JP5885158B2 - Antibodies against polypeptides containing sulfur-containing amino acid residues - Google Patents

Description

This invention is an antibody against a polypeptide comprising a sulfur-containing amino acid residues, hybridomas that produce the antibody, a kit comprising an antibody, a method of screening for how and drug candidate compounds using antibodies.

  Parkinson's disease is a neurodegenerative disease in which motor symptoms such as trembling of limbs are caused by a lack of dopamine in the brain and a relative increase in acetylcholine, and is generally diagnosed by motor symptoms such as trembling of limbs. However, when Parkinson's disease is diagnosed by motor symptoms, the symptoms have already progressed and are often difficult to treat. Therefore, there is a need for a diagnostic method for diagnosing Parkinson's disease before motor symptoms occur.

  DJ-1 was discovered as an oncogene that transforms NIH3T3 cells (see Non-Patent Document 1), and its point mutation (leucine at position 166 mutated to proline) is the cause of Parkinson's disease. It has been found that it is identical to the gene PARK7 (see Non-Patent Document 2).

  In addition, the position of the spot of DJ-1 protein two-dimensional electrophoresis moves before and after oxidative stress due to hydrogen peroxide and paraquat, and the change in the position indicates that the cause of the movement is due to oxidation of cysteine residues. It is known (see Non-Patent Document 3).

Furthermore, from the X-ray crystallographic analysis of the DJ-1 protein, the introduction of a point mutation associated with Parkinson's disease caused a change in the dimer structure, and the cysteine residue at position 106 was oxidized by X-ray irradiation, resulting in sulfin. It is known that it is changed to an acid (SO ₂ H) residue (see Non-Patent Document 4). For reference, the DNA sequence of human DJ-1 protein is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 2.

  Thus, there is a causal relationship between Parkinson's disease and the content of oxidized DJ-1 protein, and it is thought that oxidation of DJ-1 protein involves the 106th cysteine residue. . Oxidized DJ-1 protein is thought to be associated with Alzheimer's disease and arteriosclerosis in addition to Parkinson's disease. In view of this, an antibody that specifically binds to oxidized DJ-1 protein has been studied for use in diagnosis of Parkinson's disease and the like.

  For example, the inventors have developed an oxidized human DJ-1 protein in which a cysteine residue is oxidized to cysteine sulfonic acid or the like and an antibody thereto for use as an antigen (see Patent Documents 1 and 2). .) Furthermore, an antibody against oxidized DJ-1 protein has been developed using a fusion protein of a part of reduced human DJ-1 protein and a surface protein (gp64) of baculovirus as an antigen (see Patent Document 3). ).

  However, there is a problem that the antibodies against the oxidized DJ-1 protein obtained so far react with the reduced DJ-1 protein in addition to the oxidized DJ-1 protein. Therefore, in order to measure the oxidized DJ-1 protein content in a biological sample, an antibody with higher specificity has been demanded.

International Publication No. 2005-75513 JP 2007-319084 A JP 2010-183843 A

Nagakubo D, Taira T, Kitaura H, Ikeda M, Tamai K, Iguchi-Ariga M, Ariga H.J-1, a novel oncogene which transforms mouse NIH3T3 cells in cooperaion with ras.Biochem Biophys Res Commun. (1997) 231,509-13 Bonifati V, Rizzu P, van Baren MJ, Schaap O, Breedveld GJ, Krieer E, Dekker MC, Squitieri F, Ibanez P, Joosse M, van Dongen JW, Vanacore N, va Swieten JC, Brice A, Meco G, van Duijn CM, Oostra BA, Heutink P. Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism.Science. (2003) 299,256-9. Mitsumoto A, Nakagawa Y, Takeuchi A, Okawa K, Iwamatsu A, Takanzawa Y. Oxidized forms of peroxiredoxins and DJ-1 on two-dimensional gels increaed in response to sublethal levels of paraquat.Free Radic Res. (2001) 35, 301-10 . Wilson MA, Collins JL, Hod Y, Ringe D, Petsko GA. The 1.1-A resolution crystal structure of DJ-1, the protein mutated in autosomal recessive early onset Parkinson's disease. Proc Natl Acad Sci USA. (2003) 100, 9256 -61.

An object of the present invention is to provide an antibody against a polypeptide in which oxidation of a sulfur-containing amino acid residue contained is associated with a disease, and has a higher specificity for an oxidized polypeptide than before. Further, the present invention is a kit that can be more efficiently diagnosis of diseases such as Parkinson's disease, Methods, and to provide a screening method and the like.

As a result of intensive studies, the inventors have compared the reactivity with an oxidized polypeptide (eg, oxidized DJ-1 protein) to the reactivity with a reduced polypeptide (eg, reduced DJ-1 protein). High antibody. That is, the antibody of the present invention is an antibody having higher specificity for the oxidized polypeptide. The hybridoma of the present invention is a hybridoma that produces the antibody of the present invention. Furthermore, the kit of the present invention, Methods, screening method is mainly characterized by the use of antibodies of the present invention.

Antibodies of the invention, kits that use this, by way and screening methods using them, than the conventional, and discover better early and accurate patient such as Parkinson's disease, has become possible to treat.

FIG. 1 is a diagram comparing the specificity of each antibody by ELISA. FIG. 1A is a diagram schematically showing the outline of the experimental method, and FIG. 1B is a table showing the experimental results. FIG. 2 is a diagram comparing the specificity of each antibody by the Western Blot method. FIG. 3 is a diagram comparing the specificity of each antibody by two-dimensional electrophoresis. FIG. 4 summarizes the antibody screening results. FIG. 5 is a diagram showing the results of measuring the content of oxidized DJ-1 protein in a sample by the sandwich method using different types of detection antibodies. FIG. 6 is a diagram showing the results of measuring the content of oxidized DJ-1 protein in a sample by a sandwich method using D3805-2G1-110 antibody as both a capture antibody and a detection antibody. FIG. 6A is a diagram schematically showing the outline of the experimental method, and FIG. 6B is a graph showing the experimental results. FIG. 7 is a diagram showing the results of confirming the antibody recognition portion by molecular interaction analysis (Biacore method).

This invention is an antibody against a polypeptide comprising a sulfur-containing amino acid residues, a kit containing the antibody are screening methods ways and drug candidate compounds using this antibody. Therefore, these will be described in detail below.

1. Antibody The antibody of the present invention is an antibody against a polypeptide in which oxidation of the sulfur-containing amino acid residue contained is associated with a disease, and the reactivity with the oxidized polypeptide in which the sulfur-containing amino acid residue is oxidized is sulfur-containing. It is a highly specific antibody compared to a reduced polypeptide whose amino acid residues are not oxidized.

  Here, the sulfur-containing amino acid residues are specifically cysteine residues and methionine residues. In addition, as described in Patent Document 3, oxidation of sulfur-containing amino acid residues means that cysteine residues are oxidized to cysteine sulfonic acid residues, and methionine residues are oxidized to methionine sulfoxide. To be a side residue. Polypeptides include not only those containing all amino acid sequences of proteins contained in biological samples, but also polypeptides containing partial amino acid sequences near oxidized sulfur-containing amino acid residues, fusion proteins containing the same, and the like. Including. Furthermore, examples of diseases associated with oxidation of sulfur-containing amino acid residues include, but are not limited to, Parkinson's disease, Alzheimer's disease, arteriosclerosis and the like.

  In the present invention, “an antibody whose reactivity with a specific antigen is higher than that with another antigen” means an antigen-antibody reaction (binding of an antibody and an antigen) in immunology. The difference in the measured value is greatly different depending on the antigen. That is, an antibody having a large “measurement value of antigen-antibody reaction against a specific antigen” / “measurement value of antigen-antibody reaction against another antigen”.

  For example, “Reactivity with oxidized DJ-1 protein (oxDJ-1) is higher than reactivity with reduced DJ-1 protein (DJ-1)”. The value of oxDJ-1 / DJ-1 determined from the values measured by (ELISA method), Western blot method (Western blot method) and two-dimensional electrophoresis method is larger than a certain value.

  Specifically, among antibodies to the DJ-1 protein, an antibody having an oxDJ-1 / DJ-1 value of 5.6 or more determined by ELISA, an oxDJ-1 / DJ-1 value determined by Western blotting An antibody having a value of 8.0 or more and an antibody having an oxDJ-1 / DJ-1 value of 100 or more obtained from two-dimensional electrophoresis can be exemplified.

  More specifically, the monoclonal antibody D3707-13 produced by the hybridoma having the deposit number NITE P-1185, the monoclonal antibody D3708-36 produced by the hybridoma having the deposit number NITE P-1186, and the hybridoma having the deposit number NITE P-1187 And monoclonal antibody D3805-2G1-110 produced by the hybridoma of accession number NITE P-1188.

  These monoclonal antibodies are compared with the D3843-1C10-208 antibody that reacts with any of the B7411C antibody, oxidized DJ-1 protein and reduced DJ-1 protein described in Patent Document 3, as described later. It has been confirmed that the specificity to the oxidized DJ-1 protein is high.

  The antibody may be a single-chain antibody, a humanized antibody, a chimeric antibody, or the like, as well as a monoclonal antibody or a polyclonal antibody, or a labeled antibody obtained by labeling these antibodies. Alternatively, the antibody may be an antibody fused with a fluorescent protein such as green fluorescent protein (GFP).

  For labeling of the antibody, the label may be directly bonded to the antibody (directly), or the antibody of the present invention may be indirectly labeled by binding to the labeled secondary antibody as the primary antibody (indirect method). . Moreover, you may use ABC method using the fluorescence labeled avidin which has the property couple | bonded specifically with biotin instead of a secondary antibody.

Any known label used for labeling can be used. Specifically, fluorescent dyes such as FITC (fluorescein isocyanate) or tetramethylrhodamine isocyanate, radioactive substances such as ¹²⁵ I, ³² P, ¹⁴ C, ³⁵ S or ³ H, alkaline phosphatase, peroxidase, β-galactosidase or phycoerythrin Examples include enzymes and gold colloids.

2. Antibody Preparation Method The antibody of the present invention can be prepared by a known method without any particular limitation. For example, it can be prepared by cell fusion, screening, etc. after immunization using a protein as an antigen. More specific description will be given below.

(1) Antigen A polypeptide serving as an antigen is a polypeptide in which oxidation of a sulfur-containing amino acid residue contained is associated with a disease. The polypeptide may be a polypeptide containing not only the entire amino acid sequence of the protein contained in the biological sample but also a partial amino acid sequence near the sulfur-containing amino acid residue to be oxidized. Specific examples of the antigen include human oxidized DJ-1 protein obtained by the method described in Patent Document 2. In addition, the definition of a sulfur-containing amino acid residue etc. is the same as the above.

(2) Immunization Immunization is performed, for example, by immunizing a mouse by injecting the immunogen subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, or the like. The inoculation interval, inoculation amount, and the like vary depending on the inoculation method. For example, when inoculating the mouse subcutaneously, the antigen is inoculated every 3 to 4 days for about 2 weeks. Thus, antibody-producing cells such as lymphocytes are taken out from the immunized mammal and used for cell fusion operation. In immunization, it is preferable to use a suspension in which an immunogen is mixed with an adjuvant or the like. Further, if the immunogen is mixed with baculovirus and immunized, a more specific antibody can be obtained.

  Examples of mammals used in the immunization method include mice, rats, hamsters and the like. Among them, the use of mice, particularly 6-8 week old Balb / C mice, is preferred for the production of antibodies against human antigens. If a transgenic mouse expressing a baculovirus gp64 protein is used as the mammal used in the immunization method, an antibody with higher specificity can be obtained.

(3) Cell fusion Cell fusion is performed by mixing antibody-producing cells and myeloma cells in a ratio of 1: 5 to 1:20 according to a known method. For cell fusion, a fusion agent such as polyethylene glycol (PEG) or Sendai virus is used, and after cell fusion, the cells are cultured in a HAT selective medium. Thereby, only the hybridoma fused with the antibody-producing cell can be efficiently propagated.

(4) Screening method After screening hybridomas that produce the desired antibody by screening the proliferated hybridomas, the hybridomas are cloned by limiting dilution or the like and propagated in a liquid medium or in the peritoneal cavity of mammalian cells. Antibodies can be obtained. Here, as a method of screening for a hybridoma producing the target antibody, a known method such as enzyme immunoassay (EIA), radioimmunoassay (RIA), flow cytometry, or the like can be used. Among these, EIA is preferable and ELISA method is more preferable because of safety and simplicity of equipment.

  These methods may be used alone, but it is preferable to combine a plurality of methods in order to select antibodies with higher specificity. Moreover, the specificity of the antibody produced by the hybridoma obtained as a result of screening may be confirmed by combining electrophoresis and the Western Blot method.

3. Kit When detecting and measuring oxidized protein using the antibody of the present invention, secondary antibodies, color formers, buffer solutions, etc. are required, and these are purchased separately and used. May be. However, if these are combined to form a kit in advance, it is possible to easily perform detection and measurement without the need to purchase each component separately. In addition, detection and measurement can be performed more easily if a buffer solution necessary for protein extraction is also made into a kit.

4). How the method the present invention available diagnostic, the methods available for the diagnosis of diseases and oxidation and pathology changes in vivo of the sulfur-containing amino acid residue of the polypeptide is concerned with a sulfur-containing amino acid residues a is, using the antibodies of the present invention, it is a way to measure changes in oxidized DJ-1 protein content in a biological sample.

  Examples of the disease in which the sulfur-containing amino residue of a protein having a sulfur-containing amino acid residue is oxidized in vivo and the disease state is changed include Parkinson's disease, Alzheimer's disease, and arteriosclerosis. Among them, since it is highly related to the oxidized DJ-1 protein as described above, it is preferably used for diagnosis of Parkinson's disease.

  The biological sample may be a body fluid or body tissue containing protein. Specifically, blood, urine, saliva, cerebrospinal fluid, brain tissue, muscle tissue, adipose tissue, skin tissue, viscera Examples include organizations. Among these, blood that can be easily collected with little invasion to the specimen during sampling is preferable.

  Furthermore, examples of the method for measuring the amount of oxidized protein include known methods for detecting and measuring proteins in biological samples, such as EIA and RIA. Among these, EIA is preferable and ELISA method is more preferable because of safety and efficiency.

5. Method for Screening Drug Candidate Compound The method for screening a drug candidate compound of the present invention comprises (1) a step of administering a drug candidate compound to a model animal, and (2) a living body collected from the model animal using the antibody of the present invention. Measuring the change in the amount of oxidized DJ-1 protein in the sample.

Here, as a model animal, any animal that has been conventionally used for screening drug candidate compounds, such as mouse, rat, guinea pig, dog and monkey, can be used without any problem. In addition, the disease to be treated by the drug candidate compound, the biological sample to be collected, and the method for measuring oxidized DJ-1 protein are the same as those available for diagnosis.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, the claim of this invention is not restrict | limited in any meaning by a following example.

1. Preparation of antigen For use as an antigen, a reduced DJ-1 protein, an oxidized DJ-1 protein and a mutant protein in which the cysteine residue at position 106 is changed to a serine residue (hereinafter abbreviated as C106S mutant) are used. According to the description in Patent Document 2, it was prepared as follows.

  First, plasmids containing a human DJ-1 gene or C106S mutant gene to which a histidine tag was added were constructed, E. coli was transformed with these plasmids, and this E. coli was cultured in LB medium. A plurality of E. coli transformed with the human DJ-1 gene were cultured.

  Next, E. coli transformed with plasmids containing the human DJ-1 gene and the C106S mutant gene were collected and solubilized. A supernatant was obtained from the obtained solubilized fraction by centrifugation, and this supernatant was applied to a Ni-NTA column (QIAGEN). The Ni-NTA column was washed and then eluted with imidazole to obtain reduced DJ-1 protein and C106S mutant protein.

  In addition, 50 mM hydrogen peroxide was added to another E. coli transformed with the human DJ-1 gene, oxidized at 37 ° C for 15 minutes, purified in the same manner using a Ni-NTA column, and oxidized DJ. -1 protein was obtained.

2. Preparation of Antibody The obtained oxidized DJ-1 protein was suspended in pertussis adjuvant (Funakoshi) in 40 μg of oxidized DJ-1 recombinant and 40 μg baculovirus per gp64-transgenic Balb / c mouse. The immunization was carried out 2 to 4 times at 2-week intervals. After immunization, spleen cells were removed, antibody-producing cells and myeloma cells NS-1 were fused with PEG1500, and hybridomas were selected using HAT medium.

3. Primary screening of antibodies by ELISA
The antibody prepared in Example 2 was first screened by ELISA. First, an antigen solution containing oxidized DJ-1 protein (2 μg / ml carbonate buffer) was added to an ELISA plate (96 well plate) at 100 μl / well and allowed to react overnight at 4 ° C. Reacted. After completion of the solid phase reaction, discard the antigen solution on the ELISA plate, wash 4 times with washing solution (0.05% Tween 20 PBS), and then add blocking solution (0.1% Bovine Serum Albumin (BSA) PBS) at 150 μl / well. The reaction was allowed to proceed at 37 ° C for 1 hour. After the blocking reaction, the plate was washed once with a washing solution.

  A primary antibody solution containing each antibody (culture supernatant diluted with 0.05% Tween 20 PBS) was added to each well at 50 μl / well and reacted at 37 ° C. for 60 minutes. After completion of the primary antibody reaction, the ELISA plate was washed 4 times with a washing solution. A secondary antibody solution (10,000-fold diluted, 0.05% Tween 20 PBS) containing an HRP-labeled anti-mouse IgG antibody (Jacksons) was added to each well at 100 μl / well and reacted at 37 ° C. for 60 minutes.

After completion of the secondary antibody reaction, the ELISA plate was washed 3 times with a washing solution, and TMB + Substrate-chromogen (Dako Cytomstion) was added to each well as a substrate solution in a volume of 50 μl / well and allowed to react at room temperature for 30 minutes under light shielding. After completion of the substrate reaction, 1M H ₂ SO ₄ was added to each well as 50 μl / well as a reaction stop solution, and after shaking for 1 minute, absorbance at a wavelength of 450 nm was measured.

  The absorbance was measured by the same method using reduced DJ-1 protein and C106S mutant protein instead of oxidized DJ-1 protein. For comparison, the absorbance of the B7411C antibody described in Patent Document 3 was also measured by the same method. The measurement results are shown in FIG. FIG. 1A is a diagram schematically showing the outline of the experimental method, and FIG. 1B is a table showing the experimental results.

  FIG. 1 shows the difference between each clone's reaction to the reduced DJ-1 protein (Nm), the reaction to the oxidized DJ-1 protein (Ox), and the reaction to the C106S mutant protein (C106s). From this table, D3707-13 antibody, D3708-36 antibody and D3805-2G1-110 antibody have higher Ox / Nm values than B7411C antibody, that is, oxidized DJ-1 protein compared to B7411C antibody. Was found to bind specifically.

4). Secondary screening of antibodies by Western Blot method Specificity of the antibody prepared in Example 2 to oxidized DJ-1 protein, B7411C antibody described in Patent Document 3, and commercially available anti-DJ-1 antibody 3E8 (medical biology) The specificity of the laboratory for the oxidized DJ-1 protein was compared by electrophoresis and Western Blot as follows.

  Reduced DJ-1 protein, oxidized DJ-1 protein, and C106S mutant protein were each applied to a 12% polyacrylamide gradient gel at 50 ng / well and electrophoresed at room temperature for 1.5 hours. The obtained gel was applied to a membrane (PVDF membrane, Millipore) using a tank-type blotting device (BioRad) and a transfer buffer (25 mM Tris-HCl, 192 mM glycine, 10% methanol) at 4 ° C and 100 V. Blotted for 1.5 hours.

  The blot on the membrane was blocked for 2 hours with TBST buffer (Tris-HCl 10 mM, NaCl 0.15 M, Tween 20 0.5% and 5% skim milk). Each membrane such as anti-DJ-1 antibody 3E8, B7411C antibody, D3805-2G1-110 antibody, D3707-13 antibody and the membrane were reacted overnight. Subsequently, it was reacted with an HRP-labeled anti-mouse IgG antibody (Santa Cruz) and developed using Western Blotting detection reagent (Millipore). The result is shown in FIG. FIG. 2 shows the results of the D3707-13 antibody and the D3805-2G1-110 antibody among the B7411C antibody, 3E8 antibody, and the secondary screened antibodies, which are experimental controls.

  FIG. 2 shows that the D3707-13 antibody and D3805-2G1-110 antibody reacted only with the trace amounts of the reduced DJ-1 protein and the C106S mutant protein, and almost only with the oxidized DJ-1 protein. It was confirmed that In contrast, the B7411C antibody and anti-DJ-1 antibody 3E8 are mostly reacted with oxidized DJ-1 protein, but also reacted with reduced DJ-1 protein and C106S mutant protein. Was confirmed. Thus, it was found that the D3707-13 antibody and the D3805-2G1-110 antibody bind specifically to the oxidized DJ-1 protein as compared with the B7411C antibody and the anti-DJ-1 antibody 3E8.

5. Antibody tertiary screening by two-dimensional electrophoresis The substrate specificity of the antibody prepared in Example 2 and the conventional B7411C antibody and anti-DJ-1 antibody 3E8 against oxidized and reduced DJ-1 proteins Comparison was made by two-dimensional electrophoresis. Specifically, the comparison was made as follows.

(1) Sample Preparation Neuroblastoma cells SH-SY5Y were seeded in a 10 cm diameter dish containing DMEM / F12 medium (Invitrogen) and cultured in a CO ₂ incubator at 37 ° C. and 5% CO ₂ . The cultured cells were collected, washed with PBS, and subjected to two-dimensional electrophoresis.

(2) Two-dimensional electrophoresis Washed cells are dissolved in 280 μl of isoelectric focusing solution (9 M urea, 2% CHAPS, 65 mM DTE, 0.5% IPG Buffer (GE Healthcare Japan)), 15,000 rpm 20 Centrifugation was performed for a minute to remove insoluble components. Of these, 250 μl (protein mass 75 μg) was added to Immobiline Dry Strip (7 to 13 cm long, pI range 4-7, (GE Healthcare Japan)) and allowed to swell for 12 hours. After swelling, the first dimensional isoelectric focusing was performed with an IPGPhor electrophoresis apparatus (GE Healthcare Japan).

  After the first-dimensional electrophoresis was completed, the gel was equilibrated and equilibrated by immersing it in an equilibration solution (50 mM Tris-HCl (pH 6.8), 6 M urea, 30% glycerol, 2% SDS, 20 mM DTE). The dry strip was placed on top of an SDS-PAGE gel (12.5%). Set the gel in a slab electrophoresis apparatus (Bio-Rad), fill the electrophoresis buffer (25 mM Tris-HCl, 14.4 mg / ml glycine, 1 mg / ml SDS), and run at 30 mA until the bromophenol blue band is visible at the bottom of the gel did.

(3) Staining with antibodies Proteins separated by two-dimensional electrophoresis are treated with a membrane (PVDF membrane, 10% methanol) using a tank type blotting device (Bio-Rad) and a blotting buffer (25 mM Tris-HCl, 192 mM glycine, 10% methanol). (Millipore) was blotted at 4 ° C. and 100 V for 1.5 hours.

  The membrane after the transfer was immersed in a blocking buffer (20 mM Tris-HCl, 50 mM NaCl, 5% skim milk) and blocked at room temperature for 2 hours.

  After completion of blocking, the reaction was carried out with the secondary screened antibody, B7411C antibody and 3E8 antibody. Then, it was reacted with an HRP-labeled anti-mouse IgG antibody (Santa Cruz), and color was developed using a Western Blotting detection reagent (Millipore). About the result, the enlarged view of DJ-1 protein vicinity is shown in FIG. FIG. 3 shows the results of the B7411C antibody, 3E8 antibody, and the third screened antibodies D3707-13 and D3805-2G1-110, which are experimental controls.

  FIG. 3 shows that the 3E8 antibody reacts sufficiently with the reduced DJ-1 protein (native) in addition to the oxidized DJ-1 protein (ox), that is, specifically reacts with the oxidized DJ-1 protein. It was confirmed that it was not. In contrast, the D3707-13 antibody, the D3805-2G1-110 antibody, and the B7411C antibody react only with the oxidized DJ-1 protein and do not react with the reduced DJ-1 protein at all, that is, the oxidized form It was confirmed that it reacts specifically with DJ-1 protein.

  From the above results, it was found that the D3707-13 antibody and D3805-2G1-110 antibody maintained high specificity for the modified oxidized DJ-1 protein. Further, comparing the binding strengths of the antibodies, it was found that the antibodies according to the present invention, the D3707-13 antibody, and the D3805-2G1-110 antibody had higher binding strength than the conventional B7411C antibody.

The results of primary to tertiary antibody screening are summarized in FIG. Here, 1 ^st screening in FIG. 4 is a summary of the results of the ELISA method of Example 3, 2 ^nd screening is a summary of the results of Western blot method of Example 4, 3 ^rd screening of Example 5 It is the summary of the result by two-dimensional electrophoresis.

  As shown in FIG. 4, a plurality of antibodies (antibodies with high oxDJ-1 / DJ-1) having higher specificity for oxidized DJ-1 protein than the B7411C antibody described in Patent Document 3 are obtained by screening. It was.

6). Quantification of oxidized DJ-1 protein by sandwich method The antibody of the present invention was used in the sandwich method, which is a general method for quantifying proteins, and its performance was compared with conventional antibodies.

(1) Preparation of Reagent A solid phase solution (2 μg / ml, 0.1 M carbonate buffer, pH 9.6) containing D3805-2G1-110 antibody as a capture antibody was prepared. A detection antibody solution (2 μg / ml) containing an HRP-labeled antibody was prepared by the following procedure. First, the D3805-2G1-110 antibody, the B7305A antibody described in Patent Document 3, or the B7411C antibody is dialyzed against 0.2 M Na hydrogen carbonate (pH 9.5), and activated HRP (HRP is added to 0.1 M metaperiodic acid). The antibody was labeled with HRP by mixing with Na and oxidizing the sugar chain of HRP to form an aldehyde. Next, after labeling, NaBH ₄ was added to deactivate unreacted aldehyde groups. Furthermore, 0.1% BSA-PBS was added so that the concentration of oxidized DJ-1 protein prepared in Example 1 was in the range of 0 to 500 ng / ml to prepare antigen solutions having different concentrations.

(2) Preparation of plate 100 μl of the solid phase solution was dispensed into a 96-well microplate and left at 4 ° C. for 24 hours. Excess binding sites in wells were inactivated by adding 300 μl of blocking solution (0.1% BSA-PBS)

(3) Measurement of oxidized DJ-1 protein 100 μl of antigen solutions having different concentrations were added to each well of the blocked plate and allowed to react at 37 ° C. for 1 hour. After the plate was washed with 0.05% Tween-PBS, 100 μl of the detection antibody solution was added and reacted at 37 ° C. for 1 hour. After the plate was washed with 0.05% Tween-PBS, the enzyme activity on the solid phase was measured in the same manner as in Example 3. The result is shown in FIG.

  From FIG. 5, it was found that when the B7305A antibody or B7411C antibody was used as the detection antibody, no increase in absorbance was observed. This is considered to be because the B7305A antibody or the B7411C antibody was inactivated by the label. In contrast, when the D3805-2G1-110 antibody of the present invention was used, high linearity was observed between the antigen concentration and the absorbance.

  Therefore, using the same method, the D3805-2G1-110 antibody is used for both the capture antibody and the detection antibody, and the concentration of the oxidized DJ-1 protein ranges from 0 to 100 ng / ml, and the relationship between the antigen concentration and the absorbance. Investigated about. The result is shown in FIG. FIG. 6A is a diagram schematically showing the outline of the experimental method, and FIG. 6B is a graph showing the experimental results.

  From FIG. 6, it was confirmed that when the D3805-2G1-110 antibody was used as both the capture antibody and the detection antibody, there was a high correlation between the concentration of the oxidized DJ-1 protein and the absorbance. From these results, it was found that the D3805-2G1-110 antibody has high specificity for the oxidized DJ-1 protein.

7). Confirmation of recognition site by intermolecular interaction analysis with antibody and peptide
It was examined whether the D3805-2G1-110 antibody, the D3843-1C10-208 antibody, and the D3707-13 antibody recognize the vicinity of cysteine at position 106 that is most related to oxidation of oxidized DJ-1 protein. Specifically, peptide pep7 having the same amino acid sequence as the amino acid sequence at positions 103 to 109 of human DJ-1 protein or oxidized peptide pep7ox in which its cysteine residue is oxidized, D3805-2G1-110 antibody and the like The binding ability of was analyzed by Biacore (registered trademark) using the surface plasmon resonance phenomenon. Pep7 was synthesized with a peptide synthesizer, and pep7ox was obtained by adding performic acid to pep7 and oxidizing at 37 ° C. for 15 minutes.

  First, D3805-2G1-110 antibody, D3843-1C10-208 antibody, and D3707-13 antibody are immobilized on a sensor chip (Series S Sensor Chip CM5, GE Healthcare Japan), and the sensor chip on which the antibody is immobilized is Biacore ( It was inserted into a registered trademark BIACORE T-100 (GE Healthcare Japan). Next, pep7 or pep7ox solution prepared by adjusting HEPS or pep7ox to 30 μmol / l in HHBS-EP (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, pH 7.4) buffer. Was passed through the sensor chip at 10 μml / min, and the binding dissociation curve was measured. The result is shown in FIG.

  From FIG. 7, it was found that all the antibodies were bound to pep7ox more strongly than pep7. From these results, it was confirmed that the D3805-2G1-110 antibody and the D3707-13 antibody recognized the vicinity of cysteine at position 106 of the oxidized DJ-1 protein. On the other hand, in the D3843-1C10-208 antibody that reacts with oxidized DJ-1 and reduced DJ-1 used as an experimental control, no significant difference in binding strength was observed between pep7 and pep7ox. From these results, it is considered that the D3843-1C10-208 antibody has an epitope at a site other than Cys106.

Claims (8)

Monoclonal antibody D3707-13 produced by the hybridoma with accession number NITE P-1185. Monoclonal antibody D3708-36 produced by the hybridoma with accession number NITE P-1186. Monoclonal antibody D3710-62 produced by the hybridoma with accession number NITE P-1187 . Monoclonal antibody D3805-2G1-110 produced by the hybridoma with accession number NITE P-1188. Accession numbers, NITE P-1185, NITE P -1186, NITE P-1187 or NITE hybridoma is either P-1188. Kits comprising an antibody of any of claims 1-4. Using the antibody of any of 請 Motomeko 1-4, how to measure the change in oxidized DJ-1 protein content in a biological sample. A method for screening drug candidate compounds effective in treating a disease,
(1) administering a drug candidate compound to a model animal;
(2) a step of measuring a change in the amount of oxidized DJ-1 protein in a biological sample collected from a model animal using the antibody according to any one of claims 1 to 4 ;
A method for screening drug candidate compounds comprising: