JP2000329765A - Pigment labeled antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prevent a deterioration of a bonding capability of an antibody to an antigen by forming a labeled antibody chemically bonding a pigment compound between disulfide bonds of a hinge for connecting an Fab fragment in an antibody molecule to an Fc fragment. SOLUTION: A structure of an antibody is indicated by an Y-shaped structure, and constituted of totally four polypeptides of two short polypeptides of L chains and two long polypeptides of H chains 8 so that the L chains 8 and the H chains 7 become a pair so as to be associated with each other by one disulfide bond 9 and many non-covalent bonds. The L and H pairs 7 and 8 further form a dimer. Here, a region for associating three Y-shaped arms of the disulfide bond 6 between the H chains which is generally called a 'hinge 2', is divided at an upper portion of the Y shape into the Fab fragment 4 and at a lower portion of the Y shape into the Fc fragment 5 with the hinge 2 as a boundary. Thus, the pigment compound can be labeled by one to one molecule of the antibody by comparing it with the several labeled antibodies at random.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the structure of a dye-labeled antibody used in a fluorescent immunoassay.

[0002]

2. Description of the Related Art Antibodies are widely used in biochemical tests and the like as immunoassays due to their property of specifically binding to antigens. However, unlike enzymes that react chemically with substrates,
Antibodies-antigens do not perform reactions other than binding, and therefore, various approaches have been widely devised to chemically modify antibodies for the purpose of sensing the binding reaction. Among them, a method of chemically binding a fluorescent dye to an antibody surface is often used for the purpose of being used in a fluorescent immunoassay method that easily provides high sensitivity.
Usually, the functional groups on the surface of the antibody molecule used for the chemical modification of the antibody include an amino group, a thiol group, a hydroxyl group and a carboxyl group. A fluorescent dye-labeled antibody is obtained by reacting a fluorescent dye compound having a functional group that can be covalently bonded to these functional groups of the antibody molecule.

[0003] As an example of labeling a protein with a dye,
r (G. Weber, Bio)
chem. J. 51, 155, 1952). In this study, it is stated that the reaction of serum albumin with dansyl-chloride achieves the labeling of dansyl dye at a rate of 50% based on the total number of moles of serum albumin. In this case, it is considered that the reaction of the amino group of the protein with the sulfonyl chloride which is the reaction site of dansyl chloride is dominant. In this example,
The target to be modified is not an antibody, but albumin and an antibody are common in that they are proteins having the above functional groups on the surface, and the reaction in the above literature replaces the target with an antibody and is applied as it is. It is possible. Here, since the functional groups in these antibody molecules are generally hydrophilic and are present outside the antibody molecules, the dye compound introduced during the above-described labeling reaction is located outside the antibody molecules. Will be located.

[0004]

A dye-labeled antibody is used in a fluorescent immunoassay. When a large amount of a dye compound is labeled per antibody molecule for the purpose of improving the sensitivity of the measurement, the hydrophilic portion of the antibody is The imbalance of the sex part is lost and the ability of the antibody to bind to the antigen is degraded. On the other hand, since the labeled dye compound is located outside the antibody, it is affected by the surrounding environment, such as the polarity, viscosity and dissolved oxygen of the solvent, so that the fluorescence intensity is reduced and the original function is reduced. .
As described above, the conventional fluorescence immunoassay using a labeled antibody has prevented improvement in measurement sensitivity.

[0005]

Means for Solving the Problems In order to solve the above problems, in the present invention, a dye compound is chemically bonded between a disulfide bond at a hinge connecting an Fab fragment and an Fc fragment inside an antibody molecule. The present invention proposes a labeled antibody.

[0006]

DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 shows a conceptual diagram of a labeled antibody in the present invention. The structure of the antibody is shown by a Y-shaped structure, and FIG. 2 shows a distribution diagram of the antibody. It is composed of a total of four, two short polypeptides called L chain 8 and two long polypeptides called H chain 7, and the L chain 8 and the H chain 7 form a pair, and one disulfide bond 9 It is known that they associate with each other by many non-covalent bonds. The LH pairs 7 and 8 further form a dimer. Here, a region where three Y-shaped arms having a disulfide bond 6 between H chains meet, generally called a hinge portion 2, but with the hinge portion 2 as a boundary, a Y-shaped portion is formed. Fa at the top of
b fragment 4, the lower part of the Y-shape is Fc fragment 5.
It is divided as.

Here, an embodiment of the labeled antibody in the present invention will be described with reference to the production drawing of the labeled antibody shown in FIG.

The numbers in FIGS. 1 and 2 are the same as those in FIG.

The distance between the disulfide bonds of the hinge portion 2 of the antibody 6
Is chemically bound to a dye compound 3 to obtain a labeled antibody. The labeling method is to treat the antibody reductively to cleave the disulfide bond 6 of the hinge portion 2 to form a thiol group, and then sandwich the dye compound selectively reacting with the thiol group with two molecules of a half antibody. React as follows.

It is described that the reactivity of the disulfide bond is greater than the intrachain disulfide bond 9 as compared to the interchain disulfide bond 6 (Biochemical Experiment 8, Chemical modification of SH group, Gakkai Shuppan Center, pp. 121-124). And that the disulfide bond 9 between the LH chains is not cleaved by a weak reducing agent such as 2-mercaptoethylamine 10 as compared to the disulfide bond 6 between the H chains (H
ermanson BioconjugateTech
niques, AcademicPress, P463
465) based on the hinge region 2
Is specifically cleaved with, for example, 2-mercaptoethylamine 10, which is a weak reducing agent, and reacts so as to sandwich the divalent dye compound 3 with the thiol 11 of the obtained half antibody molecule. I let it. As a result, a dye-labeled antibody was obtained.

As the dye compound to be labeled so as to be included in the antibody, an aromatic hydrocarbon such as benzene, naphthalene, anthracene, tetracene, chrysene, pyrene, or fluoranthene, or a derivative thereof can be used. Furthermore, azo, quinone, triaryl,
It is a cyanine-based, phthalocyanine-based, or indigo-based dye, or a derivative having the dye skeleton as a parent. As the fluorescent dye compound, for example, a dye compound such as fluorescein, dansyl, acridine, coumarin, eosin, rhodamine, or BODIPY, or a derivative thereof can be used.

Dye compound 3 binding to thiol group of antibody
The reactive site is a functional group and or a binding site that selectively reacts with thiol, for example, a maleimide group, a halogen group, an aziridine group and a disulfide bond,
At least two or more reactive sites are present in the dye compound 3 and are oriented within the dye molecules so that two of the half antibody molecules can be crosslinked.

Next, a specific example of the labeled antibody of the present invention will be described in detail with reference to the production process diagram of FIG. In the production of the labeled antibody of the present invention, the antibody has anti-Mouse I
The gG antibody used had a dansyl skeleton as a dye.

In the first step, 2-mercaptoethylamine 10 is added as a reducing agent to the anti-Mouse IgG antibody PBS buffer solution 1 'in order to cleave the disulfide bond 6 of the hinge portion 2 of the anti-Mouse IgG antibody 1', Allowed to stir for hours. Since the cleaved thiol group 11 has high reactivity, recombination was suppressed by coexisting EDTA. After completion of the reaction, a molecular weight assay was performed by SDS-PAGE electrophoresis. Since the number of antibody molecules having a molecular weight of about 150,000 was reduced to about 75,000, only the target disulfide bond 6 of the hinge portion 2 was cleaved. Confirmed that. After the confirmation, the reaction mixture was subjected to gel filtration using Sephadex G-25 to obtain anti-Mouse.
eIgG half antibody solution 1 ″ was isolated and purified.

In the second step, in order to label the dansyl skeleton of the dye compound, 2 equivalents of dansyl-dimaleimide 3 were added to the anti-Mouse IgG half antibody 1 ″, and the mixture was stirred at room temperature for about 12 hours. Thereafter, gel filtration was performed using Sephadex G-25, and a dansyl skeleton-labeled antibody and an unlabeled anti-Mouse IgG were used.
Antibodies were each recovered.

The label was evaluated by analyzing the absorption characteristics with an absorption spectrophotometer. As a result, the dansyl-labeled anti-mouse IgG was analyzed.
The number of labels of the antibody 1 was about 1, and it was confirmed that the anti-Mouse IgG half antibody 1 ″ and dansyl-dimaleimide reacted at a ratio of 2: 1.

In the third step, the obtained dansyl-labeled anti-Mouse IgG antibody and a conventional dansyl-labeled anti-Mouse IgG antibody are used.
The respective fluorescence spectra of eIgG antibody 1 (Ex33
0 nm), as a result, as shown in FIG. 4, the relative fluorescence intensity was improved about 10 times as compared with the conventional one. From the above, the effect when the dansyl skeleton, which is a coloring compound, was placed in a hydrophobic environment was clearly exhibited.

[0018]

According to the dye-labeled antibody of the present invention, it is possible to label one dye compound per one antibody molecule, as compared with a conventional antibody labeled several times at random. It is possible to avoid deteriorating the binding ability to. In addition, since the antibody functions as an inclusion compound for the fluorescent dye, it is not affected by a hydrophilic solvent, dissolved oxygen, or the like. As a result, the original function of the fluorescent dye can be brought out. As an example, the fluorescence intensity of the dye compound of the present novel labeled antibody was improved about 10 times as compared with the dye compound in the hydrophilic solvent. This means that the fluorescence method can be applied with high sensitivity even to those which could not be measured because of the low fluorescence intensity in an aqueous solution.

[Brief description of the drawings]

FIG. 1 is a structural diagram of a dye-encapsulated labeled antibody according to one embodiment of the present invention.

FIG. 2 is an exploded structural view of the antibody.

FIG. 3 is a diagram showing a process for producing a dye-labeled antibody in one embodiment of the present invention.

FIG. 4 is a fluorescence spectrum diagram of the dye-labeled antibody.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Antibody 2 Hinge part 3 Dye compound 4 Fab fragment 5 Fc fragment 6 Disulfide bond 7 H chain 8 L chain 9 Disulfide bond 10 Mercaptoethylamine 11 Thiol group

Claims (5)

[Claims] 1. A dye-labeled antibody characterized in that a dye compound is chemically bonded between disulfide bonds in a hinge portion connecting an Fab fragment and an Fc fragment inside an antibody molecule. 2. The dye-labeled antibody according to claim 1, wherein the dye compound is a fluorescent compound. 3. The method according to claim 1, wherein the dye compound has at least two functional groups or reactive sites that selectively react with a thiol group obtained after reductively treating the antibody. Dye-labeled antibody. 4. The dye-labeled antibody according to claim 3, wherein the functional group of the dye compound is a halogen group, a maleimide group, or an aziridine group. 5. The dye-labeled antibody according to claim 3, wherein the reactive site of the dye compound is a disulfide bond connecting two or more kinds of the dye compounds.