JPH04252195A - Monoclonal antibody and hybridoma - Google Patents

Abstract

PURPOSE:To provide a novel monoclonal antibody for e.g. diagnosing Alzheimer' s disease, common to senile macula amyloid precursor protein APP 770, 751 and 695 and capable of specifically recognizing part of the section other than the sequence from the membrane penetration region to C-terminal. CONSTITUTION:Human senile macula amyloid precursor protein is boiled for 3 min in a surfactant-contg. phosphoric acid buffer solution followed by acetone treatment, and the resulting protein, in combination with an adjuvant, is administered to Balb/c mice through subcutaneous injection to immunize the mice; after the final immunization, the mice's splenocytes are collected and fused with myelolipoma strain cells followed by culture in a HAT medium, thus obtaining a hybridoma. Thence, the hybridoma is screened to select a clone capable of producing anti-human senile macula amyloid precursor protein antibody, the clone being then cloned by e.g. the limiting dilution analysis to effect monocloning. The resulting hybridoma is then incubated, thus obtaining the objective novel monoclonal antibody.

Description

[Detailed description of the invention]

0001

TECHNICAL FIELD The present invention relates to a novel monoclonal antibody and a hybridoma producing the same. Specifically, the present invention relates to a novel monoclonal antibody that specifically recognizes human senile plaque amyloid precursor protein and is useful as a diagnostic agent for neurological diseases including senile dementia. More specifically, the present invention relates to a novel monoclonal antibody that recognizes human senile plaque amyloid precursor proteins APP695, APP751, and APP770.

0002

BACKGROUND OF THE INVENTION In recent years, with the aging of the population, diseases of the brain and nervous system, including senile dementia, are becoming a social problem. Among these diseases, there are many diseases for which the cause, simple diagnosis method, or treatment method is unknown, and research and development of these diseases is strongly desired. Such diseases include Alzheimer's senile dementia and Alzheimer's disease (these two
The disease (hereinafter abbreviated as AD) is included. In the brains of AD patients, structures called senile plaques, whose main component is amyloid-β protein, are deposited in larger amounts than in normal people. The relationship has been attracting attention. Kang et al. found that senile plaque amyloid has a precursor (hereinafter abbreviated as APP) (hereinafter discovered by Kang et al.6).
APP consisting of 95 amino acids is abbreviated as APP695) was reported (Kang et al., Nature Vol. 32).
5 733-736 1987. Japanese Unexamined Patent Publication No. 63-22
No. 2693), and further research including the present inventors revealed that A
There are at least three types of PP (in addition to APP695, there are 7
APP751 consisting of 51 amino acids and APP770 consisting of 770 amino acids) were found to exist (Ponte et al., Nature Vol. 331).
525-527 1988, Tanzi et al., Natu
reVol. 331 528-530 1988,
Kitaguchi et al., Nature Vol. 33
1 530-532 1988, patent application published 2002
-501796, International Publication Number WO89/07657)
. Furthermore, senile plaque amyloid AP consisting of 714 amino acids
P714 (Golde et al. Neuron Vol.
4253-267 1990), and APP5 consisting of 563 amino acids, which does not have the senile plaque amyloid part.
63 (de Sauvage et al. ScienceV
ol. 245 651-653 1989) is also known to exist, although it is a minor component (Figure 1).

[0003] APP751 and APP770 have Kun
It was revealed that there is a region with high homology to the itz-type protease inhibitor, and that protease inhibitor activity actually exists (FIG. 1). Compared to APP695, APP751 has 56 amino acid insertions and 1 amino acid substitution. APP770 has A
There are 19 amino acid insertions and 1 amino acid substitution compared to PP751. The protease inhibitor activity is mainly responsible for this 56 amino acid inserted sequence (hereinafter abbreviated as KPI).

[0004] If the intracerebral deposition of senile plaque amyloid in AD patients is considered to be the result of intracerebral metabolic inhibition by protease inhibitors and incomplete degradation of APP, then overexpression of APP with protease inhibitor activity may result in A.
It is possible that this plays some role in the onset of D. From this point of view, the present inventors investigated the expression levels of three types of APP messenger RNA in the brains of AD patients and normal people, and found that APP770
The results showed that the expression level of messenger RNA was about twice as high. APP751 was also 1.5% higher in AD patients than in controls.
We have obtained results that are 1 to 1.3 times higher (Tana
ka et al., B. B. R. C. Vol. 157 472-
479 1988 and B. B. R. C. Vol. 16
51406-1414 1990). In addition, using human cerebrospinal fluid as a sample, protease inhibitors (KPI)
The amount of APP or its fragment containing the region was determined by trypsin ELI.
SA method (Kitaguchi et al., B.B.R.C. Vo
l. 166 1453-1459 1990 and Japanese Patent Applications Hei 1-214118 and Hei 2-220889), results showed that the AD group showed significantly higher values than the cerebrovascular dementia group ( Kitagu
Chi et al., B. B. R. C. Vol. 1661453-
1459 1990 and 31st Annual Meeting of the Japanese Society of Neurology 241 pages II-J-05
(1990). This result shows that APP770 and APP7
Protease inhibitors (K
A sandwich ELISA method (trypsin ELIS) using a rabbit antiserum that recognizes the PI region and trypsin (which binds strongly to this protease inhibitor region)
It was obtained by method A). (However, ELISA
What is Enzyme Linked Immuno?
(abbreviation for sorbent assay). However, for example, when serum or plasma is used as a test sample and trypsin is used instead of the first antibody by binding to a solid phase, trypsin binds to the protease inhibitor in the blood, so the solid phase of the assay plate In some cases, the amount of the protein to be detected (ie, APP) bound to the target protein is not sufficient. Therefore, instead of trypsin, an antibody that can recognize both APP770 and APP751, which is suitable for two-antibody sandwich ELISA, is recommended.It is also a monoclonal antibody that can industrially provide a stable supply of specific antibodies and can supply diagnostic kits of constant quality. Antibodies are desired.

[0005] In addition, APP695 and APP75 in cerebrospinal fluid
1. There is a report that the total amount of APP770 (all APPs together) is significantly increased in AD patients compared to controls (Weidemann C
ell Vol. 57 115-126, 1989
). Furthermore, APP695/APP751 in cerebrospinal fluid and blood
/APP770 ratio may be useful for AD diagnosis.

As a monoclonal antibody capable of recognizing all APP, there is 22C11, which was obtained by immunizing mice with a protein obtained by expressing a fusion protein of mouse immunoglobulin M and APP695 in Escherichia coli (Weidemann et al., Cell 57, 115). -126, 1989)
. However, 22C11 has only been used for Western blotting to detect antigens denatured with sodium dodecyl sulfate (hereinafter abbreviated as SDS) and β-mercaptoethanol, and has not been used for ELISA of non-denatured antigens.

From this point of view, APP770, AP
All three types, P751 and APP695, can be recognized, and EL
There has been a need for monoclonal antibodies suitable for ISA, particularly two-antibody sandwich ELISA.

[0008]

[Problem to be solved by the invention] Therefore, this invention
All three species, APP770, APP751, and APP695, can be recognized, and ELISA, especially 2-antibody sandwich E
An object of the present invention is to provide a monoclonal antibody suitable for LISA and a hybridoma producing the same.

[0009]

[Means for Solving the Problems] The present invention was completed as a result of extensive research by the present inventors in order to achieve the above-mentioned objects. In order to solve the above problems, the present inventors developed APP69 from which the sequence from the transmembrane region to the C-terminus was removed.
By using a peptide or protein containing 5 or more amino acids out of the 5 amino acid sequence as an antigen, APP
770, APP751, and APP695, as well as a hybridoma that produces the monoclonal antibodies, which led to the completion of the present invention. APP excluding the sequence from the transmembrane region to the C-terminus
695, e.g., APP592 described in Example 1 (Fig.
) is the C-terminal part 103 amino acids (transmembrane part and intracellular part) of APP695 deleted, and the amino acid sequence other than valine at position 289 is APP751, APP7
The amino acid sequence is the same as that of the corresponding portion of 70 (portion other than the insertion sequence unique to APP751 and APP770). Therefore, A excluding the sequence from the transmembrane region to the C-terminus
If a peptide or protein containing 5 or more amino acids of the amino acid sequence of PP695 is used as an antigen, APP
Hybridomas that produce monoclonal antibodies that can recognize all three types, ie, APP770, APP751, and APP695, can be obtained with a high probability.

[0010] In order to obtain such a monoclonal antibody, it is possible to use APP695 itself as an antigen; however, APP695 contains a sequence from the transmembrane portion to the C-terminus. On the other hand, APP695 (e.g. APP592), which excludes the sequence from the transmembrane region to the C-terminus, does not have the amino acid sequence from the transmembrane region to the C-terminus. It is easily purified because it is secreted into the serum. Furthermore, most of the APPs present in body fluids such as cerebrospinal fluid and blood do not have a sequence from the transmembrane part to the C-terminus, whereas they have a sequence from the transmembrane part to the C-terminus. APP is likely to be bound to lipid membranes, so normal ELISA
In this system, it is difficult to detect the region downstream from the transmembrane region.
Antibodies against antigens with this part deleted are more likely to have APP
APP695 and APP7 in body fluids than 695 itself
It is suitable for obtaining antibodies for detecting 51, APP770 or its fragments.

That is, the present invention provides APP770, APP7
51, a monoclonal antibody that recognizes all three types of APP695, and a hybridoma that produces the monoclonal antibody. Preparation of Antigens In general, when creating an antibody that recognizes a certain protein, it is desirable to use as an antigen a peptide having a continuous amino acid sequence of five or more residues in the amino acid sequence of the protein. Therefore, a peptide containing an amino acid sequence of five or more consecutive residues in APP695 excluding the sequence from the transmembrane region to the C-terminus can be used as an antigen. Such peptides can be produced using organic chemical techniques such as liquid phase or solid phase synthesis methods. Furthermore, it can also be produced by recombinant DNA technology. When producing APP695 with the sequence from the transmembrane region to the C-terminus removed by a recombinant DNA method using APP cDNA, if a naturally occurring restriction enzyme cleavage site is used, the N
APP592 having a sequence of 592 amino acids from the end can be easily produced. In addition, using other restriction enzyme cleavage sites, appropriate linkers, and mutators,
It is also possible to create an antigen containing all or a portion of PP695 from the N-terminus to the 623rd residue immediately before the transmembrane region.

Alternatively, immunization with a peptide or protein containing at least a part of the sequence in APP751 and APP770 that is common to APP695, excluding the sequence from the transmembrane region to the C-terminus, can be used as an antigen. APP770, APP751, APP
It is also possible to select clones that produce monoclonal antibodies that react with all 695.

[0013] When producing these antigens in animal cells and purifying the products secreted into the culture supernatant, a signal peptide (often 17 amino acids from the N-terminus) is used. For example, as described by Palmer MR et al. , Neurobio
logic Vol. 40 1028-1034, 1
990) have been removed. In the present invention, proteins used as antigens are expressed as containing this signal peptide; however, when using an antigen secreted from animal cells, the signal peptide is removed at the time of immunization.

[0014] Furthermore, the protein used as an antigen in the present invention is
It may also be a fusion protein of part or all of APP and another protein. In particular, in the case of production by recombinant DNA methods using microbial hosts such as Escherichia coli and Bacillus subtilis, such fusion proteins are preferred because they increase the production amount. As a partner protein for fusion of part or all of APP, β-galactosidase, immunoglobulin (preferably from an animal immunized with the antigen), tumor necrosis factor, etc. are used. Especially β
A fusion protein with part or all of galactosidase is preferred because it is easy to purify. An example of such a fusion protein is ZKX, described in Example 1.

[0015] For immunization, the peptide may be used as it is, and especially when the number of amino acid residues is about 20 or more, the peptide usually has sufficient antigenicity even when immunized as it is. When the number of amino acid residues is 5 to 20, the antigenicity is often not very high, and in order to further increase the antigenicity, it is preferable to use a carrier protein. Examples of carrier proteins include bovine serum albumin, immunoglobulin, thyroglobulin, KLH (Keyhol
e Limpet Hemocyanin) and the like. The method for binding the carrier protein and antigen peptide is preferably an active ester method using a carbodiimide compound or an MBS method. To explain the MBS method, cysteine is bonded to the N-terminus or C-terminus of an antigen peptide with an amide bond, and the thiol group of cysteine is reacted with meta-maleimidobenzoic acid-N-hydroxysuccinimide ester (hereinafter abbreviated as MBS). Furthermore, an antigen peptide compound and MBS are added to the amino group of the carrier protein.
This is a method of reacting conjugates of In this way, a peptide compound that can be used for antibody production according to the present invention is obtained.

[0016] APP695 itself, APP751,
Immunization with a peptide or protein containing at least part of the sequence in APP770 that is common to APP695, and the resulting monoclonal antibody
1. Among the sequences common to APP695, a method of screening for peptides and proteins that do not contain the sequence from the transmembrane region to the C-terminus can also be adopted.

Immunization Mice are immunized using the above-mentioned peptide compound or protein as an antigen in a conventional manner. For example, 10 to 50 μg of antigen per mouse at a time may be administered directly or
Denaturing treatments, e.g. Laemmli's SDS-PAGE sample buffer (Laemmli U.K. Nat.
ure Vol. 227 680-6851970
) for 3 minutes at 100°C, followed by intraperitoneal or subcutaneous immunization with Freud's complete or incomplete adjuvant, or intravenously without adjuvant. . It is better to perform denaturation treatment.
Antibody titer in serum may increase easily. The immunization interval is usually one to two weeks, and the number of immunizations is usually about 3 to 15 times. Establishment of immunity can be confirmed by collecting blood from sensitized mice and measuring the antibody titer in the blood using a standard ELISA method.

Cell fusion and hybridoma selection Next, the method for producing monoclonal antibodies and hybridomas according to the present invention will be explained below. As mentioned above, hybridomas are produced by fusing myeloma and antibody-producing cells. For antibody-producing cells, “preparation of antigen”
Use splenocytes or lymph node cells from mice, rats, etc. that have been immunized with the antigens described above. It is usually preferable to use cells from the same species of animal from which the myelocytoma and the antibody-producing cells are derived. One preferred hybridoma for practicing the invention is a hybridoma between splenocytes or lymph node cells of a mouse immunized with the antigens described above and a mouse myelocytoma. As the mouse myelocytoma, Balb/c mouse-derived myelocytoma P3X36-Ag8-653 is preferred because of its high fusion efficiency. Other myelocytomas include P3X63Ag8, P3
X63Ag8U. 8-Azagunine resistant cell lines such as 1, SP2/0-Ag14 (mouse) and YB2/0 (rat) may also be used. Generation of hybridomas and selection of target monoclonal antibody-producing hybridomas can be carried out, for example, as follows. Antibody-producing cells and myelocytoma are fused using a cell fusion promoting substance such as polyethylene glycol. The resulting hybridoma grows in a medium containing hypoxanthine, aminopterin, and thymidine (hereinafter abbreviated as HA medium). The antibody-producing cells and myelocytoma that are not fused die in the medium, and only the hybridoma produced by the fusion of both cells becomes viable. Next, hybridomas producing the antibody of interest are selected as follows. When immunizing with the antigen as is, add it as is, or when immunizing with the antigen denatured with SDS, add the hybridoma culture supernatant to a polystyrene 96-well microplate coated with the antigen denatured with SDS, and perform a regular solid-phase ELISA. practice the law In this process, antigen-specific hybridomas are selected, and further cloning is performed by limiting dilution to select hybridomas that exhibit stable antibody production ability. As the hybridoma thus obtained, for example, 1B2 (
Microtechnical Research Institute No. 11942), 3B2 (Microtechnology Research Institute No. 1
1943) and 6A3 (Feikoken Bibori No. 11944).

The desired monoclonal antibody can be recovered from the culture supernatant of the hybridoma thus selected by separation and purification operations such as salting out, ion exchange chromatography, and protein A columns, but it is difficult to obtain it in large quantities. In this case, the hybridoma may be grown in the peritoneal cavity of a histocompatible animal, nude mouse, etc., and the monoclonal antibody produced in the ascites of the animal may be purified.

Diagnostic method The monoclonal antibody according to the present invention is used below to diagnose AP.
A method for diagnosing Alzheimer's disease in which P-derived protein is detected by immunoassay will be described. Examples of the specimen include cerebrospinal fluid (CSF), plasma, serum, urine, and the like. Examples of immunoassays include enzyme immunoassay (
ELISA), radioimmunoassay (RIA), trypsin ELISA (Kitaguchi et al., B.B.R.
．． C. 166 1453-1459 1990 and Japanese Patent Application No. 1-214118, Japanese Patent Application No. 2-101017),
Examples include immunoblotting (typically Western blotting).

[0021] A method using ELISA will be described. For example, using a polystyrene microplate as a solid phase, C.
Samples such as SF, plasma, serum, urine, etc. are placed in wells together with a protein adsorption buffer, and left to stand for a certain period of time. As a buffer for protein adsorption, sodium carbonate-sodium hydrogen carbonate buffer with pH around 9.6 or PBS
(-) etc. are used. As for the conditions for standing, it is common to leave at 4° C. overnight, but it is also possible to leave at room temperature for about 2 hours, for example. The polystyrene microplate with the sample adsorbed in this way was soaked in PBS (
-), etc. After washing, a monoclonal antibody according to the present invention is added as a primary antibody and allowed to react for a certain period of time. Next, the plate is washed in the same manner as above, an enzyme-labeled anti-mouse immunoglobulin antibody is added as a secondary antibody, and the reaction is allowed to occur again for a certain period of time. Alternatively, the primary antibody may be directly labeled with an enzyme. Furthermore, after washing the plate in the same manner as above, an enzyme substrate is added and enzyme activity is measured. The enzymes used include horseradish peroxidase (HRP) and β-galactosidase (β-
Gal), alkaline phosphatase (AP), and the like.

[0022] Also, so-called two-antibody sandwich ELISA using two types of monoclonal antibodies (or a monoclonal antibody and an antiserum) that recognize different antigenic determinants
is also effective from the point of view of sensitivity. The monoclonal antibody of the present invention can be used in combination with other antibodies against APP, such as antibodies against the protease inhibitory active region present in APP770 and APP751, to form a two-antibody sandwich EL.
An ISA system can be assembled and APP can be detected with high sensitivity.

[0023] RIA can be thought of as a method in which an antibody or antigen is labeled with a radioactive isotope instead of an enzyme-labeled antibody in ELISA, and trypsin ELISA can be thought of as a method in which trypsin, anhydrotrypsin, etc. is used in place of one of the antibodies in sandwich ELISA. . When performing trypsin ELISA using the monoclonal antibody of the present invention, it may be preferable to use anhydrotrypsin or anhydrochymotrypsin, which does not have hydrolytic activity, in order to avoid digestion and removal of antigenic determinants by trypsin. When detecting APP by trypsin ELISA, APP or a fragment thereof containing a protease inhibitory active site (protease inhibitor region) can be detected.

Next, Western plotting, which is a type of immunoplotting, will be explained (Towbi
n et al., Proc. Natl. Acad. Sci. U
SAVol. 76 4350-4354 1979
). Specimens such as CSF, plasma, serum, urine, etc. can be used directly or after denaturation treatment, such as Laemmli's SDS-PAGE.
Sample buffer (Laemmli U.K.Na
ture Vol. 227680-685 197
0) for 3 minutes at 100°C, and then subjected to polyacrylamide gel electrophoresis. Subsequently, this gel is equilibrated in a transfer buffer, and then the proteins in the gel are electrically transferred to a nitrocellulose or nylon filter. After blocking this filter with BSA, gelatin, skim milk, etc. in a buffer such as Tris-HCl buffer, the monoclonal antibody according to the present invention is allowed to react with the antigen transferred onto the filter for a certain period of time. After the reaction, the filter is washed, and an enzyme-labeled anti-mouse immunoglobulin antibody is added as a secondary antibody to react with the antibody according to the present invention. After washing, a substrate for the enzyme is added and reacted on the filter. When an HRP-labeled antibody is used as the secondary antibody, 4-chloronaphthol or the like is used as the substrate. A chemiluminescent system using luminol as a substrate is also used. The amount of antigen contained in the sample can be determined by measuring the degree of color development on the filter, or in the case of chemiluminescence, the degree of blackening after exposing the film with a densitometer. It is possible to measure Furthermore, the enzyme may be directly bound to the primary antibody, or an antibody labeled with a radioisotope may be used instead of the enzyme-labeled antibody. In this case, it is possible to measure by autoradiography. It is also possible to measure the radioactivity of a specific band on the filter using a liquid scintillator, γ-well counter, etc. after cutting out that band.

[0025] Furthermore, the monoclonal antibody of the present invention can be bound to an insoluble carrier such as polyacrylamide, sepharose, agarose, etc., and used for the isolation and purification of APP or a fragment thereof.

[0026]

EXAMPLES The present invention will be explained in detail with reference to Examples below, but the present invention is not limited by these Examples. still,
The basic operations in the examples were in accordance with the following literature. T. M
Aniatis et al., Molecular Cloni
ng, A Laboratory Manual,
Cold Spring Harbor Lab
oratory (1982)D. M. Glover et al.
DNA Cloning, IRL Press (1
985)

[0027]

[Example 1] Preparation of antigen and assay protein [
Step 1] Preparation of antigen ZKX for immunization First, the production of ZKX, which is a fusion protein of a part of β-galactosidase and a fragment from the 19th residue to the 379th residue counting from the N-terminus of APP770, will be described. [Step 1-1] Expression plasmid pEX-ZKX
Construction of expression vector pEX2 (Boehringer Mannheim Yamanouchi) was digested with restriction enzymes Bsu36I (MstII) and S
A linear vector DNA fragment was obtained by cutting with alI. On the other hand, Example 1 of European Patent Application Publication No. 0304013
Plasmid pGBP2 (ATCC-67502
) was digested with KpnI and XhoI, and a DNA fragment encoding approximately 360 residues on the N-terminal side of APP770 was isolated by agarose gel electrophoresis using the Funakoshi Gene Clean Kit. Furthermore, 6 of sequence numbers 1 to 6
three synthetic linkers, 1, 2, 3, 4, 5, 6 (annealing these linkers results in Mst
II cut end and KpnI cut end, and the M of pEX2
(encodes consecutive amino acids without shifting the frame even when bound to the stII site) was synthesized using Applied Biosystems DNA Synthesizer 380A, and ligated with the two previously obtained DNA fragments using T4 DNA ligase. Ta. Figure 3 shows an overview of this.

The reaction product was prepared using Escherichia coli POP213 prepared according to the method described in the experimental book of Maniatis et al.
6 strains (Boehringer Mannheim Yamanouchi) were added to competent cells and transformed. Six ampicillin-resistant transformed colonies were selected, each plasmid was extracted, and B
When cut with amHI, all of them contained the desired plasmid. The obtained plasmid was pEX-ZKX
It was named. This plasmid expresses the fusion protein ZKX, which contains approximately 150 amino acids at the N-terminus of β-galactosidase and the sequence from residue 19 to residue 379 counting from the N-terminus of APP770, under the control of the PR promoter derived from lambda phage. I can do it. ZKX is APP7
In addition to a part of the sequence common to 70, APP751, and APP695, the protease inhibitor part and APP7
70-specific sequences, but from the transmembrane region of APP.
Does not include sequences up to the end. [Step 1-2] Escherichia coli POP2 carrying plasmid pEX-ZKX obtained in fusion protein production step 1-1
136/pEX-ZKX was cultured overnight at 30°C in L medium containing 50 μg/ml ampicillin. 1 ml of main culture solution was added to 50 μg/ml ampicillin-containing L medium 50 μg/ml.
ml and cultured at 30°C for 3 hours, then transferred to a constant temperature culture tank at 42°C and further cultured for 3 hours. At 42°C, the repressor of the lambda phage PR promoter is inactivated, and the desired fusion protein is produced by operating the PR promoter.

[0029] The culture solution is centrifuged to collect the bacterial cells, and a portion of the bacterial cells are collected by the method of Laemmli [Laemmli, U.S. K. ，
Nature Vol. 227.680-685 (1
970)], after boiling in SDS/sample buffer, using SDS using a 10% polyacrylamide gel.
- Subjected to polyacrylamide gel electrophoresis. After electrophoresis,
As a result of Coomassie staining of the gel, it was confirmed that the desired fusion protein with a molecular weight of approximately 58 kDa was present. Hereinafter, this partial β-galactosidase/partial APP770 fusion protein will be abbreviated as ZKX. [Step 1-3] In the same manner as ZKX partial purification step 1-2, E. coli POP2136/pE
X-ZKX was cultured, and bacterial cells were collected from 200 ml of the culture solution. The cultured bacterial cells were added to 20 ml of 4M urea
Suspended in mM Tris-HCl (pH 8.0) buffer,
The bacterial cells were destroyed by ultrasonic waves. Centrifuge the destroyed bacterial cells (100
00×g for 10 minutes), and the precipitate was collected. Next, this precipitate was resuspended in 20 ml of 20 mM Tris-HCl (pH 8.0) buffer containing 8 M urea, and the precipitate was dispersed by sonication. Centrifugation (10000×g,
After 10 minutes), the supernatant was collected. By the above operation, 4M
Escherichia coli proteins soluble in urea and Escherichia coli proteins insoluble in 8M urea were removed to obtain a partially purified solution of ZKX. When a part of this partially purified solution was analyzed by SDS-polyacrylamide gel electrophoresis, more than 50% of the total protein was the target fusion protein ZKX. Z
The trypsin inhibitory activity of KX was investigated using European Patent Publication No. 030.
When measured according to the method for measuring the trypsin inhibitory activity of sPI described in Example No. 4013, it showed strong inhibitory activity. Furthermore, this ZKX reacted strongly even in Western blotting using the anti-ZtPI antiserum described in Example 5 of the present invention. [Step 1-5] Preparation of anti-ZKX antiserum A rabbit was immunized with the ZKX obtained in step 1-4 to obtain an antiserum. That is, the SDS-modified APP5 described in Example 1 step 4(i) described below
Create SDS-denatured ZKX using the same method as 92, and
00 μg of SDS-denatured ZKX dissolved in 1 ml of PBS (-) was injected subcutaneously into the backs of each of two domestic rabbits together with complete Freund's adjuvant, and then injected at intervals of 2 weeks with incomplete Freund's adjuvant. 6
Two immunizations were performed. Antibody titers in serum are non-denatured and SD
This was performed by solid-phase ELISA using S-denatured ZKX (25 ng/well) as an antigen. The final titer of the antiserum is expressed as the dilution of the antiserum that shows more than twice the color development of the control (wells without antigen).
6000 times, 32000 times that of SDS-modified ZKX, and APP667, A described in Example 1 Step 2 described below.
It reacted strongly to PP592 and also to ZtPI described in Example 5 of the present invention. [Step 2] Preparation of immunization antigen APP592 APP6
A consisting of 592 amino acids (including the signal peptide), which is a protein in which 103 amino acids on the C-terminal side of 95 have been deleted.
PP592 is human senile plaque amyloid precursor A obtained by the method described in Example 1 of European Patent Publication No. 0304013.
Starting from PP695 cDNA, it was produced in monkey kidney-derived COS-1 cells by the method described in Example 5 of the above-mentioned European Patent Publication No. 0304013 and secreted into the culture supernatant,
Purified. The details will be explained below.

Plasmid pSVMT592 described in Example 5 Step 2 of European Patent Publication No. 0304013 was transformed into COS-
1 cell. That is, pSVMT592 20μ
g was applied to 1 x 106 COS-1 cells in sucrose-containing PBS (272 mM sucrose, 7 mM sodium phosphate (pH 7.4), 1 mM MgCl2) using a Bio-Rad Gene Pulser at a voltage of 400 V. Induction was performed by applying two electric pulses with a 30 second interval between 1.0 and 1.3 msec using a 3 μF capacitor. The cells after introduction were incubated at 37°C in Dulbecco's modified minimal basic medium containing 10% fetal calf serum (FCS).
, Dulbecco's phosphate buffered saline (Ca2+, Mg2+ free) (
After washing with PBS (hereinafter abbreviated as "-") to remove FCS, the medium was changed to a medium not containing FCS, and thereafter the medium was replaced every 48 hours, and the culture supernatant was collected three times in total. Further introduction 3
This was repeated 0 times to obtain a total of 900 ml of culture supernatant.

Next, (p-amidinophenyl)methanesulfonyl fluoride hydrochloride (Wako Pure Chemical Industries, Ltd.) was added to the culture supernatant of the plasmid-introduced COS-1 cells to a final concentration of 2 mM, and centriprep30
(Amicon) for concentration and further 2
Buffer exchange was performed to 0mM Tris-HCl (pH 8.0). Next, the above sample was applied to a DEAE-Sepharose column (Pharmacia), and 2
0mM Tris-HCl (pH 8.0), NaCl gradient 0
Column chromatography was performed under -1M conditions. For each chromatographic fraction, perform solid phase ELISA using the rabbit anti-ZKX antiserum obtained in steps 1-5,
The APP elution position was identified. APP592 is 0.3-
Eluted with 0.4M NaCl. The ELISA positive fraction was concentrated using Centricon 30 (Amicon), and further added to 50mM sodium phosphate (pH 7.2) in the same device.
The buffer was exchanged.

Next, affinity chromatography using a heparin column was performed. That is, DEAE-S
Partially purified fractions using an epharose column (Pharmacia) were purified using a heparin-5PW column (Tosoh Corporation).
chromatography was performed under conditions of a NaCl gradient of 0-2M. Solid-phase ELIS using rabbit anti-ZKX antiserum as well for each chromatographic fraction.
A was performed to identify the APP592 elution position. APP5
92 eluted with 1.0-1.2M NaCl. ELIS
The A-positive fraction was concentrated using Centricon TM30 (Amicon) and buffer exchanged into 20mM Tris-HCl (pH 8.0). These were added to 7.5% SDS-PAG
Coomassie staining was performed to confirm purity and molecular weight. CO introduced with pSVMT592
The protein obtained from the S-1 cell culture supernatant by the above procedure was a rather broad single band on SDS-PAGE, and the apparent molecular weight was 94.5 kDa.
It was decided. Furthermore, when the amino acid sequence of the N-terminus of this protein was analyzed using an Applied gas phase sequencer, we found that
Signal peptide (17 from the N-terminus) of APP695
1 from the N-terminus of APP695, in which the amino acid) was removed.
The amino acid sequence from the 8th Leu to the 38th Gln (also common to APP751 and APP770) was confirmed. Purified APP592 was thus obtained. [Step 3] Assay antigen APP667 and AP
In order to evaluate the prepared antibody for P648, we used 6, which is a protein in which the C-terminal 103 amino acids of each of APP751 and APP770 are deleted.
AP consisting of 48 amino acids (including signal peptide)
P648 and APP667 consisting of 667 amino acids (including signal peptide) were prepared. That is, plasmids pSVMT667 and pSVMT648 described in Example 5 Step 2 of European Patent Publication No. 0304013,
In the same manner as Step 2 of this Example, introduction into COS-1 cells, collection of culture supernatant, and purification were performed. During purification, in addition to rabbit anti-ZKX antiserum, monoclonal antibody ADJ5-3-7 (Feikoken Bacteria No. 10388), A
For PP667 and APP648, the anti-ZtPI antiserum described in Example 5 of the present invention was also used. Furthermore, APP
As for APP667 and APP648, their trypsin inhibitory activity was also used as a means of confirmation (the inhibitory activity was measured according to the method for measuring the trypsin inhibitory activity of sPI described in the Examples of European Patent Publication No. 0304013). The molecular weights of purified APP648 and APP667 were determined to be 100.5 kDa and 1 kDa, respectively, by SDS-PAGE under reducing conditions.
It was 05.5kDa. [Step 4] Creation of monoclonal antibody-producing hybridomas (i) Mouse sensitization Antigens for the creation of hybridomas were prepared as follows. APP592 obtained in Step 2 of this example was dissolved in PBS.
(-) and Laemmli's SDS-PAGE sample buffer (Laemmli U.K. Nat.
ure Vol. 227, 680-685, 1970
) for 3 minutes at 100°C, 4 times the volume of acetone was added, cooled at -20°C for 1 hour, and centrifuged at 9000 rpm for 20 minutes. In this manner, excess SDS was removed, and SDS-denatured APP592 was recovered from the precipitate, which was used as an antigen for hybridoma production. For immunization, 20 μg of SDS-denatured APP592 was added to 500 μl of PB.
Three 6-week-old Ba mice were each dissolved in S(-).
It was subcutaneously injected into lb/c female mice together with complete Freund's adjuvant, and thereafter immunized three times in total at two-week intervals. Furthermore, two subcutaneous injections were performed at one week intervals.

[0033] During the immunization process, the increase in serum antibody titer against the antigen was measured using solid-phase ELISA according to the Monoclonal Antibody Experiment Manual (written by Sakuji Toyama, Kodansha Scientific).
Confirmed by law. (ii) Cell fusion Cell fusion was performed according to the Monoclonal Antibody Experiment Manual (written by Sakuji Toyama, Kodansha Scientific). That is, 200 μl of P was administered to the mouse whose serum antibody titer was highest.
25 μg of SDS-denatured APP59 dissolved in BS(-)
2 was injected into the tail vein as a boost. Three days later, the splenocytes were removed aseptically and loosened into single cells using a stainless steel mesh, and 8-azaguanine-resistant myeloid cell line P3X63-Ag8-653 (1/5 amount of splenocytes) (from Immuno-Biological Laboratories Co., Ltd.) (obtained) and Media medium (Immunobiology Institute Co., Ltd.) were mixed, and after centrifugation,
50% PEG1500 (manufactured by Boehringer) was added to the cell pellet, and a fusion operation was performed. After that, the fused cells were centrifuged, the supernatant was removed by suction, and 30% FCS-containing HA was added.
T medium was added and plated on 11 96-well microplates. After fusion, colonies grew from about 950 wells in about 2 weeks, and these culture supernatants were treated with SDS-denatured APP592.
Solid-phase ELISA was performed using as the screening antigen,
A clone that strongly reacted with the antigen was obtained, and cloning by limiting dilution was repeated three times. Two clones showing stable antibody production after three rounds of cloning were named 3B2 and 6A3, respectively, and were entrusted to the Microbial Technology Research Institute on January 16, 1991. (3B2 is the Microtech Research Institute No. 11943, and 6A3 is the Microtechnology Research Institute No. 11
No. 944). The reactivity of monoclonal antibodies 3B2 and 6A3 produced by these hybridomas was evaluated in steps 1 and 2.
APP667, APP648, APP59 created with
When 2 was investigated, as described below, it strongly reacted with these three types of APP derivatives under both SDS denaturation and non-denaturation conditions. That is, it was found that these monoclonal antibodies can recognize any of APP770, APP751, and APP695.

[0034]

[Reference Example 1] Preparation Example 1 of anti-APP592 antiserum
A rabbit was immunized with APP592 obtained in step 2 to obtain an antiserum. That is, SDS was prepared by the method described in Example 1 step 4(i).
Create denatured APP592 and add 200 μg of SDS denatured A
Dissolve PP592 in 1 ml of PBS (-),
The vaccine was injected subcutaneously into the backs of two domestic rabbits together with complete Freund's adjuvant, and then immunized with incomplete Freund's adjuvant at two-week intervals for a total of six times.

[0035] Antibody titer in serum was determined by solid-phase ELISA using non-denatured and SDS-modified APP592 (25 ng/well) as an antigen. The final titer of the antiserum was 32,000 times that of non-denatured APP592, and 32,000 times that of SDS-denatured APP592, as shown by the dilution of the antiserum that showed more than twice the color development of the control (well without antigen). against 6
It was 4000 times more potent, and showed almost the same titer against APP667 and APP648. That is, this anti-APP59
2 antisera are APP770, APP751, APP69
It was found that all 5 can be recognized to the same degree.

[0036]

[Example 2] Creation of a monoclonal antibody-producing hybridoma using ZKX and APP592 as antigens (i) Sensitization of mice ZKX obtained in Example 1 Step 1 was used in Example 1 Step 4 (i)
Denatured as described. This SDS modified ZKX 2
0 μg dissolved in 500 μl of PBS(-) was subcutaneously injected into four 6-week-old female Balb/c mice together with complete Freund's adjuvant, and 2 weeks later, the same immunization was performed. Additionally, at 2-week intervals, SDS
Immunization of modified ZKX was performed six times using incomplete Freund's adjuvant. Two weeks later, SDS-denatured ZK
20 μg of X was injected intravenously. About a month later, this time 20μ
SDS-denatured APP592 (g) was injected subcutaneously together with Freund's complete adjuvant, and one week later, immunization with APP592 was performed in the same manner.

During the course of immunization, an increase in serum antibody titer against the antigen was confirmed by solid-phase ELISA in the same manner as in Example 1. (ii) 200 μl of PB to the mouse with the highest step value of cell fusion serum.
25 μg of SDS-denatured APP592 dissolved in S(-)
was injected into the tail vein as a boost. The subsequent operations were performed in the same manner as in Step 3 of Example 1 to obtain a clone that strongly reacted with APP592, and cloning by limiting dilution method was repeated three times. A clone showing stable antibody production after three clonings was named 1B2, and was deposited with the Microbial Technology Research Institute on January 16, 1991 (Feikoken Bacterium Deposit No. 11942). The reactivity of the monoclonal antibody 1B2 produced by this hybridoma was determined using APP667, APP648, and APP prepared in steps 1 and 2.
592, we found that SD
It reacted strongly with these three types of APP derivatives under both S-modified and non-modified conditions. That is, it was found that monoclonal antibody 1B2 can recognize any of APP770, APP751, and APP695.

[0038]

[Example 3] Characterization of monoclonal antibodies (i)
Immunoglobulin class and subclass The immunoglobulin class and subclass of monoclonal antibodies 1B2, 3B2, and 6A3 were determined using an isotyping kit (Amersham). As a result, for 1B2, IgG2b, L chain is κ, for 3B2, IgG2b, L chain
The chain was determined to be κ, IgG2a for 6A3, and the light chain to be κ. (ii) Determination of specificity The specificity of the above monoclonal antibody was determined by the test shown below.

[0039] Various secretory APPs (APP667, APP
648, APP592), non-denaturing and SDS-denaturing treatments were performed, and determination was made using a conventional solid-phase ELISA method. The above monoclonal antibody as the primary antibody, 2
The secondary antibody was β-galactosidase-labeled sheep F(ab')2 anti-mouse Ig (Amersham), and the substrate was 4-methylumbelliferyl β-D-galactopyranoside. After the enzymatic reaction was carried out for 30 minutes, the fluorescence intensity was measured using a fluorescence reader FCA (PANDEX). The results are shown in Table 1.

Monoclonal antibody according to the invention, 1B2
, 3B2, and 6A3 are all three types of APP (APP
667, APP648 and APP592),
I strongly recognized it. As a negative control, reactivity with live serum albumin (BSA) was also examined, but these three monoclonal antibodies did not react with BSA at all.

[0041]

[Example 4] Examination of solid-phase ELISA system Solid-phase ELISA was performed according to the monoclonal antibody experimental manual (written by Sakuji Toyama, Kodansha Scientific). APP592 obtained in Step 1 of Example 1 was used as the standard antigen (non-denaturing conditions Bottom), the monoclonal antibody of the present invention was used as the primary antibody at 2μ
The secondary antibody used was β-galactosidase-labeled sheep F(ab')2 anti-mouse Ig (Amersham).

[0042] As a result, all of 1B2, 3B2, and 6A3 strongly recognized non-denatured APP592 up to 3.1 ng/well.

[0043]

[Example 5] Study of two-antibody sandwich ELISA system [Step 1] Expression of β-galactosidase/APPI fusion protein in E. coli [Step 1-1] Construction of fusion protein expression plasmid sPI
(APPI-72) fused protein (ZPI)
A plasmid capable of expressing the was constructed. Specifically, expression vector pEX1 (Boehringer Mannheim Yamanouchi) was cut with restriction enzymes BamHI and PstI to obtain a linear vector DNA fragment. On the other hand, plasmid p described in Example 4 of European Patent Application Publication No. 0304013
PItrp75-1 was cleaved with BamHI and PstI, and after agarose gel electrophoresis, a 0.3 kbp fragment was isolated using the Funakoshi Gene Clean Kit. The two obtained fragments were ligated using T4 DNA ligase.

The reaction product was prepared using Escherichia coli POP213 prepared according to the method described in the experimental paper of Maniatis et al.
6 strains (Boehringer Mannheim Yamanouchi) were added to competent cells and transformed. Six ampicillin-resistant transformed colonies were selected, each plasmid was extracted, and B
When the fragments were cut with amHI and PstI and analyzed, they all contained the desired plasmid. The obtained plasmid was named pEX-ZPI. This plasmid is produced by controlling the PR promoter derived from lambda phage.
A galactosidase/APPI fusion protein can be expressed. [Step 1-2] Escherichia coli POP2 carrying plasmid pEX-ZPI obtained in fusion protein production step 1-1
136/pEX-ZPI was cultured overnight at 30°C in L medium containing 50 μg/ml ampicillin. 1 ml of main culture solution was added to 50 μg/ml ampicillin-containing L medium 50 μg/ml.
ml and cultured at 30°C for 3 hours, then transferred to a constant temperature culture tank at 42°C and further cultured for 3 hours. At 42°C, the repressor of the lambda phage PR promoter is inactivated, and the desired fusion protein is produced by operating the PR promoter.

[0045] The culture solution was centrifuged to collect the bacterial cells, and a portion of the bacterial cells were collected by the method of Laemmli [Laemmli U. et al. K.
, Nature. Vol. 227.680-68519
After boiling in SDS/sample buffer, the samples were subjected to SDS-polyacrylamide gel electrophoresis using a 10% polyacrylamide gel according to [70]. After electrophoresis, the gel was stained with Coomassie, and it was confirmed that the desired fusion protein with a molecular weight of about 120 kDa was present. Below, this β-galactosidase/APPI fusion protein is
It is abbreviated as PI. [Step 1-3] A part of the cultured bacterial cells obtained in ZPI solubilization step 1-2 was treated with urea at a final concentration of 0M.
, 2M, 4M, 6M, and 8M, respectively, were suspended in 20mM Tris-HCl (pH 8.0), the cells were disrupted by ultrasonication using an Otake sonicator (Otake Seisakusho), and insoluble matter was collected by centrifugation. The obtained insoluble matter is subjected to step 1-2.
It was treated in the same manner as above and analyzed by SDS polyacrylamide gel electrophoresis. As a result of Coomassie staining, target Z
The PI band was detected in the insoluble fractions extracted with buffers containing 0M, 2M, and 4M urea, but was not observed in the insoluble fractions extracted with buffers containing 6M and 8M urea.

From the above results, protein inclusion bodies are formed as insoluble substances in E. coli after producing the desired fusion protein.
It was found that this insoluble material was solubilized by urea of 6M or more. [Step 1-4] In the same manner as ZPI partial purification step 1-2, E. coli POP2136/pE
X-ZPI was cultured, and bacterial cells were collected from 200 ml of the culture solution. The cultured bacterial cells, 20ml containing 40ml of 4M urea
The cells were suspended in M Tris-HCl (pH 8.0) buffer and disrupted at the center. Centrifuge the destroyed bacterial cells (10,000x
g for 10 minutes) and the precipitate was collected. This precipitate was then dissolved in 20 ml of 20 mM Tris-HCl (p
H8.0) buffer, and the precipitate was dispersed by sonication. After centrifugation (10,000×g, 10 minutes), the supernatant was collected. Through the above operations, E. coli proteins soluble in 4 M urea and E. coli proteins insoluble in 8 M urea were removed, and a partially purified solution of ZPI was obtained. When a part of this partially purified solution was analyzed by SDS-polyacrylamide gel electrophoresis, it was found that 5 of the total proteins
More than 0% was the target fusion protein ZPI. [Step 1-5] Extract the protease inhibitor region 20 ml of the ZPI partially purified solution obtained in Step 1-4 was diluted with 20 mM
Dialysis was performed twice against 2 L of Tris-HCl (pH 8.0) to remove urea and at the same time restore the three-dimensional structure of the protein.

[0047] After keeping the obtained dialysate at 37°C,
TPCK-treated trypsin-immobilized agarose beads (
By adding 100 units of Sigma) and shaking at 37°C for 1 hour, the β-galactosidase region in ZPI was cleaved and at the same time the protease inhibitor region was adsorbed. This suspension was packed into a Select STM empty column manufactured by 5'Prime→3' Prime, and the agarose beads were collected. Separation of the agarose beads and buffer was performed by centrifugation at 1,000 rpm for 1 minute (collection of agarose beads in the following description was performed in the same manner). The collected beads were soaked in 0.3 M NaCl for 10 m
Elution was performed with 6 ml of an eluent consisting of M CaCl2 , 10 mM HCl (pH 2). Elution was performed three times in total, and after neutralizing each eluate with 5N NaOH, trypsin inhibitory activity was measured. The inhibitory activity was measured by the same method as the trypsin inhibitory activity measurement of sPI described in Example 4 of European Patent Publication No. 0304013.

[0048] The concentration of trypsin inhibitor activity contained in each eluate is 16.4 μg/ml, 1.5 μg/ml, and 0.27 μg in terms of BPTI in the order of the eluate fractions.
/ml. These elution fractions are referred to as ZtPI fractions. [Step 1-6] To the eluate obtained in protease inhibitor analysis step 1-5, four times the volume of acetone previously cooled to -20°C was added, and the mixture was cooled at -20°C for 1 hour. Furthermore, the mixture was subjected to cooling centrifugation (10,000×g for 15 minutes) and the precipitate was collected. 1 ml of the obtained precipitate
Redissolve in mM Tris-HCl (pH 7.5), step 1-5
Trypsin inhibitory activity was measured in the same manner as above. As a result, it was found that it contained 62 μg of trypsin inhibitor in terms of sPI as described in Example 4 of European Patent Publication No. 0304013.

A portion of the obtained inhibitor solution was added to SD
When analyzed by S-polyacrylamide gel electrophoresis, a major protein with a molecular weight of about 6 kDa was detected. [Step 1-7] Step 1-4 of creating anti-ZtPI antiserum
300 µg of ZPI obtained in 1 was incubated at 100°C in a sample buffer (containing 2% SDS, 5% β-ME, but no dye was added) used in SDS-polyacrylamide gel electrophoresis according to the Lemley method described above. After treating for a minute, 4 times the volume of acetone was added, cooled at -20°C for 1 hour, centrifuged at 9000 rpm for 20 minutes, and the precipitate was collected. The precipitate was suspended in PBS(-), mixed well with complete Freund's adjuvant (FCA), and subcutaneously injected into rabbits. FCA after 31 days
The same treatment (immunization) was performed except that Freund's incomplete adjuvant was used instead. After another 14 days, 300
μg of ZtPI obtained in steps 1-5 was added to the second ZPI
Immunization was performed in the same manner as above. After another 16 days, ZtP
The same treatment was performed using I. After a further 15 days, the SPI described in European Patent Publication No. 0304013 Example 4
150 μg was subjected to the same treatment and immunized. 50 ml of blood was collected from this rabbit to obtain antiserum. This antiserum is hereinafter referred to as anti-denatured ZtPI antiserum.

[0050] This antibody is more sensitive to SDS- than native cPI.
sPI denatured with β-ME was more strongly recognized. Furthermore, it did not react at all with urinary trypsin inhibitor (Nippon Chemical Research Co., Ltd.). [Step 1-8] Purification of anti-ZtPI antibody After inactivating 20 ml of the rabbit anti-ZtPI serum obtained in Step 1-7 at 56°C for 30 minutes, add an equal amount of PBS (-
) was added thereto to dilute the mixture, and at 4° C., a 100% saturated ammonium sulfate solution (preliminarily adjusted to pH 6.5 with 0.1N ammonia solution) was gradually added with stirring to a final concentration of 33% saturation. After the addition was complete, stirring was continued for an additional 12 hours.
Refrigerated centrifugation was performed at 3000×g for 30 minutes. The obtained precipitate was suspended in a 50% saturated ammonium sulfate solution and centrifuged again under cooling to obtain a precipitate. The precipitate was dissolved in 10 ml of 10 mM sodium phosphate buffer (pH 8.0) and dialyzed three times against 5 L of the same buffer packed in a dialysis tube. The obtained dialyzed sample was applied to a DEAE-cellulose column (DE52, Whatman) equilibrated in advance with 10 mM sodium phosphate buffer (pH 8.0), and eluted with the same buffer. Collect the flow-through fractions and concentrate with Centriprep TM30 (Amicon) to obtain 11.7 mg.
A partially purified anti-ZtPI antibody was obtained. Partially purified anti-ZtPI
Prepare antibodies using Affiprep Protein A Cartridge (
Purified rabbit anti-Zt was further purified according to the attached protocol published by Bio-Rad using
9.6 mg of PI antibody was obtained. [Step 1-9] I from ascitic monoclonal antibody
Purification of gG In order to obtain a large amount of monoclonal antibody, 1×1 each of the 1B2, 3B2, and 6A3 hybridomas obtained in Examples 1 and 2 was used.
07 cells were suspended in 0.5 ml of Medial medium (Immune Biological Laboratories Co., Ltd.) and injected into the peritoneal cavity of Balb/c mice, which had been intraperitoneally administered with 0.5 ml of pristane (Aldrich) one week beforehand. Injected. After about 2 weeks, abdominal enlargement was observed, and ascites was collected through abdominal incision. Seven mice were treated for each hybridoma, and about 10 ml of ascites was obtained from each. 5 ml of ascites derived from each hybridoma was purified using an Affiprep Protein A cartridge (Bio-Rad). Purification was performed according to the attached protocol published by Bio-Rad. 7.9 mg of purified IgG was obtained from the 1B2 clone, 4.3 mg from the 3B2 clone, and 3.3 mg from the 6A3 clone. [Step 1-10] Using 2 mg each of the 1B2, 3B2, and 6A3 purified monoclonal antibodies obtained in the antibody biotinylation step 1-9, Immunology Experimental Procedures (Vol. 9, 3539-3)
549 1982 Japanese Society of Immunology), the antibodies were biotinylated. Dialyze and buffer exchange against 0.1M NaHCO3 solution in advance to adjust the concentration to 1mg/
Purified 1B2, 3B2, and 6A3 adjusted to ml
280 μl of a dimethyl sulfoxide solution of NHS-biotin (N-hydroxysuccinimide-biotin, Vias) adjusted to a concentration of 1 mg/ml was added to the antibody, and the mixture was gently stirred at room temperature for 4 hours. Next, 3 L of PBS (
-) was dialyzed four times against biotin-labeled 1B2,
3B2 and 6A3 antibodies were obtained. [Step 1-11] Two-antibody sandwich ELISA using anti-ZtPI antibody and 1B2 monoclonal antibody 5 μg/ml of the purified rabbit anti-ZtPI antibody obtained in Step 1-8
The solution was dissolved in 0.1 M sodium carbonate-sodium hydrogen carbonate buffer (pH 9.6) at a concentration of 50 μl per well in a 96-well microplate, and left at room temperature for 2 hours. After washing the plate three times with PBS(-),
PBS (-) containing 1% bovine serum albumin (Sigma)
) was added and coated for 2 hours at 37°C. Next, a purified APP667 standard solution (2 to 2000 ng/ml) diluted in PBS (-) containing 0.1% bovine serum albumin was added, and the mixture was treated at room temperature for 1 hour with stirring. Next, 0.
After washing the plate three times with PBS (-) containing 05% Tween-20, add 50 μl of the biotin-labeled purified 1B2 IgG obtained in step 1-10 at a concentration of 2 μg/ml.
The mixture was added to each well, and the reaction was carried out at room temperature for 1 hour while stirring. PBS containing 0.05% Tween-20 (
-), β-galactosidase-labeled apidin D (Vector) was added, and the mixture was allowed to react at room temperature for 1 hour with further stirring. After washing 5 times with PBS(-) containing 0.05% Tween-20, 2mM MgCl2
4-methylumbelliferyl-β-D-galactopyranoside (1 mg/ml) dissolved in PBS(-) containing
) was added and reacted for 30 minutes at room temperature, then FCA (PAN
Fluorescence intensity was measured using DEX Co., Ltd.). In this system, 0.3n
g/well of APP667 could be detected. The results are shown in Figure 4.

[0051]

Example 6 Detection of KPI in cerebrospinal fluid Using the monoclonal antibody 3B2 of the present invention and rabbit anti-ZtPI antiserum, the method described in Example 5 (two-antibody sandwich EL
ISA), an attempt was made to detect APP or its fragments containing KPI in the cerebrospinal fluid obtained from 3 Alzheimer's disease (AD) patients and 3 patients with cerebrovascular dementia as a control group. The KPI concentration in the cerebrospinal fluid of the AD group tended to be higher than that of the target cerebrovascular dementia group.

[0052]

[Example 7] Western blotting study Western blotting was performed on human cerebrospinal fluid and various APP derivatives by the method shown below. Various APs
Regarding P derivatives, APP667 obtained in Example 1, A
1 μg each of PP648 and APP592 was used. Cerebrospinal fluid was obtained from one AD patient and one patient with cerebrovascular dementia, and 4 times the volume of methanol was added to -2
After being left at 0°C for 1 hour, centrifugation was performed at 9000 rpm for 20 minutes to collect the precipitate.
The sample is dissolved in is-hydrochloric acid (pH 8.0),
Protein quantification was performed, and 200 μg of each protein was used.

[0053] These APP and cerebrospinal fluid samples were
Emmli SDS-PAGE was boiled at 100°C for 3 minutes in sample buffer, and 7.5% SDS
-After electrophoresis on PAGE, the method of Towbin et al.
wbin et al., Proc. Natl. Acad. Sci.
USA 76 4350-4354 1979)
transfer buffer (25 Mm Tris, 192
After equilibration in 20% methanol (mM glycine, 20% methanol), electrotransfer was performed onto a nitrocellulose membrane (Bio-Rad).

This filter was added to TBS buffer (20 Mm Tris-HCl (p) containing 5% skim milk).
After blocking in H7.5), 500 mM NaCl), the monoclonal antibody 6A3 according to the invention (I
2 μg/ml) was added, reacted at 37°C for 1 hour, and after washing with TBS buffer, 37 kBq of 125
-I-labeled Protein A (Amersham) was reacted, and after washing with TBS buffer, autoradiography was performed. All three purified APP derivatives reacted strongly with 6A3. Regarding cerebrospinal fluid, approximately 100 kD is obtained from both AD and cerebrovascular dementia specimens.
Two bands were detected, a and 110 kDa, with AD giving a darker band.

[0055]

[Reference example] Purification of APP-related protein using 1B2 antibody column MAP from ascitic monoclonal antibody 1B2
Using the SII kit (BIO-RAD), the 1B2 antibody purified according to the protocol attached to the kit was combined with cyanide bromine-activated Sepharose (Pharmacia) to prepare a 1B2 antibody column. The binding method followed the protocol published by Pharmacia. 20 ml of the culture supernatant of pSVMT592-introduced COS-1 cells obtained in Step 2 of Example 1 was applied to a 1B2 column, and PBS (
-), the column bound material was eluted with 1M potassium thiocyanate. After concentration using Centriprep 10 (Amicon), 10% SDS-PAGE was performed. As a result, it was confirmed that a single band of approximately 100 kDa (Coomassie brilliant blue staining) was recovered by the 1B2 column. This band strongly reacted with monoclonal antibody 1B2 by the same Western blotting method as in Example 7. Also, PBS(-) is 2
After applying double diluted human serum and washing with PBS(-), the column bound substance was eluted with 1M potassium thiocyanate, concentrated with Centriprep 10 (Amicon), and then subjected to 10% SDS-PAGE. Approximately 11
It was confirmed that a single band of 0 Kd was recovered by the 1B2 column. This band strongly reacted with monoclonal antibody 1B2 by the same Western blotting method as in Example 7.

[0056]

[Table 1]

[0057]

[Table 2]

[0058]

Effect of the invention: According to this invention, APP770, APP
Provided are novel monoclonal antibodies that recognize all three APPs, APP 751 and APP 695, either whole or in the form of fragments thereof, and novel hybridomas that produce the same. As mentioned above, APP or a fragment thereof can be a useful diagnostic marker for Alzheimer's disease patients, so it becomes possible to diagnose Alzheimer's disease using the monoclonal antibody of the present invention. It is also useful as a research reagent for detecting, quantifying, and purifying APP fragments containing sequences common to the three types of APPs, or the three types of APPs themselves.

[0059]

[Sequence list]

Sequence number: 1 Sequence length: 39 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Symbols representing characteristics: Linker 1 Sequence TGAGGCCGAT ACTGCTGTCG TCC
CCTCAA CTGGCAGAT
39

0061
] Sequence number: 2 Sequence length: 42 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Symbols representing characteristics: Linker 2 Sequence GCACGGTTAC GATGCGCCCA TGT
ACACCAACGTAACCTAT CC
42

0062
] Sequence number: 3 Sequence length: 50 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Symbols representing characteristics: Linker 3 Sequence CATTACGGTC AATCCCGCCGT TTG
TTCCCAC GGGATCCATG AGCTCG
GTAC 50

0063
] Sequence number: 4 Sequence length: 42 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Symbols representing characteristics: Linker 4 Sequence CGAGCTCATG GATCCCGTGG G.A.A.
CAAACGG CGGATTGACC GT
42

0064
] Sequence number: 5 Sequence length: 42 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Symbols representing characteristics: Linker 5 Sequence AATGGGATAG GTTACGTTGG TGT
ACATGGG CGCATCGTAA CC
42

0065
] Sequence number: 6 Sequence length: 40 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Symbols representing characteristics: Linker 6 Sequence GTGCATCTGC CAGTTTGAGG G.G.A.
CGACGAC AGTATCGGCC
40

[Brief explanation of the drawing]

[Figure 1] Alzheimer's type senile dementia brain amyloid precursor proteins APP695, APP751, APP77
0, APP714, and APP563 are schematic diagrams. b
The blacked out part from f is a region consisting of 56 amino acids having protease inhibitor activity, the dotted part is a region consisting of 19 amino acids unique to APP770,
The shaded area indicates the senile plaque amyloid deposited in the brain. a
shows the amyloid β protein part (shaded part) of APP that is deposited in the brain and the transmembrane region. APP563 is β
It has no protein part.

[Figure 2] Secretory APP (APP667, APP648,
APP592) is a schematic diagram showing the structure of APP592). The numbers in the figure indicate the amino acid residue numbers counted from the N-terminus of each APP.

[Figure 3] Part of β-galactosidase and APP770
FIG. 2 is a schematic diagram of the construction of an expression plasmid for ZKX, which is a fusion protein with a partial protein of ZKX.

FIG. 4: APP66 by two-antibody sandwich ELISA using anti-ZtPI antibody and monoclonal antibody 1B2
7 detection results.

Claims (3)

[Claims] 1. Among the senile plaque amyloid precursor proteins, the senile plaque amyloid precursor proteins APP695, APP751, and AP
A monoclonal antibody that recognizes any P770. 2. A hybridoma producing the monoclonal antibody according to claim 1. 3. A method for detecting human senile plaque amyloid precursor protein or a fragment thereof using the monoclonal antibody according to claim 1 and subjecting a subject to immunoassay.