JPH07238096A - Antipolyubiquitin-monoclonal antibody and method for measuring polyubiquitin - Google Patents

Abstract

PURPOSE:To obtain the subject antibody for measuring a polyubiquitin, etc., derived from an antibody-forming cell by immunization of an animal against a polyubiquitinized protein formed in vitro, being specifically reacted with polyubiquitin but not with monoubiquitin. CONSTITUTION:Ubiquitin is reacted with a denatured lysozyme in 100mM tris- hydrochloric buffer solution (Ph 9.0) at 37 deg.C for 1 hour to give a polyubiquitinized protein such as polyubiquitinized lysozyme. The polyubiquitinized protein is administered to BALB/c mouse and the mouse is immunized. After the final immunization, a splenocyte is collected, subjected to cell fusion with a mouse.myeloma cell to give a hybridoma. Then, a clone capable of producing an anti-polyubiquitin is selected from the hybridoma, the clone is cloned into a monoclone. The clone is cultured in a medium to give the objective anti-polyubiquitin.monoclonal antibody which is capable of specifically reacted with a polyubiquitin, but not with monoubiquitin and measuring polyubiquitin in high sensitivity.

Description

Detailed Description of the Invention

[0001]

The present invention relates to an anti-polyubiquitin monoclonal antibody which specifically binds to polyubiquitin,
And a method for measuring polyubiquitin using the monoclonal antibody.

[0002]

2. Description of the Related Art Ubiquitin is a protein that is ubiquitously present in eukaryotes and contains 76 amino acid residues as shown in the following (Chemical formula 1). Ubiquitin is present in cells of the whole body even in humans and has an important function as described below.

[0003]

[Chemical 1]

To date, ubiquitin has been clarified to have a role as a promoter of selective proteolysis. That is, in cells, E1 (ubiquitin activating enzyme), E2 (ubiquitin conjugating enzyme), and E3
The C-terminal (Gly = glycine) of ubiquitin (Chemical Formula 1) is isopeptide-bonded to a lysine residue in a certain target protein by the action of an enzyme group called (ubiquitin discrimination protein). Then, the reaction in which the C-terminal of another ubiquitin is bound to the lysine residue in the bound ubiquitin is continuously catalyzed, so that the chain complex of ubiquitin (polyubiquitin) eventually binds to the target protein. (This reaction is called a (poly) ubiquitination reaction). The target protein to which polyubiquitin is bound in this way is immediately degraded by a proteolytic enzyme that recognizes the polyubiquitin structure.

It is considered that the E3 protein plays a role in selecting which protein the ubiquitination reaction described above targets. The important point in such a ubiquitin system is that when free ubiquitin (free ubiquitin) binds to a target protein to become polyubiquitin,
Biochemically, it functions as another substance.

Ubiquitin is known as a kind of heat shock protein in cells and organisms, and its production is generally accelerated in response to various cell invasions. The progress of
It is considered to be due to the treatment of denatured protein that increases during invasion of cells and the like. Furthermore, it was proved by the immunohistochemical method using an antibody against ubiquitin that ubiquitin is accumulated in intracellular inclusion bodies that are characteristically exhibited in Alzheimer's disease, Parkinsonism syndrome, Pick's disease, motor neuron disease, etc. (Mayer, RJ et al., B
iochem. Biophys. Acta, 1089 , 141-157, 1991). It has been recently confirmed that ubiquitin accumulated in intracellular inclusion bodies of Alzheimer's disease is bound to abnormal phosphorylated tau protein as polyubiquitin (Morishima et al., Biochemistry, 65 , 662, 1993). Based on these findings, in the above-mentioned diseases such as Alzheimer's disease, the degradation of a specific polyubiquitinated protein fails for some reason, and the possibility that it accumulates in inclusion bodies and the denatured protein It is considered possible that they will undergo polyubiquitination after being sequestered by inclusion bodies (Mayer, R.
J. et al., Biochem. Biophys. Acta, 1089 , 141-157,
1991). From this point of view, the fate of ubiquitin in inclusion bodies is expected to be in good agreement with the progression of the pathology of the above diseases. Therefore, quantifying polyubiquitin contained in tissues or body fluids is important for clinical examination of these diseases. , Has important significance.

[0007]

However, in the prior art, the analysis of polyubiquitin formation had to be carried out by using a ubiquitin labeled with a radioactive substance or a complicated measuring system using a substrate protein.

More specifically, as a conventional method for quantifying ubiquitin, [ ³ H] ATP and ubiquitin are enzymatically reacted in the presence of a ubiquitin activating enzyme (E1 enzyme) to produce an enzyme reaction product. -[ ³ H] ATP-
Bioassay for measuring ubiquitin complex (Rose,
IA, et al., Proc. Natl. Acad. Sci. USA, 84 , 14
77-1481, 1987); Immunostaining method in which ubiquitin or a ubiquitin-protein complex is immobilized on a nitrocellulose membrane, subjected to electrophoresis, and then immunostained to measure the ubiquitin-protein complex (Haas, AL and Bright. , PM, J.
Biol. Chem., 260 , 12464-12473, 1985); Radioimmunoassay using antibodies against ubiquitin; Mana
ka, H. et al., Neurosci. Lett., 13 9 , 147-149, 199
2); Competitive enzyme immunoassay (Wang, GP, e
al., Acta Neuropathol. 82 , 6-12, 1991) and the like. These assay systems have been proved to be useful for the quantification of free ubiquitin, but no study on their cross-reactivity with polyubiquitin has been made. In principle, the above-mentioned bioassay utilizes the reaction to the C-terminal of ubiquitin, and therefore polyubiquitin cannot be measured. In addition, the radioimmuth assay described above
It is also not suitable for quantification of polyubiquitin because it uses an antibody prepared by using free ubiquitin as an antigen, and the competitive enzyme immunoassay uses free ubiquitin as a competitor.

In fact, according to the studies by the present inventors, in a radioimmunoassay using an ordinary antibody (polyclonal antibody) against ubiquitin prepared using a ubiquitin-carrier protein complex as an antigen, the antibody is not polyubiquitin at all. It has been found not to intersect. This means that free ubiquitin and polyubiquitin are recognized as different substances immunologically.

There have been attempts to measure ubiquitin in body fluids of patients with various diseases using the above-mentioned conventional measuring system.
In this case, the measured values of ubiquitin obtained do not necessarily match the course of the above-mentioned diseases (Manaka, H. et al.
al., Neurosci. Lett., 139 , 147-149, 1992). According to the knowledge of the present inventor, one of the causes of such inconsistency is that the measured value of ubiquitin does not reflect the polyubiquitin concentration.

The present invention is to provide a method for measuring polyubiquitin which overcomes the above-mentioned drawbacks of the prior art.

More specifically, an object of the present invention is to provide a method for measuring polyubiquitin, which enables simple analysis of polyubiquitination reaction.

Another object of the present invention is to provide a method for measuring polyubiquitin capable of quantifying a trace amount of polyubiquitin in a sample.

Still another object of the present invention is to provide a method for measuring polyubiquitin capable of quantifying polyubiquitin with high sensitivity and reproducibility.

Still another object of the present invention is to provide a monoclonal antibody which can be suitably used in the method for measuring polyubiquitin having the above-mentioned characteristics.

[0016]

[Means for Solving the Problems] As a result of earnest research, the present inventors have found that an antibody-producing cell obtained when an animal is immunized with an artificially (in vitro) prepared polyubiquitinated protein as an antigen is used. , It was found to be extremely useful for obtaining a monoclonal antibody which has a specific binding property to polyubiquitin and does not substantially react with monoubiquitin. As a result of further studies, the present inventors have found that immunological measurement using such a monoclonal antibody having specific binding property is extremely useful for specific measurement of polyubiquitin. .

The monoclonal antibody of the present invention is based on the above findings. More specifically, it is a monoclonal antibody derived from antibody-producing cells based on immunization of a polyubiquitinated protein produced in vitro to an animal, It is characterized in that it specifically reacts with ubiquitin and does not substantially react with monoubiquitin.

According to the present invention, there is further provided a monoclonal antibody derived from an antibody-producing cell based on immunization of a polyubiquitinated protein produced in vitro with an animal,
A monoclonal antibody that specifically reacts with polyubiquitin and does not substantially react with monoubiquitin is used; utilizing the specific binding property between the antibody and polyubiquitin, polyubiquitin is immunologically determined (that is, There is provided a method for measuring polyubiquitin, which comprises measuring (using an antigen-antibody reaction).

Hereinafter, the present invention will be described in detail with reference to the drawings as necessary.

(Polyubiquitinated Protein) In the present invention, the polyubiquitinated protein that can be produced in vitro is not particularly limited, but it is usually a protein that is easily degraded in a ubiquitin-dependent manner in vivo (ie, A protein that is easily polyubiquitinated by the action of the above-mentioned enzymes E1, E2, and E3 in vivo,
Hereinafter, it is preferable to use "target protein").

More specifically, in the present invention, one or more (preferably any two of the following), especially
The protein satisfying the condition 3) can be preferably used.

Having one or more lysine residues at appropriate positions in the molecule.

The N-terminal amino acid is any one of basic amino acids such as arginine and lysine; hydrophobic amino acids such as leucine, tryptophan, phenylalanine and tyrosine; and some hydrophilic amino acids such as histidine, alanine, serine and threonine. Consist of.

"Folding state" (foldin) of protein molecules
g) is loose (ie in an unfolding state).

In the present invention, as such a protein, for example, lysozyme, ribonuclease A, hemoglobin and the like are preferably used, and particularly, lysozyme, ribonuclease A and hemoglobin which are thermally or chemically denatured are preferably used. . In addition to these, various proteins can be used in the present invention (Ciecha
nover, A. and Schwartz, AL, TIBS, 14 , 48
3-488, 1989; Rechsteiner, M., Cell, 6
6 , 615-618, 1991; Vershavsky, A., Ce.
11, 69 , 725-735, 1992).

(In vitro polyubiquitination reaction system)
In the present invention, the polyubiquitination reaction system in vitro is not particularly limited as long as the target protein can be polyubiquitinated. That is, E derived from eukaryote
A system containing 1, E2, E3 can be used. Rabbit reticulocytes (including a relatively large amount of E1, E2, and E3) or yeast can be preferably used as eukaryotic cells from the viewpoint of efficient reaction. For these eukaryotic cells, homogenates, suspensions, or hemolysates thereof may be used as they are, or the above enzymes may be purified (that is, the purity of the above enzymes may be increased) if necessary. May be.

(Animal) In the present invention, as an animal from which antibody-producing cells are to be collected, an animal capable of producing an antibody by immunization with the above polyubiquitinated protein can be used without particular limitation. More specifically, for example, cow, pig,
Mammals such as rabbits, sheep, goats, dogs, cats, rats and mice are preferably used. From the viewpoint of efficient immunization, rat or mouse is preferably used,
Mice (for example, BALB / C mice) are particularly preferably used from the viewpoint of compatibility with tumor cells (for example, plasmacytoma) which is a partner of cell fusion with antibody-producing cells.

(Method for Obtaining Monoclonal Antibody) As a method for obtaining the monoclonal antibody of the present invention, an ordinary method (for example, refer to “Monoclonal Antibody Experimental Manual”, Kodansha, 1987, edited by S. Tomiyama and Tamie Ando) is used. It is possible.

More specifically, for example, an antigen consisting of an appropriate amount (usually about 1 to 100 μg) of the above polyubiquitinated protein may be added to the above animals together with an equivalent amount of an adjuvant (for example, complete Freund's adjuvant) if necessary. The animals are immunized by intraperitoneal or subcutaneous administration. If necessary, the first immunization may be followed by one to several booster immunizations at 2-3 week intervals. Two to three days after the final immunization, splenocytes are taken out and fused with appropriate tumor cells to obtain hybridomas (fused cells). By cloning the hybridoma thus obtained, the target hybridoma producing the monoclonal antibody can be obtained.

(Reactivity of Monoclonal Antibody) The monoclonal antibody of the present invention is an antibody having a specific binding property that specifically reacts with polyubiquitin and does not substantially react with monoubiquitin.

The specificity of such a monoclonal antibody of the present invention can be evaluated, for example, as follows.

That is, the degree of competitive inhibition of unlabeled polyubiquitin and free ubiquitin (monoubiquitin) with respect to the binding between radioactive iodine ( ¹²⁵ I) -labeled polyubiquitin and a monoclonal antibody (for which specificity should be measured) is determined by radioactivity. The binding (B) of the polyubiquitin-monoclonal antibody to the binding (A) of the free ubiquitin-monoclonal antibody is measured by an immunoassay method. In this case, the relative ratio (B / A) of the above-mentioned binding properties of the monoclonal antibody of the present invention is preferably 10 ² or more, more preferably 10 ³ or more (particularly 10 ⁴ or more).

The above radioimmunoassay can be performed, for example, as follows.

The ¹²⁵ I-labeled polyubiquitin used in this assay can be obtained by modifying a part of the method for preparing a polyubiquitin sample of Example 7 described later. That is, in the method of Example 7, ubiquitin added to the reaction solution was ¹²⁵
The amount of ¹²⁵ I-labeled ubiquitin is increased 1000 times (about 50,000,000).
A ¹²⁵ I-labeled polyubiquitin fraction having high radioactivity is obtained in the same manner as in Example 7, except that the cpm is equivalent to 1 μg). 10% of this containing 1% bovine serum albumin (BSA)
mM phosphate buffer, 150 mM sodium chloride, pH
Approximately 20,0 in assay buffer consisting of 7.2 (PBS)
Dilute to 00 cpm / 0.1 ml to prepare ¹²⁵ I-labeled polyubiquitin solution.

A monoclonal antibody solution is also prepared by diluting with a similar assay buffer solution, and the dilution rate at this time is such that about 25 to 50% of ¹²⁵ I-labeled polyubiquitin binds in this assay method. And is obtained in advance.

The primary reaction of the assay was 0.1% of this antibody solution.
0.1 ml of unlabeled sample solution (polyubiquitin or free ubiquitin) having a dilution series of several tens of pg / ml to 1 μg / ml was added to ml, and ¹²⁵ I-labeled polyubiquitin solution 0.1 ml was added to the assay. 0.2 ml of buffer solution
And are added and the reaction is carried out at room temperature for 15 hours. Then, 0.05 ml of anti-mouse immunoglobulin goat serum (10-fold diluted, manufactured by Organon Technica) and 2% normal mouse serum 0.
After adding 05 ml and 0.1 ml of 7% polyethylene glycol 6000 (both of which are assay buffers), the reaction was carried out at room temperature for 1.5 hours (second reaction), and then 20
Centrifuge at 00 × g for 30 minutes to obtain a precipitate consisting of the antibody and the antigen-antibody conjugate.

The amount of ¹²⁵ I-labeled polyubiquitin bound to the antibody can be determined by measuring the radioactivity of the precipitate with a gamma counter. The binding property of a sample can be determined from the competitive inhibition curve obtained by the dilution series of the sample.

(Immunological Assay Method) Since the monoclonal antibody of the present invention has an excellent binding property to polyubiquitin as described above, immunization utilizing the antigen-antibody reaction between this monoclonal antibody and polyubiquitin The biological assay method enables selective quantification of polyubiquitin.

The immunological assay method that can be used in the present invention is not particularly limited, but various immunoassay methods (particularly labeled immunoassay methods) are generally suitable and can be used.
As the labeled substance in the labeled immunoassay, for example,
Radioactive element (radioimmunoassay; RIA), enzyme (enzyme immunoassay; EIA or ELIS)
A), a fluorescent substance (fluorescent immunoassay; FIA) can be used.

The labeling substance may be directly bound to the antigen or antibody, or may be indirectly labeled using a combination of substances having specific binding properties such as biotin-avidin. Since it is possible to reduce the cost by commonly using one labeling substance (for example, enzyme-labeled streptavidin), an indirect labeling method (for example, biotinylated antigen or antibody and enzyme-labeled streptavidin) is used. In combination, the antigen or antibody is enzymatically labeled) is preferably used.

The method of immunoassay is not particularly limited,
Any of the competitive method, the non-competitive method (sandwich method, etc.) and the two antibody method can be used. The sandwich method is particularly preferably used because it is relatively easy to increase the sensitivity.

When the immunoassay is used in the present invention, the above-mentioned monoclonal antibody having a specific binding property is usually immobilized on a solid phase and / or labeled with the above-mentioned label. For example, in the sandwich immunoassay, it is possible to use either one of the immobilized antibody or the labeled antibody as the specific monoclonal antibody of the present invention and the other as the conventional polyclonal antibody or a known monoclonal antibody with low specificity. In Although,
When importance is attached to quantification or sensitivity, both the immobilized antibody and the labeled antibody are preferably the specific monoclonal antibody of the present invention.

Details of such an immunoassay method can be found, for example, in the literature (Langone, JJ and Vunakis,
HV, eds., "Methods in Enzymology" vol.92 ', Acad
emic Press, 1983; written by Tijssen, P., translated by Eiji Ishikawa "Enzyme Immunoassay", Tokyo Kagaku Dojin, 1989;
Reference may be made to "Radioimmunoassay", Tokyo Kagaku Dojin, 1990, translated by Chard, T., Kitagawa et al.

Among the labeled immunoassay methods, the enzyme immunoassay is particularly preferably used in view of the complexity of handling and managing radioactive substances, the cost of measuring instruments (eg, spectrophotometer), and the like.

The present invention will be described in more detail below with reference to examples.

[0046]

Example 1 ( Preparation of ubiquitination-related enzymes E1, E2, and E3 proteins) Rabbit reticulocytes were mixed with 1.5 times 1 mMD.
An aqueous solution containing TT (dithiothreitol) and 4 mM MgCl ₂ was added, and the mixture was stirred for 5 minutes, left to stand on ice for 40 minutes for hemolysis, and this was centrifuged at 100,000 × g for 1 hour to remove the reticulocyte lysate in the supernatant. Recovered. 300m of this solution
l was centrifuged at 100,000 xg for 6 hours and the supernatant was
After dialyzing against 0 mM phosphate buffer, 1 mM DTT, pH 7.0, it was added to a DEAE cellulose column (200 ml) equilibrated with the same buffer. After washing the column with 10 mM phosphate buffer, 20 mM KCl, 1 mM DTT, pH 7.0, 50 mM Tris / HCl buffer, 0.5 MK
The adsorbed material was eluted with Cl, 1 mM DTT, pH 7.5. The elution fraction was concentrated to 60 mg / ml, then 50 m
MTris / HCl buffer, 0.2 mM MgCl ₂ ,
0.2 mM DTT, 0.2 mM ATP (adenosine 5
'-Triphosphate), 5% glycerol, pH 7.5, and then add ATP and MgCl ₂ to a final concentration of 5
It was adjusted to mM and 10 mM. 10 ml of this sample
50 mM Tris / HCl buffer, 10 mM MgC
10 ml of ubiquitin-immobilized agarose gel equilibrated with l ₂ , 0.2 mM DTT, 5 mM ATP, 5% glycerol, pH 7.5 was added and stirred at room temperature for 30 minutes, and the gel was packed into a column (inner diameter 16 mm, length 50 mm). It was

After washing this column with the equilibration buffer described above, 50 mM Tris / HCl buffer, 20 mM D
Elute E1 and E2 fractions at TT, pH 7.5, 50m
MTris / HCl buffer, 1MKCl, 2mMDT
The E3 fraction was eluted at T, pH 9.0. E3 fraction is
Immediately, 1M Tris / HCl buffer, pH 7.5 was added to neutralize. These elution fractions were added to 50 mM Tris /
After dialysis against HCl buffer, 1 mM DTT, 1 mM ATP, 20% glycerol, pH 7.5, ultrafiltration was used to increase the E1 and E2 fractions to 0.5 mg / ml and the E3 fraction to 1.5 mg / ml. Concentrated and stored at -70 ° C.

Example 2 (Preparation of polyubiquitinated protein)
15mg / ml E1 ・ E2 fraction, and 0.45mg / ml
In the mlE3 fraction, 2.5 mg / ml inorganic pyrophosphatase, 0.01 mg / ml creatine phosphokinase,
10 mM phosphocreatine, 1 mM DTT, 2 mMAT
100 mM Tris with P and 10 mM MgCl ₂
/ HCl buffer, pH 9.0 to a final concentration of 0.02 mg /
ml of heat-denatured lysozyme and ubiquitin (manufactured by Sigma, trade name: bovine red blood cell ubiquitin, biochemical reagent grade) having a final concentration of 6 mg / ml were added and reacted at 37 ° C. for 1 hour. The reaction was stopped by cooling with ice, and a polyubiquitinated protein (polyubiquitinated lysozyme) solution was prepared.

On the other hand, for comparison, a control reaction solution was prepared by carrying out the same reaction as above except that ubiquitin was not added.

Example 3 (Preparation of Monoclonal Antibody-Producing Cells Recognizing Polyubiquitinated Protein) 300 μl of the polyubiquitinated protein solution obtained in Example 2 was used in an amount of 300 μl of adjuvant (the first was Freund's complete adjuvant, and the second and subsequent were incomplete). (Adjuvant) 4 times in 6 weeks by intraperitoneal administration to BALB / C mice to immunize the mice, and hybridomas were prepared by a conventional method (Sakuji Tomiyama, Tamie Ando, "Monoclonal Antibody Experiment Manual" Kodansha, 1987). It was created. That is, the splenocytes of the above-mentioned mouse were collected, and mouse myeloma cells (ATCC (American Type Cuture Collectio
n) origin, P3X63-Ag8.653) and cell fusion were carried out to obtain a hybridoma.

From the hybridomas thus obtained, in order to screen for clones producing the desired polyubiquitin-specific antibody, the following (a) to
A 96-well microplate of (e) was prepared.

(A) Rabbit anti-ubiquitin polyclonal antibody was diluted to 5 μg / ml with PBS, 100 μl was added to each well of the above microplate and reacted at 4 ° C. for 12 hours to adsorb rabbit anti-ubiquitin polyclonal antibody to the wells. Let After washing the microplate with PBS, 200 μl of PBS and 3% skim milk was added to each well, and the plate was left at room temperature for 1 hour for blocking.

(B) In the well treated as in (a) above,
100 μl of each polyubiquitinated protein solution (obtained in Example 2) diluted 300 times with PBS was added, and the mixture was reacted at room temperature for 2 hours.

(C) The polyubiquitinated protein solution (obtained in Example 2) diluted 200-fold with PBS was used as described in (a) above.
Adsorption to the wells of the microplate was carried out in the same manner as in, and blocking was performed with skim milk.

(D) The control reaction solution obtained for comparison in Example 2 was diluted 200 times with PBS, and the same procedure as in (a) was repeated.
It was adsorbed to the wells of the microplate and blocked with skim milk.

(E) Ubiquitin in PBS at 1 mg / ml,
It was diluted to 5 mg / ml, 10 mg / ml and 50 mg / ml, adsorbed to wells and blocked with skim milk in the same manner as in (a).

After washing the wells subjected to the various adsorption treatments (a) to (e) with PBS, 100 μl of the culture supernatant of the hybridoma obtained above was added to each well and reacted at room temperature for 2 hours. After washing each well with PBS, 100-fold diluted biotinylated anti-mouse immunoglobulin antibody (1.5 mg / ml, Vector Laboratories) with PBS was used.
Each μl was added, and the mixture was reacted at room temperature for 1 hour.

After washing the wells with PBS, the wells were washed with PBS.
A 25-fold diluted peroxidase-labeled avidin / biotin complex (Vector Laboratories) was reacted at 100 μl / well. Then, after washing each well with PBS, 50 mM citrate buffer, pH 5.3, 0.2 mg
/ ml 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, 0.02%
H ₂ O ₂ was added at 200 μl / well, the solution in the well was developed at room temperature, and the absorbance at 405 nm was measured. The wells that developed more intense color than the wells to which a negative control was added using the mouse / myeloma culture supernatant used for the above cell fusion were defined as positive.

In this cloning, both of the wells (b) and (c) above were positive, and (a)
, (D) and (e) wells were selected as hybridomas giving a culture supernatant that was all negative.

As a result of such cloning, 6 kinds of clones (FK1, FK2, 31A, 99F, 135C, 135C, which do not react with free ubiquitin (monoubiquitin) but produce an antibody specifically reacting with polyubiquitin 16
9H) was obtained. The reactivity of each monoclonal antibody with respect to monoubiquitin and polyubiquitin, the subclass of the antibody, and the type of light chain are shown in Table 1 below.

[0061]

[Table 1]

In Table 1 above, Western blotting was performed by the method of Towbin et al. (Towbin, H., et al., Proc. Na.
tl. Acad. Sci., USA, 76 , 4350-4354, 19
79). The symbol "+" means positive and "-" means negative. A positive result indicates that the monoclonal antibody bound to the free ubiquitin or polyubiquitin transferred to the nitrocellulose membrane in the Western blotting. More specifically, peroxidase-labeled anti-mouse immunoglobulin (500-fold diluted, Dako Japan) is bound to the bound monoclonal antibody, and a color reaction (PBS, 0.05% 3,
3'-diaminobenzidine, 0.02% hydrogen peroxide,
Room temperature for 5 minutes). The more "+" signs, the stronger the color is developed with the naked eye. ELISA means the result of the enzyme immunoassay, and the more "+" signs, the greater the absorbance at 405 nm.

Example 4 (Purification of antibody for sandwich ELISA) The BALB / C mice were immunized with the FK2 cells obtained in Example 3 to obtain ascites. To 1 ml of this ascites, equal amount of 1M glicine-
0.3M NaCl (sodium chloride), pH 8.6 was added and left for 24 hours. This centrifugation supernatant is used as 0.1M borat
e-protei equilibrated with 0.15M NaCl, pH 8.5
It was added to an n-A column (trade name: ProSepA, manufactured by Bokusui Brown, 4 ml). After washing the column with the equilibration buffer, the adsorbed fraction was eluted with 0.1 M citrate pH 4.0. The eluate was immediately neutralized with 2MTris, dialyzed against PBS, and concentrated by ultrafiltration to obtain about 7 mg of highly purified IgG1 preparation (purified FK2 monoclonal antibody) per 1 ml of ascites.

Example 5 (Preparation of biotin-labeled antibody) Purified FK2 obtained in Example 4
Take a portion of the antibody solution and remove the solvent using ultrafiltration.
The concentration was changed to 0 mM bicarbonate pH 8.5 and the antibody concentration was adjusted to about 25 mg / ml. NHS-LC-biotin (Pierse) was added to this antibody solution.
20 μg was added per 1 mg of the purified antibody, and the mixture was reacted for 2 hours under ice cooling. Finally, unreacted biotin was removed by ultrafiltration.

Example 6 (Enzyme Immunoassay) Purified FK obtained in Example 5
2 Monoclonal antibody was diluted with PBS, and 100 μl of the diluted solution was added to each well of 5 μg / ml microplate.
Each of them was added and reacted at room temperature for 4 hours to adsorb the antibody to the wells. After washing with PBS containing 0.05% Tween 20 (PBS-T), 250 μl of 3% bovine serum albumin (BSA) -PBS-T was added to each well and left at 4 ° C overnight to block the wells. I went.
After washing the wells twice with PBS-T, 1% BSA-P
100 μl of sample (sample to be measured) diluted with BS-T was placed in a well and reacted at room temperature for 12 hours to immobilize FK.
2 The monoclonal antibody was bound to polyubiquitin in the sample.

After removing the excess sample, the wells were washed with PBS.
-Washed 4 times with T. Then, 1% BSA-PBS-T
8μg / ml biotinylated FK2 antibody 100μ diluted with
1 was put in a well and reacted at room temperature for 6 hours to bind polyubiquitin (in the sample) bound to the immobilized FK2 monoclonal antibody and the biotinylated FK2 antibody.

After removing the excess biotinylated antibody solution and washing the wells with PBS-T four times, peroxidase-labeled streptavidin (manufactured by DAKO) was added at about 400 ng /
1% BSA-PBS-T containing 150 ml per well
By adding μl and leaving it at room temperature for 0.5 hour, the biotinylated FK2 antibody was bound to peroxidase-labeled streptavidin by utilizing the biotin-avidin reaction.

After removing the excess labeled streptavidin solution and washing 4 times with PBS-T, 150 μl of 100
mM citric acid, 0.4 mg / ml orthophenylenediamine, 0.003% H ₂ O ₂ , pH 5.0 was added,
The reaction was carried out at room temperature for 30 minutes to carry out a color development reaction in the presence of peroxidase. After the color reaction was stopped by adding 50 μl of 4N-HCl, the absorbance at 490 nm was measured.

The optimum pH of the reaction buffer in the measurement system of this example is near neutral, and the optimum NaCl concentration is 0.0
It was confirmed to be 5 to 0.2M. In this example, the concentrations of biotinylated FK2 antibody and peroxidase-labeled streptavidin, and various reaction temperatures
It has been confirmed that the time is almost in the optimum range.

Example 7 (Evaluation of Enzyme Immunoassay) For the evaluation of the assay system used above, a polyubiquitin sample and a control sample in which the ubiquitin concentration of each type of free ubiquitin / polyubiquitin was clearly defined were prepared as follows. .

That is, 10 mM Tris / HCl, 1
0 mM NaCl, 25 mM KCl, 1.1 mM MgSO
₄ , 0.1 mM EDTA (ethylenediaminetetraacetic acid),
1 mM DTT, 0.1% CHAPS (3-[(3-cholamidop
ropyl) -dimethylammoniol] -1-propanesulfonate), 2
A DEAE cellulose column-adsorbed fraction of the rabbit reticulocyte lysate used in Example 1 (final concentration 10 mg) in a solution consisting of mM hemin and 120 μg / ml ubiquitin.
/ ml) and ATP supply reagent (final concentration 1 mMAT
P, 10 mM phosphocreatine, 20 U / ml creatine kinase) and about 50,000 cpm of ¹²⁵ I-labeled ubiquitin (corresponding to about 1 pg) were added and reacted at 37 ° C. for 4 hours to prepare a polyubiquitin sample.

On the other hand, an ATP depleting reagent (final concentration: 50 mM deoxyglucose, 2 mM) was added to a reaction solution having the same composition as above except that the reagent for supplying ATP was not added.
0 U / ml hexokinase) was added to similarly prepare a control sample.

[0073] Each of the free ubiquitin, each type ubiquitin concentration of polyubiquitin, a part of the reaction solution polyubiquitin fraction and fractionated free ubiquitin fraction and a two minutes using a gel filtration column, ¹²⁵ in each fraction It was calculated by measuring I radioactivity. As a result of such measurement, the concentrations of polyubiquitin and free ubiquitin in the polyubiquitin sample were 45 μg / ml and 55 μg / ml, respectively; the concentrations of polyubiquitin and free ubiquitin in the control sample were 0 and 0, respectively.
μg / ml and 110 μg / ml.

The concentrations of polyubiquitin and free ubiquitin in both the polyubiquitin sample and the control sample thus obtained were analyzed by the enzyme immunoassay of Example 6. The measurement results are shown in the graph of FIG. In FIG. 1, polyubiquitin samples are labeled with the polyubiquitin concentration they contain, while control samples are labeled with the free ubiquitin concentration they contain.

As shown in the graph of FIG. 1, according to the enzyme immunoassay of this Example, 0.3 ng / ml was used.
From this, it was confirmed that 20 ng / ml of polyubiquitin can be quantified. Furthermore, no cross-reactivity was observed at free ubiquitin concentrations up to 100 μg / ml,
The cross-reactivity of free ubiquitin in the above enzyme immunoassay system was 0.0001% of that of polyubiquitin.
The following was confirmed.

That is, it was found that the assay system of the present example is extremely specific to polyubiquitin, and therefore a trace amount of polyubiquitin can be quantified.

[0077]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, a monoclonal antibody derived from an antibody-producing cell based on immunization of a polyubiquitinated protein produced in vitro to an animal, which specifically reacts with polyubiquitin However, a monoclonal antibody that does not substantially react with monoubiquitin is provided.

By measuring polyubiquitin by an immunological method utilizing the specific binding property between the monoclonal antibody of the present invention and polyubiquitin, sensitivity, quantification, and / or Alternatively, it becomes possible to measure polyubiquitin with excellent selectivity.

[Brief description of drawings]

FIG. 1 is a graph showing the concentrations of polyubiquitin and free ubiquitin in a polyubiquitin sample and a control sample analyzed by an enzyme immunoassay of Example 6.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location G01N 33/577 B // (C12P 21/08 C12R 1:91) (72) Inventor Masahiro Fujimuro Hokkaido, Sapporo, Kita-ku, Kita-ku, Nishi, 6-18, Tsurumi-so 33 (72) Inventor, Hitoshi Sawada, Sapporo-shi, Higashi-ku, Kita, 13-jo, East, 15-6, 2- (72) Inventor, Yokozawa Era Chuo-ku, Sapporo South 19 West 15-3 3-15-501

Claims (10)

[Claims] 1. A monoclonal antibody derived from an antibody-producing cell based on immunization of a polyubiquitinated protein produced in vitro to an animal, which specifically reacts with polyubiquitin and does not substantially react with monoubiquitin. A monoclonal antibody characterized by the following. 2. The monoclonal antibody according to claim 1, wherein the polyubiquitinated protein is polyubiquitinated lysozyme. 3. The monoclonal antibody according to claim 1, wherein the animal is a BALB / C mouse. 4. A monoclonal antibody derived from an antibody-producing cell based on immunization of a polyubiquitinated protein produced in vitro to an animal, which specifically reacts with polyubiquitin and does not substantially react with monoubiquitin. A method for measuring polyubiquitin, which comprises using a monoclonal antibody; and measuring the polyubiquitin by an immunological method by utilizing the specific binding property between the antibody and polyubiquitin. 5. The method for measuring polyubiquitin according to claim 4, wherein the immunological method is a labeled immunoassay. 6. The method for measuring polyubiquitin according to claim 5, wherein the monoclonal antibody is labeled with a labeling substance. 7. The method for measuring polyubiquitin according to claim 5, wherein the monoclonal antibody is immobilized. 8. The method for measuring polyubiquitin according to claim 5, wherein the immunological method is an enzyme immunoassay (EIA). 9. The method for measuring polyubiquitin according to claim 5, wherein the immunological method is radio immunoassay (RIA). 10. The method for measuring polyubiquitin according to claim 5, wherein the immunological method is a fluorescence immunoassay (FIA).