JPH11236399A - Monoclonal antibody - Google Patents

Abstract

(57) [Problem] To provide a novel immunological quantification method capable of easily measuring carboxymethylated hemoglobin which is one of AGEs in a body fluid. SOLUTION: For example, using an immunoassay reagent comprising a monoclonal antibody which specifically reacts with carboxymethylated hemoglobin produced by hybridoma 2B3 (Seikagaku no. Measure methylated hemoglobin.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a monoclonal antibody that specifically recognizes carboxymethylated hemoglobin, the antibody-producing cells, an immunoassay method for carboxymethylated hemoglobin using the antibody, and an immunoassay reagent. About.

[0002]

2. Description of the Related Art In recent years, carboxymethylated proteins (hereinafter sometimes referred to as "CM-protein") are a major component of the late product of the Maillard reaction (hereinafter sometimes abbreviated as "AGE"). It is thought (Iked
a, K. , Et. al. Biochemistry, vol. 35, p8075, 1
996), These qualitative or quantitative methods include a method using a specific antibody, and carboxymethylation by gas chromatography / mass spectrometry after hydrolyzing CM-protein (hereinafter sometimes abbreviated as “CM conversion”). And the like) are known for detecting amino acids. However, the method of indirectly measuring CM-protein by gas chromatography / mass spectrometry has a problem that the operation is complicated and the sensitivity is low, so that a method using a specific antibody that is easy to measure and has high sensitivity is used. It is heavily used.

[0003] In the above-mentioned assay method using a specific antibody, CM-protein produced in a test tube (in vitro) is used for enzyme-labeled immunoassay (ELISA), radioimmunoassay (RIA), and Western blotting. The immunohistochemical staining method is available for CM-protein in the tissue. However, CM-protein in body fluids cannot be directly measured by ELISA, RIA, or Western blotting, so that detection is performed by dot blotting or CM-protein derived from body fluids is detected. Pretreat protein with alkali or proteinase K, etc.
A method has been performed in which the protein is denatured or cleaved and then detected by ELISA.

However, when measuring the CM-protein in body fluids by the dot blotting method, not only is the operation complicated, but also it takes time to learn a technique for measuring the CM-protein concentration with good reproducibility. There were drawbacks. In addition, CM-protein derived from body fluids was
When the measurement is carried out by the method, the pretreatment as described above is necessary. However, care must be taken so that the protein is not converted into CM in the pretreatment step, and the operation is complicated and time-consuming. there were.

[0005]

Among AGEs in body fluids, carboxymethylated hemoglobin (hereinafter referred to as "CM-H")
b ". ) Is strongly suggested to be associated with diabetes or diabetic complications, and measuring the CM-Hb is significant from the viewpoint of clinical tests and the like. A simple method for measuring CM-Hb has not been known so far. An object of the present invention is to develop a method for simply measuring CM-Hb in a body fluid.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, the use of a monoclonal antibody that specifically reacts only with CM-Hb makes it possible to treat a subject. It has been found that the CM-Hb concentration in blood can be easily and simply measured, and the present invention has been completed.

That is, the first invention is a monoclonal antibody that specifically reacts with CM-Hb. The second present invention is a hybridoma that produces a monoclonal antibody that specifically reacts with CM-Hb. Further, the third invention is a method for immunologically measuring CM-Hb, which comprises using a monoclonal antibody that specifically reacts with CM-Hb. This is an immunological measurement reagent for measuring CM-Hb, which comprises a specifically reacting monoclonal antibody.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The monoclonal antibody of the present invention
Reacts specifically with CM-Hb. Here, CM-Hb refers to Hb in which at least one hydrogen of the N-terminal amino group or side chain amino group of hemoglobin (hereinafter sometimes abbreviated as “Hb”) is converted to CM. The CM site may be the α chain or β chain of Hb. In addition, the term “specifically reacts with CM-Hb” means that it does not react with CM-protein other than CM-Hb, such as CM-HSA (HSA: human serum albumin), CM-collagen, and γ-globulin. Or, if it does, it reacts very weakly. More specifically, it means that the reaction with CM-Hb has an affinity 100 times or more higher than the reaction with CM-protein other than CM-Hb. In addition, the monoclonal antibody of the present invention is CM-Hb
The type (globulin class) is not particularly limited as long as it specifically reacts with, and includes any currently known globulin class. The monoclonal antibody in the present invention means not only a normal monoclonal antibody but also a partially degraded product (Fab, Fa) of the antibody.
b ', Fab'2, etc.), and the partial structure where the active fragment of the antibody (the antigen recognition site of the antibody) exists.

The method for producing the monoclonal antibody of the present invention is not particularly limited, but CM-Hb is immunized to an animal as an antigen for immunization (hereinafter sometimes referred to as "immunogen"), and then antibody-producing cells are separated from the immunized animal. A method of producing the cells by harvesting and culturing the sorted cells in a suitable medium, or by fusing the sorted antibody-producing cells with plasmacytoma cells to form a hybridoma, and culturing the hybridoma or in an animal body. It can be suitably produced by a method of transplantation, amplification and production.

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.

[0011] The hybridoma of the present invention is CM-Hb.
Although it is not particularly limited as long as it can produce a monoclonal antibody having specific reactivity to, a series of immunization of animals, cell fusion, selection of fused cells, selection of specific antibody-producing cells, and cloning in general A hybridoma prepared through the steps, which produces a monoclonal antibody having specific reactivity to CM-Hb.

First, immunization of animals will be described. CM-Hb, which is an immunogen, can be obtained by substituting hydrogen in the side chain amino group or the N-terminal amino group of the amino acid of Hb with a carboxymethyl group. As the method at this time, a known method can be used without any limitation, for example, “Shinsei Kagaku Koza 1; Protein 4” (edited by the Biochemical Society of Japan, Sections 13-16, Tokyo Chemical Dojin, published March 20, 1991). Can be carried out by the reductive alkylation method described in (1). That is, the 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 8 in the presence of a hydride reducing agent such as sodium borohydride or sodium cyanoborohydride.
It is obtained by reacting under the conditions of 10. If the pH is higher than this, proteins and the like may be denatured, and if the pH is lower than this, 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, and more preferably 0 to 10 ° C. The obtained CM-
The Hb solution is used after dialysis or ultrafiltration to remove unreacted reducing agent or aldehyde compound.

Next, the CM-
Animals are immunized with Hb as immunogen. For immunization, the immunogen to be used is not particularly limited as long as it is a solution containing purified CM-Hb, but it is preferable to use one mixed with Freund's complete adjuvant. The animal to be immunized 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. Immunization is performed several times about every 1 to 2 weeks after the initial immunization, and antibody producing cells are collected 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 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 (Nature, 25
6, 495-497 (1975)) or a method using polyethylene glycol according to the method is preferred because the operation is simple.
The method uses 30 to 50% polyethylene glycol (average molecular weight 1,000 to 4,000) at 30 to 40 ° C.
This is performed by mixing the antibody-producing cells with the myeloma cells for 1 to 10 minutes at a temperature of 1. 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.

[0016] From the hybridomas thus obtained, those that produce a monoclonal antibody that specifically reacts with CM-Hb are selected. This choice is
The culture supernatant of the well in which the hybridoma has been grown by the above culture is collected, and the presence or absence of an antibody against CM-Hb may be examined by, for example, an enzyme immunoassay such as an ELISA method. Subsequently, the hybridoma in the well in which the antibody against CM-Hb 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 a soft agarose, a single cell manipulation method, a FACS method, and the like. However, 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 to 10 ml of medium and stored frozen in liquid nitrogen. In addition, one of the hybridomas of the present invention, hybridoma 2B3
Was deposited with the Institute of Biotechnology and Industrial Technology,
On February 10, 998, "Hybridoma CM-Hb
2B3 "｛Deposit No .: Life Science Bacteria Deposit No. 16632 (FE
RM P-16632)}.

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 a protein G column,
It can be purified by using a conventional method such as an ion exchange column chromatography method, a gel filtration column chromatography method, and an electrophoresis method. Among these, affinity chromatography is preferable, and a method using a protein A column is particularly preferable.

It is preferable to confirm the specificity of the monoclonal antibody thus obtained for CM-Hb. The confirmation shows that the antibody reacts with CM-Hb, but Hb in which the N-terminus and the side chain amino group are not converted into CM is used.
(Hereinafter sometimes abbreviated as "non-CM-Hb") or C
Not reacting with CM-protein other than M-Hb.
It can be performed by examining by LISA method.
As a method for producing non-CM-Hb, an operation of flowing Hb (including CM-Hb) obtained from a sample whole blood into a column packed with a carrier carrying a polyclonal antibody recognizing CM-Hb is repeated. Non-CM-
A method for obtaining Hb and the like can be mentioned. CM other than CM-Hb
-As the protein, CM-HSA or the like created by the same method as the above-described method for creating CM-Hb can be used.

The monoclonal antibody of the present invention is CM-Hb
By using the monoclonal antibody, CM-Hb in a sample such as a body fluid can be measured by utilizing the antigen-antibody reaction.

For example, CM-Hb in a specimen can be detected by utilizing an agglutination reaction of an insoluble carrier based on the antigen-antibody reaction (immunogglutination method). In addition, radioactive substances, various dyes, colloids, enzymes and the like are bound to the monoclonal antibody or CM-Hb of the present invention to carry out an antigen-antibody reaction, and then radioactivity, enzyme activity, etc. derived from the bound substances are performed. Is measured, CM-Hb in the sample is measured.
Can be detected (labeled immunoassay). At this time, the radioactive substance that can be used includes radioactive iodine, radioactive carbon and the like. It is preferable to use fluorescent dyes such as fluorescein isothiocyanate and tetramethylrhodamine as the dye, but it is not limited thereto. Gold colloid or the like can be used as the colloid. Peroxidase, alkaline phosphatase and the like can be used as the enzyme. Furthermore, together with the monoclonal antibody of the present invention, a secondary antibody labeled with the above-mentioned dye, colloid, enzyme or the like can be used for detection of CM-Hb. In addition, the monoclonal antibody of the present invention can be used for detection of CM-Hb in combination with a monoclonal antibody or a polyclonal antibody against another CM-Hb, if necessary.

Specific examples of the immunoagglutination method include a latex agglutination method and a microtiter method as qualitative methods, and a latex quantitative method and the like as a quantitative measurement method.

Further, the specific examples of the labeled immunoassay are radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, chemiluminescence immunoassay and the like, and quantitative assays are semi-quantitative assays. Western blotting, dot blotting and the like can be exemplified.

The operation and procedure in each of these measurement methods are not particularly different from those generally employed, and can be in accordance with known non-competitive methods, competitive methods, sandwich methods and the like.

For example, a non-competitive method in a labeled immunoassay method is exemplified by reacting a monoclonal antibody of the present invention labeled with an enzyme or a radioisotope with a solid phase on which a known amount of Hb derived from a sample is immobilized. After washing, the enzyme activity or radioactivity bound to the solid phase is measured. The larger the residual activity after the reaction and the washing, the larger the amount of the monoclonal antibody of the present invention bound to the solid phase. Alternatively, the monoclonal antibody of the present invention may be immobilized on a solid phase and reacted with a known amount of Hb derived from a sample labeled with an enzyme, a radioisotope, or the like.

As another example of the competitive method, an artificially prepared standard CM-Hb labeled with an enzyme, a radioisotope, or the like and a sample are competed against a solid phase on which the monoclonal antibody of the present invention is immobilized. After the reaction and washing, the enzyme activity or radioactivity bound to the solid phase is measured. The less enzyme or radioactivity remaining on the solid phase, the more competitively the binding of standard CM-Hb was inhibited by CM-Hb in the sample.

Further exemplifying the sandwich method, CM-
A sample is brought into contact with a solid phase on which a substance that binds to Hb is immobilized, and the amount of CM-Hb in the sample bound to the solid phase is measured by a substance that binds to CM-Hb labeled with an enzyme or the like. This is done by: Here, either one of the substance that binds to CM-Hb previously adsorbed to the solid phase and the substance that binds to CM-Hb labeled with an enzyme or the like, preferably both of them specifically bind to CM-Hb. It must be a substance that binds. The monoclonal antibody of the present invention may be used for at least one of the substances that bind to CM-Hb. Other substances that bind to CM-Hb include polyclonal antibodies against CM-Hb,
A substance to which Hb can bind or adsorb, such as an anti-Hb antibody, can be used.

The form of the immunizing reagent of the present invention is not particularly limited as long as it can detect CM-Hb by utilizing a specific antigen-antibody reaction between the monoclonal antibody of the present invention and CM-Hb. For example, a form in which the monoclonal antibody and / or CM-Hb of the present invention is appropriately carried on an insoluble carrier so as to correspond to each of the above-described non-competitive method, competitive method, and sandwich method can be used. Using each of such immunological reagents according to the measurement method, CM-H
CM-Hb in a sample can be measured by detecting an antigen-antibody reaction caused by bringing b into contact with the monoclonal antibody of the present invention. Such an antigen-antibody reaction can be detected by using agglutination of an insoluble carrier or the like when the immunoreagent of the present invention is a so-called immunoagglutination reaction. When the reagent is a reagent, it can be detected as a change in a physical quantity such as colorimetry, luminescence, or fluorescence.

As specific examples of the immunoreagent of the present invention, latex agglutination reagents, microtiter reagents and the like are used as qualitative reagents, and radioimmunoassay reagents, enzyme immunoassay reagents, fluorescent immunoassay reagents and chemiluminescence are used as quantitative reagents. Examples thereof include an immunoassay reagent and a latex quantitative reagent.

The type and shape of the monoclonal antibody of the present invention or the insoluble carrier carrying the CM-Hb antigen in the sample may be appropriately selected according to the purpose of use. Examples of the material include those used as ordinary immunoassay carriers, such as glass, polysaccharides or derivatives thereof, silica gel, porous ceramics, metal oxides, red blood cells, or the synthesis of polypropylene, polystyrene, polyacrylamide, polyacrylonitrile, and the like. Resins and those obtained by introducing a reactive functional group such as a sulfone group or an amino group into the resin by a known method may be used. Examples of the shape include beads, a test plate, a sphere, a disk, a tube, and a filter. Can be illustrated.

The method for immobilizing the monoclonal antibody of the present invention or CM-Hb in a sample on an insoluble carrier may be any known method such as a physical adsorption method, a covalent bonding method, an ionic bonding method and a cross-linking method. it can. For example, when a microplate is used as the solid phase, adsorption can be performed by injecting a solution of the monoclonal antibody into the wells of the microplate and reacting for 1 to 48 hours.

When the immunoreagent of the present invention is a labeled immunoassay reagent, the basic procedure for measuring CM-Hb using the reagent (also referred to as the labeled immunoassay reagent of the present invention) is a conventional assay, for example, radio Immunoassay (RIA) method, EL
Enzyme-linked immunosorbent assay (EIA) methods such as ISA, Western blotting, and dot blotting can be used. The operations, procedures, and the like in each of these assay methods are not particularly different from those generally employed, and can be in accordance with known non-competitive methods, competitive methods, sandwich methods, and the like.
In the non-competitive method, for example, a reagent in which the monoclonal antibody of the present invention is supported on an insoluble carrier is added to the CM-Hb
(Or vice versa). In the competitive method, for example, a specific amount of an artificially prepared standard CM-Hb is supported on an insoluble carrier, while a predetermined amount of CM
After reacting -Hb with a specific amount of the monoclonal antibody of the present invention, the insoluble carrier may be contacted with the monoclonal antibody of the present invention. In the sandwich method, for example, CM-Hb in a sample is brought into contact with a reagent in which a specific amount of the monoclonal antibody of the present invention is carried on an insoluble carrier,
What is necessary is just to contact the antibody with respect to M-Hb. By these measuring methods, the CM conversion rate of the sample Hb or the amount of the CM compound in the sample Hb can be measured. Here, CM
The degree of conversion is the average degree of C
This is an index indicating whether or not it has been converted to M.

As the labeling agent in the labeled immunoassay of the present invention, radioactive substances such as radioactive iodine and radiocarbon, fluorescent substances such as fluorescein isothiocyanate and tetramethylrhodamine, enzymes such as alkaline phosphatase and peroxidase and the like can be used. Each can be exemplified.
The antigen-antibody reaction product obtained by such a method is detected using radioactivity, colorimetry, fluorescence, luminescence, and the like.

The label of standard CM-Hb is CM-H
A known method such as recognizing the Hb site of b with a biotinylated anti-Hb antibody and then labeling the enzyme with avidin is used. As a substance to be labeled, a luminescent substance, a fluorescent substance, and the like can be used in addition to the enzyme and the radioisotope. Enzymes are alkaline phosphatase, peroxidase, β-
D-galactosidase and the like are used. Enzyme activity is measured, for example, when alkaline phosphatase is used.
A known method such as reaction with a substrate such as -nitrophenyl phosphate and measurement of the amount of a reaction product generated by the enzymatic reaction by measuring absorbance is employed.

In the labeled immunoassay according to the present invention,
The amount of CM-Hb in the monoclonal antibody or the sample of the present invention carried on the insoluble carrier is generally 0.01 to 1000 μg / cm ² per surface area of the insoluble carrier. Is set to 0.
What is necessary is just to contact CM-Hb in the sample of 001 to 1000 μg or the monoclonal antibody of the present invention. In addition,
When an antibody against Hb is carried on an insoluble carrier, the monoclonal antibody of the present invention may be further contacted after contacting CM-Hb in the sample as described above. At this time, it is preferable to use a monoclonal antibody of the present invention which is not carried on an insoluble carrier and which is labeled with a labeling agent.

When the immunoreagent of the present invention is an immunoagglutination reagent, the reagent (also referred to as the immunoagglutination reagent of the present invention).
The basic operation of CM-Hb measurement in a sample using the method described above can be performed according to a usual assay method, for example, a hemagglutination method, a passive agglutination method, an immunoassay method, an immunoturbidimetry method, or the like. The operations, procedures, and the like in each of these assay methods can be based on those generally employed. The solid phase for immobilizing the monoclonal antibody is not particularly limited,
Beads or microplates made of polystyrene or the like are used. For example, for 1 g of the particulate insoluble carrier, the monoclonal antibody of the present invention is subjected to the above method in an amount of 0.001 to 0.001.
The particles carrying 100 mg (hereinafter sometimes abbreviated as “sensitizing particles”) may be dispersed in an aqueous medium so as to be 0.001 to 15% by weight and used as an active ingredient of the immunoreagent. The particle size of the insoluble carrier that carries the antibody, from the viewpoint of the ease of occurrence of aggregation after the antigen-antibody reaction and the ease of discrimination of aggregation, it is preferable to use an insoluble carrier having an average particle size of 0.05 to 10 μm. It is suitable. The sensitized particles prepared by such a method may be brought into contact with the CM-Hb antigen in the sample, and the degree of aggregation of the sensitized particles may be measured. Conventional methods such as visual observation and optical measurement can be used without limitation for the degree of aggregation of particles.

As will be shown in the examples described later, CM-total Hb derived from patients with diabetes or diabetic complications.
The proportion of Hb is significantly higher than that of healthy subjects,
There is a positive correlation between the severity of diabetic complications and the percentage of CM-Hb in the sample Hb. The proportion of CM-Hb in Hb derived from dialysis patients is also significantly higher than that in healthy subjects. Further, in Hb derived from patients who developed dialysis amyloidosis, the proportion of CM-Hb occupied by dialysis amyloidosis Is significantly higher than Hb from a dialysis patient who has not developed. Therefore, CM-H
b can be a marker for diabetes or diabetic complications in the field of clinical testing, or a marker for amyloidosis in dialysis patients. That is, C of Hb in blood
By measuring the rate of M conversion, it is possible to determine whether or not you have diabetes or diabetic complications, to predict the progress of diabetes, or to predict the onset or progress of diabetes complications This makes it possible to predict the occurrence of dialysis amyloidosis.

[0038] Therefore, the immunoassay reagent of the present invention comprises:
It is usefully used as a diagnostic reagent for diabetes, a diagnostic reagent for diabetic complications, or a diagnostic reagent for dialysis amyloidosis, and a drug efficacy evaluation reagent for a drug for treating or preventing diabetes, diabetic complications, or dialysis amyloidosis. That is, when the CM-Hb amount of antigen in the blood, which is a multi as subject to clinical testing using immunological measurement reagent of the present invention was measured serves as a diagnostic reagent, also diabetes or for diabetic complications It functions as a drug efficacy evaluation reagent when measuring the amount of CM-Hb contained in blood or the degree of decrease in the ratio due to administration of a therapeutic agent.

[0039]

EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

Example 1 (1) [Preparation of antigen] CM-Hb was produced and purified by the following methods.
Hb (SIGMA) was added to a borate buffer (0.1 M pH
9.0) to 1 mg / ml, 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.
CM-Hb was produced by stirring with 20 ml of a 1 / 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 CM-Hb.

(2) [Immunization of mice] CM-Hb purified by the above method was placed in phosphate buffered saline (hereinafter sometimes abbreviated as “PBS”) for 0.2 m.
g / ml, mixed with an equal volume of Freund's complete adjuvant, and mixed with BALB / c mice (female, 5 weeks old).
The animals were primed by intraperitoneal administration of 0.2 ml per animal. Thereafter, CM-Hb PBS three times at two-week intervals
A solution obtained by mixing an equal amount of the solution with Freund's incomplete adjuvant was intraperitoneally administered to each mouse in an amount of 0.2 ml per mouse to perform booster immunization. Two weeks after the last booster immunization, a final immunization was performed by intravenously administering 0.1 ml of a PBS solution of CM-Hb.

(3) [Cell fusion] Three days after the final immunization according to the above method, 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,
Mouse myeloma cells P3U1 were transformed with R containing 10% FCS.
The cells were cultured in PMI-1640 medium, and cells were collected during the exponential 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 mixed at 1,500 rpm for 5 minutes.
The medium was removed by centrifugation for minutes. 0.5 ml of 50% polyethylene glycol 1500 is gradually added to the cell precipitate,
The mixture was centrifuged at 1,800 rpm for 8 minutes. Then, after gradually 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 a HAT medium (RPMI-1640 medium containing 1 × 10 ⁻⁴ M hypoxanthine, 4 × 10 ⁻⁷ M aminopterin, 1.5 × 10 ⁻⁵ M thymidine, 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 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%).

(4) [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 against -Hb were selected.

The CM-Hb obtained by the above method was used in PBS.
And diluted to a concentration of 1 μg / ml, dispensed into a 96-well EIA microplate 100 μl per well, and incubated at 4 ° C. overnight. Remove the CM-Hb solution from the microplate and add 0.05% Tween
After washing three times with PBS containing n80 (hereinafter sometimes abbreviated as “T-PBS”), 250 μl of PBS containing 1% bovine serum albumin (hereinafter sometimes abbreviated as “B-PBS”) is added to each well. In addition, it was stored at 4 ° C. and used as an antigen-adsorbing plate for subsequent operations. Set the antigen adsorption plate to TP
After washing three times with BS, 100 μl of the hybridoma culture supernatant was added to each well and incubated at 37 ° C. for 1 hour. Thereafter, the medium supernatant was removed, and T-PBS was removed.
After washing three times with, the secondary antibody solution was added to each well at 100 μl.
The mixture was added at a rate of 1 l and incubated at 37 ° C for 1 hour. As a secondary antibody, peroxidase-labeled anti-mouse immunoglobulin antibody (Kappel) was diluted 500-fold with B-PBS and used. Remove the secondary antibody solution and add 3
After washing twice, 100 μl of a chromogenic substrate solution was added to each well and incubated at room temperature for 30 minutes. The coloring substrate solution was 0.01% hydrogen peroxide, 0.3 mg / ml A
A 0.1 M citrate buffer (pH 5.0) containing BTS and [2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium] 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.

(5) [Cloning of hybridomas] Hybridomas which strongly react with CM-Hb were selected by screening according to the above method, and cloned by limiting dilution. The hybridoma was diluted with RPMI-1640 medium containing 20% FCS to a concentration of 0.5 cells / 0.1 ml, and 0.1 ml was dispensed into each well of a 96-well microplate. 5% of these cells
The cells were cultured at 37 ° C. in CO ₂ . ELISA 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, and a hybridoma producing an antibody against CM-Hb was selected.
Among them, 2B3 was obtained as a hybridoma stably producing a monoclonal antibody that reacts most strongly with CM-Hb. The obtained hybridoma 2B3 was deposited as "Hybridoma CM-Hb 2B3" with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology (Deposit No.
No. 6632 (FERM P-16632)).

(6) [Immunoglobulin Class of Monoclonal Antibody] The immunoglobulin class of the antibody in the hybridoma culture supernatant was examined using a monoclonal antibody typing kit by ELISA (American Corex). 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 2B3 is IgG
Met.

(7) [Preparation of Monoclonal Antibody] Hybridoma 2B3 strain was transformed with an RPM containing 10% FCS.
The cells were cultured in the 1-1640 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, centrifuged to remove insolubles,
Loaded on an E-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-Sil, Bio-Sil).
TSK250) and chromatography to obtain a purified monoclonal antibody.

(8) [Preparation of CM-Peptide (CM-Peptide)] Peptide containing N-terminal valine residue of β-chain of Hb (amino acid sequence: Val-His-Leu-Thr-Pro-G)
lu-Glu-Cys, hereinafter sometimes referred to as “peptide βN”) or a peptide containing the lysine residue at position 66 of the β chain of Hb (amino acid sequence: Ala-His-
Gly-Lys-Lys-Val-Leu-Gly-A
la-Phe-Ser-Cys
6 ”), or α chain 61 of Hb.
Peptide containing the lysine residue (amino acid sequence: Gl
y-His-Gly-Lys-Lys-Val-Ala
-Asp-Ala-Leu-Thr-Cys (hereinafter sometimes referred to as “peptide α61”) and the like were synthesized by a solid phase method using a peptide synthesizer. For protection of the amino group of the amino acid, a t-butoxycarbonyl group (Boc) was used, and pralidoxime resin (Pam resin), which is an insoluble polymer carrier, was used as a C-terminal protecting group.

After protecting the amino group of other lysine residues by an appropriate method, the peptide is reacted with glyoxylic acid in dichloromethane at 0 ° C. for 2 hours, and further reacted with sodium cyanoborohydride for 2 hours. A valine residue at the N-terminus of the β-chain, a lysine residue at the 66th position of the β-chain, or an α-chain 61
Only the site corresponding to the amino group of the lysine residue is CM
It has become. The peptide was cleaved from the solid phase resin and treated with hydrogen fluoride at 0 ° C. for 1.5 hours to remove the protecting group, and purified by high performance liquid chromatography (HPLC). As a result, a peptide in which only the N-terminal valine residue of the β-chain of Hb is converted to CM (amino acid sequence: (CM) V
al-His-Leu-Thr-Pro-Glu-Gl
u-Cys (hereinafter sometimes referred to as “CM-peptide βN”) or a peptide in which only the lysine residue at position 66 of the β-chain of Hb has been converted to CM (amino acid sequence: Ala-
His-Gly-Lys-Lys (CM) -Val-L
eu-Gly-Ala-Phe-Ser-Cys (hereinafter sometimes referred to as “CM-peptide β66”), or a peptide in which only the lysine residue at position 61 of the α-chain of Hb has been converted to CM (amino acid sequence: Gly- His-Gly
-Lys-Lys (CM) -Val-Ala-Asp-
Ala-Leu-Thr-Cys (hereinafter sometimes referred to as “CM-peptide α61”). The purity of each CM-peptide was confirmed by mass spectrometry and amino acid analysis.

(9) [Preparation of CM-HSA] 1 mg / ml of HS dissolved in borate buffer (pH 9)
A 1 ml of 0.25 M glyoxylic acid (manufactured by SIGMA) adjusted to pH 9 was mixed with 1 ml of the mixture at 0 ° C.
Left for hours. Thereafter, 1 mg of sodium cyanoborohydride was added, and the mixture was allowed to stand for further 12 hours to convert the amino group of the protein into CM. As a control, HSA was treated in the same manner except that glyoxylic acid was not added.

The ratio of the CM group of the HSA after each of the above treatments was measured by using trinitrobenzenesulfonic acid (hereinafter sometimes abbreviated as “TNBS”) to measure the ratio of the amino group converted into the CM group and the unreacted amino group. Then, it was determined by a method as shown below.

That is, 0.5 ml of each sample of the CM-HSA and the HSA not treated with glyoxylic acid was added to 0.1 mL of each sample.
Each was added to 0.5 ml of a 0.1 M aqueous sodium hydroxide solution containing 1 M sodium tetraborate. Next, after recrystallization and washing with diluted hydrochloric acid, 20 μl of TNBS adjusted to 1.1 M with distilled water was added and stirred. 30 minutes later 1.
The reaction was stopped by adding 2 ml of 98.5 mM sodium dihydrogen phosphate containing 5 mM sodium sulfite, and after diluting 10 times with distilled water, the absorbance at 420 nm was measured. The absorbance of CM-HSA was 0.03, and the absorbance of HSA not treated with glyoxylic acid (control) was 1.
25. When the same measurement was performed in a system not containing any of the above-mentioned HSA, the absorbance was 0.03, and it was found that the conversion of CM-HSA into CM was 100%.

The CM-HSA obtained by such a method has 2
Dialysis with 0 mM PBS at 4 ° C. for 2 days to remove unreacted glyoxylic acid and sodium cyanoborohydride,
Purified CM-HSA was obtained.

(10) [Properties of Monoclonal Antibody] On a BIA-core (Pharmacia) sensor chip, CM-Hb prepared by the above method and commercially available Hb (S
IGMA) and the CM-HSA prepared by the above method was immobilized by the amine coupling method. The reagents and procedures used for the amine coupling followed the kit for BIA-core and the usual operating procedures. The amount of protein immobilized on the sensor chip surface was 50-60 ng / mm ² . The monoclonal antibody produced by hybridoma 2B3 was converted to a HEPES buffer solution (pH 7.4, 0.15 M Na
Cl) to a concentration of 50 μg / ml, and 10 μl /
30 μl of the antibody solution was injected into the sensor chip at a flow rate of min, and the affinity constant between each protein immobilized on the sensor chip and the monoclonal antibody was examined. Measurement and analysis followed the usual operating procedure in BIA-core. Table 1 shows the affinity constant between each protein immobilized on the sensor chip and the monoclonal antibody of the present invention.

[0055]

[Table 1]

As shown in Table 1, the antibody of the present invention
Specifically bound to M-Hb (affinity constant: 6.28 × 1
0 ^8). Hb (SIGMA), CM-HSA and the like do not bind, and the antibody does not react with Hb or CM-protein other than Hb that has not been converted into CM (affinity constant: both 1 × 1).
0 < ³ ).

On the BIA-core sensor chip,
CM-peptides prepared by the method of Example 1 (CM-peptide βN, CM-peptide β66, CM-peptide α6
1) and peptides having the same amino acid sequence without CM (peptide βN, peptide β66, peptide α61)
Was fixed by a thiol coupling method using a C-terminal cysteine residue. The reagents and procedures used for the 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 . The monoclonal antibody produced by hybridoma 2B3 was added to a HEPES buffer (pH 7.4, 0.15 M NaCl) at 50 μg /
Dissolve to 30 ml at a flow rate of 10 μl / min.
of the antibody solution was injected into a sensor chip, and the affinity constant between each peptide immobilized on the sensor chip and the monoclonal antibody was examined. BIA-
The usual operating procedure in the core was followed. Table 2 shows the results.

[0058]

[Table 2]

As shown in Table 2, hybridoma 2B
The monoclonal antibody produced by No. 3 reacts specifically with a peptide in which the amino group at the N-terminal of the β chain has been converted to CM (CM-peptide βN) (affinity constant: 7.84 × 10 ⁷ ), and the other CM-peptide (CM-peptide β66, CM-peptide α61) and a non-CM peptide (peptide β
N, peptide β66, peptide α61) did not react (affinity constant: less than 1 × 10 ³ ).

(11) [Goat polyclonal antibody against CM-Hb (anti-CM-Hb goat polyclonal antibody)
Creation of a goat as an animal for immunization and the CM-H created above
Immunization was performed as follows using b as an antigen.

Complete 1.5 ml of Freund's complete adjuvant was added to 1.5 ml of the antigen solution prepared to be 2 mg / ml.
Goats were injected subcutaneously into the goat. Then 2
Every week, 0.75 ml of the antigen at 2 mg / ml plus 0.75 ml of Freund's complete adjuvant was boosted. During this period, in order to confirm whether or not a polyclonal antibody against CM-Hb was produced, blood was partially collected from a goat vein once every two weeks. Six weeks later, anti-CM
-EL generation of Hb goat polyclonal antibody
All blood was collected after confirmation by the ISA method.

(12) [Preparation of affinity purification column for anti-CM-Hb goat polyclonal antibody] 25 ml of Affigel 15 was washed with 75 ml of 10 mM acetate buffer (pH 4.5), and then washed with 10 mg / ml of H.
62.5 ml of the solution b was added, and the mixture was slowly stirred at room temperature for 1 hour. Next, unreacted Hb was removed by filtration, 30 ml of 1M ethanolamine was added, and the mixture was stirred gently at room temperature to block unreacted N-hydroxysuccinimide ester. The Hb-immobilized support was packed in a column, and washed with ion-exchanged water until the absorbance at 280 nm became zero. Further, the column was equilibrated with 20 mM PBS.

(13) [Affinity purification of anti-CM-Hb goat polyclonal antibody] 1 mg of the prepared anti-CM-Hb goat polyclonal antibody
/ Ml was diluted with 20 mM PBS and applied to the affinity purification column to about 100 mg. Next, the absorbance at 280 nm is zero.
20 mM PBS until the flow rate becomes 0.5 ml / min
Flowed away. Antibody that did not bind to the column was replaced with anti-CM-Hb.
It was recovered as a goat polyclonal antibody. When the absorbance at 280 nm became 0, 20 mM PBS was replaced with 0.1 M glycine buffer (pH 3.0) to elute unnecessary proteins bound to the column.
The column was equilibrated with M PBS, the recovered antibody was applied to the column again, and the antibody not bound to the column was recovered. This operation was further repeated once to obtain an affinity-purified anti-CM-Hb goat polyclonal antibody. Using the antibody, Hb in a sample is converted into CM-Hb and non-CM-Hb.
Separated and purified by affinity purification.

(14) [Affinity Purification of Non-CM-Hb] An affinity purification column on which an anti-CM-Hb goat polyclonal antibody was immobilized was prepared in the same manner as described above.
Using the column, non-CM-Hb was separated and purified from sample Hb as the third pass fraction of the column in the same manner as described above.

(15) [Biotin labeling of anti-Hb polyclonal antibody] The biotin labeling of the antibody was performed using a protein biotinylation system (GIBCO).

A commercially available anti-Hb polyclonal antibody (Co., Ltd.)
Nippon Biotest Laboratories) was added to a solution of 1.5 mg / ml in 20 mM PBS,
M sodium carbonate buffer (pH 9.0) was added. Next, 26 μl of a 50 mg / ml CAB-NHS ester solution prepared according to the instructions was added to 6.7 ml of the antibody solution, and the mixture was slowly stirred at room temperature for 1 hour.
The reaction was stopped by adding ammonium chloride to 11 M. Thereafter, the antibody solution was desalted using a column attached to this kit. Furthermore, avidin / HAB included in the kit
When the number of moles of the introduced biotin was measured using A, 14 moles of biotin was bound to 1 mole of the anti-Hb polyclonal antibody.

(16) [Reactivity between non-CM-Hb and monoclonal antibody produced by hybridoma 2B3 obtained by the present invention] Non-CM-Hb obtained by the above method and hybridoma 2B3 obtained by the present invention Was tested for its reactivity with the monoclonal antibodies produced.

The monoclonal antibody produced by hybridoma 2B3 was added to 3 μl of sodium carbonate buffer (pH 9).
g / ml diluted to 96 wells EI
0.3 ml was added to each well of the microplate for A and allowed to stand at 37 ° C. for 1 hour for fixation. The antibody solution was removed from the microplate, and Tris-HCl buffer (pH 7.4, 0.15 M NaC) containing 0.1% Tween 20 was used.
After washing 3 times in 1), each well was filled with 20 mM MOP.
S (pH 7.4, 0.01% Tween 20 and 0.1% Tween 20).
Non-CM-Hb diluted with H.I.
0.1 ml to become 0, 50, 25, 12.5 μg
And left at 37 ° C. for 15 minutes. The solution was removed from each well, and the well was washed three times with Tris-HCl buffer (pH 7.4, 0.15 M NaCl) containing 0.5% Tween 20, and then a biotinylated anti-Hb polyclonal antibody was added to the well.
0 mM MOPS (pH 7.4, 0.15 M NaCl)
Diluted to 1 μg / ml in each well.
1 and added at 37 ° C. for 15 minutes. Remove the solution.
Tris-HCl buffer containing 1% Tween 20 (pH 7.
After washing 3 times with 4, 0.15 M NaCl), ABC
The antigen-antibody complex (2B3-CM-Hb-biotinylated anti-Hb antibody) was labeled with alkaline phosphatase via avidin according to a kit (Bexstein). The solution was removed, and Tris-HCl buffer containing 0.1% Tween 20 (pH
After washing with 7.4 (0.15 M NaCl) three times, 0.1 ml of a chromogenic substrate solution was added to each well, and the solution was added at room temperature for 4 hours.
Left for a minute. The chromogenic substrate solution is a 2-ethanolamine aqueous solution of p-nitrophenyl phosphate (BIO-RAD). After the reaction, 0.1 M of 0.5 M NaOH was added to each well to stop the reaction, and the absorbance at a wavelength of 460 nm was measured. FIG. 1 shows the measurement results of the coloring intensity derived from CM-Hb in the sample. A reaction increasing in a concentration-dependent manner was confirmed in a human whole blood sample, but no reaction was confirmed with non-CM-Hb, indicating that the antibody did not react with non-CM-Hb.

From the above findings, it was found that the monoclonal antibody produced by hybridoma 2B3 specifically reacts with Hb in which the hydrogen of the N-terminal amino group has been converted to CM.

Example 2 [Quantification of CM-Hb in Human Whole Blood by Sandwich Method Using Monoclonal Antibody] A method for quantifying CM-Hb in human whole blood using a monoclonal antibody produced by hybridoma 2B3 was examined. did. In the same manner as in Example 1 (16), sandwich E
It was examined by the LISA method. As a sample, 20 mM
MOPS (pH 7.4, 0.01% Tween 20)
And 0.15 M NaCl) at 200, 400,
The whole blood of a diabetic patient and a healthy person diluted 800 times was 0.1
Each ml was used. FIG. 2 shows the measurement results of the coloring intensity derived from CM-Hb in the sample. Specimens of diabetic patients showed significantly higher values compared to healthy subjects.

FIG. 3 shows the relationship between the severity of diabetic complications of each sample and the color intensity derived from CM-Hb in the sample. A significant correlation was confirmed between the severity of diabetic complications of each sample and the coloring intensity derived from CM-Hb in the sample.

The above results indicate that the monoclonal antibody produced by the hybridoma 2B3 obtained in the present invention can be used for immunoassay to directly measure CM-Hb, a novel marker for diabetes or diabetic complications, easily. It means you can do it.

Example 3 [Preparation of monoclonal antibody recognizing carboxymethylated lysine residue of Hb and CM-H in human whole blood by sandwich method using this antibody
Quantification of b] Using the peptide prepared in Example 1 (8) in which the lysine residue at position 66 of the β chain was converted to CM (CM-peptide β66), immunization of mice and cells in the same manner as in Example 1 fusion,
Selection of fused cells, selection of specific antibody-producing cells, cloning, and the like were performed to obtain a hybridoma (11B9) that produces an anti-CM-Hb monoclonal.

In the same manner as in Example 1 (10), the CM-peptide (CM-peptide β) prepared in Example 1 (8) was used.
N, CM-peptide β66, CM-peptide α61) and a non-CM peptide having the same amino acid sequence (peptide βN, peptide β66, peptide α61) and the monoclonal antibody produced by hybridoma 11B9 were used to determine the interaction with BIA-core. And used to calculate the affinity constant. Table 3 shows the results.

[0075]

[Table 3]

As shown in Table 3, hybridoma 1
The monoclonal antibody produced by 1B9 is a peptide (CM) in which the amino group of the lysine residue at position 66 of the β chain has been converted to CM.
-Peptide β66) (affinity constant: 1.
24 × 10 ⁸ ), other CM-peptides (CM-peptide βN, CM-peptide α61) and non-CM peptides (peptide βN, peptide β66, peptide α6)
1) did not react (affinity constant: 1 × 10
Less than ³ ).

CM-Hb, Hb (SIGMA), CM-HS chemically synthesized in the same manner as in Example 1 (10)
The interaction between A and the like and the monoclonal antibody produced by hybridoma 11B9 was examined using BIA-core, and the affinity constant was calculated. Table 4 shows the results.

[0078]

[Table 4]

As shown in Table 4, hybridoma 1
The monoclonal antibody produced by 1B9 specifically binds to CM-Hb (affinity constant: 4.56 × 10 ⁹ ), and Hb (S
IGMA) or CM-HSA, etc.
It was found that it did not react with unconverted Hb or CM-protein other than Hb (affinity constant: less than 1 × 10 ³ ).

In the same manner as in Example 1 (16), non-CM-Hb and human whole blood specimen Hb prepared in Example 1 (14) were used.
The interaction between the antibody and the monoclonal antibody produced by hybridoma 11B9 was examined by sandwich ELISA. FIG. 4 shows the measurement results. A reaction increasing in a concentration-dependent manner was confirmed in a human whole blood sample, but no reaction was confirmed with non-CM-Hb, indicating that the antibody did not react with non-CM-converted Hb.

From the above findings, hybridoma 11B9
Was found to specifically react with Hb in which the amino group hydrogen of the lysine residue at position 66 of the β chain was converted to CM.

Example 4 [Sandwich ELI Using Monoclonal Antibody Produced by Hybridoma 11B9]
Determination of CM-Hb in Human Whole Blood by SA Method] In the same manner as in Example 1 (16), hybridoma 11
By sandwich ELISA using a monoclonal antibody produced by B9, CM-Hb in dialysis patients who developed dialysis amyloidosis and those who did not developed were quantified for CM-Hb. FIG. 5 shows the measurement results of the coloring intensity derived from CM-Hb in the sample. The color intensity derived from CM-Hb in a patient sample that developed dialysis amyloidosis showed a significantly higher value than a patient sample that did not develop dialysis amyloidosis.

The above means that the immunoassay using the monoclonal antibody produced by the hybridoma 11B9 obtained in the present invention is useful for the measurement of CM-Hb, a novel marker of dialysis amyloidosis. I do.

Example 5 [Preparation of monoclonal antibody recognizing carboxymethylated lysine residue of Hb and CM-H in human whole blood by sandwich method using this antibody
Quantification of b] Immunization and cell immunization of mice by the same method as in Example 1 using the peptide (CM-peptide α61) in which the lysine residue at α-position 61 was converted into CM, prepared in Example 1 (8) fusion,
Selection of fused cells, selection of specific antibody-producing cells, cloning, and the like were performed to obtain a hybridoma (7F9) that produces an anti-CM-Hb monoclonal.

In the same manner as in Example 1 (10), the CM-peptide (CM-peptide β) prepared in Example 1 (8) was used.
N, CM-peptide β66, CM-peptide α61) and non-CM-modified peptides having the same amino acid sequence (peptide βN, peptide β66, peptide α61) and the monoclonal antibody produced by hybridoma 7F9 were used to determine the interaction with BIA-core. And used to calculate the affinity constant. Table 5 shows the results.

[0086]

[Table 5]

As shown in Table 5, hybridoma 7
The monoclonal antibody produced by F9 is a peptide in which the amino group of the lysine residue at position 61 of the α chain has been converted to CM (CM-
Specifically reacts with the peptide α61) (affinity constant: 2.5
6 × 10 ⁷ ), other CM-peptides (CM-peptide β
N, CM-peptide β66) and non-CM peptide (peptide βN, peptide β66, peptide α6)
Did not react with 1) (affinity constant: 1 × 10 ^{3 in} any case)
Less than).

CM-Hb, Hb (SIGMA), CM-HS chemically synthesized in the same manner as in Example 1 (10)
The interaction between A and the like and the monoclonal antibody produced by hybridoma 7F9 was examined using BIA-core, and the affinity constant was calculated. Table 6 shows the results.

[0089]

[Table 6]

As shown in Table 6, hybridoma 7F
The monoclonal antibody produced by 9 specifically bound to CM-Hb (affinity constant: 8.75 × 10 ⁸ ). Hb (SIG
MA), CM-HSA, etc., and it was found that the antibody did not react with non-CM-modified Hb or CM-protein other than Hb (affinity constant: less than 1 × 10 ³ ). .

In the same manner as in Example 1 (16), non-CM-Hb and human whole blood sample Hb prepared in Example 1 (14) were used.
The interaction between the antibody and the monoclonal antibody produced by hybridoma 7F9 was examined by sandwich ELISA. FIG. 6 shows the measurement results. A reaction increasing in a concentration-dependent manner was confirmed in a human whole blood sample, but no reaction was confirmed with non-CM-Hb, indicating that the antibody did not react with non-CM-converted Hb.

From the above findings, it was found that the monoclonal antibody produced by hybridoma 7F9 specifically reacts with Hb in which the hydrogen of the amino group of the lysine residue at position 61 of the α chain has been converted to CM.

Example 6 [Sandwich ELISA using monoclonal antibody produced by hybridoma 7F9]
Determination of CM-Hb in Human Whole Blood by Method A] Hybridoma 7F in the same manner as in Example 1 (16)
By the sandwich ELISA method using the monoclonal antibody produced by No. 9, CM-Hb in dialysis patients who developed dialysis amyloidosis and those who did not develop CM-Hb were quantified. FIG. 7 shows the measurement results of the coloring intensity derived from CM-Hb in the sample. The color intensity derived from CM-Hb in a patient sample that developed dialysis amyloidosis showed a significantly higher value than a patient sample that did not develop dialysis amyloidosis.

The above means that the immunoassay method using the monoclonal antibody produced by hybridoma 7F9 obtained in the present invention is useful for the measurement of CM-Hb, a novel marker of dialysis amyloidosis. I do.

Comparative Example 1 [Antigen specificity of anti-CM-HSA rabbit polyclonal antibody and quantification of CM-Hb in human whole blood by sandwich ELISA using the antibody] Anti-CM-HSA rabbit prepared by the following method Using a polyclonal antibody, the antigen specificity of the antibody was examined, and an attempt was made to construct a system for measuring CM-Hb in human whole blood by sandwich ELISA using the antibody.

(1) [Preparation of anti-CM-HSA rabbit polyclonal antibody] Rabbits weighing 2 kg or more were immunized using the CM-HSA prepared in Example 1 (9) as an antigen in the following manner.

0.5 mg of the antigen solution adjusted to 2 mg / ml was added to 0.5% of Freund's complete adjuvant.
The added ml was injected into the ear vein of the rabbit. Thereafter, 0.25 ml of the antigen at 2 mg / ml every two weeks
Was further boosted with 0.25 ml of Freund's complete adjuvant. During this period, in order to confirm whether a polyclonal antibody against CM-HSA was produced, a partial blood sample was collected from the peripheral ear vein of the rabbit once every two weeks. Six weeks later, the production of the anti-CM-HSA polyclonal antibody was confirmed by ELISA, and the whole blood was collected.

(2) [Purification of anti-CM-HSA rabbit polyclonal antibody] An affinity purification column for anti-CM-HSA rabbit polyclonal antibody having HSA immobilized on the column was prepared in the same manner as in Example 1 (12). Produced. Using this column, the anti-CM-HSA rabbit polyclonal antibody prepared by the above method was affinity-purified in the same manner as in Example 1 (13).

(3) [Biotin labeling of anti-CM-HSA rabbit polyclonal antibody] Biotin labeling of the antibody was carried out in the same manner as in Example 1 (15) using a protein biotinylation system (GIBCO). Was. 14 mol of biotin was bound to 1 mol of the anti-CM-HSA rabbit polyclonal antibody.

(4) [Antigen specificity of anti-CM-HSA rabbit polyclonal antibody] The antigen specificity of the anti-CM-HSA rabbit polyclonal antibody was examined by a competitive ELISA method as shown below.

Adjust the antibody concentration so that the antibody concentration becomes 1 μg / ml.
An anti-CM-HSA rabbit polyclonal antibody diluted with mM PBS was added to the CM-CM prepared by the method of Example 1 (9).
0.1, 1, 10, 100 μg / ml of HSA
Was added so that This solution was left at 37 ° C. for 1 hour and used as an antibody solution inhibited by CM-HSA.

The C prepared by the method of Example 1 (1)
M-Hb was added to a 1 μg / ml anti-CM-HSA rabbit polyclonal antibody solution at 0.1, 1, 10, and 10 respectively.
It was added to be 0 μg / ml. 37 ° C
For 1 hour, and used as an antibody solution inhibited by CM-Hb.

Similarly, Hb and HSA were added to a 1 μg / ml anti-CM-HSA rabbit polyclonal antibody solution at 0.1, 1, 10, and 100 μg / ml, respectively. This solution was left at 37 ° C. for 1 hour and used as an antibody solution inhibited by Hb and HSA.

In performing the competitive ELISA, the prepared CM-HSA was diluted with 20 mM PBS to 1 μg / ml. Then, the diluted CM-HSA
The solution was applied to a 96-well immunoplate (manufactured by NUNC) at 100 μl / well, left at 37 ° C. for 1 hour, and the CM-HSA was fixed to the immunoplate. After 1 hour, CM-HSA not bound to the immunoplate was removed, and 20 mM PB containing 0.5 wt% gelatin was removed.
S was applied at 100 μl per well,
After allowing to stand for a time, a portion where CM-HSA was not bound was blocked. After 1 hour, the gelatin solution was removed and 2
After washing three times with 0 mM PBS, the above concentration of CM-H
100 μl of the antibody solution inhibited by b was applied per well and left at 37 ° C. for 1 hour. After that, 20 mM
Washed 3 times with PBS, and prepared biotin-labeled anti-C
0.1 μg of M-HSA rabbit polyclonal antibody was applied per well and left at 37 ° C. for 1 hour. Further, the mixture was washed three times with 20 mM PBS, and a mixed solution of biotin and avidin labeled with alkaline phosphatase prepared according to the instruction manual (Vectastain AB manufactured by Funakoshi)
C kit) was applied at 100 μl per well, and 3
It was left at 7 ° C. for 1 hour. In addition, 0.05 wt% Tween
After washing three times with 20 mM PBS containing en20, a substrate solution prepared according to the specifications using an alkaline phosphatase substrate kit (manufactured by BIO-RAD) was added to each well at 10 / well.
0 μl was applied. After leaving at room temperature for 5 minutes, 0.4
100 μl of M sodium hydroxide solution per well
In addition, the alkaline phosphatase reaction was stopped, and
The absorbance at 5 nm was measured. FIG. 8 shows the results. From these results, the reaction between the anti-CM-HSA rabbit polyclonal antibody and CM-HSA was inhibited not only when CM-HSA but also CM-Hb was used as the inhibitor. It was found that the polyclonal antibody also showed reactivity with CM-Hb.

In the same manner as described above, the reaction specificity of the anti-CM-HSA rabbit polyclonal antibody with various proteins and CM-proteins was examined. Table 7 shows compounds that specifically react with the anti-CM-HSA rabbit polyclonal antibody and compounds that do not react.

[0106]

[Table 7]

The results suggested that the anti-CM-HSA rabbit polyclonal antibody binds to the CM-converted protein and CM-modified product.

(5) [C in human whole blood by a sandwich method using an anti-CM-HSA rabbit polyclonal antibody]
Quantification of M-Hb] Sandwich ELISA using an anti-CM-HSA rabbit polyclonal antibody in the same manner as in Example 1 (16)
CM-Hb in human whole blood was quantified by the method. However, it was not possible to determine CM-Hb in human whole blood by this method.

From the above results, it was possible to measure CM-Hb in the sample using the ELISA system using the monoclonal antibody produced by hybridoma 2B3 (Example 1).
ELISA using M-HSA rabbit polyclonal antibody
In the A system, CM-Hb in the sample could not be measured (Comparative Example 1). Thus, antibodies that recognize various CM-proteins can detect CM-Hb in blood by ELISA.
It could not be detected without pretreatment of the sample by Method A.

[0110]

The use of the monoclonal antibody of the present invention makes it possible to measure CM-Hb in a body fluid specifically and simply without the need for complicated pretreatment. This means that CM-Hb, a novel marker for diabetes, diabetic complications, dialysis amyloidosis, etc., can be easily and directly measured, and its industrial significance is great.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing the results obtained by examining the reactivity of a monoclonal antibody produced by the hybridoma 2B3 of the present invention with a sample and non-CM-Hb by a sandwich ELISA method.

FIG. 2 shows that each of the diabetic patient and the healthy subject has 10
The amount of CM-Hb in the sample was determined by sandwich ELISA using a monoclonal antibody produced by hybridoma 2B3.
It is a figure showing the result of having measured by method A. In the figure, the vertical axis represents the unit amount (U) of CM-Hb contained per 1 mg of Hb in the sample, and the horizontal axis represents the distinction between diabetic patients and healthy subjects.

FIG. 3 is a diagram showing the results of examining the correlation between the amount of CM-Hb contained in a unit sample Hb of a diabetic patient and the severity of diabetic complications of each sample. In the figure, the vertical axis represents the unit amount (U) of CM-Hb contained per 1 mg of Hb in the sample, and the horizontal axis represents the severity of diabetic complications.

FIG. 4 is a diagram showing the results of examining the reactivity of a monoclonal antibody produced by the hybridoma 11B9 of the present invention with a specimen and non-CM-Hb by a sandwich ELISA method.

FIG. 5 is a graph showing 10 dialysis patients who developed dialysis amyloidosis and 10 patients who did not.
The amount of CM-Hb in the sample was determined by sandwich ELI using a monoclonal antibody produced by hybridoma 11B9.
It is a figure showing the result measured by the SA method. In the figure, the vertical axis represents CM-CM contained per mg of Hb in the sample.
The unit amount (U) of Hb is represented, and the horizontal axis represents the distinction between patients who developed dialysis amyloidosis and patients who did not.

FIG. 6 is a diagram showing the results of examining the reactivity of a monoclonal antibody produced by the hybridoma 7F9 of the present invention with a specimen and non-CM-Hb by a sandwich ELISA method.

FIG. 7 is a graph showing 10 patients who developed dialysis amyloidosis and 10 patients who did not.
The amount of CM-Hb in the sample was determined by sandwich ELISA using a monoclonal antibody produced by hybridoma 7F9.
It is a figure showing the result of having measured by method A. In this figure, the vertical axis indicates CM-contained per mg of Hb in the sample.
The unit amount (U) of Hb is represented, and the horizontal axis represents the distinction between patients who developed dialysis amyloidosis and patients who did not.

FIG. 8 shows the results of examining the antigen specificity of the prepared anti-CM-HSA antibody by competitive ELISA. In this figure, the vertical axis represents the absorbance at 405 nm, and the horizontal axis represents the amount of each inhibitor added (the amount of added protein (inhibitor) per well).

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // C12P 21/08 C12N 15/00 C (72) Inventor Keisuke Miura 1-1-1, Mikage-cho, Tokuyama-shi, Yamaguchi Prefecture Tokuyama Corporation Inside

Claims (4)

[Claims] 1. A monoclonal antibody that specifically reacts with carboxymethylated hemoglobin. 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.