JPH04291155A - Enzyme labeled antibody - Google Patents

Abstract

PURPOSE:To obtain a better enzyme labeled antibody by a method wherein a specified subclass of antibody is treated by a reducing agent to isolate a sulfhydryl group and caused to react with a maleimide group introduced enzyme subjected to a specified treatment. CONSTITUTION:IgG2a and IgG2b in a mouse monoclonal antibody IgG fraction are treated by a reducing agent without being digested by pepsin to obtain a half-molecular antibody wherein a sulfhydryl group is isolated. In parallel, a bivalent bonding reagent to be bonded to an amino group and a sulfhydryl group is caused to react with an amino group of an enzyme to prepare a maleimide group introduced enzyme. The half molecular antibody wherein the sulfhydryl group is isolated is mixed with the enzyme into which the maleimide group is introduced in a phosphate buffer liquid with pH of 6.0-7.0 to react at 4-37 deg.C for 1-24 hours. After the reaction, the reactant is refined by a gel filtering chromatography to obtain an enzyme-labeled antibody.

Description

[Detailed description of the invention]

0001

FIELD OF INDUSTRIAL APPLICATION The present invention relates to enzyme-labeled antibodies, which are the main constituent reagents of enzyme immunoassays, which are widely practiced in the basic fields of life science and medicine as well as in clinical tests.

0002

[Prior Art] Enzyme-labeled antibodies that have been used conventionally are those prepared by a method that utilizes the amino group of the antibody or a method that reduces the antibody and binds it to an enzyme using the liberated sulfhydryl group. Broadly classified.

[0003] A method for obtaining an enzyme-labeled antibody using the amino group of an antibody uses glutaraldehyde, which is a bivalent binding reagent with two reactive groups in the molecule, to bind the amino group of the antibody and the amino group of the enzyme. glutaraldehyde method, in which horseradish peroxidase is combined with periodic acid to oxidize and cleave the sugar chain in the enzyme to liberate an aldehyde group, and periodate oxidation, in which this aldehyde group is combined with the amino group of the antibody. The law is known.

[0004] A divalent binding reagent is a reagent that has two reactive groups in its molecule and reacts with functional groups such as amino groups and sulfhydryl groups of proteins to form intramolecular or intermolecular crosslinks of proteins. be. Glutaraldehyde, 1,5-difluoro-2,4-dini
trobenzen, etc., and those that bridge between sulfhydryl groups include N,N'-o-phenylened.
imalimide, N, N'-ethylene-
bis-(iodoacetoamide) and the like. In addition, as a heterofunctional divalent binding reagent for binding an amino group and a sulfhydryl group,
cinimidyl-6-maleimidohexa
noate, N-succinimidyl-4-(N
-maleimidomethyl)-cyclohe
There are xane-1-carboxylate and the like.

However, since the above-mentioned method utilizes an unspecified number of amino groups present on the antibody, the enzyme is bound to an unspecified position on the antibody. As a result, antibody activity is often significantly reduced due to direct binding of the enzyme to the antigen-binding site of the antibody or steric hindrance of the enzyme bound to the vicinity of the antigen-binding site. In addition, especially in the glutaraldehyde method, bonding between enzymes and antibodies also occurs, so the resulting enzyme-labeled antibodies have variable molecular weights, low activity, and high blank values, making them unsuitable for high-sensitivity enzyme immunoassays. It was inappropriate.

On the other hand, as a method using the sulfhydryl group of an antibody, a rabbit antibody is digested with pepsin and F(ab'
) After reducing the fractions with a reducing agent to obtain a Fab' fraction in which sulfhydryl groups are released, this sulfhydryl group and the amino group or sulfhydryl group of the enzyme are combined with a divalent binding reagent N-succinimidyl- 4-(
N-maleimidomethyl)-cycloh
exane-1-carboxylate or N,N'
A method is known in which an enzyme-labeled antibody is obtained by binding with -o-phenylene dimaleimide.

[0007] Since the enzyme-labeled antibody obtained by this method binds to the enzyme at a hinge site remote from the antigen-binding site of the antibody, there is little decrease in antibody activity and is of relatively high quality.

[0008] Conventionally, polyclonal antibodies such as rabbit and goat antibodies have been widely used. However, in recent years, mouse monoclonal antibodies have become widely used due to their high specificity and the ability to stably produce products of constant quality, and the importance of enzyme-labeled antibodies using mouse monoclonal antibodies has increased. There is.

[0009]

Problems to be Solved by the Invention: Mouse monoclonal antibody IgG fractions contain IgG1, IgG2a, and IgG2b.
, IgG3 subclass. Among these, IgG1
and IgG3 subclass antibodies can be easily converted to F(ab')2 by pepsin digestion, further reduced to release the sulfhydryl group, and combined with an enzyme to obtain an enzyme-labeled antibody.

[0010] However, it is known that when subclass antibodies of IgG2a and IgG2b are digested with pepsin, decomposition progresses to low-molecular-weight peptides, making it impossible to obtain an F(ab')2 fraction having antibody activity. Therefore, in general, as a monoclonal antibody against the antigen to be measured, I
Antibodies of the gG1 or IgG3 subclass have been selected and used. However, it is not easy to obtain mouse monoclonal antibodies suitable for enzyme-linked immunosorbent assays that have high specificity and reactivity, and when only IgG2a or IgG2b were available, it was unavoidable to use a method that utilizes the amino groups of antibodies to obtain enzyme-linked antibodies. We have prepared labeled antibodies and have been content with measurements with poor sensitivity.

[0011]

[Means for Solving the Problems] The present invention provides an enzyme-labeled antibody that is linked to an enzyme through the sulfhydryl group of the mouse monoclonal antibody IgG2a and IgG2b subclasses. The IgG2b subclass has become available for highly sensitive enzyme-linked immunosorbent assays.

That is, antibodies of these subclasses are treated with a reducing agent without being digested with pepsin to obtain half-molecule antibodies with free sulfhydryl groups. In parallel with the antibody reduction treatment, a maleimide group-introduced enzyme is prepared using a bivalent binding reagent. Next, half of the antibody with a free sulfhydryl group and an enzyme with a maleimide group introduced are mixed and allowed to react. After the reaction is completed, the target enzyme-labeled antibody can be obtained by purification by gel filtration.

[0013] For reducing the antibody, reagents having a reducing action such as 2-mercaptoethylamine, 2-mercaptoethanol, dithiothreitol, etc. can be used. pH5.0-7.
The antibody is dissolved in 0.1M acetate buffer or phosphate buffer in the range of 0.0 to 0.0, and a reducing agent is added in the range of 5 to 20mM. Incubate at 25-37°C for 30-90 minutes to reduce. Since the conditions differ somewhat depending on the reducing agent used, the properties of the antibody clone, etc., it is necessary to set the optimal conditions through preliminary experiments.

A maleimide group-introducing enzyme can be prepared by reacting an amino group of the enzyme with a divalent binding reagent that binds to an amino group and a sulfhydryl group. Specifically, the aforementioned N-succinimidyl-6
-maleimidohexanoate, N-suc
cinimidyl-4-(N-maleimidom
ethyl)-cyclohexane-1-carb
oxylate and its derivative N-sulfosu
ccinimidyl-4-(N-maleimido
methyl)-cyclohexane-1-car
N,N-dimethylfor boxylate etc.
mamide (10-100mM) and added to 10 times the volume of enzyme solution (0.5-10mg/0.1M phosphate buffer, pH 7.0) at 25-37℃, 20-9
After 0 minutes of reaction, unreacted binding reagents are removed by gel filtration.

[0015] As the enzyme in this case, many enzymes commonly used in enzyme immunoassay methods can be used, such as horseradish peroxidase, glucose oxidase, alkaline phosphatase, and β-galactosidase. In addition, β-galactosidase derived from the large intestine has a free sulfhydryl group, and this sulfhydryl group is bound to N,N'-o-phenylened, which is a divalent binding reagent.
By reacting with imalimide, maleimide group-introduced β-galactosidase can be obtained.

[0016] The antibody with the free half of the sulfhydryl group thus obtained and the enzyme into which the maleimide group has been introduced are mixed in a phosphate buffer solution, pH 6.0 to 7.0, and
React at 7°C for 1 to 24 hours. After the reaction, the enzyme-labeled antibody is obtained by purification by gel filtration chromatography.

[0017]

[Example] Examples and reference examples such as the preparation of enzyme-labeled antibodies according to the present invention and enzyme immunoassay using the same will be described below, but these are not intended to explain the present invention in more detail. However, the present invention is not limited to these examples.

[0018]

[Example 1 Mouse monoclonal antibody, IgG2a
Preparation of enzyme-labeled antibodies using subclasses] 0.05M phosphate buffer, pH 6.5, containing 0.1M NaCl;
Anti-myosin mouse monoclonal antibody IgG dissolved in
2a subclass (clone number M705, 1 mg/0.
2 ml) was added with 5 μl of 0.5M 2-mercaptoethylamine, heated at 37°C for 90 minutes, and then 0.1M
Reduced antibodies were obtained by passing through a Sephadex G-25 column equilibrated with phosphate buffer, pH 6.0. Meanwhile, in parallel, horseradish peroxidase (2 mg/0.3 ml) dissolved in 0.1 M phosphate buffer, pH 7.0, and 50 mM N-succimidyl-4-(N- m
aleimidomethyl)-cyclohexa
Add ne-1-carboxylate and incubate at 30°C for 1
Allowed time to react. After the reaction, the enzyme was passed through a G-25 column in the same manner to obtain an enzyme into which a maleimide group had been introduced. These reduced antibodies and maleimide enzyme were mixed and left at 4°C for 24 hours, then T
Purification was performed by gel filtration through SK gel and G3000SW (Tosoh) to obtain a horseradish peroxidase-labeled mouse monoclonal antibody of the IgG2a subclass.

[0019]

[Example 2 Mouse monoclonal antibody, IgG2b
Preparation of enzyme-labeled antibodies using subclasses] Instead of the monoclonal antibodies used in Example 1, anti-myosin mouse monoclonal antibodies produced by different fusion cell lines, I
A gG2b subclass antibody (clone number M904) was enzyme-labeled in the same manner as in Example 1 to obtain a horseradish peroxidase-labeled mouse monoclonal antibody of the IgG2b subclass.

[0020]

[Reference Example 1 Preparation of enzyme-labeled antibody by periodate oxidation method] Horseradish peroxidase aqueous solution (4 mg/m
l) 300 μl of 0.2 M sodium metaperiodate aqueous solution was added to 0.5 ml and left at room temperature for 20 minutes. Thereafter, it was dialyzed against 1mM acetate buffer, pH 4.5. After adding 0.2M sodium bicarbonate buffer, pH 9.5 to the dialysate solution and adjusting the pH to 9, anti-myosin mouse monoclonal antibody (clone number M705 or 904) solution (4 mg/0.5 ml) was added. The mixture was left to react at room temperature for 2 hours. After the reaction, 60 μl of sodium borohydride aqueous solution (4 mg/ml) was added, and after reduction treatment, G30
The peroxidase-labeled antibody was obtained by purification through a 00SW column.

[0021]

[Example 3 Measurement of myosin light chain by enzyme immunoassay] 50 μl of standard myosin solution and 0.1 M phosphate buffer (pH 6.5 , 0.15M NaCl, 0.
An enzyme-labeled antibody diluted with 5% bovine serum albumin (containing 5% bovine serum albumin) was added and allowed to react at room temperature for 1 hour. After reaction, zero the plate.
．． Washed 4 times with Tris-HCl buffered saline containing 1% Tween-20, 2,2'-amino-bis-3-
100 μl of a chromogenic enzyme substrate solution consisting of ethylbenzothiazoline-6-sulfonic acid and hydrogen peroxide was added, and the mixture was allowed to react at room temperature for 5 minutes. After adding 100 μl of 1% oxalic acid to stop the reaction, the absorbance at 414 nm was measured and a calibration curve was created. The results are shown in Table 1.

[0022]

[Table 1]

As shown in Table 1, the calibration curves obtained using the enzyme-labeled antibodies obtained in Examples according to the present invention and Example 2 were both steep curves despite the fact that the blank was low. Sensitive and highly accurate measurements were possible. On the other hand, the calibration curve obtained using the enzyme-labeled antibody obtained in the reference example had a high blank and a curve with a small slope, and was not suitable for enzyme immunoassay.

[0024]

Effects of the Invention As explained above, according to the present invention, IgG2a and IgG2b subclass antibodies of mouse monoclonal antibodies were used. Enzyme-labeled antibodies suitable for highly sensitive enzyme-linked immunosorbent assays are now available. Thus, the significance of increasing the utility value of IgG2a and IgG2b subclass antibodies of mouse monoclonal antibodies in the field of immunoassay is extremely significant.

[Table 1]
A414, average value of duplicate measurements Enzyme-labeled antibody standard myosin concentration (ng/ml)
0
12.5 25 50
100 Example 1
0.003 0.285 0.546
0.905 1.295 Example 2
0.002 0.19
8 0.368 0.579 0.
881 Reference example 1

Claims (2)

[Claims] 1. An enzyme-labeled antibody obtained by binding an enzyme to an antibody, which is obtained by reducing the antibody to liberate a sulfhydryl group, and then binding an enzyme using the sulfhydryl group. 2. An enzyme-labeled antibody characterized in that the antibody according to claim 1 is an IgG2a or IgG2b subclass of the IgG fraction of a mouse monoclonal antibody.