JPH0674278B2 - Glucito-lysine derivative - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel glucitol lysine derivative.

2. Description of the Related Art In recent years, under physiological conditions, especially in diabetic patients, a non-enzymatic binding reaction between sugar and protein, that is, a high concentration of glucose in the blood, the aldehyde group of glucose, and a relatively half-life in vivo. A non-enzymatic binding reaction with the amino groups of the N-terminal and side chains of the long protein of Escherichia coli was clarified, and the aldimine (Schiff's base) generated by this binding further produces a stable ketoamine by Amadori rearrangement and becomes a glycosylated protein. In addition, the measured value of this glucosylated protein correlates well with the fasting blood glucose level, and the value becomes a high value in diabetic patients, which is clearly distinguished from healthy subjects. It was clarified that the value is effective as a blood glucose control index, and its measurement enables diagnosis of diabetes mellitus, elucidation of pathological condition, investigation of prognosis, and the like.

As a method for measuring the above-mentioned glycosylated protein, the conventional TBA method (Tiobarbituric acid) method, that is, by adding a weak acid to serum and heating it, 5-hydroxymethylfurfural (5
-HMF) is released and colorimetric determination of the color development due to its binding with thiobarbituric acid (TBA) and borate affinity chromatography, that is, utilizing the cis-diol group of the sugar bound to the protein, aminophenyl It is known that the acid column is applied to a gel column fixed to a suitable carrier such as Sepharose CL-6B, and the glucosylated protein adsorbed on the gel is eluted and separated, and the colorimetric measurement is performed by ultraviolet absorption or the Lowry method. ing.

In addition, in order to facilitate operability and perform quick measurement, a technique for measuring the above-mentioned glycosylated protein by an immunoassay has been studied, and some antibodies for that purpose have been proposed.

However, in the existing measurement technology, in addition to operational problems such as complexity of operation and long time required for measurement,
Especially, in terms of accuracy, reproducibility, etc., there is still no one that meets the demand of the field (“Clinical examination” MOOK, No. 18, p60-68).
(1984), JP-A-59-119264.

In such a current situation, the present inventors previously replaced the existing method for measuring glucosylated protein with higher specificity, and made it possible to accurately grasp non-enzymatic glucosylation in a subject. Has been developed successfully, which has excellent reproducibility and reproducibility, can perform simple and rapid measurement, and is suitable as a screening means (Japanese Patent Application No. 61-10758).
issue).

That is, according to the non-enzymatic coupling reaction of protein and glucose,
It is considered that the ε-amino group of the lysine residue in the protein is glycosylated to form an aldimine and a ketone, and such a reaction also occurs in vivo. The measurement of the ketoamine makes it possible to know the degree of non-enzymatic gelcosylation, which is carried out by the use of an antibody which selectively binds to the ketoamine.

However, the above ketoamines have different conformations in solution at equilibrium in the solution, namely closed ketose and α- and β- of both 6-membered pyranose and 5-membered furanose structures.
It is known to exist in an anomer. Therefore, even if an attempt is made to adopt a method using a monoclonal antibody that recognizes ketoamine in order to carry out a highly sensitive and highly accurate immunoassay that meets the needs of the field, the monoclonal antibody is extremely highly specific in terms of its recognition. , The desired effect is difficult to hope for.

On the other hand, according to the study of the present inventors, the reduction treatment of the above-mentioned alumindin and ketoamine produces a glucitol lysine adduct (glucitol protein) which is also conformationally stable, and the reduced body fluid is used as a sample. It has been confirmed that a measurement technique making full use of the advantages of the monoclonal antibody can be provided by using the monoclonal antibody, and thus the use of a monoclonal antibody having the above-mentioned glucitol lysine (reduced glycosylated lysine) residue as an epitope. A new immunoassay technique characterized by was established.

An object of the present invention is to provide a novel glucitol lysine derivative useful as a standard antigen (standard) in the immunoassay method based on the above research results.

Composition of the invention According to the invention, the formula A glucitol lysine derivative represented by

The compound of the present invention is useful as a standard in the method for measuring glucosylated protein in body fluid described in detail below, and the use of the compound meets the requirements of the art,
It is capable of excellent measurement of glucosylated proteins, and is extremely useful for diagnosis and maintenance of diabetes.

The compound of the present invention is produced, for example, by subjecting a known non-enzymatic coupling reaction product of 6-amino-2- (benzoylamino) acetylamino-hexanoic acid and glucose to a reduction treatment. The above-mentioned non-enzymatic coupling reaction can be carried out according to a conventional method, for example, in a suitable solvent such as a common buffer solution, about 20
Incubate at about 37 ° C for about 72 to 180 hours. The reduction treatment is preferably performed at the same time as this non-enzymatic coupling reaction, and the reduction treatment can be carried out according to a reaction method generally used for reduction of Schiff's base or reduction of carbonyl group to hydroxyl group. A specific example thereof is a reduction method using a hydrogenation reducing agent such as NaBH ₄ or NaCNBH ₄ . These reducing agents are usually used in an equimolar amount to 22 times the molar amount of glucose used in the reaction, and the reaction can be carried out at about 20 to 37 ° C. for 72 to 180 hours. After completion of the reaction, the compound of the present invention can be isolated and purified according to a usual method, for example, by subjecting it to distillation and then subjecting it to a purification operation such as column chromatography.

Hereinafter, a technique for measuring a glucosylated protein using the compound of the present invention will be described in detail.

The method is a measurement technique using a monoclonal antibody that specifically recognizes a glucitol lysine residue.

The above-mentioned antibody used in the method is a hybridoma prepared by fusing mammalian plasma cells (immune cells) immunized with an immunogen having a glucitollysine residue with mammalian plasmacytoma cells. ) Is prepared, a clone producing an antibody recognizing the glucitol lysine residue is selected therefrom, and the desired antibody (monoclonal antibody) is obtained from the clone.

In the above, as the immunogen to be used, a reduced glucosylated lysine (glucitol lysine) or a reduced glucosylated protein (glucitol protein) in which a glucitol residue is bound to lysine or to a carrier protein via the lysine residue is used. ) Is generally used, but as long as it has a glucitol lysine structure, it is not limited to the above, and various kinds can be used. As typical examples thereof, for example, reduced glucosylated β-lipoprotein (Glc-LDL), reduced glucosylated polylysine (Glc-PL), reduced glucosylated bovine serum albumin (Glc-BSA), glucitosyl lysine ( Glc-L) and the like can be exemplified, but of course, the compound of the present invention can also be used as the immunogen.

The production of these antigens can be carried out by a conventional method. For example, a conjugate (aldimine or ketoamine) obtained by a non-enzymatic coupling reaction of lysine or a carrier protein with glucose may be subjected to a reduction treatment. It may be the same as that in the production of the compound.

The mammal to be immunized with the immunogen in the above method is not particularly limited, but it is preferably selected in consideration of compatibility with plasmacytoma cells used for cell fusion, and in general, mouse and rat are used. Etc. are advantageously used.

Immunization is carried out by a general method, for example, by administering the above-mentioned immunizing antigen to a mammal by intravenous administration or intraperitoneal injection. More specifically, the immune antigen is PBS
It is preferable to dilute the mixture to a suitable concentration with the above method and to administer it to the animal several times every 2 to 14 days so that the total dose will be about 1 to 100 μg / mouse. As the immune cells, splenocytes extracted about 3 days after the final administration are preferably used.

Further, as the mammalian plasmacytoma cell as the other parent cell to be fused with the above-mentioned immune cell, various already known cell lines such as p3 (p3 / x63-Ag8) [Nature, 256 , 495-49
7 (1975)), p3-U1 (Current Topics in Microbiology
and Immunolmgy, 81 , 1-7 (1978)], NS-1 [Eur.J.
Immunol., 6 , 511-519 (1976)], MPC-11 [Cell, 8 , 4
05-415 (1976)], SP2 / 0 (Nature, 276 , 269-270 (197
8)), FO [J. Immunol. Meth., 35 , 1-21 (1980)] x 63.
6.5.3. [J. Immunol., 123 , 1548-1550 (1979)], S194
[J.Exp.Med., 148 .313-323 (1978)] and the like, R210 [Nature, 277, 131-133 (1979)] in the rat bone marrow tumor cells such as are used.

The fusion reaction between the immune cell and the plasmacytoma cell is basically a known method, for example, the method of Milstein et al. [Method in Enzymology, Vol.73, pp3 (1981)].
And so on. More specifically, the fusion reaction is carried out in an ordinary nutrient medium in the presence of a fusion promoter, for example. As the fusion accelerator, those usually used,
For example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like are used, and if desired, an auxiliary agent such as dimethyl sulfoxide can be added and used in order to enhance the fusion efficiency. The ratio of immune cells to plasmacytoma cells used is the same as in the usual method, and for example, immune cells may be used in an amount of about 1 to 10 times that of plasmacytoma cells. The medium at the time of the fusion, for example, RPMI-1640 medium as used for the growth of the plasmacytoma cell line, MEM medium,
In addition, various ordinary media used for this type of cell culture can be used, and it is usually preferable to remove a serum replacement solution such as fetal calf serum (FCS). Fusion is a well-mixed predetermined amount of the immune cells and plasmacytoma cells in the medium, a PEG solution preheated to about 37 ° C., for example, an average molecular weight of about 1000 ~ 6000, in a normal medium It is carried out by adding and mixing at a concentration of 30 to 60 W / V%. After that, a desired hybridoma is formed by repeating the operation of sequentially adding an appropriate medium, centrifuging and removing the supernatant.

Isolation of the desired hybridoma obtained is carried out by culturing in a usual selection medium, for example, HAT medium (medium containing hypoxanthine, aminopterin and thymidine). Culturing in the HAT medium may be carried out for a time sufficient to kill cells other than the target hybridoma (unfused cells etc.), usually for several days to several weeks. The hybridoma thus obtained is subjected to a conventional limiting dilution method to search for a target antibody-producing strain and to perform monocloning.

The production strain can be searched by, for example, the ELISA method (Engvall, E., Meth.
nzymol., 70 , 419-439 (1980)], plaque method, spot method, agglutination reaction method, Ouchterlony
Method, radioimmunoassay (RIA) method, and various other methods generally used for antibody detection ["Hybridoma method and monoclonal antibody", R & D Planning, Inc., pp30-53, March 5, 1982]. Day]. The search may be carried out using the above-mentioned immunogen, and in particular, the immunogen used as an immunogen to eliminate an antibody that may bind to an antigenic determinant other than the glucitol-lysine residue. The use of different immunizing antigens is desirable.

Thus, the desired hybridoma producing an antibody that recognizes a glucitollysine residue is obtained. They can be subcultured in an ordinary medium and can be easily stored in liquid nitrogen for a long period of time.

The desired monoclonal antibody can be collected from the hybridoma by culturing the hybridoma according to a conventional method, and as a culture supernatant thereof, or by administering the hybridoma to a mammal compatible therewith and proliferating it, as ascites fluid thereof. The method of obtaining it is adopted. The former method is suitable for obtaining highly pure antibody, and the latter method is suitable for mass production of antibody. Further, the antibody obtained as described above is further subjected to salting out,
It can also be purified by an ordinary purification means such as an absorption method, a gel filtration method, or affinity chromatography.

The thus obtained monoclonal antibody has extremely high specificity for the glucitol lysine residue, and therefore, the use of the antibody can accurately grasp the degree of non-enzymatic glycosylation in a subject. You can Further, this enables more accurate diagnosis of diabetes and blood glucose control.

The present invention provides a method for measuring a non-enzymatic glycosylated protein in a body fluid of a subject using the above-mentioned specific antibody, and a measuring kit for the measuring method, wherein the compound of the present invention is used as a standard. It also provides a new technology that

The measurement of the above-mentioned glycosylated protein requires the use of the compound of the present invention as a standard, and its basic operation may follow a usual immunoassay method such as RIA method and enzyme immunoassay method (EIA). it can. The operations, procedures and the like in each immunoassay are not particularly different from those generally adopted, and can be based on, for example, the well-known competitive method, Sangertisch method and the like.

In the above method (hereinafter referred to as the method of the present invention), a reduction-treated body fluid can be used as a sample. Such body fluids include blood, urine, cell tissue fluid, lymph fluid, pleural effusion,
Ascites fluid, amniotic fluid, gastric juice, pancreatic juice, cerebrospinal fluid, sleep fluid and the like can be exemplified.
The reduction treatment of isotopic fluid can be carried out according to the method described, and it is usually 30% by using a reducing agent at a concentration of about 10 to 100 mM.
It can take from about a minute to 180 hours. It is most preferable to use these samples after the proteinaceous fraction in them has been collected (or glucose removed) in advance before the reduction treatment. The fractions are collected by a method known per se, for example, a treatment method using an organic solvent such as acetone, methanol, ethanol, propanol, dimethylformamide (DMF) as a protein precipitant, ammonium sulfate, sodium sulfate, sodium phosphate, etc. The treatment method using a salting-out agent, the ultrafiltration treatment method using a dialysis membrane, a flat plate membrane, a hollow fiber membrane and the like, and a combination thereof. In addition, according to the above-mentioned known method, when purified to albumin or lipoprotein fractions and used as a sample after reduction treatment, instead of the total amount of non-enzymatic glycosylated protein, specific protein specific to The amount of non-enzymatic glycosylation can be known.

Further, the above method can also be based on an insolubilization method (method using an insolubilized antigen or an insolubilized antibody). In this case, the insolubilized antigen and the insolubilized antibody are produced by chemically or physically reacting the compound of the present invention or the immunogen or the antibody with an insoluble carrier according to a conventional method. Examples of the insoluble carrier include cellulose powder, cefadex, sepharose, polystyrene, paper, carboxymethyl cellulose, ion exchange resin, dextran, plastic film, plastic tube, nylon, glass beads, silk, polyamine-methyl vinyl ether-maleic acid co-polymer. Polymers, amino acid copolymers, ethylene-maleic acid copolymers and the like can be used. The insolubilization is carried out by the covalent bond method such as diazo method, peptide method (acid amide derivative method, carboxychloride resin method, carbodiimide resin method, maleic anhydride derivative method, isocyanate derivative method, cyanogen bromide activated polysaccharide method, cellulose carbohydrate method. Chemical methods such as nato derivative method, method using condensation reagent, etc., alkylation method, carrier binding method using cross-linking reagent (glutaraldehyde, hexamethylene isocyanate, etc. are used as cross-linking reagent), carrier binding method by Ugi reaction, etc. Reaction; or an ionic bonding method using a carrier such as an ion exchange resin; and a physical adsorption method using porous glass such as glass beads as a carrier.

As the labeled antigen or labeled antibody, those obtained by labeling the compound of the present invention or the immunogen or the antibody with various labeling agents such as usual radioactive substances, enzyme-labeled substances and fluorescent substances are used. As the radioactive substance as the labeling agent,
Radioactive iodine such as ¹²⁵ I is used as a fluorescent substance as fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), and substituted rhodamine isothiocyanate (XRI).
TC), rhodamine B / isothiocyanate, dichlorotriazine fluorescein (DTAF), etc., as enzyme labeling substances, peroxidase (POX), microperoxidase, chymotrypsinogen, procarboxypeptidase, glyceroaldehyde-3-phosphate Examples thereof include dehydrogenase, amylase, phosphorylase, D-nase, P-nase and the like. The labeling method by these can also follow a conventional method [J. Biol. Chem., 254 , 9349-935.
1 (1979); Nature, 194 , 495 (1962); Fluorescent Antibody Method, Medical Chemistry Laboratory No. 4, 263-270; Acta. Endocrinol. Suppl, 168 ,
206 (1972);. Proc.Nat.Acad.Sci, USA, 57, 713 (1967)
Etc.].

The method for measuring and quantifying the test substance (reduced glucosylated lysine residue amount) in the sample in the method of the present invention is the insolubilized antigen, insolubilized antibody, using the compound of the present invention as a standard.
It is carried out by immunoreacting with either a labeled antigen or a labeled antibody. At that time, the solvent used in the measurement system is a usual solvent that does not adversely affect the reaction, for example, a buffer having a pH of about 4 to 8 such as a citrate buffer solution, a phosphate buffer solution, a Tris-hydrochloric acid buffer solution, an acetic acid buffer solution or the like. The liquid can be exemplified as a preferable one. Further, the immune reaction conditions at the time of measurement are not particularly limited, and may be the same as the usual measuring method of this kind. That is, the immunoreaction is generally carried out at a temperature of 45 ° C. or lower, preferably about 4 to 40 ° C. for 1 to 40 hours. Conjugates and educts (B-
The separation of F) also follows a known method, for example, when the insolubilization method is adopted, the solid phase-liquid phase can be separated by a separation means such as centrifugation, separation, washing, decantation and the like. In other cases, conventional methods such as the dextran-activated carbon method and the second antibody method may be used.

Hereinafter, the method of the present invention will be described by taking an insolubilization method with a simple operation as an example. (1) First, a test substance in a sample to be measured and a certain amount of an insolubilized antigen are fixed to a labeled antibody. React competitively with the amount,
Then, BF separation is performed, and the labeling agent activity of either one is measured to quantify the amount of the test substance. (2) First, the test substance and a fixed amount of the labeled antigen are mixed with a fixed amount of the insolubilized antibody. A competitive reaction is carried out, followed by BF separation, and the activity of either labeling agent is measured to quantify the amount of the test substance. (3) The test substance and the insolubilized antibody are reacted to form an immune complex. Then, a certain amount of the labeled antibody is reacted with this complex, the activity of the labeling agent of the labeled antibody bound to the complex is measured, and the amount of the test substance is quantified. Can be carried out by using

Thus, the amount of reduced glucosylated lysine residue in the sample can be measured, which sensitively reflects the degree of non-enzymatic glucosylation in the subject. By the research of the present inventors, the above-mentioned measuring method specific to a specific protein in a body fluid has been developed. Taking albumin as an example, the method is carried out by the following method and is extremely simple.

(4) By adding an insolubilized anti-human serum albumin antibody to a sample to form an immune complex, reacting the complex with a labeled antibody, and measuring the labeling activity of the labeled antibody bound to the complex, The degree of non-enzymatic glucosylation specific to albumin can be grasped.

The degree of non-enzymatic glucosylation measured by the above various methods, by using the compound of the present invention as a standard, for example, a unit protein amount or a specific, for example, albumin molecule, of the glucitol lysine residue It can be expressed as the number of moles. Furthermore, the compound of the present invention can be used as a reference substance in terms of number of moles of other standards whose reaction amount is unknown.

A particularly convenient way to carry out the above measurement method is to use a kit. It is essential for such a kit to contain the compound of the present invention as a standard, and it is important to further contain the monoclonal antibody as an antibody reagent. Stabilizers and / or preservatives such as glycerol and bovine serum proteins can be added to this antibody reagent. Preferably, this antibody reagent is freeze-dried, and the kit may contain a water-soluble or water-miscible solvent. Furthermore, a buffer solution for keeping the reconstituted reagent system at a constant pH and / or a preservative and / or stabilizer for preventing deterioration of the sample before use may be added to the antibody reagent. it can. The standard is usually in the form of a solution dissolved in an appropriate buffer or the like. The buffer solution is not considered to be an essential component of the kitting reagent, but when the assay method of the present invention is carried out, the pH is adjusted to 4
It is preferable to use a material having a thickness of about 8 or so. The reconstitution agent preferably contains water, but part or all of the water can be replaced with a solvent miscible with water.
Solvents miscible with water are well known to those skilled in the art, and for example, glycerin, alcohols, glycols, glycol ethers and the like can be used.

Effects of the Invention According to the present invention, a glucitol lysine derivative having a glucitol lysine residue and useful as a standard is provided. The derivative has excellent immunoreactivity, and by utilizing it, the degree of non-enzymatic glucosylation can be accurately grasped, and it is possible to favorably perform diabetes diagnosis and blood glucose control in a subject.

Examples Hereinafter, examples and reference examples will be given to describe the present invention in more detail.

Example 1 Production of glucitol lysine derivative of the present invention N-2- (N-benzoylglycyl) -L-lysine (Protein Research Promotion Society) 200 mg and D-glucose 129 mg were mixed with water and dioxane (1: 1). Dissolve in 10 ml and add NaB
100 mg of CNH ₃ was added, and the mixture was kept at room temperature for 3 to 4 days. Next, acetic acid was added to the reaction system to stop the reaction, and after the distillation, methanol was added and the mixture was distilled.

Purify this by column chromatography using TSK120T column (manufactured by Toyo Soda Co., Ltd.) [solvent A; 90% acetonitrile, internal standard 50 mM TFA, solvent B; 5% acetonitrile, gradient A10% + B90% → A60% + B40%]. Then, the compound of the present invention, that is, N2- (N-benzoylglycyl) -N6-D-glucitol-L-lysine was obtained at a retention time of 9.93 minutes (2 ml / min). Yield 58.2%.

The PMR analysis chart of the obtained compound of the present invention is shown in FIG. The main peaks are as follows.

_{PMR (DMSO-d 6, 2.50ppm} ): δ (ppm) (400MH) = 8.69 (t, J = 6.0), 8.30 (m) 8.15 (d, J = 7.9) 7.87 (d, J = 7.6) 7.54 ( t, J = 7.6) 7.48 (t, J = 7.6) 4.23 (ddd, J = 8.3,7.9,4.6) 3.98 (dd, J = 16.7,6.0) 3.87 (dd, J = 16.7,6.0) 3.67 (dd, J = 4.8,1.2) 3.60 (dd, J = 10.4,2.8) 3.40 to 3.51 (m) 3.07 (dd, J = 12.5,3.7) 2.94 (dd, J = 12.5,8.0) 2.88 (m), 1.74,1.61 (M), 1.34 (m) Further, the compound of the present invention obtained above was hydrolyzed with 6N-HCl (120 ° C., 20 hours), and then analyzed with an amino acid analyzer (Hitachi 835).

As a result, glycine and glucitollysine were confirmed in almost equimolar amounts.

Reference Example 1 Production of immune antigen 100 mg of β-lipoprotein (LDL, Sigma) was added to 50 mM
Dissolve in 10 ml of PBS (pH 7.4) and add D-glucose 7
Added 00mg and Na CNBH ₄ 200 mg, was held 4-7 days at room temperature. Next, acetic acid is added to the reaction system to stop the reaction,
It was dialyzed against distilled water. The dialysate was freeze-dried to obtain the desired reduced glycosylated protein (Glc-LDL).

In the above, instead of β-lipoprotein, polylysine (PL, Protein Research Encouragement Society), BSA, lysine (L) and human serum albumin (HSA) were used, respectively, and reduced glucosylated protein and reduced glucosylated amino acid were similarly prepared. (Glc-PL, Glc-BSA, Glc-L and Glc-HSA) were obtained.

Each of the glycosylated proteins obtained above was hydrolyzed with 6N HCl (120 ° C, 20 hours), and then amino acid analysis was performed with an amino acid analyzer to determine Gl for total lysine (Lysine).
The content ratio of c-L was determined.

As a result, it was 29.89% for Glc-LDL and 2 for Glc-PL.
4.10%, Glc-BSA 15.56%, Glc-HSA
Was about 10% (this is referred to as "10% Glc-HSA"). In addition, in the same manner as above, the same content ratio of 61.6% Glc-H
SA (this is designated as "61.6% Glc-HSA") was also obtained.

Production of Monoclonal Antibody -1 Glc-LDL obtained above was applied to Balb / c mouse at 5 μm.
It was administered subcutaneously at a dose of g / mouse. The above administration was repeated 5 times every other week for immunization, and 3 days after the final immunization, the spleen was removed,
Splenocytes were washed 3 times with RPMI-1640 medium.

Mouse osteoma cell line P3-U1 [Current Topics in Microb
[Biology and Immunology, 81, 1-7 (1978)] was washed in the same manner, and 1 × 10 ⁶ thereof and 1 × 10 ^{7 of the} above spleen cells were put into a 50 ml centrifuge tube and mixed. After centrifugation at 200 xg for 5 minutes, the supernatant was removed with a Pasteur pipette. Polyethylene glycol 2000 (Wako Pure Chemical Industries, Ltd.) kept at 37 ℃ 35 W / V% RPMI-1
1 ml of 640 solution was added dropwise, and the mixture was gently mixed for 10 minutes.
RP containing 15% FCS and 1 mM pyruvate kept at 37 ℃
5 ml of MI-1640 (hereinafter referred to as "complete RPMI") was added for 10 minutes, the same amount of complete RPMI was added for 4 minutes, and then 5 ml of complete RPMI was added dropwise and stirred for 1 minute each. After centrifuging at 200 xg for 5 minutes, the supernatant was removed, and after repeating this procedure again, suspend the cells in complete RPMI kept at 37 ° C to give 1 x 10 ⁷ cells / ml. A plate (Falcon) was inoculated with 1 ml each and cultured at 37 ° C. in a 5% carbon dioxide incubator. 24 hours later Complete RPMI medium containing 1.0 × 10 ^-4 M hypoxanthine, 4.0 × 10 ^-7 M aminopterin and 1.6 × 10 ^-5 M thymidine (hereinafter referred to as "HAT medium") 1
ml was added to each well. After that, half of the supernatant is
On day 5 and 5 each, a new HAT medium was replaced, and half of the supernatant was mixed with 1.0 × 10 ⁻⁴ M hypoxanthine and
The complete RPMI medium containing 1.6 × 10 ⁻⁵ M thymidine (hereinafter referred to as “HT medium”) was used. Similarly, on days 6, 7 and 9, half of the supernatant was replaced with HT medium and on day 10 half of the supernatant was replaced with complete RPMI medium. Thereafter, the growth and maintenance were carried out in this complete RPMI medium.

The hybridoma thus obtained was cloned by the limiting dilution method. That is, 2.5 × 10 hybridomas / ml,
Balb / c mouse complete with thymocytes at 4 × 10 ⁶ cells / ml
RPMI medium was prepared, and this was seeded on a 200-well plate so that the number of hybridomas was 5 cells / well, and the cells were cultured.
The growing hybridomas were further cloned in the same manner as 0.25 hybridomas / well.

The clones that produce the desired antibody can be searched by the immobilization method using goat anti-mouse immunoglobulin (Katsupel) labeled with ¹²⁵ I or an enzyme using the plate or beads on which the immunogen (Glc-BSA) is immobilized. Done.

Thus, a desired hybridoma producing a monoclonal antibody represented by clone No. OAL-M-10 and having the specific reactivity described below was obtained.

-2 Clone No. OAL-M-10 obtained in -1 above
Was cultured in a complete RPMI medium in a 5% carbon dioxide incubator at 37 ° C. for 48 hours. Centrifuge the culture (3000
rpm, 10 minutes) to obtain a culture supernatant containing the desired monoclonal antibody.

The above antibody belonged to the IgG ₂ a subclass. This is a rabbit antibody (Litton.Bionetico.Inc.Kensington.MD20795) against various mouse immunoglobulin classes and ¹²⁵
The method of Yeh et al. Using I-labeled protein A (Ming-Yang Yeh et al., Proc. Natl. Acad. Sci., USA, Vo
l.76, No. 6, 2927-2931 (1979)].

-3 1 × 10 ⁶ clone No. OAL-M-10 obtained in -1 above was suspended in 0.5 ml of RPMI-1640 medium and intraperitoneally administered to Balb / c mice. After 2-3 weeks, the accumulated ascites was collected to obtain 2-5 ml / mouse of the ascites containing the target antibody. The concentration of this antibody was about 0.2 to 1 mg / ml. PBS5 in 5 ml of this ascites
ml and 10 ml of saturated ammonium sulfate were added, and the mixture was gently stirred at 0 ° C. The precipitate obtained by centrifugation (10000 rpm × 30 minutes, 4 ° C.) was applied to a gel filter on a Sephadex G-25 column (manufactured by Pharmacia) equilibrated with 0.05 M Tris-HCl (pH 8.6). The fraction eluted near the void volume was applied to Protein A-Sepharose CL-4B (manufactured by Pharmacia) equilibrated with the same buffer as above, to adsorb the IgG fraction, and then pH.
After thoroughly washing with 50mM citrate buffer 5.5, to elute IgG ₂ a in acetate buffer pH 4.3, to obtain a purified antibody OAL-M-10.

Preparation of insolubilized antibody The purified antibody obtained in -3 above was treated with 0.15M NaCl and 0.05% Na.
The amount of protein was adjusted to 20 μg / ml with 50 mM PBS (pH = 7.4) containing N ₃ .

Polystyrene beads (Precision Plastic Co. Ltd., US
A, diameter 6.4 mm) 10,000 pieces are thoroughly washed with diluted "Mama Lemon" (Lion Co., Ltd., undiluted solution 1.5 ml / l distilled water),
Further, it was washed with distilled water. Next, this was immersed in a 0.5 M aqueous sodium hydroxide solution for 3 days, and then washed with distilled water until the washing liquid had a pH of about 6.

To 1000 ml of the above antibody solution, 800 beads were added, and the mixture was allowed to stand for 2 hours under reduced pressure with occasional stirring, and then left at 4 ° C. overnight. After passing the beads and washing with physiological saline,
50 mM PBS (pH = 7.4) containing 5% plasma BSA (Seikagaku Corporation)
The mixture was left under reduced pressure for 2 hours and then at 4 ° C. overnight. The beads were removed and washed thoroughly to obtain an insolubilized antibody.

Preparation of Labeled Antigen -1 A solution of 50 μg of the immunogen Glc-BSA obtained above in 200 μ of 0.1 M borate buffer (pH = 8.2) was added to Na ¹²⁵ I.
(NEN) 1 mCi was added. Iodogen (Pierc)
e company) 40 μg / 20 μ dichloromethane solution was put into a glass glass test tube, the solvent was blown off under a nitrogen gas stream, and dried.
The above antigen solution was added to this test tube, and the mixture was kept at 0 ° C. for 5 minutes.
The reaction was carried out with gentle stirring. After transferring this reaction product to another test tube and stopping the reaction, gel filtration (Sefakureel S200, 1 x 38 cm, eluent = 0.2 mM gelatin containing 50 mM
The albumin fraction corresponding to the radioactivity peak was collected with PBS (pH 7.4) to obtain ¹²⁵ I-labeled antigen.

In addition to the above, the chloramine T method [Nature, 194 , 496 (19
62)] and the Bolton-Hunter method [Biochem. J., 89 , 114.
(1963)], good ¹²⁵ I-labeled antigens were obtained.

-2 An enzyme-labeled antigen was prepared using the lysine residue of peroxidase. That is, 10 mg / ml peroxidase was dissolved in 50 mM phosphate buffer, and 70 mg of glucose was added to this.
And NaBH _{4 (} 20 mg) were added, and the mixture was reacted at 4 ° C. for 7 days. After dialysis, purification by gel filtration and affinity chromatography was performed to obtain the desired labeled antigen.

Preparation of labeled antibody Using the purified antibody obtained in -3 above, ¹²⁵ I-labeled antibody was obtained in the same manner as in -1 above.

Preparation of insolubilized antigen Using the immunizing antigen Glc-PL obtained above, an insolubilized antigen was obtained in the same manner as above.

Specificity of antibody Among the immunogens obtained above, soluble Glc-BSA and Glc-L
DL and Glc-L were used as standards. As controls, β-lipoprotein, D-glucose, D-sorbitol, lysine and polylysine were used.

The 12-fold diluted solution 100μ of the antibody obtained in the above-mentioned -2, 100μ of the above standard or control of a serial dilution series, and 100μ of the ¹²⁵ I-labeled antigen obtained in the above-mentioned -1 (about 20000cpm),
Atsushi buffer (0.15M NaCl, 0.1% gelatin and
It was added to 300 µ of 50 mM sodium phosphate buffer (pH 7.4) containing 0.02% NaN ₃ and incubated overnight at 4 ° C. Anti-mouse IgG goat antibody (× 40, manufactured by Japan Antibody Research Institute) 100μ, normal mouse serum (× 400) 100μ
And 12.5% polyethylene glycol 200μ were added and incubated at room temperature for 30 minutes. Centrifuge (30
00 rpm × 30 minutes) and decantation were carried out to perform BF separation, and the radioactivity of both was measured.

Results are shown in FIG. In the figure, the vertical axis represents B / B ₀ (%), the lower horizontal axis represents the standard concentration (μg / ml), and the upper horizontal axis represents the control concentration (μg / ml). Also in the figure (1)
Is Glc-BSA, (2) is Glc-LDL, (3) is Glc-L,
(4) shows the results for β-lipoprotein, (5) for D-glucose, (6) for D-sorbitol, (7) for lysine, and (8) for polylysine.

From the above FIG. 2, it is clear that the above antibody has extremely high specific reactivity with the glucitol lysine residue.

Standard curve (calibration curve) In the above, a standard curve was prepared by using the compound of the present invention as a standard.

Results are shown in FIG. In the figure, the vertical axis represents B / B ₀ (%) and the horizontal axis represents the concentration of standard (ng / ml). In the figure, (1) shows the results using the compound of the present invention as a standard,
Also, (2) shows the results using 61.6% Glc-HSA as a standard.

From the above, 61.6% Glc-HSA shows that 61.6% lysine residues are glycosylated from the results of the amino acid analysis, but by using the compound of the present invention as a standard, 61.6% Gl
It was confirmed that Glc-Lys residues involved in the immune reaction of c-HSA accounted for 28.6% of all lysine residues.

-1 Serum was collected and separated from diabetic patients and normal persons.

To each 50 μm of this serum, 80% ethanol (2 m) was added and mixed, followed by centrifugation (3000 rpm × 15 minutes) to obtain a precipitate. This was dissolved in 500 mM of 50 mM PBS containing 10 mM NaBH ₄ and left for 30 minutes. The reaction was stopped by adding 10 μ of 5% acetic acid, 500 μ of 50 mM PBS containing 0.1% gelatin was further added, and the thus prepared sample was used as a “sample”.

(2) 100 µ of each of the above-mentioned samples and 100 µ (about 40,000 cpm) of the ¹²⁵ I-labeled antibody obtained above were incubated at 37 ° C for 1 hour. One insolubilized antigen (bead) obtained above was added to this, and further incubated at 37 ° C. for 1 hour. After washing the beads with distilled water, their radioactivity was measured.

As a result, there is a clear distinction between normal and diabetic patients,
It was confirmed that the degree of non-enzymatic glycosylation was high in diabetic patients.

-3 To 250 μm of the above sample, one insolubilized anti-human serum albumin antibody (bead) was added, and incubated at 37 ° C. for 1 hour. After washing with distilled water, ¹²⁵ I-obtained above
About 40,000 cpm of labeled antibody was added and incubated at 37 ° C for 1 hour. After washing with distilled water, its radioactivity was measured.

This measurement result can be displayed as the number of moles of glucitollysine lysine residues from the standard curve shown in FIG. 3, and the degree of non-enzymatic glucosylation in diabetic patients was accurately grasped. .

[Brief description of drawings]

FIG. 1 is a PMR analysis diagram of the compound of the present invention, FIG. 2 is a graph for determining the specificity of the antibody, and FIG. 3 is a standard curve using the compound of the present invention as a standard.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location G01N 33/577 9015-2J

Claims (1)

[Claims] 1. A formula A glucitol lysine derivative represented by: