JPH0843397A - Monoclonal antibody combinable with human aldose reductase and its usage - Google Patents

Abstract

PURPOSE:To measure conveniently and accurately micro-quantity of human aldose reductase existing in human blood, urine, tissue, etc., and use it in such as clinical examination. CONSTITUTION:Human aldose reductase is measured by using monoclonal antibody combinened with human aldose reductase but not combined with cow, dog guinea pig, rat or rabbit aldose reductase.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel monoclonal antibody characterized by specifically binding to human aldose reductase and a method for measuring human aldose reductase using the antibody. By using the monoclonal antibody and the antibody fragment thereof that specifically bind to human aldose reductase of the present invention, the function and structure of human aldose reductase can be analyzed. Also,
Using a monoclonal antibody that specifically binds to the human aldose reductase of the present invention, the content of human aldose reductase in tissue, blood or urine is measured, and grasping the trend of the aldose reductase is understood in basic medicine and clinical medicine. It has a very important meaning in the field.

[0002]

2. Description of the Related Art Alditol reductase [Alditol: NAD (P)
+ 1-oxidoreductase, EC 1.1.1.21] is an enzyme that converts glucose into sorbitol, a sugar alcohol (polyol), using NADPH as a coenzyme. Sorbitol is converted to fructose by sorbitol dehydrogenase,
This pathway forms the polyol metabolic pathway, which is the alternative pathway for glucose metabolism. Aldose reductase is present in many tissues of mammals, and its physiological role was considered to be involved in the production of fructose as an energy source of sperm in the seminal vesicles in the old days. Recently, it has been suggested that the role of osmolyte in the formation of osmolytes in the renal medulla is involved in the formation of polyol. However, there are still many unclear points about its role. Recently, it is considered that aldose reductase is involved in cell damage in the early stage of diabetes onset. That is, in the hyperglycemic state of diabetes, intracellular glucose concentration increases in tissues that perform insulin-independent glucose uptake, for example, the lens, retina, peripheral nerve, renal glomerulus, and this excess glucose is It is converted to sorbitol by aldose reductase. Sorbitol has high polarity and is difficult to diffuse to the outside of the cell, and its conversion rate to fructose is slow, so it is accumulated inside the cell, and as a result, intracellular osmotic pressure is increased, water inflow occurs, cell swelling, Causes cell membrane degeneration. It is considered that this is one of the onset factors of diabetic complications such as neurosis, retinopathy, nephropathy, and cataract. Currently, aldose reductase inhibitors are actively developed as a preventive or therapeutic drug for diabetic complications. (Gabbay KH, Merola LO and Field RA,
Science, 151, 209-210 (1966), Robison
WG Jr., Kador PF and Kinoshita JH, Scienc
e, Vol. 221, pp. 1177-1179 (1983), Engerman RL.
and Kern TS, Diabetes, Volume 33, pp. 97-100 (1984
), Nishimura C., Lou MF and Kinoshita JH,
J. Neurochem., 49, 290-295 (1987), Kino.
shita JH and Nishimura C., Diabetes Metab. Re
v., Volume 4, pp. 323-337 (1988), Kador RF, Nak.
ayama T., Sato S., Smar M. and Miller, DD, “We
iner H. and Flynn TG eds Progressin Clinicala
nd Biological Research Vol. 290, Enzymology and Mo
lecular Biology of Carbonyl Metabolosm 2, ”237-250
P. (1989), Alan R. Liss, Inc., New York).

Recently, the protein structure of aldose reductase derived from human placenta, retina and muscle has been reported (Bohren K.
M., Bullock B., Wermuth B. and Gabbay KH, J. Bi
ol. Chem., 264, 9547-9551 (1989), Nishi.
mura C., Matsuura Y., Kokai Y., Akera T., Carper
D., Morjana N., Lyons C. and Flynn TG, J. Biol.
Chem., Vol. 265, pp. 9788-9792 (1990)), it has been shown that the primary structures are exactly the same even if the tissues of origin are different. In addition, human aldose reductase has been shown to have about 80% homology with rat, bovine and rabbit aldose reductase (Carp
er DA, Wistow G., Nishimura C., Graham C., Wata
nabe K., Fujii Y., Hayashi H., Hayaishi. O., Exp.
Eye Res., Vol. 49, pp. 377-388 (1989), Schade S.
Z., Early SL, Williams TR, Kezdy FJ, Hein
rikson RL, Grimshaw CE, Doughty CC, J. Bi
ol. Chem., Vol. 265, pp. 3628-3635 (1990), Garc.
ia-Perez A., Martin B., Murphy HR, Uchida S., M
urer H., Cowley BD Jr., Handlar JS, Burg M.
B., J. Biol. Chem., Volume 264, 16815-16821 (1989
Year)). Furthermore, a method for producing human aldose reductase by genetic engineering has been reported (Nishimura C., Yamaoka T.,
Mizutani M., Yamashita K., Akera T., Tanimoto T.,
Biochim. Biophys. Acta, 1078, 171-178 (1
991)).

An antibody that specifically recognizes human aldose reductase has been developed, and the distribution of human aldose reductase is examined by immunostaining, or human aldose reductase such as each tissue, blood or urine is measured by immunoassay. If possible, elucidate the onset mechanism of diabetic complications based on the increased activity of polyol metabolism and serve as an index for the administration of aldose reductase inhibitors used as therapeutic drugs for diabetic complications. It is considered to have very important significance in the field of medicine. Thus, development of an antibody that specifically binds to human aldose reductase and development of a method capable of specifically and easily measuring human aldose reductase in a sample with high sensitivity have been desired. We have already developed an antibody that binds to human aldose reductase and a method for measuring human aldose reductase using the antibody (JP-A-5-317083), but the monoclonal antibody used in the invention of JP-A-5-317083 is However, it may be possible to recognize biological substances other than human aldose reductase present in human-derived samples, that is, it was insufficient in terms of specificity for human aldose reductase.

[0005]

SUMMARY OF THE INVENTION It is an object of the present invention to solve the above-mentioned drawbacks of the prior art and develop a method for specifically and easily measuring human aldose reductase in a sample with high sensitivity. Is.

[0006]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that they bind to human aldose reductase and can bind to bovine, dog, guinea pig, rat or rabbit aldose reductase. By an immunoassay using a monoclonal antibody characterized by not binding to, it was found that human aldose reductase in a human-derived sample can be measured more specifically and easily with high sensitivity. Invented. That is, the present invention binds to human aldose reductase and does not bind to bovine, dog, guinea pig, rat or rabbit aldose reductase, a monoclonal antibody, a hybridoma producing the antibody, and the antibody. At least 1
Kindly, it relates to an immunoassay method for human aldose reductase to be used.

Hereinafter, the present invention will be described in detail. Monoclonal antibody is a cell fusion method by Köhler and Milstein (G. Kohler Milstein, Nature (London), Vol. 256, 4
It can be obtained by a conventional method of culturing and secreting a hybridoma prepared by the method described on page 95-497 (1975)) and separating it from the culture solution. That is, after immunizing a mammal with human aldose reductase, antibody-producing cells of this animal are fused with myeloma cells to obtain a hybridoma. The animal used for immunization is not particularly limited, and various mammals such as mouse, rat, guinea pig, rabbit, sheep, goat, cat, dog and the like can be used. Of the above-mentioned immunized animals, Balb / c mice are generally used as the monoclonal antibody for reasons such as ease of handling, but mice of other strains can also be used. At that time, the concentration of the antigen used for immunization should be selected so that a sufficient amount of antigen-stimulated lymphocytes are formed. For example, a physiological saline solution containing 1 to 100 μg of the antigen is made into a suspension of complete Freund's adjuvant or incomplete Freund's adjuvant, and is administered to animals by intraperitoneal injection or the like. Alternatively, the antigen may be directly injected into the spleen. The administration is performed once to several times every 2 to 4 weeks, and the final immunization is usually performed by intravenously administering a physiological saline solution containing 1 to 100 μg of the antigen. A few days after the final immunization, antibody-producing cells such as lymphocytes, preferably spleen cells, are extracted from the immunized animal for cell fusion and fused with myeloma cells to obtain hybridomas.

The human aldose reductase used as an antigen is not particularly limited as long as it is of human origin.
From the viewpoint of easy availability, those derived from human placenta are preferable, and from the viewpoint that a large amount of highly pure ones can be obtained, the genetic engineering manufacturing method is used. That is, the cDNA of human aldose reductase was used to clone the cDNA of human aldose reductase using a baculovirus expression vector into which the cDNA of human aldose reductase was inserted.
Human aldose reductase can be obtained by obtaining a recombinant baculovirus contained in DNA, infecting insect (Spodoptera frugiperda) cells with this recombinant virus, and expressing human aldose reductase. When a peptide or the like having the amino acid sequence of human aldose reductase is used as an antigen, it can be obtained by chemical synthesis or genetic engineering techniques, but its origin is not limited.

In the cell fusion, 3 to 4 days after the final immunization, spleen cells prepared from spleens aseptically taken out and appropriate myeloma cells are fused in the presence of a fusion promoter. The myeloma cells used for the fusion may be derived from mammals, but in general, those derived from animals of the same species as the animals used for immunization are preferable, for example, SP-2 / 0-Ag14 in mice.
, NS-1-Ag4 / 1, P3-X63-Ag8-U1, P3-NS1-1-Ag1, P3-X6
3-Ag8, FO, X-63-Ag8-6.5.3, 210.RCY3.Ag1.2.3, S194 /
5XXO.BU.1, SKO-007, GM15006TG-A12 etc. are used,
Y3.Ag1.2.3 is used in rats. As the fusion method, the HVJ method using Sendai virus, the electric fusion method, or the PEG method using polyethylene glycol having an average molecular weight of 1000 to 6000 can also be used. In this case, the mixing ratio of spleen cells and myeloma cells at the time of fusion is generally 10: 1 to
A range of 2: 1 is preferred.

Isolation of hybridomas from the fused cells is accomplished by killing the unfused cells in a selective medium in which the unfused spleen cells, unfused myeloma cells and a mixture of fused cells cannot survive the unfused myeloma cells. It can be performed by culturing for an appropriate time (about 1 week). When HGPRT-deficient myeloma cells are used as the fusion parent strain, the selection medium is, for example, HAT medium (hypoxanthine,
A medium containing aminopterin and thymidine), HA2 (medium containing hypoxanthine and azaserine) and the like are used.
In this selection medium, unfused myeloma cells die, and unfused spleen cells are non-neoplastic cells that die after a period of time (about 1 week), so select viable cells. Thus, a hybridoma can be obtained. Hybridomas that produce the desired antibody can be searched for and cloned by a conventional limiting dilution method. The hybridoma producing the monoclonal antibody of the present invention thus obtained can be grown in a medium suitable for growth and can be easily stored for a long period of time in an ultralow temperature freezer or liquid nitrogen. The hybridoma thus obtained can produce an antibody by growing in a nutrient medium or in the abdominal cavity of a mammal, and the produced monoclonal antibody is purified from the culture supernatant or the ascites or serum of the mammal. can do. The antibody can be purified by a biochemical method used for ordinary protein purification, such as centrifugation, dialysis, salting out with ammonium sulfate, ion exchange chromatography with DEAE column, gel filtration, affinity chromatography, etc. .

The thus obtained monoclonal antibody that binds to human aldose reductase contains several antibodies that bind to different sites of the antigen used for immunization, and is specific to human aldose reductase. Bovine,
Contains antibodies that do not bind to dog, guinea pig, rat or rabbit aldose reductase, as well as antibodies that bind to both human aldose reductase and bovine, dog, guinea pig, rat or rabbit aldose reductase. . In addition, an antibody that binds to human aldehyde reductase, which is a closely related enzyme of human aldose reductase and has a very similar amino acid sequence, may be included. We have already developed an antibody that binds to human aldose reductase and a method for measuring human aldose reductase using the antibody (Japanese Patent Laid-Open No. 317083/1993) .However, these antibodies are effective for bovine, dog, guinea pig, rat or rabbit. It is an antibody that also binds to aldose reductase. It is very difficult to obtain an antibody that specifically binds to human aldose reductase and does not bind to bovine, dog, guinea pig, rat or rabbit aldose reductase. In order to obtain a monoclonal antibody that specifically binds to human aldose reductase of the present invention, as a hybridoma screening method, an antibody that does not compete with ARmAb25 is produced from among anti-human aldose reductase monoclonal antibody-producing hybridomas. It was found that a hybridoma producing a monoclonal antibody that specifically binds to human aldose reductase can be efficiently selected by screening the hybridomas. ARmAb25 is Japanese Patent Laid-Open No. 5-31
The monoclonal antibody against human aldose reductase described in 7083, which is produced by hybridoma AR25. This hybridoma AR25 was deposited on July 4, 1994 as FERM P-14417 at the Patent Microorganism Depositary Center, Institute of Biotechnology, Institute of Industrial Science and Technology.

The monoclonal antibody that binds to human aldose reductase of the present invention includes the antibody itself and a fragment of the antibody that binds to human aldose reductase. Examples of such antibody fragments include F (ab ') ₂ , Fab', Fab and the like.
Such an antibody fragment is, for example, in the case of F (ab ′) ₂ or Fab ′, the antibody obtained as described above is used in a known method,
For example, it can be obtained by digesting the antibody with pepsin to give an F (ab ') ₂ fragment, or by further reducing the F (ab') ₂ fragment to give a Fab 'fragment (Eiji Ishikawa, enzyme-labeled. Law, Biochemistry Experimental Method 27, Academic Publishing Center, 1991). Furthermore, the monoclonal antibody of the present invention is a mouse antibody, modified human antibody, mouse-human.
Chimeric antibodies and the like are also included.

Further, an enzyme immunoassay (EI
A), fluorescence immunoassay (FIA) or radioimmunoassay
By carrying out (RIA), it became possible to specifically and accurately measure a trace amount of human aldose reductase. For example, as the EIA, a method known per se described in "Enzyme Immunoassay (Biochemical Experimental Method 11)" (1st edition, P. Tijssen, translated by Eiji Ishikawa, Tokyo Kagaku Dojin, 1989), etc. Can be used. Here, the EI based on the sandwich method and the competitive method, respectively.
A is briefly explained, but the same principle is applied to RIA and FIA as in EIA. In EIA by the sandwich method, an antibody is immobilized on a carrier (immobilized primary antibody), an antigen is added to this, and the antigen is bound to the immobilized primary antibody by an antigen-antibody reaction. Next, an enzyme-labeled antibody (enzyme-labeled secondary antibody) is added, and the enzyme-labeled secondary antibody is bound to the antigen bound to the above-mentioned immobilized primary antibody by an antigen-antibody reaction. After that, the enzyme-labeled secondary antibody that did not bind to the antigen was removed, an enzyme reaction was performed by adding a substrate, and from the calibration curve showing the relationship between the known amount of the antigen and the enzyme activity labeled to the secondary antibody Determine the amount of antigen. In order to simplify the operation, a method (single-step sandwich method EIA) in which the antigen and the enzyme-labeled secondary antibody are simultaneously added to the immobilized primary antibody is also used. Usually, in the sandwich method, the primary antibody and the secondary antibody are used in combination with antibodies having different recognition sites from each other, and when the monoclonal antibody of the present invention is used as the primary antibody or the secondary antibody, human aldose reductase Specific measurement is possible. In this case, the other antibody may be a polyclonal antibody as long as it binds to human aldose reductase, or may be any type of monoclonal antibody as long as it has different recognition sites.

The polyclonal antibody can be obtained by a commonly used method, for example, the method described in "Biochemistry Society of Japan, Shinsei Chemistry Laboratory Course 12, Tokyo Kagaku Dojin, 1992". Immunized animals include, but are not limited to, horses,
Goat, sheep, rabbit, guinea pig, mouse, chicken and the like can be mentioned. When a rabbit is used as an immunized animal, the antigen is diluted to an appropriate concentration with physiological saline or the like to prepare a suspension of complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum hydroxide adjuvant, etc., at 10 to 1000 μg / time. Inject cubs, then 2-4 more
After one week, booster injections are given 1 to 3 times to obtain antiserum.
Injections are preferably given subcutaneously at multiple sites. The polyclonal antibody can be prepared from the antiserum by the same method as in the purification of the above monoclonal antibody.

In EIA by the competitive method, an antigen-antibody reaction is performed with an antigen and a fixed amount of antibody, and then an antibody that has not bound to the antigen is allowed to undergo an antigen-antibody reaction with the immobilized antigen. The amount of antibody bound to the immobilized antigen is measured with an enzyme-labeled antibody.
Alternatively, after performing an antigen-antibody reaction with an antigen and an antibody labeled with a predetermined amount of enzyme (hereinafter, labeled antibody), the labeled antibody remaining without binding to the antigen and the solid-phase antigen are allowed to undergo an antigen-antibody reaction. Then, the amount of enzyme bound to the solid phase is measured. The enzyme amount can be measured by an ordinary method, but in any case, the antigen amount in the sample can be calculated from a calibration curve prepared using a known amount of labeled antigen.
Alternatively, a fixed amount of a mixture of an enzyme-labeled antigen and a sample is brought into contact with the immobilized antibody to cause an antigen-antibody reaction, and the labeled antigen and the unlabeled antigen compete with each other to bind to the antibody. Then, the amount of the enzyme-labeled antigen bound to the immobilized antibody is added to the substrate of the enzyme and measured, and the amount of the antigen is calculated from the calibration curve prepared using a known amount of the labeled antigen.

In each of the sandwich method and the competition method, F (ab ') ₂ , obtained by digesting the antibody with pepsin,
Fab 'obtained by reducing F (ab') ₂ and an antibody fragment that binds to an antigen such as Fab obtained by digesting the antibody with papain are used as antibodies, and these are also used as labeled labeled antibodies. You can do it. Enzymes for labeling antibodies and antigens include peroxidase and β
-D-galactosidase, alkaline phosphatase,
There are glucose oxidase, etc., and "monoclonal antibody" (Tatsuo Iwasaki et al., Kodansha Scientific
(1984)), `` Enzyme labeling method (Biochemical Experimental Method 27) '' (No.
1st edition, Eiji Ishikawa, Academic Publishing Center, 1991). On the other hand, as the solid phase, silicon, nylon, plastic, glass sticks, beads, microplates or test tubes can be used. The human aldose reductase antibody of the present invention can also be used for separation and purification of human aldose reductase from tissues, blood, urine and the like. Hereinafter, the present invention will be described more specifically by way of examples, but the present invention is not limited thereto.

[0017]

Example 1 Polyclonal antibody that binds to human aldose reductase
(1) Preparation of antigen used for immunization Nishimura et al. [Nishimura et al., Biochim. Bio
Phys. Acta, 1078, 171-178 (1991)], 15 mg of human aldose reductase (hereinafter sometimes referred to as the present antigen) was obtained. The protein quantification of human aldose reductase was carried out by the Bradford method using bovine gamma globulin as a standard. (2) Preparation of polyclonal antibody Japanese white rabbits (female, 2.0 kg) were subcutaneously immunized with 500 μg of human aldose reductase together with complete Freund's adjuvant, and after immunization for 3 weeks, the same amount of human aldose reductase and FCA was boosted. did. The same booster immunization was performed twice every two weeks, and 10 days after the final immunization, blood was collected from the ear artery. After allowing the blood to stand at room temperature for several hours, overnight at 4 ° C, centrifuge at 5000 xg for 10 minutes, add NaN ₃ to the supernatant to a final concentration of 0.02%, and add serum I got the goods. Protein A column (Ampure
PA Kit, Amersham) using immunoglobulin G (IgG)
The fraction was purified to obtain a rabbit anti-human aldose reductase polyclonal antibody (hereinafter referred to as a polyclonal antibody).

(3) Specificity of Polyclonal Antibody When the human-derived Hela cell line and human tissue extracts were analyzed by Western blotting using 1 μg / ml of this antibody, human aldose reduction was observed. A protein that reacts with the antibody was found at the same molecular weight position as the enzyme. Furthermore, the cross-reactivity of this antibody with aldehyde reductase, which is present in a large amount in human tissues as a related enzyme of aldose reductase, was examined by Western blotting. When human aldose reductase 0.1 μg and human kidney aldehyde reductase 0.1 μg and 0.2 μg were subjected to SDS polyacrylamide gel electrophoresis and analyzed by Western blotting using antibodies, human aldose reductase was detected. However, human kidney aldehyde reductase was not detected.

Example 2 Monoclonal antibody binding to human aldose reductase
(1) Preparation of antigen used for immunization Human aldose reductase 11 was prepared in the same manner as in (1) of Example 1.
to obtain mg. (2) Preparation of immune spleen cells 9-week-old male Balb / c mice were intraperitoneally injected with 40 μg of human aldose reductase and complete Freund's adjuvant (Complete Freund's adjuvant).
und's adjyuvant). After 4 weeks, an emulsion of 30 μg of human aldose reductase and Freund's incomplete adjuvant was additionally administered intraperitoneally. After another 7 days, human aldose reductase 30μ
A physiological saline solution containing g was intravenously administered. Of final immunity
After 3 days, the mice were sacrificed and their spleens were removed and used for cell fusion. (3) Preparation of hybridoma Cells obtained by loosening the spleen with tweezers are used in GIT medium.
(Manufactured by Nippon Pharmaceutical Co., Ltd.) and passed through a nylon mesh to obtain a single cell suspension. Add this single cell suspension to 0.
83% ammonium chloride solution (9 parts by volume) and 0.17M Tris
(Hydroxymethyl) aminomethane hydrochloric acid buffer (pH 7.6)
It was treated with a mixed solution of (1 part by volume) at 4 ° C for 1 minute to destroy the red blood cells in the single cell suspension and removed by centrifugation. Ten%
Logarithmic growth of mouse myeloma cells SP-2 cultured in GIT medium supplemented with fetal bovine serum (hereinafter referred to as 10% FCS-GIT medium)
It was washed twice with GIT medium. The spleen cells and the mouse myeloma cells SP-2 were suspended in GIT medium at a ratio of 7.5: 1 and homogenized, and the cells were recovered by centrifugation, and the cells were heated to 37 ° C in an aqueous solution.

2.0 ml of a 50% polyethylene glycol solution having an average molecular weight of 1,500 (manufactured by Boehringer Mannheim) preheated to 37 ° C. was gradually added to the cells over 1 minute, and a cell fusion reaction was further performed for 1 minute. 10 mL of GIT medium was added dropwise to this mixed solution over 4 minutes to stop the cell fusion reaction, and this was centrifuged to obtain cells. 5 × 10 ⁶ cells / cell were prepared using GIT medium (hereinafter referred to as 5% HCF-GIT medium) containing 5% of an additive reagent “Blyclone” (manufactured by Dainippon Pharmaceutical Co., Ltd.) for producing a hybridoma containing a hybridoma growth factor. The suspension was suspended at a concentration of ml, and 100 μl of this suspension was dispensed into each well of a 96-well microwell plate (manufactured by Corning). After incubating this plate for 1 day at 37 ℃ in an atmosphere containing 5.5% carbon dioxide, 5% containing 4 × 10 ^-7 M aminopterin, 1.6 × 10 ^-5 M thymidine and 1 × 10 ^-4 M hypoxanthine.
HCF-GIT medium (hereinafter referred to as HAT-5% HCF-GIT medium) 100
μl was added to each well. After one day, aspirate half of the medium from each well and then add 10% HAT-5% HCF-GIT medium.
0 μl was added. After that, half of the medium was replaced with fresh medium every 2 or 3 days, and the culture was continued. 10 days after cell fusion, proliferation of hybridoma was observed.

(4) Selection of monoclonal antibody-producing hybridoma that binds to human aldose reductase Screening for antibodies that bind to human aldose reductase in the hybridoma culture supernatant uses a microplate on which human aldose reductase is immobilized. By EIA. Immobilization of human aldose reductase was carried out in a 96-well immunomicroplate (Nunc) at a concentration of 0.5 μg / ml in each well with 0.15 M NaCl in 0.01 M phosphate buffer (pH 7.4).
100 μl of human aldose reductase solution dissolved in (hereinafter referred to as PBS) was added and allowed to adsorb at room temperature for 2 hours. After washing this plate 5 times with PBS, add PBS solution containing 0.2% bovine serum albumin and 0.05% Tween 20 (hereinafter referred to as BSA-Tween 20-PBS) to each well to prevent nonspecific adsorption of proteins. Each well was completely blocked.

In each well, 100 μl of a diluted solution of the hybridoma culture supernatant obtained as described in (3) above in PBS.
Was added and reacted at room temperature for 1 hour to bind the monoclonal antibody to the enzyme immobilized in the well. For detection of the monoclonal antibody bound to the enzyme, Vectastin ABC kit (manufactured by Vector Laboratories) was used. In other words, the plate was replaced with PBS containing 0.05% Tween20
5 times), and 100 μl of biotinylated goat anti-mouse immunoglobulin antiserum in BSA-Tween20-PBS solution.
Add, allow to react for 1 hour, then wash with Tween 20-PBS, then add 100 μl of avidin-peroxidase BSA-Twee
The n20-PBS solution was added and reacted for 20 minutes. After washing the plate 5 times with Tween 20-PBS, each well was treated with an equal mixture of 0.03% hydrogen peroxide and 1 mg / ml orthophenylenediamine dihydrochloride in 100 mM citrate buffer (pH 5.0).
0 μl was added and incubated at room temperature for 10 minutes. Then, 100 μl of 2N sulfuric acid was added to each well to stop the reaction, and the absorbance at 490 nm of the reaction product solution was measured using ImmunoReader NJ-2000 (manufactured by Intermed). Of 384 wells in total containing the fused cells, 156 wells showed proliferation of hybridomas, and 43 wells of antibody production that bind to human aldose reductase were positive.

(5) Screening of monoclonal antibody-producing hybridomas that do not compete with ARmAb25 Monochrome that specifically binds to human aldose reductase
As a method for efficiently screening a hybridoma producing a null antibody, a hybridoma producing a monoclonal antibody that does not compete with ARmAb25 was selected by EIA using a microplate on which human aldose reductase was immobilized. A total of 43 types of human aldose reductase binding antibody hybridoma cultures were incubated with HAT-
24-well flat bottom microplate containing 5% HCF-GIT medium
(Made by Corning). A part of the grown hybridoma was transferred to a 24-well flat-bottom microplate containing GIT medium, and the culture supernatant was collected 3 days later. The immobilization of human aldose reductase on the microplate was performed in Example 2.
According to (4), a human aldose reductase solution dissolved in each well at a concentration of 0.1 μg / ml was added and adsorbed. After blocking each well with BSA-Tween 20-PBS, add 100 μl of serially diluted BSA-Tween 20-PBS solution to the resulting hybridoma culture supernatant, react at 4 ° C overnight, and fix to the wells. A monoclonal antibody was bound to the humanized aldose reductase. Plate 300 μl Tw
Wash 5 times with een 20-PBS and add HRP of ARMAb25 to each well.
100 μl of a labeled Fab '(described in JP-A-5-317083) in BSA-Tween 20-PBS solution (20 ng / ml) was added and reacted for 1 hour. After washing the plate 5 times with 300 μl Tween 20-PBS,
100 μl of substrate containing ortho-phenylenediamine (1.0 mg /
ml orthophenylenediamine, 0.015% hydrogen peroxide) was added and reacted at room temperature for 20 minutes. Add 10% 4N sulfuric acid to this well.
The reaction was stopped by adding 0 μl and the immunoleader NJ-2000
Was used to measure the absorbance of the reaction product solution at 490 nm. 43
Hybridomas were cloned in a total of 17 antibody-producing positive wells that did not compete with ARmAb25 and those that did compete slightly.

(6) Cloning of Monoclonal Antibody-Producing Hybridomas that Bind Human Aldose Reductase A culture solution of a total of 17 hybridomas having antibody activity that binds human aldose reductase was added to HAT-5% HCF-GIT.
The medium was transferred to a 24-well flat bottom microplate (made by Corning) containing the medium. Hybridomas that have grown are HAT-5
Cloning was performed by the limiting dilution method using a 96-well microplate containing% HCF-GIT medium. The cloning operation was performed twice to obtain a total of 13 clones. (7) Purification of monoclonal antibody binding to human aldose reductase Balb / c mice were intraperitoneally administered with 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane) 0.5 ml each 10 days before. Monoclonal antibody-producing hybridoma cells that bind to the human aldose reductase obtained in (6) above
2-5 × 10 ⁶ were transplanted. Approximately 1 week later, ascites was collected from the abdominal cavity of the mouse, and monoclonal antibody was collected from the ascites.
A crude antibody salted out with a 25 to 50% saturated ammonium sulfate solution was obtained. This crude antibody was dissolved in a small amount of PBS, separated using the Affigel Protein A -MAPS kit (Bio-Rad), and the antibody solution was dialyzed against PBS.
Stored at ° C. 1.5 to 10 mg of purified antibody was obtained per 1 ml of ascites.

Example 3 Monoclonal antibody binding to human aldose reductase
Characteristics (1) Monochrome - mice immunoglobulin subclasses of antibodies were identified purified immunoglobulin subclass of the monoclonal antibody monochrome - were identified using monoclonal antibody isotyping kit (Amersham). The results are shown in Table 1.

[0026]

[Table 1] Table 1 ─────────────────────────── Monoclonal antibody Immunoglobulin subclass ─────────── ───────────────── ARmAb43 IgG ₁ ARmAb47 IgG ₁ ARmAb49 IgG ₁ ARmAb50 IgG ₁ ARmAb51 IgG ₁ ARmAb52 IgG ₁ ARmAb53 IgG ₁ ARmAb54 IgG ₁ ARmAb55 IgG ₁ ARmAb56 IgG ₁ ARmAb57 IgG ₁ ARmAb58 IgG ₁ ARmAb60 IgG ₁ ─────────────────────────── all monochrome - monoclonal antibody was IgG _1.

(2) Reactivity with Human Aldose Reductase and Human Aldehyde Reductase The reactivity between the monoclonal antibody that binds to human aldose reductase and human aldose reductase and human aldehyde reductase is as follows. Using a microplate fixed with aldehyde reductase
Checked by EIA. To immobilize human aldose reductase or human aldehyde reductase on a microplate,
The same procedure as in (4) of Example 1 was performed, and BSA-Tw was added to this.
Monoclonal antibody (0.1 μg / ml) in een20-PBS
(~ 100 μg / ml) was added, and the mixture was incubated at room temperature for 1 hour. The detection of the antibody bound to the microplate on which human aldose reductase or human aldehyde reductase was immobilized was carried out by the same method as (4) of Example 2 using Vectastin ABC kit. Table 2 shows the results of adding 0.1 μg / ml of the monoclonal antibody to the microplate on which human aldose reductase was immobilized, and the results of adding 100 μg / ml of the monoclonal antibody to the microplate on which human aldehyde reductase was immobilized. Show.

[0028]

[Table 2] Table 2 ─────────────────────────────────── 490nm Absorbance Monoclonal antibody ── ─────────────────────── Aldose reductase Aldehyde reductase (AR) (ALR) ─────────────── ──────────────────── ARmAb43 0.84 0.25 ARmAb47 2.48 0.00 ARmAb49 1.98 0.00 ARmAb52 2.26 0.14 ARmAb55 1 .47 0.00 ARmAb56 2.08 0.00 ──────────────────────────────────── ARMAb47, ARmAb49, ARmAb55 and ARmAb56 are antibodies that bind human aldose reductase but not human aldehyde reductase. Was Tsu. On the other hand, ARMAb43, ARMAb52
Was a small amount of an antibody that binds to human aldehyde reductase.

(3) Differentiation of binding site for human aldose reductase of monoclonal antibody In order to determine the difference of binding site of antibody for human aldose reductase, various mouse anti-human aldose reductases described in JP-A-5-317083 Monoclonal antibody (ARmAb 2, 7
, 19, 21, 35 and 36) were labeled with horseradish peroxidase (hereinafter referred to as HRP), and the labeled antibody and ARMAb4
3, 47, 49, 52, 55, 56 were allowed to undergo a competitive reaction with each unlabeled antibody, and the presence or absence of inhibition was examined to determine the difference in binding site of each antibody to human aldose reductase. HRP to antibody
Is labeled with the periodate oxidation method (enzyme immunoassay method 3
Edition, Eiji Ishikawa et al., Ikusho Shoin, 1987). Human aldose reductase in PBS (0.2 μg / ml) was added to each well of the immunomicroplate.
100 μl of was added and allowed to adsorb at room temperature for 2 hours. After washing the plate 3 times with PBS, add BSA-PBS to each well and
The wells were completely blocked to prevent non-specific protein adsorption to the wells. Add 100 HRP-labeled antibody to each well.
Tween20-PBS containing μl and 0.2% BSA (BSA-Tween20-PBS
The unlabeled antibody (0-1 mg / ml) serially diluted with
After 0 μl was added at the same time and an antigen-antibody reaction was performed at room temperature for 1 hour, the plate was washed 4 times with Tween 20-PBS. Add 100 μl of ortho-phenylenediamine-containing substrate (hydrogen peroxide and ortho-phenylenediamine dihydrochloride) to each well in 100 mM citrate buffer (pH 5.0) so that the respective concentrations are 0.015% and 1 mg / mL. (Dissolved liquid, the same below) is added and allowed to react for 30 minutes at room temperature.
The reaction was stopped by the addition of μl. The absorbance at 490 nm of the reaction solution was measured with ImmunoReader NJ-2000. The results are shown in Table 3.

[0030]

[Table 3] ARmAb55 does not compete with any other labeled antibody, and the other five monoclonal antibodies do not compete with ARmAb7, 19, 21, 35.
It did not compete with ARmAb2, 36. From the above results, ARMA
b43, ARMAb47, ARmAb49, ARmAb52 and ARmAb56 are antibodies that bind to the same or very close sites to the binding sites for ARmAb7, 19, 21, 35 as aldose reductase, respectively, while ARmAb55 is ARmAb2, 7, 19, 2
It was a monoclonal antibody that binds to a site different from 1, 35 and 36.

(4) Reactivity with various animal aldose reductases (preparation of various animal lens homogenates) In order to examine the reactivity of the monoclonal antibody obtained in the present invention with aldose reductases of animals other than humans, A homogenate containing aldose reductase was prepared from an animal lens. The lens was extracted from the eyeballs of cattle, dogs, guinea pigs, rats, and rabbits. Each of these has 3-5 volumes of 0.
It was immersed in 01M phosphate buffer (pH 7.0), homogenized at 4 ° C., and centrifuged at 15,000 × g for 30 minutes. (Purification of aldose reductase) After diluting the homogenate with 10 mM phosphate buffer (pH 7.0), Matrix gel orange
An A affinity column was applied and an elution fraction was obtained with the above buffer containing 0.6 M NaCl. The AR active fraction was concentrated by ultrafiltration (Centricut U-10, Kurabo Industries). The obtained sample was subjected to activity measurement and Western blotting. (Measurement of aldose reductase activity) The activity of aldose reductase is measured by (Nishimura C., Yamaoka T., Mizutani M.,
Yamashita K., Akera T., Tanimoto T., Biochim. Biop
hys. Acta, 1078, 171-178 (1991)). That is, 0.1 M phosphate buffer (pH 6.2), 150 μ
A 1 ml reaction solution containing M NADPH, 10 mM DL-xylose and a sample was measured for a decrease in absorbance at a wavelength of 320 nm for 3 minutes from the time when NADPH was added, and the amount of NADPH oxidized by aldose reductase was determined. The absorbance is measured by Hitachi Spectr
It was carried out at 25 ° C. using an ophotometer U-2000. As a blank, the nonspecific decrease in absorbance when no sample was added was measured, and the activity of aldose reductase in the sample was measured from the following formula 1.

[0032]

[Formula 1] Enzyme activity (unit / mL) = (△ AT set / min－ △ AB
lank / min) × 1.0 / ε / sample solution volume (μl) △ A Tset / min: Decrease in absorbance per minute when adding sample △ A Blank / min: Decrease in absorbance per minute in blank ε: NADPH Micromolar extinction coefficient (ε
= 6.22 μmol ^-1 × cm ^-1 ) As a result of measuring the aldose reductase activity in the various animal lens homogenates thus prepared and the purified aldose reductase, any homogenate or purified product showed aldose reductase activity. Had. (Western blotting) The purified aldose reductase was subjected to Western blotting analysis using 10 μg / ml of the monoclonal antibody of the present invention.
The results are shown in Table 4.

[0033]

[Table 4] +: Western blotting reaction product was observed at the same position as each aldose reductase −: Western blotting reaction product was not found at the same position as each aldose reductase ARmAb55 was not human It was a human aldose reductase-specific monoclonal antibody that did not bind to any aldose reductase. ARmAb43, ARmAb47, ARmAb49,
ARmAb52 and ARmAb56 were monoclonal antibodies that also bind to aldose reductase from other animals.
ARmAb55 is produced by the hybridoma AR55.
This hybridoma AR55 is FE on July 4, 1994.
It has been deposited as RM P-14418 at the Patent Microorganism Depositary Center, Institute of Biotechnology, Institute of Industrial Technology. Also,
When any of the homogenates was used as a sample and similarly subjected to Western blotting, no reaction product with the antibody was observed except at the same position as the aldose reductase.

Example 4 Polyclonal antibody binding to human aldose reductase
And mono that specifically bind to human aldose reductase
Measurement of human aldose reductase using clonal antibody
(Using polyclonal and monoclonal antibodies
( EIA based on the sandwich method ) From the result of (3 ) in Example 3, it is possible to specifically measure human aldose reductase by using ARMAb55 as the primary antibody or the secondary antibody. (Preparation of HRP labeled Fab ') 10m PBS solution containing 2mg / ml of ARMAb55
1 M citrate buffer (pH 3.2) was added to 1 to adjust the pH to 4.0. To this, 250 µg of porcine gastric mucosal pepsin (manufactured by Sigma) was added and incubated at 37 ° C for 12 hours to digest the antibody. 3M Tris-HCl buffer (pH 8.6) was added to adjust the pH to 7.0, and after concentrating to 2 ml, the concentrate was adjusted to 0.
Superrose 12 equilibrated with 1M phosphate buffer (pH 6.0)
Pass it through an HR16 / 50 column (Pharmacia) to obtain F (ab ') ₂
Fractions were obtained. The amount of F (ab ') ₂ recovered was 5.2 mg. The F (ab ') ₂ fraction was concentrated to 2 mL, to which 2-mercaptoethylamine and EDTA were added at 10 mM and 0.5 mM, respectively.
F (ab ′) ₂ was reduced by adding M so that it would be M and incubating at 37 ° C. for 90 minutes. Add this to 0.1M containing 5mM EDTA
Superrose 12 equilibrated with phosphate buffer (pH 6.0)
The Fab 'fraction was obtained by passing it through an HR 16/50 column.
Fab 'of g was recovered.

Introduction of the maleimide group into the HRP was carried out with 10 mg HR
After dissolving P in 0.1 M phosphate buffer (pH 7.0),
Add 150 μL of a solution of 10 mg of N- (ε-maleimidocaproyloxy) -succinimide (manufactured by Siben Chemical Co.) in 1 ml of N, N-dimethylformamide at 30 ° C.
It was carried out by reacting for 60 minutes. Add this reaction mixture to 0.
HR with maleimide group introduced was passed through a PD10 column (Pharmacia) equilibrated with 1M phosphate buffer (pH 6.0).
P fraction was obtained. A concentration of 1.5 mg / ml prepared by the above
1.2 ml of maleimide group-introduced HRP having a concentration of 3.6 mg / ml was added to 3 ml of Fab 'solution and reacted at 30 ° C. for 60 minutes. This reaction solution was concentrated to 1.5 ml, and this concentrate was equilibrated with 0.1 M phosphate buffer (pH 6.5) Super Rose 12HR 16/50
After passing through the column, 10 ml of HRP-labeled Fab 'fraction was obtained. In the prepared HRP-labeled Fab ', the binding molar ratio between HRP and Fab' was about 1: 1.

(A) Two-stage sandwich EIA (hereinafter poly-
Mono-two-step method) Add 150 μl of 5 μg / ml polyclonal antibody diluted with 50 mM sodium bicarbonate buffer (pH 9.6) to each well of a 96-well immunomicroplate (Nunc) at 25 ° C.
The antibody was solid-phased by allowing it to stand overnight at. Then plate 30
After washing 5 times with 0 μl Tween20-PBS, BSA-Tween20-PBS
Was added to each well in an amount of 300 μL and blocked at room temperature for 1 hour. Next, add 100 μl of human aldose reductase diluted with BSA-Tween20-PBS to the wells and incubate at room temperature for 90 minutes, then plate this plate with 300 μl of Tween20-PBS.
Washed twice. Next, add HRP of ARmAb55 above to each well.
100 μl of labeled Fab 'in BSA-Tween20-PBS solution (150 ng / ml)
l, and reacted for 30 minutes. Add 300 μl of this plate
After washing 5 times with Tween 20-PBS, 100 μl of a substrate containing ortho-phenylenediamine (0.5 mg / ml ortho-phenylenediamine, 0.0075% hydrogen peroxide) was added and reacted at room temperature for 30 minutes. The reaction was stopped by adding 100 μl of 2N sulfuric acid to this well, and the absorbance at 490 nm of the reaction product solution was measured using ImmunoReader NJ-2000. The results are shown in Table 5.

[0037]

[Table 5] The human aldose reductase concentration and absorbance showed linearity. (b) One-step method sandwich EIA (hereinafter referred to as poly-mono one-step method) Polyclonal antibody was added at 5 µg / in 50 mM carbonate buffer (pH 9.5).
After adding 150 μl to each well of the immunomicroplate and adsorbing it overnight at room temperature, wash the immunomicroplate 5 times with PBS.
Each well was filled with BSA-PBS and blocked at room temperature for 1 hour to prepare an antibody-immobilized plate. The HRP-labeled Fab 'of ARmAb55 prepared as described above was diluted with BSA-Tween20-PBS to 150 ng / ml, and 50 μl was added to each well.
l, and 50 μl of human aldose reductase solution serially diluted with BSA-Tween20-PBS to each well,
The mixture was reacted at room temperature for 90 minutes, and then washed 5 times with Tween20-PBS. 100 μl orthophenylenediamine-containing substrate (0.5 mg / ml orthophenylenediamine, 0.0075
% Hydrogen peroxide) and allow the reaction to proceed for 30 minutes at room temperature, then
Add 100 μl of 2N sulfuric acid to the plate to stop the reaction,
490 nm of the reaction product solution using the ImmunoReader NJ-2000
The absorbance of was measured. Human aldose reductase concentration and 49
Table 6 shows the relationship of the absorbance at 0 nm.

[0038]

[Table 6] The human aldose reductase concentration and absorbance showed linearity.

Example 5 Monochrome binding specifically to human aldose reductase
Of human aldose reductase using a null antibody
EIA based on Deutsch method) Based on the result of (4) in Example 3, human aldose reductase was specifically measured by using ARMAb55 as a primary antibody (an antibody that binds to a solid phase) or a secondary antibody (an enzyme-labeled antibody). However, when a monoclonal antibody is used as both the primary antibody and the secondary antibody, it cannot be substantially measured with the same antibody or a combination of antibodies having similar antigen binding sites. is there. From the results of the antibody competitive inhibition experiment of (3) in Example 3, a monoclonal antibody that can be combined with ARMAb55 can be determined. Here, ARMAb5
An example using 5 and ARMAb25 is shown. (a) Two-step sandwich EIA (hereinafter referred to as mono-mono two-step method) Prepare ARmAb55 at 5 μg / ml with PBS, add 150 μl to each well of the immunomicroplate, and adsorb overnight at room temperature. After that, the wells were washed 5 times with PBS, and the wells were blocked with BSA-PBS at room temperature for 1 hour so that non-specific adsorption did not occur. Next, 100 μl of human aldose reductase diluted with BSA-Tween 20-PBS was added to this well, reacted at room temperature for 90 minutes, and this plate was washed 5 times with Tween 20-PBS. Then, in each well, in the same manner as in Example 4,
HRP-labeled ARMAb25 with BSA-Tween 20-PBS at 150 ng / m
Dilute to 100 μl, add 100 μl to each well and incubate for 30 minutes at room temperature. Then, add 5 μl of BSA-Tween 20-PBS.
Washed twice. To this, 100 μl of substrate containing ortho-phenylenediamine (0.5 mg / ml ortho-phenylenediamine, 0.
(0075% hydrogen peroxide) was added and reacted at room temperature for 30 minutes. Stop the reaction by adding 100 μl of 2N sulfuric acid to this well,
490 nm of reaction product solution using ImmunoReader NJ-2000
The absorbance of was measured. The results are shown in Table 7.

[0040]

[Table 7] The human aldose reductase concentration and absorbance showed linearity. (b) One-step sandwich EIA (hereinafter referred to as mono-mono one-step method) ARmAb55 was prepared to 50 μg / ml with 50 mM carbonate buffer (pH 9.5) and 250 μg was added to each well of the immunomicroplate.
After adding l of each and adsorbing overnight at room temperature, wash this immunomicroplate 5 times with PBS, and add BSA-PB to each well.
The plate was filled with S and blocked at room temperature for 1 hour to prepare an antibody-immobilized plate. ARMAb2 prepared as described above
5 HRP-labeled Fab 'at 150 ng / ml in BSA-Tween20-P
Dilute with BS and add 100 μl to each well.Add 100 μl of human aldose reductase solution diluted with BSA-Tween20-PBS to each well and leave it at room temperature for 90 minutes, then with Tween20-PBS. Washed 5 times. 2 in each well
00 μl Ortho-phenylenediamine-containing substrate (0.5 mg / ml
Ortho-phenylenediamine, 0.0075% hydrogen peroxide) was added and the reaction was carried out at room temperature for 30 minutes.
Add 100 μl of sulfuric acid to stop the reaction, and
The absorbance of the reaction product solution at 490 nm was measured using NJ-2000. Table 8 shows the relationship between the concentration of human aldose reductase and the absorbance at 490 nm.

[0041]

[Table 8] The human aldose reductase concentration and absorbance showed linearity. Example 6 Monoclonal antibody that binds to human aldose reductase
And aldose reduction using polyclonal antibody
Enzyme measurement (monoclonal antibody and polyclonal
EIA based on sandwich method using antibody) (Preparation of HRP-labeled Fab ') Rabbit anti-human aldose reductase polyclonal antibody sodium acetate buffer containing 2 mg / ml
250 μg of porcine gastric mucosal pepsin (manufactured by Sigma) was added to 10 ml of (pH 4.5), and the antibody was digested by incubating at 37 ° C. for 20 hours. 3M Tris-HCl buffer (pH 8.6) was added to adjust the pH to 7.0, and after concentrating to 2 mL, this concentrated solution was adjusted to 0.
Superrose 12 equilibrated with 1M phosphate buffer (pH 7.0)
Pass through an HR16 / 50 column (Pharmacia) to remove F (ab ') ₂
Fractions were obtained. The amount of F (ab ') ₂ recovered was 8.2 mg. The F (ab ') ₂ fraction was concentrated to 2 ml and added with 2-mercaptoethylamine and EDTA at 10 mM and 0.1 mM, respectively.
F (ab ′) ₂ was reduced by adding M so that it would be M and incubating at 37 ° C. for 90 minutes. Add this to 0.1M containing 5mM EDTA
Superrose 12H equilibrated with phosphate buffer (pH 6.0)
Fab 'fraction was obtained by passing through R 16/50 column, 6.5 mg
Fab 'was recovered. To introduce a maleimide group into HRP, 10 mg of HRP was dissolved in 0.1 M phosphate buffer (pH 7.0), and then 10 mg of N- (ε-maleimidocaproyloxy) -succinimide (manufactured by Siben Chemical Co. ) 1 ml
It was carried out by adding 150 μl of a solution dissolved in N, N-dimethylformamide and reacting at 30 ° C. for 60 minutes.
This reaction product solution was equilibrated with 0.1 M phosphate buffer (pH 6.0) and passed through a PD10 column (Pharmacia) to obtain a maleimide group-introduced HRP fraction. The concentration of 4.0 mg / ml was added to 3.2 ml of the Fab 'solution of 2.0 mg / ml prepared above.
Add 1.3 ml of HRP containing maleimide group of
The reaction was performed for 60 minutes. The reaction product was concentrated to 1.5 ml,
Pass this concentrate through a Superrose 12HR 16/50 column equilibrated with 0.1M phosphate buffer (pH 6.5) to obtain 10 ml of HRP.
A labeled Fab 'fraction was obtained. In the prepared HRP-labeled Fab ', the binding molar ratio between HRP and Fab' was about 1: 1. (a) Two-step method sandwich EIA (hereinafter referred to as mono-poly two-step method) 5 μg / diluted with 50 mM sodium hydrogen carbonate buffer (pH 9.6) into each well of a 96-well immunomicroplate (Nunc) 150 μl of ml of ARMAb55 was added and left at 25 ° C. overnight to immobilize the antibody. Then plate the plate with 300 μl Tw
After washing 5 times with een 20-PBS, 300 μl of BSA-Tween 20-PBS was added to each well and blocking was performed at room temperature for 1 hour. Next, 100 μl of human aldose reductase diluted with BSA-Tween 20-PBS was added to the wells, reacted for 90 minutes at room temperature, and then this plate was washed 5 times with 300 μl of Tween 20-PBS. Then, in each well, BSA-Tw of HRP-labeled Fab 'of the rabbit anti-human aldose reductase polyclonal antibody described above was added.
100 μl of een 20-PBS solution (150 ng / ml) was added and reacted for 30 minutes. Plate the plate with 300 μl Tween 20-PBS.
After washing twice, 100 μl of substrate containing ortho-phenylenediamine (0.5 mg / ml ortho-phenylenediamine, 0.0075
% Hydrogen peroxide) was added and reacted at room temperature for 30 minutes. The reaction was stopped by adding 100 μl of 2N sulfuric acid to this well, and the absorbance at 490 nm of the reaction product solution was measured using ImmunoReader NJ-2000. The results are shown in Table 9.

[0042]

[Table 9] The human aldose reductase concentration and absorbance showed linearity. (b) One-step sandwich EIA (hereinafter referred to as mono-poly one-step method) ARmAb55 was prepared at 5 μg / ml with 50 mM carbonate buffer (pH 9.5), and 150 μm was added to each well of the immunomicroplate.
After adding l of each and adsorbing overnight at room temperature, wash this immunomicroplate 5 times with PBS and add to each well.
The plate was filled with BSA-PBS and blocked at room temperature for 1 hour to prepare an antibody-immobilized plate. Rabbit anti-human aldose reductase polyclonal antibody prepared as described above
HRP-labeled Fab 'at 150ng / ml in BSA-Tween20-PBS
50 μl of this solution was added to each well, and 50 μl of human aldose reductase solution serially diluted with BSA-Tween20-PBS was added to each well and left at room temperature for 90 minutes.
Then, the plate was washed 5 times with Tween20-PBS. 100 μ in each well
l Add orthophenylenediamine-containing substrate (0.5 mg / ml orthophenylenediamine, 0.0075% hydrogen peroxide),
After performing the reaction for 30 minutes at room temperature, add 2N sulfuric acid to the plate.
Add 100 μl to stop the reaction and immunoreader NJ-2000
Was used to measure the absorbance of the reaction product solution at 490 nm. Table 10 shows the relationship between the concentration of human aldose reductase and the absorbance at 490 nm.

[0043]

[Table 10] The human aldose reductase concentration and absorbance showed linearity. Example 7 Monochrome that specifically binds to human aldose reductase
Of aldose reductase in human erythrocytes using null antibody
Male five constant healthy 23-58 year old, whole blood 0.5 to 1.0 ml in an equal volume of ACD solution heparinized blood from the vein of women five (acid-cit
rate-dextrose solution; 23 mM citric acid, 44.9 mM sodium citrate, 81.7 mM dextrose) was added, and the mixture was stored at 4 ° C and used as a sample. For each sample,
Add 10 ml of ice-cold PBS, mix, centrifuge at 1,500 xg for 10 minutes at 4 ° C to remove the supernatant, then add 10 ml of ice-cold PBS and repeat centrifugation under the same conditions twice.
20 mM phosphate buffer (pH 7.0) was added to (red blood cells) until the volume was the same as that of whole blood, and the mixture was frozen and stored at -80 ° C. Next, the frozen red blood cell sample was thawed and re-frozen with dry ice acetone was performed twice to completely lyse the red blood cells.
After that, centrifugation is carried out to obtain hemolyzed blood, and then, Example 5 (a)
The amount of aldose reductase contained in hemolysis was measured by the enzyme immunoassay method of (b). Each hemolysate is BSA-Tween20
-Measured by 200-fold dilution with PBS. The amount of hemoglobin contained in the hemolyzed blood was measured by the sodium lauryl sulfate method (hemoglobin-Test Wako II). The results are shown in Table 11.

[0044]

[Table 11] Human aldose reductase in erythrocytes could be measured by both the two-step method and the one-step method.

Example 8 Polyclonal antibody binding to human aldose reductase
Alternatively, a model that specifically binds to human aldose reductase
Aldose reduction fermentation in human blood using a noclonal antibody
Measurement of elemental 0.5 to 1.0 ml of whole blood collected from veins of 5 healthy 25-45-year-old males and 5 females were cryopreserved at -80 ° C.
Next, the operation of thawing the frozen sample and refreezing was performed twice, and the red blood cells in the blood were completely lysed, followed by centrifugation to obtain hemolyzed blood. Example 5 (b) (mono-mono one-step method) and enzyme immunoassay using polyclonal antibody and ARMAb25 (described in JP-A-5-317083) (poly-mono two-step method)
The amount of aldose reductase contained in the hemolyzed blood was measured by. Each hemolysate was diluted 200 times with BSA-Tween20-PBS and measured. The amount of hemoglobin contained in hemolyzed blood was determined by the sodium lauryl sulfate method (hemoglobin-Test Wako II).
Was measured by The results are shown in Table 12.

[0046]

[Table 12] Poly-mono: Poly-mono two-step method (ARmAb25) Mono-mono: mono-mono one-step method (ARmAb55, ARmAb25) The amount of human aldose reductase in whole blood can be determined by either the poly-mono method or the mono-mono method. Although the measurement was possible, the value of the mono-mono method without ARmAb55 was smaller than that of the poly-mono method without the monoclonal antibody ARmAb55 of the present invention. This suggests that the assay method using the monoclonal antibody ARmAb55 of the present invention has higher specificity for human aldose reductase.

Example 9 Monochrome binding specifically to human aldose reductase
Null antibody (mono-mono one-step method) and conventional human aldo
Various animal lenses using antibodies that bind to glucose reductase
Measurement of Ludose Reductase Using various animal purified aldose reductases obtained in Example 3 (4) and the purified human aldose reductase obtained in Example 1 (1) as samples, the mono-of Example 5 (b) was used. Mono one-step method,
The amount of aldose reductase contained in each sample was measured by the mono-poly one-step method of Example 6 (b) and the enzyme immunoassay (49-56 method) using the monoclonal antibody ARmAb49 and the monoclonal antibody ARmAb56. The enzyme immunoassay using ARmAb49 and ARmAb56 was performed as follows. ARmA
b49 was adjusted to 5 μg / ml with 50 mM carbonate buffer (pH 9.5),
Add 250 μl to each well of the immunomicroplate.
Each time, the cells were adsorbed overnight at room temperature, and this immunomicroplate was washed 5 times with PBS, and BSA-PBS was added to each well.
The plate was filled with and blocked at room temperature for 1 hour to prepare an antibody-immobilized plate. ARMA prepared as in Example 4
HRP-labeled Fab 'of b56 was adjusted to 150 ng / ml with BSA-Tween2
Dilute with 0-PBS, add 100 μl to each well, and add 100 μl of each animal purified aldose reductase solution diluted with BSA-Tween20-PBS to each well.
It was left for a minute and then washed 5 times with Tween 20-PBS. 200 μl orthophenylenediamine-containing substrate (0.
(5 mg / ml orthophenylenediamine, 0.0075% hydrogen peroxide) was added and the reaction was carried out at room temperature for 30 minutes, then 100 μl of 4N sulfuric acid was added to the plate to stop the reaction, and Immunoreader NJ-2000 was used. The absorbance of the reaction product solution at 490 nm was measured. Each sample was measured by 20-fold dilution with BSA-Tween 20-PBS. The results are shown in Table 13.

[0048]

[Table 13] Table 13 ──────────────────────────────────── Animal species Absorbance at 490 nm ─── ───────────────────────────────── Mono-mono method Mono-poly 49-56 method ─────── ────────────────────────────── Human 1.056 1.254 0.903 Bovine 0.011 0.005 0.827 Dog 0.002 0.003 0.374 guinea pig 0.003 0.001 0.168 ─────────────────────────────── ────── Mono-mono method: Mono-mono one-step method (ARmAb55, ARmAb25) Mono-poly method: Mono-poly one-step method (ARmAb55) 49-56 method: Mono-mono one-step method (ARmAb49, ARmAb56) Human Specific to aldose reductase Black-and-white to bind to -
In the mono-mono method and the mono-poly method using the null antibody ARmAb55, when various aldose reductases were used as samples, no color was observed at 490 nm in samples other than human aldose reductase. It was a specific enzyme immunoassay.

Example 10 Monochrome binding specifically to human aldose reductase
Human aldose reductase assay using a null antibody (EIA based on competitive method) 200 μl of human aldose reductase solution dissolved in PBS at 0.5 μg / ml in each well of 96-well immunomicroplate (Nunc) Was added and allowed to adsorb at room temperature for 2 hours. This plate was washed 5 times with PBS containing 0.05% Tween20, and then BSA-Tween20-PBS was added to each well to completely block each well so that non-specific adsorption of proteins did not occur. On the other hand, human aldose reductase solution serially diluted with BSA-Tween20-PBS and BSA-Tween2 as test samples
ARMAb55 diluted to 50 ng / ml with 0-PBS and 150 mM each.
μl was added, and the mixture was reacted at room temperature for 1 hour. This reaction liquid 200
μl was added to each well of the plate on which the above-mentioned human aldose reductase was immobilized, and the mixture was reacted at room temperature for 1 hour. A vector stain ABC kit (manufactured by Vector Laboratories) was used to detect the monoclonal antibody bound to the enzyme. That is, the plate was washed 5 times with PBS containing 0.05% Tween20 (hereinafter referred to as Tween20-PBS), and 100 μl of a biotinylated goat anti-mouse immunoglobulin antiserum BSA-Tween20-PBS solution was added and reacted for 1 hour. , Then Tween2
After washing 5 times with 0-PBS, 100 µl of a solution of avidinylated peroxidase in BSA-Tween20-PBS was added and reacted for 20 minutes. After washing the plate 5 times with Tween20-PBS, 100 mM citrate buffer (pH 5.0) containing 0.03% hydrogen peroxide and 1 mg / ml orthophenylenediamine dihydrochloride in each well.
100 μl of an equal volume mixture of and was added and incubated at room temperature for 10 minutes. Then, 100 μl of 2N sulfuric acid was added to each well to stop the reaction, and the absorbance at 490 nm of the reaction product solution was measured using an immunoleader NJ-2000 (manufactured by Intermed). The results are shown in Table 14.

[0050]

[Table 14] As shown in Table 14, a calibration curve depending on the aldose reductase concentration was obtained.

[0051]

INDUSTRIAL APPLICABILITY According to the present invention, by using a monoclonal antibody that specifically binds to human aldose reductase, a trace amount of aldose reductase in human blood, urine or tissue can be simply and specifically expressed. It became possible to measure.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12P 21/08 9358-4B G01N 33/53 N // C12N 15/00 (C12P 21/08 C12R 1 : 91) (72) Inventor Sadaharu Urakami 2-5-2 Marunouchi, Chiyoda-ku, Tokyo Sanryo Gas Chemical Co., Ltd.

Claims (3)

[Claims] 1. A monoclonal antibody which binds to human aldose reductase and does not bind to bovine, dog, guinea pig, rat or rabbit aldose reductase. 2. A hybridoma producing a monoclonal antibody which binds to human aldose reductase but does not bind to bovine, dog, guinea pig, rat or rabbit aldose reductase. 3. An immunoassay for human aldose reductase using at least one monoclonal antibody which binds to human aldose reductase but does not bind to bovine, dog, guinea pig, rat or rabbit aldose reductase.