme-bioactive agent conjugate.

In the present invention, a 1-fluoro-2,4-dinitrobenzene / ethanol solution or the like is used as a reagent for blocking free amino groups of the enzyme itself in order to prevent self-condensation of the enzyme. In the present invention, an ethylene glycol aqueous solution, glycerin, or the like is used as the stop solution.

As a specific example, horseradish peroxidase (HRP) is dissolved in a 300 mM sodium bicarbonate solution (pH 8.1), and a 1-fluoro-2,4-dinitrobenzene / ethanol solution is added to the solution. Add and react at 20 ° C. for 1 hour. Then, the reaction solution was shielded from light, a 0.05 M aqueous NaIO ₄ solution was added, and the mixture was added at 20 ° C.
And react for 30 minutes.

In the present invention, the aldehyde group is reacted with a free amino group of a physiologically active substance to generate a Schiff base. The reaction is generally carried out at room temperature for 2-3 hours in 0.01 M sodium carbonate buffer, pH 9.5.

In the present invention, the Schiff base is then treated with a reducing agent to form a secondary amine, and the enzyme is bound to a physiologically active substance. Examples of the reducing agent include sodium borohydride. These concentrations are obtained by directly adding several mg (about 5 mg) of a solid and reacting at room temperature for 30 to 40 minutes or by reacting at 4 ° C. overnight. In the case where the solid is dissolved and adjusted immediately before addition, 0.1 to 0.2 ml of pure water having a concentration of about 4 mg / ml is added, and the reaction is performed at 4 ° C. for 2 hours.

These physiologically active substances to which the enzyme is bound are usually separated and purified from the reaction solution by gel chromatography in the final step. For example, Sephadex G
200 or Ultrogel AcA44 (LKB) or the like is used.

In order to measure the activity of the enzyme bound to the physiologically active substance, a general enzyme activity method can be applied.

In one embodiment of the present invention, peroxidase is oxidized with periodate to convert a hydroxyl group of a sugar chain in the enzyme into an aldehyde group, and the aldehyde group is replaced with a free amino group of a physiologically active substance; The reaction is performed in a buffer having a buffer concentration of 50 mM or more and the pH of the buffer is 10 or more to generate a Schiff base, and then the Schiff base is treated with sodium borohydride to form a secondary amine. To bind the enzyme to the biologically active substance.

In another embodiment of the present invention, the enzyme and the physiologically active substance are separately dialyzed, and then both dialysates are directly mixed to form a free amino group of the physiologically active substance and an aldehyde group derived from the sugar chain of the enzyme. Generate a Schiff base under basicity. In the present invention, the concentration of the dialysis buffer is conventionally 10 mM, but is set to be 50 mM or more, and the pH is conventionally 9.
At pH 5, by maintaining the pH at 10 or more, the average amount of enzyme binding per molecule of the physiologically active substance is 2 or more, usually,
Approximately three can be bonded and can be stably bonded.

The physiologically active substance bound with the enzyme of the present invention is
It is effectively used as a labeling substance in an immunological method for detecting a biological component. Examples of the immunological method include a sandwich measurement method using a solid phase and a competitive method. The solid phase includes a porous membrane, polystyrene beads, 9
There is a 6-well plate and the like, and the method of measuring the enzyme which is a label follows each enzyme measurement method.

In the enzyme-labeled product of the present invention, in particular, non-specific binding of the labeled antibody to the solid phase is small, and the signal by the enzyme is large, so that high-sensitivity measurement becomes possible. For example, the enzyme-labeled antibody according to the present invention can be widely used for immunoassays and assays, such as hormones, peptides, tumor markers, various hepatitis viruses, and various biological substances. For example, CEA, AFP, and TSH can be mentioned as measurement targets. It can also be used for use as an enzyme-labeled antibody for immunostaining of tissue sections.

[0024]

Next, the present invention will be described in detail with reference to Reference Examples and Examples. In the examples, the number of perosidase bonds to the separated and purified physiologically active substance was generally calculated from the absorbance measurements at 280 nm and 403 nm according to the following formula.

[0025]

(Equation 1) Here, using the value of ε (403, HRP) = 2.1 RZ (HRP) = 3.0 ε (280, IgG) = 1.4, it can be summarized by the following equation.

[0026]

(Equation 2)

The measurement sensitivity (S / N ratio) is S, that is, the signal indicates a signal at a certain concentration of each measurement item, and N is the noise, and is the background of the measurement item. . That is, the higher the S / N ratio, the higher the measurement sensitivity.

REFERENCE EXAMPLE 1 Conventional HRP cross-linking method 5 mg of peroxidase (HRP) derived from horseradish was added to a 0.3 M sodium bicarbonate solution (pH 8.1) 1.0 m
L, and 0.1 mL of a 1% 1-fluoro-2,4-dinitrobenzene / ethanol solution is added to the solution,
The reaction was performed at 20 ° C. for 1 hour. Then, the reaction solution was shielded from light, and 1.0 mL of a 0.05 M NaIO ₄ aqueous solution was added.
The reaction was carried out at 20 ° C. for 30 minutes. Further, the reaction solution was added to the reaction solution at 0.1.
1.0 mL of 16 M ethylene glycol aqueous solution was added, and 1
Stir slowly for hours. This results in excess NaIO ₄
Oxidation was stopped. Next, the oxidized aldehyde-containing peroxidase solution was dialyzed overnight at 4 ° C. using a dialysis membrane against 10 mM sodium carbonate buffer pH 9.5 (J. Histochemistry and Cytochemistry).
22,1084,1974).

On the other hand, CEA is used as a target to be bound.
5 mg each of the antibody, AFP antibody or CK-MM antibody was dialyzed against the same 10 mM sodium carbonate buffer (pH 9.5) at 4 ° C. overnight using a dialysis membrane. After completion of the dialysis, the obtained antibody (labeled substance) was mixed with 5 mg of the above-mentioned aldehyde-modified peroxidase at 20 ° C. for 3 hours to generate a Schiff base. Further, 5 mg of solid sodium borohydride was added and stirred for 30 minutes to reduce the Schiff base to obtain a stable secondary amine. Then, 0.01
The secondary amine was dialyzed against M phosphate buffered saline pH 7.2 (hereinafter abbreviated as PBS) at 4 ° C. overnight.
After the dialysis, the dialysate was subjected to Sephadex G200 column chromatography to ablate the unreacted HRP and HR.
The P-labeled antibody was separated to obtain a purified HRP-labeled product.

[0030] In the same manner as in Example 1 Reference Example 1, horseradish peroxidase and (HRP) 5 mg was dissolved in 0.3M sodium bicarbonate solution (pH 8.1) 1.0 mL, 1% in solution 1
0.1 mL of an ethanol solution of -fluoro-2,4-dinitrobenzene was added, and the mixture was reacted at 20 ° C for 1 hour. Next, the reaction solution was shielded from light, 1.0 mL of a 0.05 M NaIO ₄ aqueous solution was added, and the mixture was reacted at 20 ° C. for 30 minutes.
Further, 1.0 mL of a 0.16 M ethylene glycol aqueous solution was added to the reaction solution, and the mixture was stirred slowly for 1 hour. This stopped the oxidation due to excess NaIO ₄ . Further, the aldehyde-modified peroxidase was converted to 100 mM sodium carbonate buffer pH 10.0 using a dialysis membrane,
Dialysis was performed overnight at 4 ° C.

At the same time, using a separately prepared dialysis membrane, an anti-human CEA monoclonal antibody (OY
Dialysis was performed overnight at 4 ° C against 5 mg of 100 mM sodium carbonate buffer (pH 10.0). The next day, the two were mixed at 20 ° C. for 3 hours to prepare a Schiff base. Next, in the same manner as in Reference Example 1, 5 mg of solid sodium borohydride was added, and the mixture was stirred for 30 minutes to reduce the Schiff base. After the reduction was completed, the peroxidase-labeled anti-CEA monoclonal antibody was dialyzed against 0.01 M phosphate buffered saline. After completion of the dialysis, the dialyzed product was ablated by Sephadex G200 column chromatography to separate unreacted HRP and HRP-labeled anti-human CEA monoclonal antibody (labeled product) to obtain a purified HRP-labeled anti-human CEA monoclonal antibody.

Using the obtained monoclonal antibody, human CEA in the sample was measured by the measurement system described in Reference Example 2. For comparison, the HRP-labeled human CEA antibody prepared in Comparative Example 1 was used in the same manner. The absorbance at 280 nm and 403 nm were measured, respectively, and the results of the enzyme binding ratio calculated by applying the above formula are shown in Table 1 below. In addition, the measurement sensitivity (S / N ratio) is shown.

Reference Example 2 An actual human CEA assay antibody solution prepared by preparing an anti-human CEA monoclonal antibody (Clone 5909 manufactured by OY Medix) in a 10 mM sodium carbonate buffer (pH 8.5) to a concentration of 10 μg / mL. Was dispensed at a rate of 100 μL into a 96-well microtiter plate (manufactured by Coaster), and 3 mL was added at room temperature.
After standing for a period of time, a 0.01 M phosphate buffered saline solution (pH
7.2) Washing with liquid. Thereafter, the solid phase was immersed in 100 μL of 0.01 M phosphate buffered saline (pH 7.2) containing 1% bovine serum albumin (hereinafter abbreviated as BSA) for 1 hour to perform a block treatment to obtain a solid phase.

A standard solution containing human CEA was added to a 96-well microtiter plate prepared with 100 μL, and reacted at room temperature for 1 hour. Then, after washing with 0.01 M phosphate buffered saline, purified HRP-labeled anti-human CEA monoclonal antibody (Clone 5905, manufactured by OY Medix)
100 μL was added, reacted for 1 hour, washed with 0.01 M phosphate buffered saline (pH 7.2), and then 0.3 g / L 3,3 ′, 5,5 ′ containing hydrogen peroxide. 100 μL of a tetramethylbenzidine solution was added and reacted for 30 minutes. Next, the absorbance at 660 nm was measured with a spectrophotometer, and a standard curve was created. At that time, CEA80
CEA at a concentration at which the absorbance at the ng / mL concentration is the same
0 ng / mL (that is, the background level) was compared with Reference Example 1.

[0035]

[Table 1]

As is evident from Table 1, when the peroxidase-labeled human CEA antibody of the present invention is used, the enzyme binding ratio is higher and the measurement sensitivity is higher than the conventional labeled antibody.

Example 2 An HRP-labeled human AFP antibody was prepared in the same manner as in Example 1 using the anti-human AFP monoclonal antibody of Example 1. Using the obtained antibody, human AFP was measured by the measurement system described in Reference Example 3. For comparison, the HRP-labeled human AFP antibody prepared in Comparative Example 1 was used.
Further, the absorbance at 280 nm and 403 nm were measured, respectively, and the results of the enzyme binding ratio calculated by applying the formula are shown in Table 2 below.

Reference Example 3 An actual human AFP measurement system anti-human AFP monoclonal antibody (clone 47-5, manufactured by Fujisawa Pharmaceutical Co., Ltd.) was added to a 10 mM sodium carbonate buffer (pH 8.5) at a concentration of 10 μg / mL. 100 μL each of the prepared antibody solution was dispensed into a 96-well microtiter plate (manufactured by Coaster), left at room temperature for 3 hours, and washed with 0.01 M phosphate buffered saline.
It was immersed in 100 μL of phosphate buffered saline (pH 7.2) containing 1% bovine serum albumin (hereinafter abbreviated as BSA) for 1 hour to perform a block treatment to obtain a solid phase.

100 μL of a standard solution containing human AFP was added to the prepared microtiter, and reacted at room temperature for 1 hour. After washing with 0.01 M phosphate buffered saline (pH 7.2), 100 μL of purified HRP-labeled anti-human AFP monoclonal antibody (Clone 20-7, manufactured by Fujisawa Pharmaceutical Co., Ltd.)
And reacted for 1 hour, washed with 0.01 M phosphate buffered saline (pH 7.2), and washed with 0.3% hydrogen peroxide.
100 μL of a g / L 3,3 ′, 5,5 ′ tetramethylbenzidine solution was added and reacted for 30 minutes. The absorbance was measured at 660 nm using a spectrophotometer, and a standard curve was created.
At that time, AFP 0 ng / mL (that is, background level) at a concentration at which the absorbance at the AFP 100 ng / mL concentration became the same was compared and examined.

[0040]

[Table 2]

Example 3 An HRP-labeled human CK-MM antibody was prepared in the same manner as in Example 1 using an anti-human CK-MM monoclonal antibody. Using the obtained antibody, human CK-MB was measured by the measurement system described in Reference Example 4. For comparison, the HRP-labeled human CK-MM antibody prepared in Comparative Example 1 was used. Further, the absorbance at 280 nm and 403 nm were measured, respectively, and the results of the enzyme binding ratio calculated by applying the formula are shown in Table 2 below.

Reference Example 4 Actual human CK-MB assay system anti-human CK-BB monoclonal antibody (Dako Co., Ltd.)
(Clone 10-F, Co., Ltd.) was adjusted to a concentration of 10 ug / mL in 10 mM sodium carbonate buffer (pH 8.5), and 100 μL of the antibody solution was dispensed into a 96-well microtiter plate (Costar). After leaving at room temperature for 3 hours, 0.01M phosphate buffered saline (pH 7.
After washing in 2), phosphate buffered saline (pH 7.2) containing 1% bovine serum albumin (hereinafter abbreviated as BSA) 100
The plate was immersed in μL for 1 hour to perform a block treatment to obtain a solid phase.

100 μL of a standard solution containing human CK-MB was added to the prepared microtiter, and the mixture was reacted at room temperature for 1 hour. 0.01 M phosphate buffered saline (pH 7.
After washing with 2), purified HRP-labeled anti-human CK-MM monoclonal antibody (clone 9F, manufactured by Dako) 10
After addition of 0 μL, the mixture was reacted for 1 hour, washed with phosphate buffered saline (pH 7.2), and then washed with 0.1 ml containing hydrogen peroxide.
100 μL of a 3 g / L 3,3 ′, 5,5 ′ tetramethylbenzidine solution was added and reacted for 30 minutes. The absorbance at 660 nm was measured with a spectrophotometer, and a standard curve was created. At that time, CK-MB at 0 ng / mL (ie, background level) at a concentration at which the absorbance at CK-MB at 40 ng / mL became the same was compared and examined.

[0044]

[Table 3]

Example 5 The peroxidase-labeled human CEA antibody prepared in Example 1 was used as a labeled antibody for an immunoassay reagent using Immunopora CEA (manufactured by Toyobo Co., Ltd.) using a porous membrane as a solid support. For comparison, measurement sensitivity was compared using a labeled antibody prepared by a conventional method (Reference Example 1).
As a reagent used for the measurement, a block solution, a diluting solution, a washing solution, and a coloring solution included in the kit were used. 100 ng / m of standard solution
When the measurement sensitivity (S / N) at L and 0 ng / mL was determined, it was 300 for the enzyme-labeled antibody of the present invention and about 10 for the conventional method. As is clear from the above results, the method of the present invention exhibits a remarkable effect in a short-time measurement method using a porous membrane as a solid phase.

[0046]

The label produced by the method of the present invention does not need to cleave the sugar chain of the enzyme with a high concentration of periodate and has a low ratio of aldehyde conversion, so that the non-specificity of the label to the solid phase is low. Less specific binding, that is, a smaller background blank, which enables highly sensitive measurement, which can be used for immunological assay methods and immunological assay kits. became. Since the enzyme-labeled physiologically active substance according to the present invention, particularly the enzyme-labeled antibody, has a small cleavage of the sugar chain and a large amount of bound enzyme, the nonspecific binding of the labeled antibody to the solid phase is small, and the signal by the enzyme is low. Due to the large number, high-sensitivity measurement became possible.

Claims (6)

[Claims] 1. An enzyme is oxidized to convert a hydroxyl group of a sugar chain in the enzyme into an aldehyde group, and the aldehyde group is reacted with a free amino group of a physiologically active substance to generate a Schiff base. In a method of treating a base with a reducing agent to generate a secondary amine and binding the enzyme to a physiologically active substance, the concentration of a buffer during the reaction between the aldehyde group and the amino group is set to 50 mM or more. A method for binding an enzyme to a physiologically active substance, wherein the pH of the buffer is 10 or more. 2. The method according to claim 1, wherein the enzyme is peroxidase or glucose oxidase. 3. The method according to claim 1, wherein the physiologically active substance is a polyclonal antibody, a monoclonal antibody or a fragment thereof. 4. The method according to claim 1, wherein the oxidizing agent is periodic acid. 5. The method according to claim 1, wherein the reducing agent is sodium borohydride. 6. The method according to claim 1, wherein the buffer is sodium carbonate-sodium bicarbonate, hydrochloric acid-sodium carbonate or a good buffer.