WO2002050121A1

WO2002050121A1 - Novel antibody

Info

Publication number: WO2002050121A1
Application number: PCT/JP2001/010919
Authority: WO
Inventors: Takeshi Iwatsubo; Hideo Fujiwara; Masato Hasegawa
Original assignee: Taisho Pharmaceutical Co.,Ltd.
Priority date: 2000-12-13
Filing date: 2001-12-12
Publication date: 2002-06-27
Also published as: JPWO2002050121A1; JP4055579B2; AU2002221132A1

Abstract

An antibody binding specifically to a protein having an amino acid sequence derived from the sequence of SEQ ID NO:1 by phosphorylation of one or more Ser; and drugs for detecting synucleinopathy lesions characterized by containing this antibody.

Description

Specification

New antibody technology

The present invention relates to an antibody that specifically binds to α-synuclein aggregates such as Levibody, a Levibody containing the antibody, an agent for detecting a synucleinopathy (synucleinopathy) lesion, and a Lewy body. And a method for detecting synucleinopathic (synucleinopathy) lesions. Background art

Degenerative neurons such as Parkinson's disease (PD) and Lewy body dementia (DLB) accumulate «-synuclein in the form of Lewy bodies (LB) [Am. J. Pathol, 152 879 (1998)]. -Synuclein is also known to accumulate in glial cells of multiple system atrophy, a type of spinocerebellar degeneration, LB in Hallervorden-Spatz disease, degenerative neurites, etc. in addition to 、 D and DLB. It is collectively called one (synucleinopathy). Furthermore, the discovery of mutations in a-synuclein in familial PD families [Science 276: 2045 (1997)] has highlighted the role of α-synuclein in PD and in synucleinopathy.

However, the relationship between ΡD and DL Β and ——synuclein, including the fact that L 、 is not formed only by overexpressing α-synuclein in cells, and the fact that α-synuclein mutation is not observed in non-familial PD There are still many unclear points, and the elucidation at the molecular level is awaited.

Various anti-synuclein antibodies have been prepared for α-synuclein research.However, it is known that these antibodies bind not only to Levi bodies, but also to the histonuclein present in normal brain. Had been.

On the other hand, Ser of nuclein can be phosphorylated by G protein-coupled receptor kinase (GRK), casein kinase (CK1, CK2), etc. [J Biol Chem 275, 26515 (2000), J Biol Chem 275, 390] (2000)], but under physiological conditions There are no reports that the above-mentioned Lewy bodies are phosphorylated under pathological conditions such as PD or other synucleinopathies. Disclosure of the invention

Means for Solving the Problems As a result of earnest studies to achieve the above object, the present inventors have found that a part of the constituent amino acids is phosphorylated in hynuclein, which forms an aggregate such as Levi body. Furthermore, they have found that an antibody prepared using the synthetic peptide having the phosphorylated amino acid as an antigen specifically binds to α-synuclein aggregates, thereby completing the present invention.

That is, the present invention is an antibody that specifically binds to a protein in which one or more Ser in SEQ ID NO: 1 is phosphorylated.

Further, the present invention is an antibody that specifically binds to a protein in which any one of Ser9, Ser42, Ser87 or Serl29 in SEQ ID NO: 1 is phosphorylated.

Further, the present invention is an antibody that specifically binds to a protein in which Serl 29 is phosphorylated in SEQ ID NO: 1.

Further, the present invention is an antibody that specifically binds to a peptide in which Ser6 is phosphorylated in SEQ ID NO: 2.

The present invention is also an antibody that specifically binds to a protein in which Ser87 is phosphorylated in SEQ ID NO: 1.

Further, the present invention is an antibody that specifically binds to a phosphorylated Ser6 peptide in SEQ ID NO: 3.

Further, the present invention is a drug for detecting a lesion of synucleinopathy, which comprises any one of the above antibodies.

Further, the present invention provides a method for detecting α-synuclein in which Serl29 or Ser87 is phosphorylated in SEQ ID NO: 1, which comprises using any of the above antibodies. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the results of Western blot analysis of -synuclein extracted from DLB brain. Represent. The fraction obtained by solubilizing DLB brain with 50 mM Tris buffer (Tris HC1), 1% Triton-X, 1% SarkosyK 8 M urea (Urea) was used for anti-human α-synuclein antibody LB509. Western blot analysis. C represents normal control human brain, and D represents DLB patient brain. The molecular weight marker position (in kilodaltons) is shown on the left. The α-synuclein protein band is positive at 15 kilodaltons.

FIG. 2 shows the results of staining of DL L brain tissue with an anti-synuclein antibody and a phosphorylation-specific anti-synuclein antibody. After fixing DLB cerebral cortex to formalin, slice into 50-micron thickness, and use avidin-biotin complex method with anti-human spleen synuclein antibody LB509 (Α) and Ser 129 phosphorylation specific α-synuclein antibody (Β). And stained with diaminobenzidine to develop a brown color. In Α, LB shows a positive circle (arrow), and normal -synuclein shows a diffuse, fine-granular, positive staining throughout the neuropil, but a negative neuronal cell body (*) . In B, in addition to L B (arrow), a short shrunken Lewy neuri te (arrow head) is clearly depicted.

FIG. 3 shows the results of Western blot analysis using anti-synuclein specific anti-synuclein antibody extracted from DLB brain. The fraction obtained by solubilizing DLB brain with 50 mM Tris buffer (Tris HC1), 1% Triton-X, 1% SarkosyK 8 M urea (Urea) was used for Serl29 phosphorylation-specific α-synuclein antibody. Was used for Western blot analysis. C represents normal control human brain, and D represents DLB patient brain. The position of the molecular weight marker (displayed in mouth Dalton) is shown on the left. A band of phosphorylation α-synuclein protein is positive at a position of 15 kDa specifically for the urea-soluble fraction.

FIG. 4 shows an ELISA study of the specificity of Ser 129 phosphorylated α-synuclein-specific antibody after affinity purification. The amount of Serl29 phosphorylated peptide shown on the horizontal axis (sequence is as in Example 2) or recombinant full-length human synuclein is fixed to the microwell plate, and reacted with Ser129-phosphorylated human synuclein-specific antibody. Color was developed using TMB Microwell Peroxidase Substrate (Funakoshi). Serl29 phosphorylation α-synuclein-specific antibody reacts specifically with Serl29 phosphorylated peptide, but does not react with non-phosphorylated recombinant human synuclein.

FIG. 5 shows an ELISA study of the specificity. Serl29 phosphorylation in the amount indicated on the horizontal axis (The sequence is as in Example 5.) Alternatively, the total length of the recombinant << ■

After fixing to a microwell plate, and reacting with a Serl 29 phosphorylation α-synuclein-specific antibody, color was developed using TMB Microwel II Peroxidase Substrate (Funakoshi). Serl 29 phosphorylated synuclein-specific antibody reacts specifically with Serl 29 phosphorylated peptide, but does not react with non-phosphorylated recombinant-synuclein.

FIG. 6 shows the results of Western blot analysis of 0! -Synuclein extracted from DLB brain. Western blot was performed using the fraction obtained by solubilizing DLB brain with 50 mM Tris buffer (Tris HC1), 1% Triton-X, 1% SarkosyK 8 M urea (Urea), and SDS. Lot analysis was performed. The position of the molecular weight marker (in kilodaltons) is shown on the left. The band of a-synuclein protein is positive at 15 kilodaltons.

FIG. 7 shows the EUSA study of the specificity of Ser87 phosphorylated α-synuclein-specific antibody after affinity purification. The amount of Ser87 phosphorylated peptide indicated on the horizontal axis (sequence is as in Example 8) or recombinant full-length synuclein is immobilized on a microwell plate, and after reaction with Serl 29 phosphorylated sph-synuclein-specific antibody, TMB Mi crowel The color was developed using l Peroxi dase Substrate (Funakoshi). Ser87 phosphorylated <<-synuclein specific antibody reacts specifically with Ser87 phosphorylated peptide, but does not react with non-phosphorylated recombinant α-synuclein. BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, "a protein in which one or two or more Sers in SEQ ID NO: 1 is phosphorylated" refers to a protein in which any of Ser, 9, 42, 87 or 129 in SEQ ID NO: 1 is phosphorylated The phosphorylated Ser may be present at one site or at multiple sites.

Serl 29 means Ser which is the 129th amino acid from the N-terminus in the amino acid sequence, Ser 6 means Ser which is the 6th amino acid from the N-terminus in the amino acid sequence, and Ser87 means the amino acid Ser means the amino acid located at the 87th position from the N-terminus in the sequence.

The term “specifically binds” means that it binds (or reacts) to human synuclein in which Ser of SEQ ID NO: 1 is phosphorylated, but binds (or reacts) to non-phosphorylated human synuclein. Means no reaction). For example, in SEQ ID NO: 1, it binds to a protein in which Serl29 is phosphorylated, but does not bind to a protein in which Serl29 is not phosphorylated.

Specifically, Ser-phosphorylated peptides or full-length α-synuclein give a positive reaction by ELISA or Western blot, but do not react with non-phosphorylated Ser or peptide. Means that.

An “antibody” is a polyclonal or monoclonal antibody that specifically binds to a fragment containing synuclein and phosphorylated Ser consisting of a full-length molecule, or a partial fragment of these antibodies (eg, papain or It means a fragment (Fab or F (al ') 2 or Fab') obtained by digestion with pepsin, and can be produced according to a known production method as described later.

“Synucleinopathic lesion detection drug” refers to the immunohistological study of the brain tissue of synucleinopathy that exhibits LB or other α-synuclein-positive lesions (eg, degenerative neurites such as GCI and Lewy neurite). Antibodies, reagents or drugs that show a positive reaction or specifically react with insoluble / accumulated human synuclein by Western blot analysis of brain tissue.

For detection of synucleinopathic lesions, use an antibody that binds to Serl29-phosphorylated protein but does not bind to Serl29-unphosphorylated protein in SEQ ID NO: 1 from the viewpoint of detection sensitivity. Is preferred.

The antibody according to the present invention can be produced according to the following production method.

(1) Preparation of antigen

The DLB brain is fractionated into soluble and insoluble fractions using various surfactants such as Triton-X and Sarkosyl. Further, the insoluble fraction is dissolved in urea, and purified using an anion exchange column, for example, a Q-sepharose column. After digesting the purified product with cyanogen bromide and separating the peptide fragment by HPLC, the Ser phosphorylated target product can be obtained. Also, synthetic peptides containing phosphorylated Ser, for example,

_{_{① CAYEMPS (P0 3 H 2)}} EEGYQ ( obtained by adding a cysteine to the N-terminus of SEQ ID NO: 2)

_{_{② CVEGAGS (P0 3 H 2)}} IAAAT ( obtained by adding a cysteine to the N-terminus of SEQ ID NO: 3) Can be synthesized by a solid phase method. The synthesized peptide uses N- (6- maleimidocaproy loxy) succ inimide as a condensing reagent, binds to Keyhole Lympet Hemocyanin, and uses it as an antigen. Incidentally, ① and ② are amino acid is a peptide represented by one letter notation, especially S (P0 ₃ ¾) represents Rinsani匕serine.

(2) Preparation of antibody

Immunization is carried out by administering the antigen peptide solution to a warm-blooded animal by itself or with a carrier or diluent about 2 to 10 times in total. The warm-blooded animals used include, for example, egrets, dogs, guinea pigs, mice and rats. It is preferable that a sample blood be taken at the time of subcutaneous immunization four to six times to measure the antibody titer. The measurement of the antibody titer in the serum can be carried out by fixing the peptide used as the antigen to a 96-well microtiter plate and ELISA. After confirming that the antibody titer has risen sufficiently, the whole blood can be collected and the antibody can be separated and purified by a commonly used method. Examples of the purification method include a gel filtration method and a purification method using an active adsorbent such as protein A. Specificity for phosphorylated α-synuclein can be improved.

(3) Preparation of monoclonal antibody

Monoclonal antibody-producing cells are prepared by selecting individuals with antibody titers from warm-blooded animals immunized with the antigen, collecting spleen or lymph nodes 2 to 5 days after the final immunization, and producing the antibody contained in them. By fusing the cells with myeloma cells, monoclonal antibody-producing eight hybridoma cells can be prepared. The fusion operation can be performed according to a known method, for example, the method of Kohler et al. (Nature, 256, 495 (1975)). Examples of myeloma cells include PAI and P3U1. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus (HVJ), preferably PEG having a molecular weight of 1,000 to 6,000. Cell fusion can be performed efficiently by adding at a concentration of about 10 to 80% and incubating at 20 to 40 ° C.

Selection of the monoclonal antibody can be performed according to a known method. Usually in animal cell culture medium supplemented with HAT (hypoxanthine, aminopterin, thymidine) Done. As a selection and breeding medium, for example, RPMI 1640 medium containing 10 to 20% fetal bovine serum can be used. The cultivation is usually performed under 5% CO 2 at a culture temperature of 20 to 40 for 5 days to 3 weeks.

The culture supernatant is collected from the wells in which the hybridoma cells have been cultured, and antibodies that react with the antigen peptide are selected by ELISA (enzyme-linked immunosorbent assay). First, the antigen peptide is placed on a 96-well plate, allowed to adsorb to the bottom overnight, and then blocked with calf serum. After reacting the supernatant of the hybridoma cells for 1 hour at 37:

Incubate Immunoglobulins / HRP (DAK0) at 37 ° C for 1 hour, and develop color using orthophenylenediamine as a substrate. After terminating the reaction with acid, measure the absorbance at 490 nm, select an antibody with a value of about 3 and perform cloning by limiting dilution.

The desired hybridoma cells thus obtained are cultured, and a monoclonal antibody can be obtained from the culture supernatant. Alternatively, the eight hybridoma cells can be transformed into, for example, a mouse (

Balb / c) can be administered intraperitoneally to obtain monoclonal antibodies from the ascites fluid. Purification of the monoclonal antibody can be carried out in the same manner as ordinary separation and purification of a polyclonal antibody. Industrial applicability

According to the present invention, it is possible to provide an antibody that specifically binds to human synuclein aggregates detected in a living body such as Lewy bodies, and it is possible to detect Levi bodies, pathological diagnosis of synucils including PD and DLB, etc. Useful for Example 1 Western blot analysis of <<-synuclein extracted from DLB brain

Gray matter was cut out from the cerebral cortex of the DLB brain and the normal brain, and was solubilized stepwise using various surfactants as follows. First, the cerebral cortex was treated with Tris A solution pH 7.5 [50 mM Tris (Gibco BRL), 1 mM EGTA (Wako Pure Chemical Industries), 0.5 mM PMSF (Boehringer Mannheim),

1 g / ml ant ipain (SIGMA), 1 xg / ml peps tat in (SIGMA), 1 g / ml leupept in (Wako Pure Chemical), 50 mM imidazole (Kanto Chemical), 25 mM i6-glycerophosphate (Kanto) chemistry), 20 mM NaF (Kanto chemical), homogenized in _{_{_{10 mM Na 4 P 2 0 7}}} ( Kanto chemical)] Then, the mixture was centrifuged at 1,000 X g for 5 minutes at 4 ° C in a centrifuge (Hitachi Machine). The obtained supernatant is 1000 g sup, and the obtained precipitate is lOOOOg ppt. The lOOOOg sup was centrifuged in a centrifuge at 4 ° (:, 15 minutes, 350,000 X g. The supernatant was used as the Tris-soluble fraction.

1000g ppt is homogenized in Triton-X solution [Tris A solution, 1% Triton-X 100 (Wako Pure Chemical), 10% sucrose (Kanto Chemical), 0.5 M NaCl (Kanto Chemical)], and centrifuged at 4 ° C, centrifuged at 350,000 × g for 15 minutes. The supernatant was a Triton-X soluble fraction.

The resulting precipitate was homogenized in a Sarkosyl solution [50 mM Tris pH7.5, ImM EGTA, 1% Sarkosyl (Wako Pure Chemical Industries)], and centrifuged at 350,000 Xg at 25 ° C for 15 minutes with a centrifuge. . The supernatant was a Sarkosyl soluble fraction.

The precipitate thus obtained was again homogenized in Sarkosyl solution, and then centrifuged at 350,000 X g at 25 ° C for 15 minutes in a centrifuge. The Sarkosyl-insoluble precipitate obtained here was homogenized in Tris A solution pH 7.5, and centrifuged at 350,000 X g for 25 to 15 minutes using a centrifuge. The resulting precipitate is sonicated in a urea solution [50 mM Tris pH 7.5, ImM EGTA, 8 M urea (nacalai tesque)] using an ultrasonic crusher (Brans on) and then placed in a 37 ° C water bath. Let stand for 30 minutes. Then, it was centrifuged at 350,000 X g at 25 ° C for 15 minutes. This supernatant is used as the urea-soluble fraction.

The Tris-soluble fraction, Triton-X-soluble fraction, Sarkosyl-soluble fraction, and urea-soluble fraction thus obtained were electrophoresed using SDS-PAGE (SDS polyacrylamide gel electrophoresis), and immobilon membrane ( MILLIPORE) and the immunoblotting method [Proc. Natl. Acad. Sci. USA, 94] using the monoclonal antibody LB509 [Am. J. Pathol, 152 879 (1998)] that specifically recognizes human α-synuclein. 2025 (1997)], normal soluble human synuclein was recovered in DLB brain, Tris-soluble fraction of normal brain, and Triton-X soluble fraction. On the other hand, in the urea-soluble fraction, insoluble hisuclein with a migration pattern almost identical to that of normal α-synuclein was detected at the position of 15 kilodaltons (kDa) on the Wesson blot specifically for DLB brain (Fig. 1). ).

Normal human synuclein was purified as follows. DL Add ammonium sulfate (Kanto Chemical) to the Tris-soluble fraction obtained from the cerebral cortex of the normal and normal brains to a final concentration of 50%, leave on ice for 30 minutes or more, and centrifuge for 4 minutes. Centrifuge at 350,000 X g for 15 minutes at ° C Released. Remove the supernatant, suspend the precipitate in Tris B solution pH 7.5 [50 mM Tris, ImM EGTA, 1% 2-mercaptoethanol (Kanto Chemical), 0.5 M NaCl], and heat treat [100 ° C, 5 minutes] Then, the supernatant was recovered by centrifugation. After removing the salt contained in the supernatant with a PD-10 column (Pharmacia Biotech), purification was performed using a DEAE Cellulose column (Whatman), and soluble tissin nuclein was eluted in about 0.1 M NaCl fraction. After concentration with a lyophilizer (Tomy), fractionation was performed by high-performance liquid chromatography (Hewlett Packard) using a TSKgel SuperSW3000 column (4.6X600 marshal, Tosoh). The fraction with a molecular weight of about 15 kDa, which was confirmed to contain α-synuclein by Western blotting using SDS-PAGE, was subjected to reverse phase HPLC using an Aquapore RP300 column (2.1 × 30 mm, Applied Biosystems). Was separated into fractions with acetonitrile (Kanto Chemical) concentration of about 60%.

Α-synuclein contained in the urea-soluble fraction derived from DLB brain was purified as follows. First, the protein contained in the urea-soluble fraction was adsorbed on a Q-sepharose column (Pharmacia Biotech) and adsorbed stepwise with a urea solution containing 0 M, 0.1 M, 0.2 M, 0.3 M, and 0.5 M NaCl. When the protein was eluted, synuclein eluted in the 0.3 M NaCl fraction. This fraction was fractionated by reverse-phase HPLC using an Aquapore RP300 column. —Synuclein was recovered in a fraction with an acetonitrile concentration of about 60%.

The Hishi nuclein contained in the Tris-soluble fraction and urea-soluble fraction purified using HPLC in this manner was dried with a freeze dryer and suspended in a 70% formic acid (Wako Pure Chemical) solution. Peptide bonds were chemically cleaved with 0.2% cyanogen bromide (Nacalai tesque). After the reaction, the solution was diluted about 10-fold, dried with a freeze dryer, and suspended in an 8 M aqueous solution of guanidine hydrochloride (Nacalai Tesque). The peptide fragments thus obtained were separated and fractionated by reversed-phase HPLC using a Superspher Select B column (2.1 × 125 thigh, Merck).

The separated fractions were analyzed with a TOF (time of flight) mass spectrometer (PerSeptive Biosystem) and an amino acid sequence analyzer (Applied Biosystems) [J. Biol. Chem. 267 17047 (1992)]. As a result, from the Tris-soluble fraction, a fraction with an acetonitrile concentration of about 31% was added to a fraction with a mass number of 1232 corresponding to the C-terminal part of 117 to 127 of the synuclein, With a mass number of 1515 corresponding to the end portion of 128 to 140 A signal was detected. From the urea-soluble fraction, in addition to these signals, a signal with a mass number of 1595, in which one phosphoric acid was added to the most C-terminal part of synuclein, was detected in the fraction with an acetonitrile concentration of about 31%. Was done. Further, the peak with a mass number of 1595 was subjected to MS / MS analysis by the nano'electrospray method [J. Neurochem., 712465 (1998)] to confirm the phosphorylation of Serl29. Example 2 Preparation of Antibody (anti-PSerl29)

It includes a 124-134 sequences of a- synuclein, and peptidyl-de containing Serl29 phosphorylated, synthesized by solid phase method CAYEMPS (P0 ₃ ¾) EEGYQ (Peptide Institute), KLH (Keyhol e Lympet Hemocyanin ) And conjugated with Conjugation with KLH followed standard practice. 1 ml of lmg / ml antigen peptide physiological saline solution and 1 ml of Freund's complete adjuvant (SIGMA) were emulsified by sonication, and immunized by dividing the heron (Japanese white, weight 2.7 kg, female) into at least 10 places on the back . One month later, 0.5 ml of 1 mg / m 1 antigen-peptide physiological saline solution and 1 ml of Freund's incomplete adjuvant (SI GMA) were emulsified by sonication and boosted in the same manner. A booster immunization was performed with 0.5 ml of lmg / ml antigen peptide physiological saline solution and 1 ml of Freund's incomplete adjuvant emulsified by sonication. Blood was collected one week after immunization, and the collected blood was mixed well with a Pasteur pipette, kept at room temperature for 1 hour, then left at 4 ° C, centrifuged at 5,000 X g for 10 minutes to remove antiserum. Obtained.

In order to purify the antibody, about 21111 of Affi-gel 10 (810-1 ^ 0 company) was reacted with about 7.5 mg of recombinant human synuclein (prepared using [FEBS Lett., 436 309 (1998)]). Column was prepared. After inactivated by treating the anti-serum 56 ° C, 10 _{_{minutes, PBS (8iM Na 2 HP0 4}} ( Wako Pure Chemical), 2mM Na¾P0 ₄ (Wako Pure Chemical), 131 mM NaCl) was diluted 5-fold with After passing through a 5 m filter (MILLIP0RE) and circulating through this column for 10 hours or more, the antibody not adsorbed to the column was designated as Ser 129 phosphorylated a-synuclein-specific antibody. Example 3 Staining of pathological tissue and Western blot analysis Serl29 phosphorylation—Synuclein-specific antibody (anti-PSerl 29) prepared in Example 2 was used to stain PD and DLB brains.

After fixing DLB cerebral cortex to formalin, cut into 50-micron-thick sections, and avidin-biotin complex method using anti-human human synuclein antibody LB509 (A) and Serl29 phosphorylation-specific human synuclein antibody (B). It was immunostained and developed brown with diaminobenzidine. As a result, related lesions such as LB and Lewy neurite showed a strong positive reaction in the midbrain substantia nigra and cerebral cortex, but normal α distributed finely in neuropils recognized by conventional anti-α-synuclein antibodies. —Synuclein [Am. J. Pathol, 152 879 (1998)] (FIG. 2A) was negative for this antibody (FIG. 2B). Glial cytoplasmic inclusions (GCI), which appeared in the brain of patients with multiple system atrophy (MSA), also showed a strong positive reaction to this antibody, similar to conventional anti-synuclein antibodies.

In Example 1, fractions obtained by gradually solubilizing the cerebral cortex of DLB brain and normal brain using various surfactants were analyzed by Western blotting using SDS-PAGE. The human synuclein-specific antibody does not react with the normal human synuclein contained in the Tris-soluble fraction and the Triton-X soluble fraction, but specifically with only α-synuclein contained in the urea-soluble fraction. Reacted (Figure 3). Example 4Specificity of antibody (anti_PSerl29)

It was confirmed by ELISA (enzyme-linked immunosorbent assay) that the single synuclein antibody prepared in Example 2 was a Serl 29-phosphorylated α_synuclein-specific antibody.

After adsorbing 5 zg / wel 1 of recombinant Hsinuclein or antigen peptide on the bottom of a 96-well plate (Greiner) according to a standard method, dilute the Hsinuclein antibody prepared in Example 2 100-fold and react. When the color was developed using a conventional method, it showed no binding to the recombinant ο; -synuclein, but showed strong reactivity with the antigen peptide (Fig. 4).

In the Western blotting method using SDS-PAGE, this antibody reacted strongly with histine nuclein, a urea-soluble fraction derived from DLB brain. The reaction was lost by reacting Escherichia coli alkaline phosphatase (SIGMA) at 10 units / mK at 65 ° C for 2 hours [J. Biol. Cheni. 267 17047 (1992)].

Furthermore, in immunohistochemical studies, the reactivity of the antibody to the antigen was absorbed by mixing the antibody with the antigen peptide [Am. L Pathol., 152 879 (1998)]. Reactivity of related lesions such as LB and Lewy neurite disappeared. Example 5 Preparation of Antibody (mAb PSerl29)

Facial - it comprises a sequence of 124-134 of synuclein, and peptides containing the Ser 129 phosphorylated, CAYEMPS (P0 ₃ H ₂₎ was KLH conjugated EEGYQ, and an antigen. 0.5% SDS was added to 100 1 of a 1 mg / ml antigenic peptide physiological saline solution, emulsified with Freund's complete adjuvant, and immunized on the back of a mouse (Balb / c, 6 weeks old). Two weeks later, a booster immunization was performed with 500 lmg / ml antigen peptide physiological saline solution, 0.5% SDS, and Freund's incomplete adjuvant emulsified by ultrasonic treatment, and thereafter, booster immunization was performed every week. On day 40 after immunization, the spleen was removed, and lymphocytes were removed in RPMI 1640 medium (containing penicillin and streptomycin), and subjected to erythrocyte treatment with 0.17 M ammonium chloride. The extracted lymphocytes were fused with the mouse myeloma-derived myeloma PAI strain by the polyethylene glycol method (PEG4000) to prepare hybridoma cells. The hybridoma cells were suspended in a HAT medium containing one feeder cell, dispensed into a 96-well plate (Greiner), and cultured for 15 days. Example 6 Screening of monoclonal antibody (mAb PSerl29)

The culture supernatant was collected from the wells in which the hybridoma cells were cultured, and mAb PSerl29, which reacts with the antigenic peptide, was selected by ELISA (enzyme-linked immunosorbent assay). First, 40 ^ 1 of 10 g / ml antigenic peptide was inoculated on a 96-well plate, adsorbed to the bottom of the plate at 4 ° C, and blocked with IOOI 10% calf serum at 37 ° C for 30 minutes. After reacting the supernatant 50 of the hybridoma cells for 37 hours, 37 hours, and then diluting Mouse I maraudal unoglobulins / HRP (DAK0) 1000 times, reacting at 37 ° C for 1 hour, and using orthophenylenediamine as a substrate. And developed. Stop the reaction with 25 ^ 1 8N sulfuric acid and measure absorbance at 490nm As a result, mAb PSerl29 with a value of about 3 was selected and cloned by the limiting dilution method.

Mice (Balb / c) to which 0.5 ml of pristane was intraperitoneally administered 7 days and 3 days before, respectively, were intraperitoneally injected with the selected mAb PSerl 29 hybridoma cells, and about 10 days later, ascites was collected. The collected ascites was left at room temperature for 30 minutes, left still at 4 ° C, centrifuged at 15K rpm for 10 minutes, and the supernatant was collected. Example 7 Specificity of Monoclonal Antibody (mAb PSerl29)

After adsorbing antigen-peptide or recombinant α-synuclein to the bottom surface of a 96-well plate, one-synuclein antibody (ascites) was diluted 1000-fold and allowed to react to develop color, but it did not bind to recombinant α-synuclein. It reacted specifically and strongly with the antigen peptide (Fig. 5).

DL Β The cerebral cortex of the brain was solubilized stepwise using various surfactants. The fractions analyzed by Western blotting using SDS-PAGE showed that Serl29 phosphorylated «-synuclein antibody was Tris It did not react with normal α-synuclein contained in the soluble fraction and Triton-X soluble fraction, but specifically reacted with histological nuclein contained in the urea soluble fraction (FIG. 6). Example 8 Preparation of Antibody (anti-PSer87)

Facial - comprises 82- 92 array of synuclein, and polypeptides comprising Ser87 phosphorylated _{[KLH] - CVEGAGS (P0 3} ¾) were synthesized IAAAT (Peptide Institute) was the antigen.

1 ml of lmg / ml antigen peptide physiological saline solution and 1 ml of Freund's complete adjuvant (SIGMA) were emulsified by sonication, and divided into 10 or more backs of egrets (Japanese white, weight 2.7 kg, female) I was immunized. One month later, 0.5 ml of lmg / ml antigenic peptide physiological saline solution and 1 ml of Freund's incomplete adjuvant (SIGMA) were emulsified by sonication for the second immunization. Then, 0.5 ml of a 1 mg / ml physiological saline solution of an antigen peptide and 1 ml of Freund's incomplete adjuvant were emulsified by sonication and boosted. 1 week after immunization The collected blood was mixed well with a Pasteur pipette, left at room temperature for 1 hour, allowed to stand at 4 ° C, and centrifuged at 5,000 X g for 10 minutes to obtain an antiserum.

To purify the antibody, about 7.5 mg of recombinant Hi-synuclein (prepared with [FEBS Lett., 436 309 (1998)]) was used for about 2 ml of Affi-gel 10 (BIO-RAD). A reacted column was prepared. After inactivated by treating the anti-serum 56 ° C, 10 _{_{minutes, PBS (8mM Na 2 HP0 4}} ( Wako Pure Chemical), 2mM Na¾P0 ₄ (Wako Pure Chemical), 131 mM NaCl

), And then passed through a 5μπι filter (MILLIPORE), and circulated through this column for 10 hours or more. The antibody not adsorbed to the column was defined as a Ser 87-Phissis nuclein-specific antibody. Example 9 Specificity of Antibody (anti-PSer87)

It was confirmed by ELISA (enzyme-linked immunosorbent assay) that the α-synuclein antibody prepared in Example 8 was an antibody specific to Ser87 phosphorylation α-synuclein.

After adsorbing 1.25 gell of recombinant α-synuclein or an antigen peptide (used for antibody production) on the bottom of a 96-well plate (Greiner), the human synuclein antibody prepared in Example 8 was diluted 100-fold. When the reaction was carried out, no binding was observed with the recombinant α-synuclein, but it was strongly reactive with the antigen peptide.

Claims

The scope of the claims

1. An antibody that specifically binds to a protein in which one or more Ser in SEQ ID NO: 1 is phosphorylated.

2. An antibody that specifically binds to a protein in which any one of Ser9, Ser42, Ser87 or Serl29 in SEQ ID NO: 1 is phosphorylated.

3. An antibody that specifically binds to a protein in which Serl 29 is phosphorylated in SEQ ID NO: 1.

4. An antibody that specifically binds to a phosphorylated Ser6 peptide of SEQ ID NO: 2.

5. The antibody according to claim 3 or 4, wherein Serl 29 in SEQ ID NO: 1 does not bind to a protein that is not phosphorylated.

6. An antibody that specifically binds to a protein in which Ser87 is phosphorylated in SEQ ID NO: 1.

7. An antibody that specifically binds to a phosphorylated Ser6 peptide in SEQ ID NO: 3.

8. The antibody according to claim 6 or 7, wherein Ser87 in SEQ ID NO: 1 does not bind to a protein that is not phosphorylated.

9. The antibody according to any one of claims 1 to 8, which is a monoclonal antibody.

10. The antibody according to any one of claims 1 to 8, which is a polyclonal antibody.

11. A drug for detecting a synucleinopathy lesion, comprising the antibody according to any one of claims 1 to 10.

12. A method for detecting synuclein, wherein Serl29 or Ser87 is phosphorylated in SEQ ID NO: 1, wherein the antibody according to any one of claims 1 to 10 is used.