JP2002000268A - Monoclonal antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for the immunological determination of carboxymethylated hemoglobin which is one of AGE in humor, especially a hemoglobin carboxymethylated at the side chain amino group of the lysine residue confirmed to have a relationship to nephropathy in high sensitivity, accuracy and reproducibility. SOLUTION: The carboxymethylated hemoglobin in humor is determined by using an immunological determination reagent containing a monoclonal antibody produced e.g. by hybridoma 9H6 (NIBH deposit No.773) or hybridoma 7B8 (NIBH No.774), having a dissociation constant of <=10-8 M and a dissociation rate constant of $10-2 s-1 to a peptide having a specific amino acid sequence and carboxymethylated at the side chain amino group of the 5th lysine residue from the N-terminal or to a peptide having another specific amino acid sequence and carboxymethylated at the side chain amino group of the 5th lysine residue from the N-terminal.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a monoclonal antibody which specifically recognizes carboxymethylated hemoglobin and has a specific dissociation constant and a specific dissociation rate constant, the antibody-producing cells, and carboxymethylation using the antibody. The present invention relates to a method for measuring hemoglobin immunologically and a reagent for immunological measurement.

[0002]

2. Description of the Related Art In recent years, AGE (Advanced Glycation End Products) has been used for the severity of diabetic complications (Brownlee, M., et al, 1988, N. En.
gle. J. Med., 318: 1315-1321) and aging (Araki, N. et.
al, 1992, J. Biol. Chem., 267: 10211-10214), chronic kidney disease (Miyata, T. et al, 1996, J. Am. Soc. Neph.
rol., 7: 1198-1206), Alzheimer's disease (Smith, M.
A., et al, 1994, Prox. Natl. Acad. Sci. USA, 91: 5
710) and so on.

[0003] The main component of AGE is a carboxymethylated protein in which the side chain amino group of a lysine residue is carboxymethylated (hereinafter, carboxymethylation is simply referred to as “CM”).
, And the protein may be abbreviated as “CM-modified protein”. ), And carboxymethylated lysine (hereinafter sometimes abbreviated as “CML”) that constitutes a CM protein is considered to be a major epitope of AGE (Ikeda, K., et al., 1996, Biochemistry, 3
5: 8075-8083) Therefore, AGE measurement is mainly based on CML
Is determined.

Conventionally, instrumental analysis such as high performance liquid chromatography or gas chromatography / mass spectrometry has been used for the quantification of CML constituting the CM protein. However, the operation is complicated and the sensitivity is low. .
Therefore, recently, the operation is simple and the sensitivity is high.
Techniques using immunological measurement methods such as method A (enzyme-labeled immunoassay) and dot blotting are often used.

In recent years, carboxymethylated hemoglobin (hereafter, hemoglobin is simply abbreviated as “Hb”, and carboxylated hemoglobin is sometimes abbreviated as “CM-Hb”), which is one of AGEs, has recently been used for diabetes. Relationship with the development of sexual complications (Japanese Patent Application Laid-Open No. 10-185917) and relationship with dialysis amyloidosis (Motomiya,
Y. et al, 1998, Kidney Int. 54: 1357-1366), which has attracted attention as a useful index for the diagnosis, treatment, prevention, or evaluation of the efficacy of diabetic complications or dialysis amyloidosis. .

[0006] The measurement of the CM-modified hemoglobin (CM-Hb) is also performed by ELISA or dot blotting (Motomiya,
Y. et al, 1998, Kidney Int. 54: 1357-1366).
When the anti-CM protein polyclonal antibody used in the conventional CML measurement is used, the specificity is low due to the recognition of all the CM-protein, and the sensitivity is low and the measurement accuracy is low. was there.

[0007] Such a problem is described in Japanese Patent Application Laid-Open No. H11-2363.
This problem has been solved by using a monoclonal antibody that specifically reacts with CM-modified hemoglobin as disclosed in JP-A-99-99.

However, CM-Hb to be measured includes a plurality of types of CM-Hb having different sites to be converted into CM, specifically, those in which a valine residue at the N-terminal of the β chain of hemoglobin is converted to CM (hereinafter, referred to as “CM-Hb”). Is also referred to as CMV-Hb.), Those in which the lysine residue of the hemoglobin β chain is converted to CM, and those in which the lysine residue of the hemoglobin α chain are C
M-ized (and generically C
Also called ML-Hb. ) Etc., and C of the above type
Although the M-Hb can be measured without any particular problem using the monoclonal antibody disclosed in the above publication, the above-mentioned and the above types of CM-Hb (CML-H
As for the measurement b), it was found that there was a problem in the reproducibility of the measurement, and as a result, there was a problem that sufficient measurement sensitivity and measurement accuracy could not be obtained.

[0009] CM-Hb of the above type (CMV-H
b) is generated by autoxidation of sugar, and its relevance to diabetes has been confirmed. On the other hand, CM-Hb (CML-Hb) of the above-mentioned type and (2) It is produced by peroxidation and has been confirmed to be related to nephropathy and the like, and it is significant to measure the amount of CML-Hb with high accuracy and high sensitivity.

[0010]

Accordingly, the present invention provides a C
Among the M-hemoglobins, the present invention provides a monoclonal antibody capable of detecting CML-Hb with high accuracy and high sensitivity, and using the monoclonal antibody to precisely detect CML-Hb,
It is an object of the present invention to provide a method for measuring with high sensitivity and a measuring reagent.

[0011]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, have the amino acid sequence represented by SEQ ID NO: 1 and a lysine residue at the fifth position from the N-terminal. Has the amino acid sequence represented by SEQ ID NO: 2 or a peptide in which the side chain amino group is CM-modified,
Succeeded in obtaining a novel monoclonal antibody that reacts with a specific dissociation constant and a specific dissociation rate constant for a peptide in which the side chain amino group of the lysine residue is CMated,
By using the monoclonal antibody, CML
The inventors have found that it is possible to measure -Hb with high accuracy and high sensitivity, and have completed the present invention.

That is, a first aspect of the present invention is a peptide having the amino acid sequence of SEQ ID NO: 1 wherein the side chain amino group of the fifth lysine residue from the N-terminal is carboxymethylated, or the amino acid of SEQ ID NO: 2. A peptide having a sequence and a carboxymethylated side chain amino group of the fifth lysine residue from the N-terminal has a dissociation constant of 10 ^-8 M or less and a dissociation rate constant of 10 ^-2 s ^-1 or less A monoclonal antibody characterized in that:

[0013] The monoclonal antibody of the present invention can be prepared, for example, using the hybridoma Mb66-9H6 (FERM P-179) deposited at the National Institute of Bioscience and Human Technology.
00) or hybridoma Mb68-7B8 (FERM
P-17901).

A second aspect of the present invention is a hybridoma producing the monoclonal antibody of the present invention.

A third aspect of the present invention is a method for immunologically measuring carboxymethylated hemoglobin, characterized by using the above-mentioned monoclonal antibody of the present invention. It can be suitably carried out by using the above-mentioned "reagent for measuring carboxymethylated hemoglobin comprising the monoclonal antibody of the present invention."

According to the measuring method of the present invention, CM-Hb, particularly CML-Hb, present in a sample such as a hemolyzed sample can be easily measured with high sensitivity and high accuracy. In the method, such an excellent effect is exhibited by using the monoclonal antibody of the present invention showing not only the dissociation constant but also the dissociation rate constant with a specific low value for the antigen to be measured. .

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION The monoclonal antibody of the present invention
A peptide having the amino acid sequence of SEQ ID NO: 1 and a CM of the side chain amino group of the fifth lysine residue from the N-terminal (hereinafter, a peptide having the amino acid sequence of SEQ ID NO: 1 is referred to as “peptide β66”; The peptide converted into CM is referred to as “C
M-peptide β66) or a peptide having the amino acid sequence of SEQ ID NO: 2 in which the side chain amino group of the fifth lysine residue from the N-terminus has been converted to CM (hereinafter, SEQ ID NO: 2)
A peptide having the amino acid sequence of "peptide α61"
Said CM-peptide is referred to as “CM-peptide α
61 ").

The amino acid sequence of SEQ ID NO: 1 corresponds to the 62nd to 73rd amino acid sequence from the N-terminal of the β-chain of hemoglobin (Hb), and the CM-peptide β6
In No. 6, the side chain amino group of the lysine residue at the position corresponding to the 66th position from the N-terminus of the β chain of Hb is converted to CM. In addition, the amino acid sequence of SEQ ID NO: 2 is
In the CM-peptide α61, the side chain amino group of the lysine residue at the position corresponding to the 61st position from the N-terminus of the α-chain of Hb is CM. Has been

The CM-peptide β66 and CM-peptide α61 may be chemically synthesized from amino acids or the like, or may be fragments derived from proteins or longer-chain peptides. When these peptides are chemically synthesized, they can be synthesized using a known method such as a solid phase method or a liquid phase method. When these peptides are chemically synthesized, the extension reaction of the peptide may be performed in advance using CML, or the extension reaction of the peptide may be performed using amino acids as a raw material.
The fifth lysine side chain from the N-terminus and the fifth from the N-terminus in CM-peptide α61 may be converted to CM. In addition, CML
Although there is no particular limitation on the method for synthesizing CML, for example, after reacting lysine with glyoxylic acid, it is reduced with a reducing agent such as sodium borohydride or sodium cyanoborohydride to synthesize CML, and then cation exchange chromatography. CML in which the side chain amino group has been converted to CM can be separated by chromatography. In the case where the specific lysine residue is converted into CM after the extension reaction of the peptide using amino acids as a raw material, the peptide β66 and the peptide α61
Since each of these contains two lysine residues, it is necessary to devise a method for selectively converting one of them into CM. For example, when synthesizing by a solid phase method, the lysine used for the elongation reaction of the peptide can be deprotected with a base such as piperidine at the ε-amino group as lysine to be converted into CM.
Using a compound having a fluorenylmethoxycarbonyl (Fmoc) group introduced therein, a 2-chlorobenzyloxycarbonyl (CIZ) group capable of deprotecting an ε-amino group as a non-CM lysine with a strong acid such as hydrofluoric acid, etc. In addition, a protecting group that can be deprotected with a weak acid such as a t-butoxycarbonyl (Boc) group is introduced into the α-amino group of lysine, and amino acids other than lysine may be used as necessary. An appropriate protecting group that is not deprotected by a base is introduced into the side chain functional group, and a protecting group that can be deprotected by a weak acid such as a t-butoxycarbonyl (Boc) group is introduced into the α-amino group in the same manner as lysine. For example, it is necessary to devise a method such that a specific amino group is protected according to the reaction conditions. After performing an elongation reaction of the peptide by a known method using the amino acid into which these protecting groups have been introduced, the protecting group of the ε-amino group in the lysine residue to be converted into CM using a base such as piperidine. I do. Next, after the ε-amino group of lysine deprotected by the above method is converted to CM, final deprotection may be performed using a strong acid such as hydrofluoric acid.

The CM- of the monoclonal antibody of the present invention
It is necessary that the dissociation constant (K) for peptide β66 or CM-peptide α61 is 10 ⁻⁸ M or less, and the dissociation rate constant (k ₋ ) is 10 ⁻² s ⁻¹ or less. By satisfying such conditions, CML-Hb can be reliably captured, and CML-Hb can be measured with high sensitivity and high reproducibility. Here, CML-Hb has a side chain amino acid of lysine residue CM
And the valine residue of the CML-Hb may be converted to CM.

Here, the dissociation constant (K) is defined by the following reaction formula in which an antigen binds to a monoclonal antibody to form a complex.

[0022]

Embedded image

[0023] is the equilibrium constant for the equilibrium reaction represented by the association rate constant (k ₊₎ and dissociation rate constant _- with the following formula K = k (k) - those defined by _/ k ₊ (M) Is an index that relatively indicates the ease of binding between the antibody and the antigen.

For example, for a reaction in which a complex is formed by an antigen-antibody reaction between CM-peptide β66 and a monoclonal antibody, the concentrations of CM-peptide β66, the monoclonal antibody, and the complex are determined by [Pe], [MAb],
And [MAb · Pe], the dissociation constant K can be expressed by the following equation using the Michaelis-Menten equation: K = k ₋ / k ₊ = [Pe] × [MAb] / [MAb · Pe] Chemistry Experiment Course 1: Protein V
(Tokyo Kagaku Dojin, 1st edition, 1991, pp. 280-282), such as a Scatchard plot or Hill plot. Recently, surface plasmon resonance (hereinafter referred to as BIA-core (Amersham Pharmacia))
It may be described as “SPR”. SPR: surface pl
asmon resonance) relatively simple operation with a dissociation rate constant by the use of the inter-molecules by applying the principle interaction analysis apparatus or the like (k _-), and binding rate constants (k ₊₎ will be able to measure the It is also possible to easily determine the dissociation constant (K) based on the above equation. Below, SP
An apparatus for analyzing the interaction between molecules using the principle of R will be described. When light incident on the triangular prism of glass is totally reflected at the interface, if a metal thin film is in contact with the reflecting surface, incident light at a certain angle is absorbed by surface plasmon resonance (SPR), and the reflected light corresponding to this is absorbed. Strength decreases. The reflection angle of the attenuated reflected light depends on the refractive index of the medium layer in contact with the outside of the metal thin film. The molecular interaction analyzer has a metal thin film corresponding to the above-mentioned metal thin film-medium layer-a dextran thin layer on a sensor chip.
When the analyte binds to the ligand immobilized on the dextran thin layer, the refractive index of the dextran thin layer-ligand layer changes. Since the refractive index of the dextran thin layer-ligand layer depends on the binding amount of the analyte to the ligand, the binding amount of the analyte to the ligand is:
This is reflected in the reflection angle of the attenuated reflected light with respect to the light incident on the glass triangular prism adjacent to the sensor chip. That is, by sensing the reflection angle of the attenuated reflected light, the amount of binding between the ligand and the analyte can be measured. Therefore, by reacting the analyte with the sensor chip on which the ligand is immobilized and measuring the binding amount in real time by the intermolecular interaction analyzer,
The binding rate constant (k ₊ ) between the ligand and the analyte can be determined. Furthermore, after binding of the ligand and the analyte,
Ligand - the dissociation rate constant by measuring the dissociation of the ligand and the analyte is contacted with a buffer without analyte in real time to the analyte complex (k _-) can also be obtained. In BIA-core (Amersham Pharmacia) intermolecular interaction analysis device sold, such as the protocol is almost automated, association rate constant (k _+), and dissociation rate constant (k _-) is also easily calculated by dedicated software It is possible to In the present invention, the monoclonal antibody of the present invention is immobilized on a sensor chip by using a peptide such as peptide CM-β66 or CM-peptide α61 as the ligand, and the monoclonal antibody of the present invention is used as the analyte. The association rate constant (k ₊ ) and the dissociation rate constant (k ₋ ) can be easily obtained.

The monoclonal antibody CM-peptide β6
6 and the dissociation constant K for CM-peptide α61 is 10
^-8 M or the dissociation rate constant k _- is 10 ^-2 s ^-1
In the case of exceeding CM, it becomes difficult to measure CM-Hb with good reproducibility because the binding between the antibody and these peptides becomes unstable. In particular, when the dissociation constant K exceeds 10 ^-8 M, the binding ability between the monoclonal antibody and the peptide decreases, resulting in a decrease in measurement sensitivity. C in terms of effect
The dissociation constant K and the dissociation rate constant k ₋ for M-peptide β66 or CM-peptide α61 are 6 × 10
^It is preferably at most ^-9 M and at most 5 × 10 ^-3 s ^-1 .

The type (globulin class) of the monoclonal antibody of the present invention is not particularly limited as long as it specifically reacts with CM-peptide β66 or CM-peptide α61. Also includes those of the globulin class. The monoclonal antibody in the present invention includes not only ordinary monoclonal antibodies but also partially degraded products of the antibodies (Fab, Fab ', Fab'2, etc.), and active fragments of the antibodies (antigen recognition sites of the antibodies). Also includes a partial structure in which is present.

The method for producing the monoclonal antibody of the present invention is not particularly limited, but after immunizing an animal with an antigen for immunization (hereinafter also simply referred to as "immunogen"), CM-peptide β66 or CM- A method for selectively sorting cells producing a specific antibody against peptide α61 and culturing the sorted cells in a suitable medium to produce the antibody; or hybridizing the sorted antibody-producing cells with plasmacytoma cells to produce a hybridoma The hybridoma can be suitably produced by culturing or transplanting it into an animal and amplifying it.

At this time, CM-Hb or CM-peptide β66 or CM-peptide α61 was used as the immunogen.
And a hapten complex can be used. Examples of the hapten used at this time include proteins such as human serum albumin and bovine serum albumin. CM-Hb can be obtained by substituting the hydrogen of the amino acid side chain amino group or N-terminal amino group of Hb with a carboxymethyl group. Known methods can be used for this CM conversion without any limitation. For example, “Shinsei Kagaku Koza 1, Protein 4” (edited by the Japanese Biochemical Society,
It can be carried out by the reductive alkylation method described in 1991, Tokyo Chemical Dojin, p13-p16). That is, an aldehyde compound and Hb are dissolved in an aqueous solution such as a borate buffer or a phosphate buffer, and the pH is adjusted to 8 to 10 in the presence of a hydride reducing agent such as sodium borohydride or sodium cyanoborohydride.
The reaction can be carried out under the following conditions to produce CM. Under a higher pH condition, proteins and the like may be denatured. Under a lower pH condition, the hydride reducing agent becomes unstable. In order to allow the reaction to proceed specifically and quantitatively, the reaction temperature is preferably room temperature or lower,
More preferably, it is good to carry out at 0-10 degreeC. The obtained CM-Hb solution is used after dialysis or ultrafiltration to remove unreacted reducing agent or aldehyde compound.

Hereinafter, a method for producing the monoclonal antibody of the present invention using a hybridoma capable of producing the monoclonal antibody of the present invention (hereinafter, also referred to as “hybridoma of the present invention”) will be specifically described. Here, a hybridoma is a hybrid cell formed by artificially fusing two different types of cells (parent strain), for example, a plasmacytoma cell established so that it can be cultured, and an antibody taken from a living body. It can be produced by fusing with a production cell. The hybridoma can be cultured and has an antibody-producing ability.

The hybridoma of the present invention can be generally prepared through a series of steps such as animal immunization, cell fusion, selection of fused cells, selection of specific antibody-producing cells, and cloning.

The animal is immunized by immunizing the animal with CM-Hb or the like obtained as described above as an immunogen. The form of the immunogen used for immunization is not particularly limited, but it is preferable to use one mixed with Freund's complete adjuvant in order to enhance the immunization effect. The immunized animal is preferably a mammal, for example, a mouse, a guinea pig, a rat, a rabbit, a sheep, a chicken, etc., and a mouse is particularly preferable in terms of ease of handling. The immunization method can be carried out by appropriately combining known appropriate methods. Usually, this is carried out by injecting the immunogen into at least one subcutaneous, intramuscular, or intraperitoneal cavity of an animal. More preferably, the immunogen is injected into the lymph nodes. About 1 from the first immunization
Immunization is performed several times every 2 weeks, and antibody producing cells are separated from the immunized animal about 3 to 5 days after the final immunization. As antibody-producing cells, cells having antibody-producing ability such as spleen cells and lymph node B cells may be extracted.

Next, steps after the cell fusion will be described. In general, myeloma (myeloma) cells are used as the plasmacytoma cells, which are the other parent strain required for producing a hybridoma by cell fusion. Examples of myeloma cells include mouse-derived P3U1, 65
3, SP2, X63-Ag8, NS-1, MPC-11
AG1, AG2, AG3, RCY3 derived from rats, etc.
Human-derived SKO-007, such as 210, can be used, but mouse-derived SKO-007 is preferable, and P3U1, 653 is particularly preferable from the viewpoint of easy availability and results. Furthermore, it is preferable that the antibody-producing cells and the myeloma cells are derived from the same animal.

For cell fusion, a method using polyethylene glycol, a method using Sendai virus, an electric fusion and the like can be used, and the method of Kohler and Milstein (1975, Natu
re, 256: 495-497) or a method using polyethylene glycol according to this method is preferred because the operation is simple. The method is carried out by mixing antibody-producing cells and myeloma cells for 1 to 10 minutes using 30 to 50% polyethylene glycol (average molecular weight: 1,000 to 4,000) at a temperature of 30 to 40 ° C. Will be Hybridomas obtained by cell fusion can be selected by culturing in a selective medium in which only hybridomas can grow. For example, HAT (hypoxanthine, aminopterin,
A thymidine) medium and a Haz (hypoxanthine-azacerin) medium are preferred. For example, HA
The T selection method is effective when using myeloma cells that are HGPRT (hypoxanthine-guanine phosphoribosyl transferase) -deficient strains as described above for cell fusion. By culturing the cells in a HAT medium after cell fusion, the growth of myeloma cells of the HGPRT-deficient strain is inhibited by aminopterin, and only the aminopterin-resistant hybridomas can be selectively grown.

From the hybridomas thus obtained, they specifically react with CM-peptide β66 or CM-peptide α61 and have a dissociation constant (K) of 10 ^−8.
Those that produce a monoclonal antibody having a dissociation rate constant (k ₋ ) of 10 ⁻² s ⁻¹ or less are selected. This selection is made as follows. CM-peptide β66,
Alternatively, for specificity to CM-peptide α61, the culture supernatant of a well in which hybridoma growth was observed by the above culture was collected, and the presence or absence of an antibody to CM-peptide β66 or CM-peptide α61 was determined by, for example, an ELISA method. It may be examined by an enzyme immunoassay or the like. The dissociation constant (K) and the dissociation rate constant (k ₋ ) are as follows: CM-peptide β66 or C-peptide.
Using the culture supernatant of the well of the hybridoma strain in which the reactivity to the M-peptide α61 has been confirmed, the measurement may be performed using an intermolecular interaction analyzer such as BIA-core as described above. Subsequently, CM-peptide β66 or C
The hybridoma in the well in which the antibody against M-peptide α61 is present may be cloned. Cloning is to convert the above-mentioned specific antibody-producing cells into a uniform cell population derived from one clone. For example, a limiting dilution method, a method of picking up colonies on soft agarose, a single cell manipulation method, FACS
The limiting dilution method is simple and preferred.

A single monoclonal antibody-producing cell can be selected by the above method. The selected hybridoma cell line can be grown in 2-10 ml of medium and stored frozen in liquid nitrogen. The hybridoma 9H6, which is one of the hybridomas of the present invention and produces a monoclonal antibody specific to CM-peptide β66, was deposited with the National Institute of Bioscience and Biotechnology, National Institute of Advanced Industrial Science and Technology. Mb6
6-9H6 "｛Deposit No .: Life Science Bacteria Deposit No. 773 (FE
RM P-17900)}. Also, one of the hybridomas of the present invention, CM-peptide α
Hybridoma 7B8, which produces a monoclonal antibody specific to 61, was deposited with the National Institute of Bioscience and Biotechnology, National Institute of Advanced Industrial Science and Technology, and
Mb68-7B8 "{Deposit No .: Deposit No. 774 (FERM P-17901)}.

As a method for preparing the monoclonal antibody of the present invention using the hybridoma of the present invention, any conventionally known method for preparing an antibody using the hybridoma can be used without any limitation. For example, a monoclonal antibody can be prepared by transplanting a hybridoma into the peritoneal cavity of an animal, amplifying the hybridoma, and collecting ascites. The antibody thus obtained can be purified and used as needed. Examples of the purification method include ammonium sulfate fractionation, polyethylene glycol fractionation, ethanol fractionation, affinity chromatography using a protein A column or protein G column, ion exchange column chromatography, gel filtration column chromatography. It can be purified by using a conventional method such as a method and an electrophoresis method. Among these, affinity chromatography is preferable, and a method using a protein A column is particularly preferable.

Using the thus obtained monoclonal antibody of the present invention, it is possible to measure CM-Hb, particularly CML-Hb, in a sample such as a body fluid with high sensitivity and high accuracy by utilizing an antigen-antibody reaction. it can. The method for measuring CM-Hb using the monoclonal antibody of the present invention is not particularly limited, but an immunological assay reagent for measuring CM-Hb containing the monoclonal antibody of the present invention (hereinafter referred to as the immunological assay of the present invention) ).

The immunological assay reagent of the present invention contains the monoclonal antibody of the present invention, and the antibody and CML-H contained in the sample.
There is no particular limitation as long as CML-Hb in a sample can be qualitatively or quantitatively measured using an antigen-antibody reaction with b, and various forms may be used depending on various epidemiological measurement methods. Usually, (i) a monoclonal antibody of the present invention or a radioactive substance, a monoclonal antibody of the present invention labeled with a labeling substance such as various dyes, colloids, enzymes, etc. supported on an insoluble carrier; An antigen that reacts with the monoclonal antibody of the present invention or a combination of the antigen supported on an insoluble carrier and the labeled monoclonal antibody of the present invention or the labeled monoclonal antibody of the present invention; A monoclonal antibody, or (iv) a combination of the monoclonal antibody of the present invention and a labeled secondary antibody.

For example, when a so-called immunoagglutination method for detecting CML-Hb in a sample utilizing an agglutination reaction of an insoluble carrier based on an antigen-antibody reaction is employed, the above-mentioned (i) or (ii)
Of the present invention. When a so-called labeled immunoassay for detecting CML-Hb in a sample is performed by measuring physical properties such as radioactivity and enzyme activity derived from a labeling substance after performing an antigen-antibody reaction, It can be carried out by using the immunological measurement reagents of the present invention (ii) to (iv).

Examples of the insoluble carrier include glass used as a usual immunoassay carrier; polysaccharides or derivatives thereof; silica gel; porous ceramics; metal oxides; erythrocytes; polypropylene, polystyrene, polyacrylamide, and polyacrylonitrile. And the like, or those into which a reactive functional group such as a sulfone group or an amino group is introduced by a known method, and the shape of the resin may be a bead, a test plate, a sphere, a disk, or the like.
Examples include a tube shape and a filter shape. In general, the average particle size of the reagent used in the immunoagglutination method has a mean particle size of 0. 0 from the viewpoint of the ease of occurrence of aggregation and the easiness of discrimination of aggregation.
An insoluble carrier of 05-10 μm is used.

As a method for carrying (immobilizing) the monoclonal antibody of the present invention or its antigen on these insoluble carriers, known methods such as a physical adsorption method, a covalent bonding method, an ionic bonding method and a cross-linking method are used without any limitation. Can be used. For example, when a microplate is used as an insoluble carrier (solid phase),
The monoclonal antibody solution is injected into the wells of the microplate and allowed to react for 1 to 48 hours to allow the antibody to be supported by adsorption.

The amount of the substance to be carried, such as the monoclonal antibody of the present invention, carried on the insoluble carrier slightly varies depending on the form of the reagent. For example, in a reagent used in an immunoagglutination method, usually, 0.001 to 100 mg of the monoclonal antibody of the present invention is supported on 1 g of an insoluble carrier. In the labeled immunoassay reagent, a substance to be supported according to the reagent form is generally supported at a rate of 0.01 to 1000 μg / cm ² per surface area of the insoluble carrier.

As the labeling substance used in the immunoassay reagent of the present invention, radioactive iodine or radiocarbon can be used as a radioactive substance; fluorescent dyes such as fluorescein isothiocyanate and tetramethylrhodamine can be used as dyes. Gold colloid or the like can be used as the colloid; and peroxidase, alkaline phosphatase or the like can be used as the enzyme. Such a labeling substance is detected using radioactivity, colorimetry, fluorescence, luminescence, etc. according to each type.

The method of labeling with these labeling substances is not particularly limited as long as it does not adversely affect the antigen-antibody reaction, and known methods can be employed without any limitation. For example, a method of directly labeling a peptide or protein with an enzyme or recognizing and binding CML-Hb with a biotinylated anti-Hb antibody and then labeling the enzyme with avidin is used.

Specific examples of the type of the immunological assay reagent of the present invention include a latex agglutination reagent and a qualitative reagent.
Microtiter reagents and the like are quantitative reagents such as radioimmunoassay reagents, enzyme immunoassay reagents, fluorescent immunoassay reagents, chemiluminescence immunoassay reagents,
Latex quantitative reagents and the like.

The method of measuring CML-Hb in a sample using the immunological measurement reagent of the present invention is characterized in that the immunological measurement reagent of the present invention is used as a measurement reagent, and that CML-Hb such as blood is used as a sample. There is no particular difference from the conventional immunological measurement method except that a substance that can contain Hb is used.

For example, when employing the immunoagglutination method,
Depending on the purpose, in the case of qualitative analysis, latex agglutination method,
A general procedure such as a microtiter method may be used, and a quantitative analysis may be performed according to a general procedure such as a latex quantitative method. That is, the basic operation is performed by a usual assay method, for example, a hemagglutination reaction method, a passive agglutination reaction method, an immunospecific wax method,
According to immunoturbidimetry, etc., the sensitized particles having the monoclonal antibody of the present invention supported on an insoluble carrier are 0.001 to 15% by weight.
After contacting a reagent containing an active ingredient, which is dispersed in an aqueous medium, with a specimen so as to obtain the above, the degree of aggregation of the sensitized particles may be measured by visual observation, optical measurement, or the like.

In the case of performing a quantitative analysis on the labeled immunoassay, a radioimmunoassay (RIA)
Method), enzyme immunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc., and semi-quantitative analysis is performed according to general procedures such as Western blotting, dot blotting, etc. Or a competition method, a sandwich method, or the like.

For example, a non-competitive method in a labeled immunoassay is exemplified. A solid phase on which an Hb component derived from a sample containing a known amount of Hb is immobilized is reacted with a labeled monoclonal antibody of the present invention, and washed. Thereafter, a method for measuring physical properties derived from the labeling substance bound to the solid phase can be exemplified. In the method, CM is used in the immobilized Hb component.
-Only CM-Hb containing the sequence of peptide β66 or CM-peptide α61, that is, CML-Hb, reacts with the monoclonal antibody of the present invention. Since the amount of the monoclonal antibody of the present invention is large, the amount of CML-Hb in the sample can be measured by measuring the residual activity and the like.

As another example of the competitive method, a known amount of artificially produced labeled CML-Hb (hereinafter referred to as standard CM-HB) is immobilized on a solid phase on which the monoclonal antibody of the present invention is immobilized.
Also called Hb. ) And a sample are competitively reacted, washed, and then the solid phase is measured for physical properties derived from a labeling substance such as enzyme activity and radioactivity.
In this method, the lower the value of these measured physical property values,
This means that the binding of the above standard CML-Hb was competitively inhibited by CML-Hb in the sample, and that the sample contained a large amount of CML-Hb.

As another example of the sandwich method, an anti-CML-Hb antibody such as the monoclonal antibody of the present invention (anti-CM
Also referred to as L-Hb antibody 1. ) Was contacted with a solid phase having a known amount of immobilized thereto, and further labeled anti-CML-
A method for measuring physical properties derived from a labeling substance by contacting with an Hb antibody (also referred to as anti-CML-Hb antibody 2) can be exemplified. In this method, since the amount of the anti-CML-Hb antibody 2 corresponding to the amount of CML-Hb in the sample is immobilized, the amount of CML-Hb in the sample can be known by measuring the above physical property values. . In this method, one of the anti-CML-Hb antibodies 1 and 2 may be the monoclonal antibody of the present invention, but it is preferable that both are the monoclonal antibodies of the present invention from the viewpoint of measurement accuracy and measurement sensitivity. It is.

[0052]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited to these examples.

Example 1 [The dissociation constant for CM-peptide β66 is 10 ⁻⁸ M or less and the dissociation rate constant is 10 ⁻² s
The monoclonal antibody] (1) Preparation of antigen] following the method of the invention in which ^-1 was generated and purification of CML-Hb. Hb (SIGMA) was added to a borate buffer (0.1 M, pH
9.0), and 200 ml of this solution was added at room temperature for 2 hours to a 1M glyoxylic acid solution 1
After stirring with 80 ml, 10 mg was further added at room temperature for 2 hours.
CML-Hb was produced by stirring with 20 ml of a 20 ml / ml sodium cyanoborohydride solution. Furthermore, after dialysis using a dialysis membrane for 20 hours in a phosphate buffer (3.3 mM, pH 7.4), ultrafiltration was performed to obtain purified CML-Hb.

(2) [Preparation of CM peptide] Peptide β66 and peptide α61 were synthesized by a solid phase method using a peptide synthesizer. The amino group of the amino acid was protected with a t-butoxycarbonyl group (Boc), and as a C-terminal protecting group, pralidoxime resin (Pam resin), which is an insoluble polymer carrier, was used. An appropriate protecting group was introduced into the side chain functional group of the amino acid according to each functional group. It corresponds to the 66th Hbβ chain (fifth from the N-terminus of the amino acid sequence 1 of the present invention) to be subsequently subjected to CM or the 61st Hbα chain (the fifth from the N-terminus of the amino acid sequence 2 of the present invention). To protect the ε-amino group of lysine, a 9-fluorenylmethocarbonyl group (Fmoc) is
The 2-chlorobenzyloxycarbonyl group (CIZ) was used for protection of the lysine ε-amino group that had not been subjected to lysine. 0
After removing the 9-fluorenyl methoxycarbonyl group (Fmoc) by treating with a 20% piperidine / dichloromethane solution at 1.5 ° C. for 1.5 hours, the solid phase resin was treated with hydrogen fluoride at 0 ° C. for 1.5 hours. The peptide was further cleaved off and the remaining protecting groups were removed, and then purified by high performance liquid chromatography (HPLC) to obtain peptide β66 and peptide α61.

The CM-peptide β66 is a peptide β66
After performing an elongation reaction of the peptide using an amino acid into which an appropriate protecting group has been introduced in the same manner as described above, the reaction is carried out at 0 ° C. for 1.5 hours.
9% piperidine / dichloromethane solution
-The fluorenylmethocarbonyl group (Fmoc) was removed.
Then, after reacting with glyoxylic acid at 0 ° C. for 2 hours in dichloromethane, and further reacting with sodium cyanoborohydride for 2 hours, only the site corresponding to the ε-amino group of the lysine residue at position 66 of the Hbβ chain of the peptide is obtained. Was commercialized. By treating with hydrogen fluoride at 0 ° C. for 1.5 hours, the peptide was separated from the solid phase resin, and the protecting group was removed.
Purification by high performance liquid chromatography (HPLC) gave CM-peptide β66. CM-peptide β6
The purity of 6 was confirmed by mass spectrometry and amino acid analysis.

For CM-peptide α61, CM-peptide α61
Hbα chain 61 of the peptide in the same manner as peptide β66
Only the site corresponding to the ε-amino group of the lysine residue
The CM-peptide α61 converted into CM was obtained.

A peptide containing an N-terminal valine residue of the β chain of Hb (amino acid sequence: Val-His-Leu-Thr-Pro-Glu-Glu-Cy
s, hereinafter also referred to as “peptide βN”)
It was synthesized by a solid phase method using a peptide synthesizer. The protection of the amino group of the amino acid is performed by the t-butoxycarbonyl group (Bo
Using c), pralidoxime resin (Pam resin), which is an insoluble polymer carrier, was used as a C-terminal protecting group. An appropriate protecting group was introduced into the side chain functional group of the amino acid according to each functional group. In addition, valine corresponding to the N-terminal of the β chain of Hb has an α-amino group as a t-butoxycarbonyl group (Bo
The one not protected in c) was used. After the peptide elongation reaction, the peptide was cleaved from the solid phase resin by removing the remaining protecting groups by treating with hydrogen fluoride at 0 ° C. for 1.5 hours, and then subjected to high performance liquid chromatography (HP
LC) to give peptide βN.

A peptide in which only the N-terminal valine residue of Hb was converted to CM (amino acid sequence: (CM) Val-His-Leu-Thr-Pr
o-Glu-Glu-Cys (hereinafter also referred to as “CM-peptide βN”) undergoes a peptide elongation reaction in the same manner as peptide βN, and then is converted into CM in the same manner as CM-peptide β66 and CM-peptide α61. And the Hb of the peptide
Only the site corresponding to the α-amino group of the N-terminal valine residue
CM-peptide βN converted into CM was obtained.

(3) [Immunization of mice] CML-Hb purified by the above method was adjusted to 0.2 mg / ml in phosphate buffered saline (hereinafter sometimes abbreviated as “PBS”). After dilution, the mixture was mixed with an equal volume of Freund's complete adjuvant, and the first immunization was performed by intraperitoneal administration of 0.2 ml per BALB / c (female, 5 weeks old). Thereafter, a solution obtained by mixing an equal volume of a PBS solution of CML-Hb with incomplete Freund's adjuvant was intraperitoneally administered at 0.2 ml per mouse three times at two-week intervals for booster immunization. Two weeks after the last boost, CML
The final immunization was performed by intravenously administering 0.1 ml of a PBS solution of -Hb.

(4) [Cell fusion] Three days after the final immunization according to the method described above, the spleen was excised from the immunized mouse, and the spleen cells were replaced with 10% fetal bovine serum (FC).
The suspension was suspended in RPMI-1640 medium containing S). on the other hand,
RP containing mouse myeloma cell P3U1 at 10% FCS
The cells were cultured in MI-1640 medium, and cells were collected during the logarithmic growth phase and used for cell fusion. The mouse spleen cells and P3U1 were each washed three times with a serum-free RPMI-1640 medium, mixed at a ratio of 5: 1, and centrifuged at 1,500 rpm for 5 minutes to remove the medium. 0.5 ml of 50% polyethylene glycol 1500 is gradually added to the cell precipitate,
Centrifugation was performed at 1,800 rpm for 8 minutes. Then, after slowly adding 20 ml of serum-free RPMI-1640 medium,
The supernatant was removed by centrifugation at 1,500 rpm for 5 minutes. The precipitated cells were transferred to HAT medium (1 × 10 ⁻⁴ M hypoxanthine, 4 × 10 ⁻⁷ M aminopterin, 1.5 × 10 ⁻⁵ M thymidine, RPMI-1640 medium containing 20% FCS).
The suspension was suspended in 50 ml, and 0.1 ml was dispensed into each well of a 96-well microplate. 5% C
The cells were cultured at 37 ° C. in O ₂ . One day after the cell fusion, 0.1 ml of HAT medium was added to each well. After 7 to 10 days, colonies of hybridomas that grew were observed. The total number of wells in which hybridomas had grown was 235 wells (53%).

(5) [Screening] The supernatant of the well in which the hybridoma has sufficiently grown is collected,
By performing the ELISA method as follows, CM
-Hybridomas producing antibodies to peptide β66 were selected.

The CM-peptide β6 obtained by the above method
6 was diluted with PBS to a concentration of 5 μg / ml, dispensed 50 μl per well into a 96-well EIA microplate, and incubated at 4 ° C. overnight. Remove the CML-Hb solution from the microplate and add 0.05%
After washing three times with PBS containing Tween 80 (hereinafter sometimes abbreviated as “T-PBS”), 1% was added to each well.
PBS containing bovine serum albumin (hereinafter "B-PBS")
Was stored at 4 ° C., and used as an antigen-adsorbing plate in the subsequent operations. The antigen-adsorbing plate was washed three times with T-PBS, and 100 μl of the hybridoma culture supernatant was added to each well, and the mixture was added at 37 ° C.
For 1 hour. Thereafter, the medium supernatant was removed, and the cells were washed three times with T-PBS. Then, 100 μl of the secondary antibody solution was added to each well, followed by incubation at 37 ° C. for 1 hour. Peroxidase-labeled anti-mouse immunoglobulin antibody (Kappel) was used as a secondary antibody.
A 500-fold dilution with BS was used. After removing the secondary antibody solution and washing three times with T-PBS, 100 μl of a chromogenic substrate solution was added to each well and incubated at room temperature for 30 minutes. The chromogenic substrate solution is 0.01% hydrogen peroxide,
0.1 M citrate buffer (pH 5.0) containing 0.3 mg / ml ABTS, [2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium].
0) was used. After confirming an appropriate amount of color development, 1% SDS was added to each well to stop the reaction, and the absorbance at a wavelength of 410 nm was measured.

(6) [Cloning of Hybridoma] The CM-peptide β
Hybridomas that strongly reacted with 66 were selected and cloned by the limiting dilution method. 20 hybridomas
0.5 / RPM-1640 medium containing% FCS
The solution was diluted to a concentration of 0.1 ml and dispensed 0.1 ml to each well of a 96-well microplate. The cells were cultured at 37 ° C., 5% CO ₂ . An ELISA method was performed on the culture supernatant of the well in which the hybridoma had grown in a single colony in the same manner as described above,
Hybridomas producing antibodies to CM-peptide β66 were selected. Among them, CM-peptide β66
9H6 was obtained as a hybridoma stably producing a monoclonal antibody that reacts most strongly with. The obtained hybridoma 9H6 is referred to as "Hybridoma Mb66-9H".
No. 6 "was deposited with the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology.
17900)}.

(7) [Immunoglobulin Class of Monoclonal Antibody] The immunoglobulin class of the antibody in the hybridoma culture supernatant was examined using a monoclonal antibody typing kit (American Corex) by ELISA. This kit uses a rabbit IgG antibody specific to each class and subclass of mouse immunoglobulin to examine the immunoglobulin class by a method according to the above-described ELISA method. The immunoglobulin class of the monoclonal antibody produced by hybridoma 9H6 is IgG
Met.

(8) [Preparation of monoclonal antibody] Hybridoma 9H6 strain, RPMI containing 10% FCS
Cultured in -1640 medium. An equivalent amount of saturated ammonium sulfate was added to the culture supernatant of the hybridoma, and the precipitate was collected by centrifugation. This precipitate was dissolved in a small amount of 10 mM Tris-HCl buffer (pH 8.5) and dialyzed against the same buffer. After dialysis, centrifugation was performed to remove insolubles, which was then subjected to DEAE-
Loaded on a cellulose column. After washing with a buffer solution, elution was performed with a salt concentration gradient, and an IgG fraction was collected. This fraction was subjected to a gel filtration HPLC column (Bio-Rad, Bio-Sil TSK).
250) and chromatographed to obtain a purified monoclonal antibody.

(9) [Properties of Monoclonal Antibody] Various peptides (peptide β) prepared by the above method were placed on a sensor chip of BIA-core (Amersham Pharmacia).
66, peptide α61, peptide βN), and
Various CM-modified peptides (CM-peptide β66, CM-peptide α61, and CM-peptide β
N) was immobilized by a thiol coupling method utilizing the C-terminal cysteine residue. The reagents and procedures used for thiol coupling followed the kit for BIA-core and the usual operating procedures. The amount of the peptide immobilized on the sensor chip surface was 5 to 6 ng / mm ² . Hybridoma 9H
6 was dissolved in a HEPES buffer (pH 7.4, 0.15 M NaCl) to a concentration of 50 μg / ml, and 10 μl / min. At a flow rate of 30 μl, the antibody solution was injected into the sensor chip, and the dissociation rate constant and dissociation constant between each of the peptides immobilized on the sensor chip and the monoclonal antibody were examined. Measurement and analysis followed the usual operation procedure in BIA-core. Table of results
Shown in 1.

[0067]

[Table 1]

As shown in Table 1, hybridoma 9H
6 produced CM-peptide β
66, and its dissociation constant K is 5.46 × 1
0 ^-9 M and the dissociation rate constant k _- were 2.37 × 10 ^-3 s ^-1 . Other CMs (CM-peptide α61, CM-peptide βN) and unCMed peptides (peptide β
66, peptide α61 and peptide βN) (dissociation constant: 1 × 10 ⁻³ M or more; all, dissociation rate constant: 1 × 10 ⁻² s ⁻¹ or more).

Example 2 [CM-in a sample using the latex agglutination reagent containing the monoclonal antibody obtained in Example 1]
Measurement of Hb] (1) Preparation of Monoclonal Antibody-Supported Latex Suspension (A) Prepared in Example 1 Average particle size 0.32 μm, latex concentration 1% (w / v)
The polystyrene particles (manufactured by Fujikura Kasei Co., Ltd.) were suspended in a 20 mM phosphate buffer solution having a pH of 7.2. 1 ml of this particle suspension was mixed with 1 ml of the monoclonal antibody (9H6) solution obtained in Example 1 diluted to 0.3 mg / ml with 20 mM phosphate buffer at pH 7.2, and mixed at 37 ° C. Shake for 1 hour. Then, 0.3% (w
/ V) bovine serum albumin (Oriental Yeast Co., Ltd.)
2 ml of 20 mM phosphate buffer at pH 7.2 containing fraction V) was added, and the mixture was further shaken at 37 ° C. for 2 hours. Next, the precipitate obtained by centrifugation was mixed with 100 mM sodium chloride and 0.1% sodium azide at pH 7.5.
100 mM Tris buffer (hereinafter, this mixed solution is
Abbreviated as S. 4) 4 ml was added and suspended to prepare an instep preparation.

(2) Preparation of Sample (Hemolyzed Sample) Blood was collected from a diabetic patient, and 20 μL of whole blood containing CML-Hb was collected.
l, hemolysis was performed by adding 980 μl of purified water containing 2% sodium dodecyl sulfate (SDS), and then 1 ml of purified water was added.
A 1: 5000 dilution was used to prepare a 1: 5000 hemolysis sample.
This hemolyzed sample was used as a sample for the following measurements.

(3) Preparation of Sample Charge Dilution Solution (Otsurugi) 1.7% (w / v) polyethylene glycol, 100 m
M sodium chloride, 6% choline chloride, 0.1% (w /
v) 0.1 M Tris buffer containing sodium azide (pH
7.5) was prepared to prepare a second preparation.

(4) Measurement of CM-Hb in Sample A sample (hemolysis sample) (6 μl) was mixed with 220 μl of a second agent in a glass cell and stirred to further reduce the hemolysis sample to 3 /
Diluted to 113. After leaving still at 37 ° C. for 5 minutes, 100 μl of the former was added and stirred, and the change in optical density at a wavelength of 804 nm from 10 seconds to 200 seconds (sensitivity, ΔOD 804)
nm). In the above operation, the sample was set as a sample, R1 was used as an agent, and R2 was used as an instep, and the automatic analyzer TBA-30 was used.
R (manufactured by Toshiba Medical Co., Ltd.) and measured. For the measurement of the sample, simultaneous reproducibility was performed by performing simultaneous measurement at n = 10,
Further, daily reproducibility was measured at n = 5. Table 2 shows the results.
It was shown to. Note that CM-Hb measured in this example is
CML-Hb.

[0073]

[Table 2]

As shown in Table 2, hybridoma 9
In the measurement using the monoclonal antibody produced by H6,
Both simultaneous reproducibility and daily reproducibility are C.I. V. The value was 5% or less, showing very good results. The sensitivity (ΔOD 804 nm) was also good.

Comparative Example 1 [The dissociation constant for CM-peptide β66 was 10 ⁻⁸ M or less and the dissociation rate constant k ₋ was 10 ^−2.
Measurement of CM-Hb in Sample Using Latex Aggregation Reagent Containing Monoclonal Antibody Exceeding s ^-1 ] Hybridoma 11B9 showing reactivity to CM-peptide β66 was obtained in the same manner as in Example 1, and a monoclonal antibody was obtained from the hybridoma. I got In addition, the hybridoma 1
1B9 is the same as hybridoma 11B9 disclosed in JP-A-11-236399.

The monoclonal antibody obtained was examined for its immunoglobulin class in the same manner as in Example 1 (7). The immunoglobulin class was IgG. Further, in the same manner as in Example 1 (9), BIA-co
When examined using re, the dissociation constant and dissociation rate constant were as shown in Table 3.

[0077]

[Table 3]

As shown in Table 3, the monoclonal antibody used in this comparative example specifically reacted with CM-peptide β66, and the dissociation constants were 8.06 × 10 ⁻⁹ M and 10 ⁻⁸ M or less. However, the dissociation rate constant k _- is 3.28 × 10 ^-2 s ^-1
And ^exceeded 10 ^-2 s ^-1 .

Next, CM-Hb in the sample was measured in the same manner as in Example 2 except that the above monoclonal antibody was used. Table 4 shows the sensitivity, simultaneous reproducibility, and daily reproducibility at that time.

[0080]

[Table 4]

As shown in Table 4, in the measurement using the above monoclonal antibody, the sensitivity was good, but both the simultaneous reproducibility and the daily reproducibility were C.I. V. The value was as bad as 10% or more, and CM-Hb in the sample could not be measured with good reproducibility.

Comparative Example 2 [The dissociation constant for CM-peptide β66 exceeded 10 ⁻⁸ M and the dissociation rate constant k ₋
^-2 s ^-1 Measurement of CM-Hb in a sample using a latex agglutination reagent containing a monoclonal antibody of ¹ or less) Except for selecting a hybridoma that reacts weakly to CM-peptide β66 in the cloning of the hybridoma of Example 1, Hybridoma 11G2 in the same manner as in Example 1.
To obtain a monoclonal antibody produced by the hybridoma. The immunoglobulin class of the antibody is IgG
Met.

Further, in the same manner as in Example 1 (9), various C
Interaction with M-peptide and peptide before CM-formation
When examined using BIA-core, the dissociation constant and dissociation rate constant were as shown in Table 5.

[0084]

[Table 5]

As shown in Table 5, the monoclonal antibody specifically reacted with CM-peptide β66.
Dissociation constant K was over 5.72 × 10 ^-8 M and 10 ^-8 M. The dissociation rate constant k _- was 7.24 × 10 ^-3 s ^-1 , which was 10 ^-2 s ^-1 or less.

Next, in the same manner as in Example 2 except that the monoclonal antibody was used, CM-Hb in the sample was measured, and the sensitivity, simultaneous reproducibility, and daily reproducibility were determined. Table 6 shows the results.

[0087]

[Table 6]

As shown in Table 6, in the measurement using the above monoclonal antibody, the simultaneous reproducibility and the daily reproducibility were both C.I. V. The value was as poor as 10% or more, and CM-Hb could not be measured with good reproducibility. Also, the sensitivity was low.

Comparative Example 3 [The dissociation constant for CM-peptide β66 exceeded 10 ⁻⁸ M and the dissociation rate constant k ₋
^-2 s ^-1 measured for CM-Hb in a sample using a latex agglutination reagent comprising a monoclonal antibody in excess of was repeated except for selecting the weakly reactive hybridoma in Example 1 Cloning of hybridomas CM- peptide β66, like Thus, hybridoma 16B4 was obtained, and a monoclonal antibody produced by the hybridoma was obtained. The immunoglobulin class of the antibody was IgG.

Next, the interaction with various CM peptides and the peptide before CM conversion was examined using BIA-core in the same manner as in Example 1 (9). The dissociation constant and dissociation rate constant were as shown in Table 7. It became the value shown.

[0091]

[Table 7]

As shown in Table 7, the monoclonal antibody specifically reacted with CM-peptide β66.
The dissociation constant K exceeds 7.19 × 10 ⁻⁷ M and 10 ⁻⁸ M,
The dissociation rate constant k _- is also 8.35 × 10 ^-2 s ^-1 and 10
^-2 s ^-1 was exceeded.

Next, CM-Hb in the sample was measured in the same manner as in Example 2 except that the monoclonal antibody was used, and the sensitivity, simultaneous reproducibility, and daily reproducibility were determined. Table 8 shows the results.

[0094]

[Table 8]

As shown in Table 8, in the measurement using the above monoclonal antibody, both the simultaneous reproducibility and the daily reproducibility were C.I. V. The value was as poor as 10% or more, and CM-Hb could not be measured with good reproducibility. Further, the sensitivity was low.

Example 3 [Dissociation constant for CM-peptide α61 is 10 ⁻⁸ M or less and dissociation rate constant is 10 ⁻² s
Monoclonal antibody of the present invention of ^-1 or less] Using the CM-peptide α61 prepared in Example 1 (2), immunization of mice, cell fusion, selection of fused cells, and specificity in the same manner as in Example 1. Selection of antibody-producing cells, cloning, and the like were performed to obtain 7B8 as a hybridoma stably producing a monoclonal antibody that reacts most strongly with CM-peptide α61, and a monoclonal antibody produced by the hybridoma was obtained. In addition, hybridoma 7B8
Was deposited as "Hybridoma Mb68-7B8" with the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology {Deposit No .: No. 774 (FERM p-17901)}.
The immunoglobulin class of the above antibody was IgG.

When the interaction of the above monoclonal antibody with various CM-peptides and the peptide before CM-formation was examined using BIA-core in the same manner as in Example 1 (9), the dissociation constant and the dissociation rate constant were found to be: The values shown in Table 9 were obtained.

[0098]

[Table 9]

As shown in Table 9, hybridoma 7B
8 produced CM-peptide α
61 and a dissociation constant K of 1.26 × 1
At 0 ^-9 M, the dissociation rate constant k _- was 1.34 × 10 ^-3 s ^-1 .

Example 4 [CM-in a sample using the latex agglutination reagent containing the monoclonal antibody obtained in Example 3]
Measurement of Hb] CM-Hb in a sample was measured in the same manner as in Example 2 except that the monoclonal antibody obtained in Example 3 was used, and the sensitivity, simultaneous reproducibility, and daily reproducibility were determined. Table 10 shows the results.

[0101]

[Table 10]

As shown in Table 10, in the measurement using the above monoclonal antibody, the simultaneous reproducibility and the daily reproducibility were both C.I. V. The value is very good at 5% or less, and the sensitivity (Δ
OD 804 nm) was also good.

Comparative Example 4 [Dissociation constant for CM-peptide α61 was 10 ⁻⁸ M or less and dissociation rate constant k ₋ was 10 ^−2.
Measurement of CM-Hb in Sample Using Latex Agglutination Reagent Containing Monoclonal Antibody Exceeding s ^-1 ] Hybridoma 1A5 showing reactivity to CM-peptide α61 is obtained in the same manner as in Example 3, and the hybridoma is produced. Monoclonal antibody (Immunoglobulin class: I
gG) was obtained.

Next, when the interaction with various CM-peptides and the peptide before CM-formation were examined using BIA-core in the same manner as in Example 1 (9), the dissociation constant and the dissociation rate constant were as shown in Table 11. It became the value shown.

[0105]

[Table 11]

As shown in Table 11, the above monochromator
Antibody specifically reacts with CM-peptide α61,
The separation constant K is 9.21 × 10^-9M, but the dissociation rate constant
Number k _-Is 2.42 × 10^-2s^-1And 10^-2s^-1Beyond
Was.

Next, in the same manner as in Example 2 except that the monoclonal antibody was used, CM-Hb in the sample was measured, and the sensitivity, simultaneous reproducibility, and daily reproducibility were determined. Table 12 shows the results.

[0108]

[Table 12]

As shown in Table 12, in the measurement using the above monoclonal antibody, the sensitivity was good, but both the simultaneous reproducibility and the day-to-day reproducibility were C.I. V. The value was as poor as 10% or more, and CM-Hb could not be measured with good reproducibility.

Comparative Example 5 [The dissociation constant for CM-peptide β66 exceeded 10 ⁻⁸ M and the dissociation rate constant k ₋
-Measurement of CM-Hb in Sample Using Latex Agglutination Reagent Containing Monoclonal Antibodies Exceeding ^-2 s ^-1 ) Thus, a hybridoma 7F9 was obtained, and a monoclonal antibody (immunoglobulin class of IgG) produced by the hybridoma was obtained. The hybridoma 7
F9 is the same as hybridoma 7F9 disclosed in JP-A-11-236399.

Next, when the interaction with various CM-peptides and the peptide before CM-formation were examined using BIA-core in the same manner as in Example 1 (9), the dissociation constant and the dissociation rate constant were as shown in Table 13. It became the value shown.

[0112]

[Table 13]

As shown in Table 13, the monoclonal antibody specifically reacted with the CM-peptide α61, but the dissociation constants K exceeded 3.91 × 10 ⁻⁸ M and 10 ⁻⁸ M, and the dissociation rate constants k _-is also 3.62 × 10 ^-2 s ^-1 and 10 ^-2
s ^-1 was exceeded.

Next, in the same manner as in Example 2 except that the monoclonal antibody was used, CM-Hb in the sample was measured, and the sensitivity, simultaneous reproducibility, and daily reproducibility were determined. Table 14 shows the results.

[0115]

[Table 14]

As shown in Table 14, in the measurement using the above monoclonal antibody, the simultaneous reproducibility and the day-to-day reproducibility were both C.I. V. The value was as poor as 10% or more, and CM-Hb could not be measured with good reproducibility. Further, the sensitivity was not sufficient.

[0117]

EFFECT OF THE INVENTION By using the monoclonal antibody of the present invention, CML-Hb, which was conventionally difficult to measure with high sensitivity and high precision, can be obtained with high sensitivity and high precision by an immunological measurement method with simple operation. It became possible to measure with good reproducibility.

[0118]

[Sequence list]

SEQ ID NO: 1 Sequence length: 12 Sequence type: amino acid Tropoie: Linear Sequence type: Peptide SEQ ID NO: 2 Sequence length: 12 Sequence type: amino acid Tropoie: linear Sequence type: peptide

────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] February 8, 2001 (2001.2.8)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0018

[Correction method] Change

[Correction contents]

Sequence Listing Free Text The amino acid sequence of SEQ ID NO: 1 is the same as that of hemoglobin (H
b) the amino acid sequence at positions 62 to 73 from the N-terminus of the β-chain, and the side chain of a lysine residue at the position corresponding to the 66th position from the N-terminus of the β-chain of Hb in the CM-peptide β66. The amino group has been converted to CM. The amino acid sequence of SEQ ID NO: 2 corresponds to the amino acid sequence at positions 57 to 68 from the N-terminus of the α chain of Hb. In the CM-peptide α61, the amino acid sequence corresponds to the 61st position from the N-terminus of the α chain of Hb. The side chain amino group of the lysine residue at the corresponding position is converted to CM.

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name]

[Correction method] Change

[Correction contents]

[0118]

[Sequence List] <110> TOKUYAMA CORPORATION; A & T CORPORATION <120> Monoclonal Antibody <140> JP / 2000-194075 <141> 2000-06-28 <160> 2 <210> 1 <211> 12 <212> PRT < 213> Artificial Sequence <220> <223> The amino acid sequence which correspond to between 62th and 73th from N-terminal on hemoglobin beta-chain <400> 1 Ala His Gly Lys Lys Val Leu Gly Ala Phe Ser Cys 1 5 10 < 210> 2 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence which correspond to between 57th and 68th from N-terminal on hemoglobin beta-chain <400> 2 Gly His Gly Lys Lys Val Ala Asp Ala Leu Thr Cys 1 5 10

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/53 C12N 15/00 ZNAC 33/577 5/00 B (72) Inventor Koichiro Hirata Tokuyama, Yamaguchi Prefecture No. 1-1 Mikage-cho F-term in Tokuyama Corporation (reference) 4B024 AA11 BA44 GA03 GA18 HA15 4B064 AG27 CA10 CA20 CC24 DA13 4B065 AA91X AA92X AC14 BA08 CA25 CA46 4H045 AA11 AA30 BA16 CA40 DA76 DA86 EA50 FA72 GA06 GA10 GA22

Claims (4)

[Claims] 1. A peptide having the amino acid sequence of SEQ ID NO: 1 and having a carboxymethylated side chain amino group of the fifth lysine residue from the N-terminal, or having the amino acid sequence of SEQ ID NO: 2, Having a dissociation constant of 10 ^-8 M or less and a dissociation rate constant of 10 ^-2 s ^-1 or less for a peptide in which the side chain amino group of the fifth lysine residue is carboxymethylated. antibody. 2. A hybridoma producing the monoclonal antibody according to claim 1. 3. An immunoassay for carboxymethylated hemoglobin, which comprises using the monoclonal antibody according to claim 1. 4. An immunoassay reagent for measuring carboxymethylated hemoglobin, comprising the monoclonal antibody according to claim 1.