JPH04252955A - Measuring method, reagent and kit for protein - Google Patents

Abstract

PURPOSE:To achieve handiness and a higher sensitivity of measurement by using an antibody for recognizing an amyloid precursor protein APP770 of Alzheimer's disease in at least one combination thereof. CONSTITUTION:Two kinds of antibodies bonded respectively to two different antibody recognizing parts on an antigen by a sandwich ELISA(enzyme Linked Immunosorbent Assay) method to detect the presenCE of an antigen or an amount thereof. In this case, an antibody herein used for the detection of APP770 is, for example, a 7E11 monoclonal antibody to meet that one thereof is an antibody for recognizing APP770 specifically. Other antibodies therein used are an antibody which can recognize various APPs such as APP695, APP751 and APP770 and a antibody which recognizes an antigen determinant differing from a first antibody among those recognizing an array intrinsic to the APP770.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring protein, a reagent, and a kit. More specifically, Alzheimer's disease amyloid precursor protein APP77 is obtained by a sandwich ELISA method characterized in that an antibody that specifically recognizes Alzheimer's disease amyloid precursor protein APP770 is used in at least one of the combinations.
The present invention relates to immunological assay methods, reagents, and kits for 0 or fragments thereof.

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 causes, simple diagnostic methods, and treatments have not been established, and research and development of these diseases is strongly desired. Such diseases include Alzheimer's type senile dementia and Alzheimer's disease (hereinafter these two will be collectively abbreviated as AD). In the brains of AD patients, structures called senile plaques, whose main component is senile plaque amyloid-β protein (hereinafter abbreviated as senile plaque amyloid), are deposited in larger amounts than in normal people, so it is possible to distinguish between senile plaque amyloid and AD. The relationship between disease onset and onset has been attracting attention. Kang et al. reported that senile plaque amyloid has a precursor (hereinafter abbreviated as APP) (hereinafter the APP discovered by Kang et al. is abbreviated as APP695) (Kang et al., Nature 325 733-736 1).
987, Japanese Unexamined Patent Publication No. 63-222693), and further research by the present inventors has shown that APP has at least three
Types (in addition to APP695, APP751 and APP7
70) has been shown to exist (Ponte et al.
Nature 331 525-527 198
8, Tanzi et al., Nature 331 528
-530 1988, Kitaguchi et al., Nat.
ure 331 530-532 1988, European Patent Publication No. 0304013, Patent Application Publication
Publication No. 2-501796, International Publication Number WO89/
07657). Furthermore, Alzheimer's disease amyloid precursor protein APP714 (Gold
e et al., Neuron 4 253-267 19
90), and no senile plaque amyloid portion, 563
APP563 consisting of amino acids (de Sauvage et al. Science 245 651-653
1989) is also known to exist, although it is a minor component (Figure 1). Kun for APP751 and APP770
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). 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, overexpression of APP, which has protease inhibitor activity, may play a role in the development of AD. It is possible that this is the case. 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 the expression level of APP770 messenger RNA was about twice as high in AD patients. Ta. Furthermore, the amount of APP751 messenger RNA was found to be 1.1 to 1.3 times higher in AD patients than in controls (Tanaka et al., B.B.R.C. 157 47
2-479 1988 and B. B. R. C. 16
5 1406-1414 1990). APP77
The difference between AD patients and controls was clearer for 0 messenger RNA, suggesting that specific detection of APP770 protein provides important information for the diagnosis of AD.

[0003] In addition, using human cerebrospinal fluid as a specimen, protease inhibitors (KPIs; KPIs are APP7
It refers to 56 residues from Glu at position 289 to Ala at position 344 counting from the N-terminal side of APP751 and APP751, and has inhibitory activity against various proteases. ) The amount of APP or its fragments containing the region was determined by trypsin ELIS.
Method A (Kitaguchi et al., B.B.R.C.166
1453-14591990 and Patent Application Hei 1
-214118, Japanese Patent Application No. 2-220889), it was found that the AD group showed significantly higher values than the cerebrovascular dementia group (Kitaguchi et al.
et al., B. B. R. C. 166 1453-1459
1990, and the 31st Annual Meeting of the Japanese Society of Neurology, Abstracts 241 pages II-J-05 1990). This result was demonstrated using trypsin E (which strongly binds to this protease inhibitor region) and a rabbit antiserum that recognizes the protease inhibitor (KPI) region commonly present in APP770 and APP751.
It was obtained by the LISA method. (However, ELI
SA stands for Enzyme Linked Immuno
(abbreviation for nosorbent assay). However, for example, when serum or plasma is used as the sample and trypsin is bound to a solid phase and used instead of the first antibody, trypsin binds to the trypsin inhibitor in the blood, so the solid phase of the assay plate is The amount of analyte protein (ie, APP) bound to the sample may not be sufficient. Therefore, APP7 is an alternative to the trypsin ELISA method.
A highly sensitive detection method for 51 and APP770 is desired. Furthermore, considering that the difference in the expression level of messenger RNA between AD patients and controls was particularly remarkable for APP770 as mentioned above, there is a strong desire for a method to specifically and highly sensitively detect the APP770 protein. There is. However, there are currently no reports on a method for specifically detecting APP770 protein.

KPI sequences, namely APP770 and APP
Regarding antibodies that recognize 751, antibodies prepared using synthetic peptides as antigens have been reported (Anders et al.
on et al., EMBO Journal 8 362
7-3632, 1989, Palmer et al., Neur.
ology 40 1028-10341990)
, these are all used only for Western blotting and not for ELISA. In the Western blotting method, the sample is concentrated, desalted, and denatured with a surfactant or reducing agent, and then the proteins are separated by electrophoresis.
For this, an antigen-antibody reaction must be performed. Therefore, there are problems in that the operation is complicated, it is difficult to measure a large number of specimens, and the detection sensitivity is significantly affected by the amount of contaminant proteins other than APP contained in the test sample. Furthermore, the antibodies used in these Western blotting are APP770 and APP751.
cannot be distinguished. In addition, the C-terminal side of APP (
Sequence common to APP695, 751, and 770. APP6
95, residues 667-676),
There is also a report that there was no significant difference between AD/control when APP concentration in serum was examined by radioimmunoassay (hereinafter abbreviated as RIA) (Rumble et al.
New England J. Med. 320, 1
446-1452, 1989). However, most of the APPs present in body fluids such as cerebrospinal fluid and blood do not have a sequence on the C-terminal side of the transmembrane region (for example, Palmer et al.,
roc. Natl. Acad. Sci. USA 86
, 6338-6342, 1989, Palmer et al.
Neurology 40, 1028-1034, 1
990, van Nostrand et al., Science
e 248, 745-748, 1990, Cole et al., Biochem. Biophys. Res. Comm
．． 170, 288-295, 1990, etc.). Under these current circumstances, there has been a need to establish a specific and rapid quantitative method for APP770 in clinical practice.

[0005]

SUMMARY OF THE INVENTION Accordingly, the present invention provides a measurement method, reagent, and kit that are easy to perform measurement operations and sample preparation, and are capable of measuring APP770 and its fragments with high sensitivity. The purpose is to

[0006]

[Means for Solving the Problems] In order to achieve the above object, the present inventor has conducted extensive research and found that at least one type of antibody that specifically recognizes APP770 was used as a method for quantifying APP770 and its fragments. Antibody sandwich E
The present invention was completed by discovering that the LISA method is possible, and by using this method, a reagent and a kit that are easy to perform measurement operations and sample preparation and can quantify these with high sensitivity have been completed.

The present invention will be explained in detail below. What is the immunoassay method using sandwich ELISA method?
This is a method of measuring the presence or absence of an antigen or its amount using two types of antibodies that each bind to two different antibody recognition sites (referred to as antigenic determinants) on an antigen. One of the antibodies for detecting APP770 used in the present invention must be an antibody that specifically recognizes APP770, and the other antibody must be an antibody that specifically recognizes APP770.
Various A such as PP695, APP751, or APP770
It may be a combination of an antibody that can recognize PP or an antibody that recognizes a sequence unique to APP770 and that recognizes an antigenic determinant different from that of the first antibody.

As an antibody that specifically recognizes APP770, for example, the 7E11 monoclonal antibody described in Example 1 of the present invention (FERM
P-11633) produced by the hybridoma P-11633). The other antibody used is APP695
, APP751, or APP770, or an antibody that recognizes a sequence unique to APP770 and that recognizes an antigenic determinant different from that of the first antibody. Furthermore, most of the APP in cerebrospinal fluid and serum does not contain the sequence from the transmembrane region to the C-terminus.
Polyclonal antibodies or monoclonal antibodies that recognize APP derivatives obtained by removing the transmembrane region of 695, APP751, or APP770 from the carboxyl terminal are also useful. For example, A
A is an APP695 derivative in which the C-terminal 103 amino acids (transmembrane region and intracellular region) of PP695 are deleted.
Rabbit anti-APP592 polyclonal antibodies etc. and mouse anti-AP obtained by immunization with PP592 (see Examples)
Examples include P592 monoclonal antibody. As an example of monoclonal antibodies,
FERM P-11942, monoclonal antibody 1B
2), No. 11943 (FERM P-1
FERM P-11944 (FERM P-11944, which produces monoclonal antibody 6A3), and the like. Alternatively, it may be a combination with an antibody that recognizes KPI present on APP770 and APP751. Such an example includes the anti-ZtPI polyclonal antibody described in Example 1 of the present invention.

[0009] These antibodies can of course be used as intact antibodies, but also as antibody fragments that maintain their essential binding ability, such as Fab, Fab', (Fab').
It can also be used as 2 etc. The test sample in the present invention is not particularly limited, but specifically, cerebrospinal fluid,
Examples include blood (plasma and serum), urine, and the like. Furthermore, extracts of tissues and cells such as the brain can also be used.

Examples of the insoluble carrier used in the present invention include polystyrene, polyethylene, polypropylene, polyester, fluororesin, glass, metal, and combinations thereof. Furthermore, the present invention can be carried out with various shapes of the insoluble carrier, such as a tray shape, a spherical shape, a fiber shape, a rod shape, a disk shape, a container shape, a cell, a test tube, and the like. A 96-well microplate or the like is preferable in that a large number of test samples can be measured simultaneously.

[0011] In the present invention, the antibody used as the second antibody may be directly labeled with an enzyme, a radioisotope, or a fluorescent substance such as FITC. Alternatively, a labeled antibody against such second antibody may be used as the third antibody. Alternatively, the second antibody is labeled with a marker compound such as biotin, and a substance that specifically binds to this marker compound (in the case of biotin, streptavidin or avidin) is labeled with an enzyme, fluorescent dye, radioisotope, etc. A method of combining is also used.

[0012] In the present invention, the final detection means is not particularly limited.
It is necessary to use an antibody that specifically recognizes APP770 as one of the two types of antibodies in the set, and any detection means may be used after recognition and immobilization by such a combination. However, a typical detection method that can be used in the present invention is a detection method using a second antibody labeled with an enzyme, a radioisotope, a fluorescent substance, or other binding substance.

[0013] In the detection method using an enzyme, enzymes include β-D-galactosidase (β-Gal), horseradish peroxidase (HRP), alkaline phosphatase (AP), etc., and the colorimetric method corresponding to each enzyme is used. A substrate or fluorescent substrate is added and the product is quantified using a colorimeter or fluorometer. Typical substrates for the above enzymes include o-
Nitrophenyl-β-D-galactoside (colorimetric substrate),
4-Methylumbelliferyl-β-D-galactoside (
Examples of the substrate for HRP include o-phenylenediamine (a colorimetric substrate), and p-nitrophenyl phosphate (a colorimetric substrate) as a substrate for AP. As a detection method using radioisotope, 125-I
Using a second antibody labeled with etc., radioactivity is measured using a γ-well counter or the like. In addition, the second antibody is biotin-labeled and further reacted with an avidin-labeled enzyme,
Detection can also be performed by performing the same reaction as in the case of detection using an enzyme. In the method using a fluorescent substance, examples of the fluorescent substance include fluorescein isothiocyanate (FITC). These are not limited to those exemplified, but other types can be used as long as they are used in immunological assay methods.

[0014] It is also possible to construct RIA using the antibodies used in the present invention. AP as one method of RIA
P770 or a fragment thereof is labeled with a radioactive isotope to form a labeled antigen, and this and the antigen (unlabeled antigen) in the sample are
Examples include a method in which the antibody used in the present invention is competitively bound to an insoluble carrier, and the amount of antigen in the sample is measured based on the degree of binding of the labeled antigen. That is, if the amount of antigen in the sample is large, the amount of labeled antigen bound to the insoluble carrier will decrease, and the amount of free labeled antigen will increase. The opposite is true if the amount of antigen in the sample is low. Other RIA methods include, as described above, a method in which the second antibody used in the present invention is labeled with a radioisotope and the amount of the labeled antibody bound to the antigen in the sample is measured.

Next, a method for measuring Alzheimer's disease amyloid precursor protein APP770 according to the present invention will be explained in detail. Antibody that specifically recognizes APP770 (
The first antibody) is immobilized on a suitable insoluble carrier, such as a 96-well polystyrene microplate (hereinafter referred to as "immobilized antibody"). As the fixation buffer, sodium carbonate-sodium hydrogen carbonate buffer (pH 9.
6), or a phosphate buffered saline solution (hereinafter abbreviated as PBS(-)), etc. may be used. For fixation, it is common to leave it at 4°C overnight, but it is also possible to leave it at room temperature for about 2 hours, for example. The microplate on which the first antibody is immobilized in this way is washed with PBS(-) or the like. Next, in order to avoid non-specific binding between the insoluble carrier and the reagent to be measured or the test sample, a suitable substance such as 1-3% bovine serum albumin or skim milk/PBS is added.
(−) The surface of the insoluble carrier is coated with the solution. The coating is generally carried out at 37° C. or room temperature for about 2 hours while standing or stirring with a plate mixer or the like. After coating, the microplate is washed again with PBS(-) or the like. The thus obtained insoluble carrier on which the first antibody is immobilized is brought into contact with the test sample for a certain period of time and at a certain temperature to cause a reaction. The reaction is generally carried out overnight at 4°C with stirring using a plate mixer, but it is generally carried out at room temperature or 37°C overnight.
It is also possible to do this for about 2 hours. During this time, the immobilized antibody (first antibody) and APP770 or its fragment in the test sample bind. After washing with PBS(-), etc., the cells were labeled with an appropriate labeling substance, for example, an enzyme such as β-galactosidase, horseradish peroxidase, or alkaline phosphatase, or a radioactive isotope such as 125-I, as described above. An antibody that can recognize APP770 (
A solution of the second antibody) is contacted with APP770 or a fragment thereof bound to the immobilized antibody on an insoluble carrier for a certain period of time and at a certain temperature, for example, overnight at 4°C, or for 1 to 2 hours at room temperature to react with the second antibody. let This is washed again with PBS(-) or the like, and then the amount of the labeling substance labeled with the second antibody present on the insoluble carrier is measured. In the detection method using an enzyme, β-D-galactosidase (β-G
al), horseradish peroxidase (HRP
), alkaline phosphatase (AP), etc., and a colorimetric or fluorescent substrate corresponding to each enzyme is added, and the product is quantified using a colorimeter or fluorometer. Typical substrates for the above enzymes include o-nitrophenyl-β-D-galactoside (colorimetric substrate), 4-methylumbelliferyl-β-D as a substrate for β-Gal;
-As a substrate for HRP, such as galactoside (fluorescent substrate),
Examples of AP substrates include o-phenylenediamine (colorimetric substrate) and p-nitrophenyl phosphoric acid (colorimetric substrate). As a detection method using a radioisotope, a second antibody labeled with 125-I or the like is used to detect γ-
Measure radioactivity using a well counter or the like. In addition, by utilizing the high affinity of avidin for biotin,
Detection can also be carried out by labeling the antibody with biotin, reacting it with an enzyme labeled with avidin or streptavidin, and performing the same reaction as in the case of detection using an enzyme. On the other hand, instead of the test sample, standard products of various concentrations, such as purified Alzheimer's disease amyloid precursor protein or its fragments produced by recombinant DNA methods, or standard cerebrospinal fluid, blood, urine, etc. By preparing a calibration curve and comparing it with the measured values of the test sample, the amount of Alzheimer's disease amyloid precursor protein in the test sample can be calculated and quantified. APP77 as the first antibody
Although an antibody that specifically recognizes 0 and an antibody that recognizes APP as a second antibody have been described, the combination of the first antibody and the second antibody may be reversed. Alternatively, the first antibody and the second antibody may be a combination of antibodies that recognize different antigenic determinants on the sequence specific to APP770.

The measurement reagent in the present invention is composed of a first antibody, one of which is bound to an insoluble carrier, and the other of which is a second antibody. Furthermore, in order to utilize this reagent efficiently and easily, a kit can be formed containing various auxiliary agents in addition to these antibodies. Such auxiliary agents include, for example, standards for preparing calibration curves, lysing agents for dissolving reagents, detergents used for washing insolubilized carriers, enzymes used as labeling substances for antibodies, enzyme activity Examples include those commonly used as kits for immunological measurement reagents, such as substrates and reaction terminators for measuring .

[0017]

EXAMPLES The present invention will be explained in detail with reference to Examples below, but the present invention is not limited by these Examples. Example 1 Preparation of antigen, assay protein and antibody [Step 1] Preparation of hybridoma 7E11 producing monoclonal antibody specific to APP770 (i) Sensitization of mouse Antigen for preparation of hybridoma producing antibody specific to APP770 APPI-88 (Patent Application Hei 2-2424
In No. 69, it is listed under the name of IJ. ) was prepared as follows. APPI-88 is APP77
It contains the amino acid sequence from the 289th Glu residue to the 369th Ala residue of 0. Kitaguchi et al. (Kitaguchi et al., Bio
chim. Biophys. Acta. 1038 1
05-113 1990, and patent application Hei 2-24246
APPI-88 was expressed in animal cells by the method described in No. 9), and 500 μg of purified APPI-88 was obtained. This purified APPI-88 was dissolved in PBS(-), and La
emmli SDS-PAGE sample buffer (L
aemmli U. K. Nature 227 6
80-685 (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. Excess amount of SDS was removed and SDS-denatured APPI was extracted from the sediment.
-88 was recovered and used as an antigen for hybridoma production. Immunization with 50 μg of SDS-denatured APPI
-88 dissolved in 500 μl of PBS(-) was injected into the peritoneal cavity of three 6-week-old Balb/c female mice together with Freund's complete adjuvant, and then injected with Freund's incomplete adjuvant at two-week intervals. Total 4
Two immunizations were performed. During the immunization process, the rise in serum antibody titer against the antigen was measured using solid-phase EL according to the Monoclonal Antibody Experiment Manual (written by Sakuji Toyama, Kodansha Scientific).
Confirmed by ISA method.

(ii) Cell fusion Cell fusion was performed according to the monoclonal antibody experimental manual (Kodansha Scientific, written by Sakuji Toyama). The mouse with the highest serum antibody titer was given 200 μl of PBS (
-), 50 μg of SDS-denatured APPI-88 was injected through the tail vein, and 3 days later, the splenocytes were aseptically removed and loosened into single cells with a stainless steel mesh. Resistant myeloma cell line P3X
63-Ag8-653 cells (obtained from Immunobiological Laboratories Co., Ltd.) and MediaITM medium (Co., Ltd.)
After centrifugation, 50% PEG1500 (manufactured by Boehringer) was added to the cell pellet to perform a fusion operation. After that, the fused cells were centrifuged, the supernatant was removed by suction, and HAT medium containing 30% FCS was added.
It was plated on 11 6-well microplates. After fusion, colonies grew from 600 wells in about 2 weeks, and solid-phase ELISA was performed on these culture supernatants using SDS-denatured APPI-88 as a screening antigen.
A clone that strongly reacted with PI-88 was obtained, and cloning by limiting dilution method was repeated three times. One clone showing stable antibody production after three rounds of cloning was selected as 7E11.
and was deposited with the Microbial Technology Research Institute on July 27, 1990 (Feikoken Bibori No. 11633).

The reactivity of monoclonal antibody 7E11 produced by this hybridoma was evaluated using APP667 and AP prepared in steps 2 (i) and (ii) of this example described below.
When examining P648 and APP592, as described later, APP
It reacted strongly only with 667. That is, it was found that monoclonal antibody 7E11 specifically recognized APP770.

[Step 2] Preparation of APP (i) Preparation of APP592 APP592 (Figure 2), which consists of 592 amino acids (including a signal peptide), which is a protein in which the C-terminal 103 amino acids of APP695 are deleted, is a European patented protein. Publication number 03
Starting from the human senile plaque amyloid precursor APP695 cDNA obtained by the method described in Example 1 of No. 0413, 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. It was secreted into the culture supernatant and 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 10 COS-1 cells in sucrose-containing PBS (272 mM sucrose, 7 mM sodium phosphate (pH 7.4), 1 mM MgCl2) at a voltage of 400 V using Bio-Rad's Gene PulsarTM. , a capacitor of 3 μF, 1.0 to 1.3 msec, and the introduction was performed by applying two electric pulses with an interval of 30 seconds. The cells after introduction were incubated at 37°C in Dulbecco's modified minimal basic medium containing 10% fetal calf serum (FCS).
After culturing for 24 hours under 5% CO2, Dulbecco's phosphate buffered saline solution (Ca2+, Mg2+ free) (hereinafter P
After washing with BS (abbreviated as -) to remove FCS,
The medium was replaced with an S-free medium, and thereafter the medium was replaced every 48 hours, and the culture supernatant was collected three times in total. The introduction was further carried out 30 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 Centriprep™
30 (Amicon), and buffer exchange into 20mM Tris-HCl (pH 8.0) was performed in the same device. Next, DEAE-Sepharose
The above sample was applied to a column (Pharmacia), and 20mM Tris-hydrochloric acid (pH 8.0), NaCl
Column chromatography was performed with a gradient of 0-1M. For each fraction of chromatography, APP expressed in Escherichia coli in Reference Example 2 of the present invention, as described below.
Rabbit anti-AP created against 19-379 (ZKX)
Solid-phase ELIS using P19-379 (ZKX) antiserum
A was performed to identify the APP elution position. (ZKX is A
A polypeptide containing from the 19th amino acid residue to the 379th amino acid residue from the N-terminus in PP770 and β
- It is a fusion protein with galactosidase, expressed in E. coli, and purified by solubilization with 6M urea. The purification method was detailed in Reference Example 2. ZKX is AP
In addition to the sequences common to P770, APP751, and APP695, the protease inhibitor portion and APP7
70-specific sequence, but does not include the sequence from the transmembrane region to the C-terminus of APP). APP592 is 0.
It was eluted with 3-0.4M NaCl. The ELISA positive fraction was concentrated using Centricon TM30 (Amicon), and further added with 50mM sodium phosphate (pH
7.2) Buffer exchange was performed.

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 ELISA using rabbit anti-APP19-379 (ZKX) antiserum was similarly performed on each chromatographic fraction to identify the APP592 elution position. APP592 is 1.0-1.2MNaC
It eluted at 1. ELISA positive fraction to Centricon™
After concentration using 30 (Amicon), 20mM Tris
- Buffer exchanged with hydrochloric acid (pH 8.0). These were subjected to 7.5% SDS-PAGE and Coomassie
Staining was performed to confirm purity and molecular weight. pSVM
The protein obtained by performing the above procedure from the culture supernatant of COS-1 cells introduced with T592 was subjected to SDS-P.
It was a rather broad single band on AGE, and the apparent molecular weight was determined to be 94.5 kDa. Furthermore, when the amino acid sequence of the N-terminus of this protein was analyzed using an Applied gas phase sequencer, it was found that the signal peptide (17 amino acids from the N-terminus) of APP695 was removed.
Amino acid sequence from the 18th Leu to the 33rd Gln from the N-terminus of P695 (APP751, APP77
0) was confirmed. Thus, purified APP59
2 was obtained.

(ii) Preparation of APP648 and APP667 APP648 (Fig. 2) consisting of 648 amino acids (including signal peptide) and 667 amino acids, which are proteins in which the C-terminal 103 amino acids of APP751 and APP770 are deleted. APP667 (Fig. 2) consisting of
Plasmid pSVMT66 described in No. 13, Example 5, Step 2
7, and pSVMT648 in step 2(i) of this example.
In the same manner as above, introduction into COS-1 cells, collection of culture supernatant, and purification were performed. During purification, rabbit anti-APP19-3 was used to confirm each fraction in chromatography.
79 (ZKX) antiserum, the monoclonal antibody ADJ5-3-7 (FEI accession number FE) obtained in Example 9 of JP-A-2-138995 was used for APP667.
RM P-10388) was also used. Furthermore, APP
As for APP667 and APP648, their trypsin inhibitory activity was also confirmed as one of the confirmation methods, and strong inhibitory activity was confirmed for both of them (inhibitory activity measurement was carried out in accordance with European Patent Publication No. 0
This was carried out according to the method for measuring the trypsin inhibitory activity of sPI described in Example No. 304013). Purified APP648
, the molecular weight of APP667 is determined by SDS-PA under reducing conditions.
GE, 100.5kDa and 105.5kDa, respectively.
Met.

[Step 3] Preparation of anti-APP antibody [Step 3-1] Preparation of anti-APP monoclonal antibody-producing hybridomas 3B2 and 6A3 (i) Mouse sensitization Antigen for hybridoma preparation was prepared as follows. did. Purified APP59 obtained in step 2(i) of this example
2 in PBS(-) and Laemmli's SDS-
PAGE sample buffer (Laemmli U.
K. Nature227 680-685 (19
After boiling at 100° C. for 3 minutes in 70)), 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 way, an excess amount of SDS
was removed, and SDS-denatured APP592 was recovered from the sediment.
This was used as an antigen for hybridoma creation. For immunization, 500 μl of 20 μg of SDS-denatured APP592
was dissolved in PBS(-) and injected subcutaneously into three 6-week-old female Balb/c mice together with complete Freund's adjuvant, and immunizations were then performed a total of three times at two-week intervals. Furthermore, two subcutaneous injections were performed at one week intervals.

During the immunization process, the increase in serum antibody titer against the antigen was measured by solid-phase ELISA according to the Monoclonal Antibody Experiment Manual (Kodansha Scientific, written by Sakuji Toyama).
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 I 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 cloning three times were named 3B2 and 6A3, respectively, and were deposited with the Microbial Technology Research Institute on January 10, 1991. (The accession number for 3B2 is FAIKEN BYO 11943, and that for 6A3 is NO. 11944). The reactivity of monoclonal antibodies 3B2 and 6A3 produced by these hybridomas was determined using APP66, which was prepared in steps 2(i) and (ii) of this example.
7, APP648, and APP592 were examined, and as described later, they strongly reacted with these three APP derivatives under both SDS denaturation and non-denaturation conditions. That is, these monoclonal antibodies are APP770, APP7
51 and APP695 can be recognized.

[Step 3-2] Preparation of anti-APP monoclonal antibody-producing hybridoma 1B2 (i) After sensitization of mice ZKX obtained in Step 1-3 of Reference Example 2 was transferred to Step 3-3 of Example 1 as described above. 1(i). 500 μg of this SDS-denatured ZKX
1 of PBS(-) was injected subcutaneously into four 6-week-old female Balb/c mice together with complete Freund's adjuvant, and 2 weeks later, the same immunization was performed. Furthermore, immunization with SDS-denatured ZKX was performed six times at two-week intervals using incomplete Freund's adjuvant. Two weeks later, 20 μg of SDS-modified ZKX was intravenously injected. After about a month, 20 μg of SDS-denatured A
PP592 was injected subcutaneously together with Freund's complete adjuvant, and one week later, immunization with APP592 was performed in the same manner.

During the immunization process, the increase in serum antibody titer against the antigen was measured using solid-phase ELIS in the same manner as in step 3-1 of this example.
Confirmed by method A. (ii) 200 μl of PB to the mouse with the highest antibody titer in the cell fusion serum.
25 μg of SDS-denatured APP592 dissolved in S(-)
was injected into the tail vein as a boost. The subsequent operations are performed in the same manner as in step 3-1(ii) of this example, and the APP
A clone that strongly reacted with 592 was obtained, 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 10, 1991 (Feikoken Bacterium Deposit No. 11942). The reactivity of monoclonal antibody 1B2 produced by this hybridoma was determined by A
When PP667, APP648, and APP592 were examined, as described below, they strongly reacted with these three APP derivatives under both SDS denaturation and non-denaturation conditions. That is, monoclonal antibody 1B2 is APP770
, APP751, and APP695. [Step 4] Characterization of monoclonal antibodies (i) Determination of immunoglobulin classes and subclasses Isotyping kit (
Amersham). As a result, for 7E11, IgG1, L chain is κ, 1
IgG2b for B2, κ for L chain, IgG2b for 3B2, κ for L chain, IgG2a for 6A3
, the L chain was determined to be κ.

(ii) Determination of specificity The specificity of the above monoclonal antibody was determined by the test shown below. Various APPs (APP667, APP648
, APP592), non-denaturing and SDS-denaturing treatments were performed, and determination was made using a conventional solid-phase ELISA method. The above monoclonal antibody was used as the primary antibody at 2 μg/
ml of IgG, the secondary antibody was β-galactosidase-labeled sheep F(ab')2 anti-mouse Ig (Amersham), and the substrate was 4-methylumbelliferyl-
After performing an enzymatic reaction for 30 minutes using β-D-galactopyranoside, the fluorescence intensity was measured using a fluorescence reader FCA (PANDEX). The results are shown in Table 1.

Monoclonal antibody 7E11 is APP66
It reacted only with 7. 1B2, 3B2, and 6A3 are all three types of APP (APP667, APP648 and A
I strongly recognized all of PP592). As a negative control, reactivity with bovine serum albumin (BSA) was also examined, but these four monoclonal antibodies did not react with BSA at all.

[Step 5] Purification of IgG from ascites-infused monoclonal antibodies In order to obtain a large amount of monoclonal antibodies, 1×10 7 hybridomas of each of 7E11 obtained in step 1 and 1B2, 3B2, and 6A3 obtained in step 3 of this example were used. , MediaI medium (
Immunobiological Research Institute Co., Ltd.) 0.5ml and suspended in 0.5ml.
It was injected into the peritoneal cavity of a Balb/c mouse that had previously been intraperitoneally administered with 0.5 ml of pristane (Aldrich) one week before. After about 2 weeks, abdominal enlargement was observed, and ascites was collected through abdominal incision. Seven mice were treated for each hybridoma, and approximately 10 ml of ascitic fluid was obtained from each. 5 ml of the obtained 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. 5.7 mg from 7E11 clone, 7.9 mg from 1B2 clone, 4.3 mg from 3B2 clone, 3.3 mg from 6A3 clone
of purified IgG was obtained.

[Step 6] Preparation and purification of anti-ZtPI (anti-KPI) polyclonal antibody [Step 6-1] Expression of β-galactosidase/KPI fusion protein in E. coli (i) Construction of fusion protein expression plasmid Expression of E. coli β-galactosidase sPI (28 of APP770) at the N-terminus
It includes the 8th Arg residue to the 359th Arg residue. European patent application 88113 for the production of sPI
No. 283.1, Example 7. Also, Bioc
Him. Biophys. Acta 1038 1
A plasmid capable of expressing a fusion protein (ZPI) fused with APPI-72 was constructed. Specifically, the expression vector pEX1 (Boehringer Mannheim Yamanouchi)
was digested with restriction enzymes BamHI and PstI to obtain a linear vector DNA fragment. On the other hand, European patent application 881
Plasmid pPItrp 75-1 described in Example 4 of No. 13283.1 (Publication No. 0304013, corresponding Japanese patent application No. 63-201998) was added to BamHI.
and PstI, and after agarose gel electrophoresis, 0.
A 3 kbp fragment was isolated using the Funakoshi Gene Clean Kit. The two obtained fragments were transformed into T4DN.
Ligation was performed using A ligase.

[0033] The reaction product was prepared according to Maniatis et al.'s experimental book (T
．． Maniatis et al., Molecular Clo
ning. A Laboratory Manual
l, Cold Spring Harbor L
In addition to E. coli strain POP2136 (Boehringer Mannheim Yamanouchi) competent cells prepared according to the method described in the laboratory (1982),
Transformed. Six ampicillin-resistant transformed colonies were selected, each plasmid was extracted, and BamHI and P
When cut with stI and analyzed, all of them contained the desired plasmid. The obtained plasmid was pEX-Z
It was named PI. This plasmid can express a β-galactosidase/KPI fusion protein under the control of the PR promoter derived from lambda phage.

(ii) Production of fusion protein E. coli POP2136/pEX-ZPI carrying the plasmid pEX-ZPI obtained in step 6-1 (i) of this example was added to 50μ
The cells were cultured overnight at 30°C in L medium containing g/ml ampicillin. 1 ml of the main culture solution was added to 50 ml of L medium containing 50 μg/ml ampicillin, and after culturing at 30°C for 3 hours, the culture was transferred to a constant temperature culture tank at 42°C and further cultured for 3 hours. At 42°C, lambda phage P
The repressor of the R promoter is inactivated and the PR promoter is activated to produce the desired fusion protein.

[0035] 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, 227.680-685 (1970)]
After boiling in SDS-PAGE sample buffer, SDS-PAGE was run using a 10% polyacrylamide gel according to
It was subjected to polyacrylamide gel electrophoresis. After electrophoresis, the gel was stained with Coomassie, and it was confirmed that the desired fusion protein with a molecular weight of about 120 Kd was present. Below, this β-galactosidase/KPI fusion protein is referred to as ZP.
Abbreviated as I.

(iii) ZPI solubilization Step 6 of this example
A part of the cultured bacterial cells obtained in (ii) of -1 was added to 20mM containing urea at final concentrations of 0M, 2M, 4M, 6M, and 8M, respectively.
The cells were suspended in Tris-HCl (pH 8.0), the cells were disrupted by ultrasonic waves using an Otake sonicator (Otake Seisakusho), and insoluble materials were collected by centrifugation. The obtained insoluble matter was treated in the same manner as in step 6-1 (ii) of this example, and analyzed by SDS polyacrylamide gel electrophoresis.

As a result of Coomassie staining, a band of the target product ZPI 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. There wasn't. From the above results, the target fusion protein forms protein inclusion bodies as insoluble matter in E. coli after production.
It was found that this insoluble material was solubilized by urea of 6M or more.

(iv) Partial purification of ZPI This example step 6
-1, in the same manner as (ii), E. coli POP2136/
pEX-ZPI was cultured, and bacterial cells were collected from 200 ml of culture solution. The cultured cells were suspended in 40 ml of 20 mM Tris-HCl (pH 8.0) buffer containing 4M urea, and the cells were disrupted by ultrasonication. Centrifuging the destroyed bacterial cells (1
0000×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 (10000x
g, 10 min) and then the supernatant was collected. By the above operations,
E. coli proteins soluble in 4 M urea and E. coli proteins insoluble in 8 M urea were removed to obtain a partially purified solution of ZPI. 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 ZPI.

(v) Extraction of protease inhibitor region 20 ml of the partially purified ZPI solution obtained in step 6-1 (iv) of this example was added to 2 L of 20 mM Tris-HCl (pH 8.0).
The protein was dialyzed twice to remove urea and at the same time restore the three-dimensional structure of the protein. The obtained dialysate was
After incubating at ℃, 100 units of TPCK-treated trypsin-immobilized agarose beads (Sigma) were added,
By shaking at ℃ 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 0.
3M NaCl, 10mM CaCl2, 10m
Elution was performed with 6 ml of an eluent consisting of M 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.

[0040] The trypsin inhibitor activity concentrations contained in each eluate were 16.4 μg/ml, 1.5 μg/ml, and 0.27 in terms of BPTI in the order of elution fractions.
It was μg/ml. These elution fractions were combined with ZtPI
Fraction. (vi) Analysis of protease inhibitors The eluate obtained in step 6-1 (v) of this example was added to -20°C in advance.
4 times the volume of acetone cooled to 20° C. was added, and the mixture was cooled at −20° C. for 1 hour. Furthermore, refrigerated centrifugation (10,000 x g
15 minutes), and the precipitate was collected. 1 ml of the obtained precipitate
The sample was redissolved in 20mM Tris-HCl (pH 7.5), and the amount of trypsin inhibitory activity was measured in the same manner as in step 6-1 (v) of this example. As a result, European Patent Publication No. 03
62μ in terms of sPI described in Example 4 of No. 04013
It was found that it contained trypsin inhibitor equivalent to 1.

A part 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 Kd was detected. [Step 6-2] Preparation of anti-ZtPI antiserum Step 6 of this example
300 μg of ZPI obtained in step (iv) of -1 was added to the sample buffer (2% SDS,
After processing at 100°C for 5 minutes in a solution containing 5% β-ME (but no dye was added), 4 times the volume of acetone was added, cooled at -20°C for 1 hour, and heated at 9000 rpm for 20 minutes.
A centrifugation operation was performed for 1 minute, and the precipitate was collected. The precipitate is P
The suspension was suspended in BS(-), mixed well with complete Freund's adjuvant (FCA), and subcutaneously injected into rabbits. After 31 days, the same treatment (immunization) was performed except that incomplete Freund's adjuvant was used instead of FCA. After another 14 days, 300 μg of ZtPI obtained in step 6-1 (v) of this example was treated in the same manner and immunized. After another 16 days, the same treatment was performed using ZtPI. After another 15 days, European Patent Publication No. 0304013 Example 4
150 μg of sPI obtained by the method described in 1. 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-ZtPI antiserum. Antibody titers in serum were measured using non-denatured and SDS-denatured sPI (25
ng/well) as the antigen. The final titer of the antiserum was 64,000 times that of undenatured sPI, and 64,000 times that of SDS denatured sPI, as shown by the dilution of the antiserum that showed more than three times the color development of the control (well without antigen). It was 128,000 times as much. Furthermore, it did not react at all with urinary trypsin inhibitor (Nippon Chemical Research Co., Ltd.). That is, this anti-ZtPI antiserum is KP
It was found to have high specificity and antibody titer for the I sequence. [Step 6-3] Purification of anti-ZtPI IgG 20 ml of the anti-ZtPI serum obtained in step 6-2 of this example was purified at 56°C for 3
After 0 min incubation to inactivate, equal volume of PBS
(-) and dilute with 100% saturated ammonium sulfate solution (
(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 completed, stirring was continued for an additional 12 hours, and then refrigerated centrifugation was performed at 3000 xg 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), packed into a dialysis tube, and dialyzed three times against 5 L of the same buffer. The obtained dialyzed sample was treated with DEA equilibrated in advance with 10 mM sodium phosphate buffer (pH 8.0).
It was applied to an E-cellulose column (DE52, Whatman) and eluted with the same buffer. The flow-through fractions were collected and concentrated using Centriprep TM30 (Amicon) to obtain 15.7 mg of partially purified anti-ZtPI antibody. The partially purified antibody was further purified using Affiprep™ Protein A cartridge (Bio-Rad) according to the attached protocol published by Bio-Rad, and purified anti-ZtP was obtained.
12.7 mg of I IgG was obtained.

[Step 7] Biotinylation of antibodies Using 2 mg each of the 7E11, 1B2, 3B2, and 6A3 purified monoclonal antibodies obtained in step 5 of this example and the purified anti-ZtPI antibody obtained in step 6-3, the immunization experiment procedure was performed. (Volume 9, 3539-
3549 1982 edited by the Japanese Society of Immunology), the antibodies were biotinylated. Dialyze and buffer exchange against 0.1M NaHCO3 solution in advance to obtain a concentration of 1mg.
Purified 7E11, 1B2, 3B2 adjusted to /ml
, 6A3, and anti-ZtPI antibody were added with 280 μl of a dimethyl sulfoxide solution of NHS-biotin (N-hydroxysuccinimide-biotin, Pierce) adjusted to a concentration of 1 mg/ml, and the mixture was gently stirred at room temperature for 4 hours. Next, dialysis was performed four times against 3 L of PBS(-) at 4°C to obtain biotin-labeled 7E11, 1B2, 3B2, 6A3 and anti-ZtPI antibody.

Example 2 Two-antibody sandwich ELISA using a combination of anti-ZtPI antibody and 7E11 monoclonal antibody 5 μg of the purified anti-ZtPI antibody obtained in step 6-3 of Example 1
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 (-), PB containing 1% bovine serum albumin (Sigma) was added.
S(-) was added and coated at 37°C for 2 hours. Next, 0.
Purified APP667 standard solution (2-2000 ng/ml) diluted in PBS(-) containing 1% bovine serum albumin
was added, and the mixture was treated at room temperature for 1 hour with stirring. Next, after washing the plate three times with PBS (-) containing 0.05% Tween-20, 1 μg/ml of the biotin-labeled purified 7E11 IgG obtained in step 7 of Example 1 was added.
50 .mu.l of the solution was added to each well at a concentration of 50 .mu.l, and the reaction was carried out at room temperature for 1 hour with stirring. 0.05% Tween-2
After washing three times with PBS(-) containing 0, β-galactosidase-labeled avidin D (Vector) was added, and the mixture was allowed to react at room temperature for 1 hour with further stirring. 0.05% Tw
After washing 5 times with PBS (-) containing een-20, 2 m
4- dissolved in PBS(-) containing M MgCl2
After adding methylumbelliferyl-β-D-galactopyranoside (1 mg/ml) and reacting at room temperature for 30 minutes,
Fluorescence intensity was measured using FCA (PANDEX). As shown in FIG. 3, the detection limit was about 0.5 ng/well.

Example 3 Two-antibody sandwich ELISA using a combination of 1B2 monoclonal antibody and 7E11 monoclonal antibody Two-antibody sandwich ELISA using a combination of 1B2 monoclonal antibody and 7E11 monoclonal antibody using the same method as in Example 2
Antibody sandwich ELISA was investigated. As shown in FIG. 4, the detection limit was about 30 ng/well.

Example 4 Two-antibody sandwich ELISA using a combination of 3B2 monoclonal antibody and 7E11 monoclonal antibody Two-antibody sandwich ELISA using a combination of 3B2 monoclonal antibody and 7E11 monoclonal antibody using the same method as in Example 2
Antibody sandwich ELISA was investigated. As shown in FIG. 5, the detection limit was about 20 ng/well.

Example 5 Two-antibody sandwich ELISA using a combination of 6A3 monoclonal antibody and 7E11 monoclonal antibody Two-antibody sandwich ELISA using a combination of 6A3 monoclonal antibody and 7E11 monoclonal antibody using the same method as in Example 2
Antibody sandwich ELISA was investigated. As shown in FIG. 6, the detection limit was about 20 ng/well.

Reference Example 1 AP by RIA (radioimmunoassay) method using 7E11 monoclonal antibody
Detection of P667 (i) Buffer A 50 mM sodium phosphate buffer (pH 7.0) containing 150 mM NaCl, 0.1% gelatin (Bio-Rad) and 0.02% NaN3 was prepared and designated as buffer A.

(ii) Preparation of 125I-APP667 The purified APP667 obtained in Example 1 Step 2 (ii) was purified by the Bolton-Hunter method (Bolton et al., Biochem).
．． J. 133, 529-539, 1973) to obtain 125I-APP667. (iii) Preparation of APP667 standard solution The purified APP667 obtained in Example 1 step 2(ii) was diluted with 20mM Tr.
It was dissolved in is-HCl (pH 8.0) and diluted with buffer A to prepare APP667 standard solutions at various concentrations.

(iv) RIAAPP of APP667
667 standard solution 1μg/m with buffer A
Purified 7E11 IgG prepared to a concentration of 1 (Example 1)
200 μl of solution (purified in step 5), 125I-labeled APP
667 buffer A solution 100 μl (10000 cp
m) and 200 μl of buffer A and mix, 4
C. for 18 hours. Dextran-carbon liquid (Dextran T-70 (Funakoshi Pharmaceutical Co., Ltd.),
Add 200 μl of 0.0025% solution and activated carbon Norit A (Sigma) 0.25% solution), let stand at 4°C for 18 hours, and centrifuge at 2.500 x g for 20 minutes. Then, the radioactivity of the supernatant was measured using a gamma well counter. The measurement limit was approximately 20 ng/assay.

Example 6 Preparation of APP770 detection kit Anti-ZtPI antibody as the first antibody, biotin-labeled 7E11 monoclonal antibody as the second antibody, purified APP667 as the standard APP, β-galactosidase-labeled streptavidin as the labeled detection reagent, 4 -Methylumbelliferyl-β-D-galactopyranoside, dimethyl sulfoxide as substrate solubilizer, PBS as buffer
(-), Tween-20 as a detergent, PBS containing 1mM MgCl2 as an enzyme reaction buffer (-)
, using bovine serum albumin as a coating agent, APP77
A 0 detection kit was prepared (Table 2). APP770 was detected using this kit.

[0051] Powder PBS (-) (manufactured by Nissui Pharmaceutical Co., Ltd.)
9.6 g was dissolved in purified water to make 1 liter of PBS(-) solution. 0.05 using Tween-20 as a cleaning agent
A PBS(-) solution containing % Tween-20 was prepared. 1 g of bovine serum albumin as a coating material for 100 m
It was dissolved in 1 liter of PBS(-) to make a 1% bovine serum albumin solution. The first antibody was prepared by dissolving 1 mg of anti-ZtPI antibody in 1 ml of PBS (-) as a 200-fold concentrated solution. The second antibody was prepared by dissolving 1 mg of biotin-labeled 7E11 monoclonal antibody in 1 ml of PBS (-) and making a 1000-fold concentrated solution. β-galactosidase labeled-streptavidin 1 mg in 1 ml PBS
(-) was dissolved and prepared as a 1000-fold concentrated solution. As a substrate for β-galactosidase, 10 mg of 4-methylumbelliferyl-β-D-galactopyranoside was dissolved in 100 μl of dimethyl sulfoxide.
It was prepared as a 00-fold concentrated solution at the time of use. Standard APP (purified A
PP667 (lyophilized product of 1 ml of PBS (-) solution containing 10 μg and 10 mg of bovine serum albumin) is 1
ml of purified water.

[0052] With each prepared reagent, AP was prepared according to Example 2.
Detection of P770 was performed and similar results were obtained. Example 7 APP77 in human cerebrospinal fluid using this kit
APP770 in the cerebrospinal fluid was quantified using the kit described in Example 6 of the present invention using cerebrospinal fluid obtained from two Alzheimer's disease patients as samples. The method was the same as in Example 2. The results were 28.2ng/ml and 34.8ng/ml.

Reference Example 2 Preparation of Immunization Antigen ZKX Protein and Anti-ZKX Antibody [Step 1] Preparation of Immunization Antigen ZKX First, a part of β-galactosidase and 19th to 379th residues counting from the N-terminus of APP770 were prepared. The production of ZKX, which is a fusion protein with a fragment up to a residue, will be described.

[Step 1-1] Expression plasmid pEX
- Construction of ZKX The expression vector pEX2 (Boehringer Mannheim Yamanouchi) was digested with restriction enzymes Bsu36I (MstII) and SalI to obtain a linear vector DNA fragment. On the other hand, European Patent Application No. 88113283.1 (Publication No. 0304013, the corresponding Japanese application is Japanese Patent Application No. 88113283.1 (Publication No. 0304013)
1998), plasmid pGBP2 described in Example 1 of
(ATCC-67502) was cut with KpnI and XhoI, and the N of APP770 was analyzed by agarose gel electrophoresis.
A DNA fragment encoding about 360 residues on the terminal side was isolated using a Gene Clean kit manufactured by Funakoshi. Additionally, six synthetic linkers of SEQ ID NOs: 1-6, 1, 2, 3
, 4, 5, 6 (When these linkers are annealed, they have an MstII cut end and a KpnI cut end as shown in Figure 7, and even when bound to the MstII site of pEX2, they do not move out of frame and encode consecutive amino acids. ) was synthesized using Applied Biosystems DNA Synthesizer 380A, and ligated with the two previously obtained DNA fragments using T4 DNA ligase. Figure 7 shows an overview of this.

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 cut with amHI and analyzed, all of them contained the desired plasmid. The obtained plasmid was pEX-
It was named ZKX. 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
In addition to part of the sequence common to APP770, APP751, and APP695, it contains a protease inhibitor portion and a sequence specific to APP770, but does not contain the sequence from the transmembrane region to the C-terminus of APP.

[Step 1-2] Fusion protein production reference example 2 Escherichia coli POP2136/pEX-ZKX carrying the plasmid pEX-ZKX obtained in step 1-1 was incubated at 50%
The cells were cultured overnight at 30°C in L medium containing μg/ml ampicillin. 1 ml of the main culture solution was added to 50 ml of L medium containing 50 μg/ml ampicillin and cultured at 30° C. for 3 hours, then transferred to a constant temperature culture tank at 42° C. and cultured for an additional 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.

[0057] The culture solution was centrifuged to collect the bacterial cells, and a portion of the bacterial cells were collected using the Laemmli method [Laemmli U. et al. K.
, Nature 227 680-685 (1
970)] and then subjected to SDS-polyacrylamide gel electrophoresis using a 10% polyacrylamide gel after boiling in SDS-PAGE sample buffer. After electrophoresis, the gel was stained with Coomassie, and the molecular weight was approximately 58k.
It was confirmed that the desired fusion protein of Da existed. Below, this partial β-galactosidase/partial APP
770 fusion protein is abbreviated as ZKX. [Step 1-3] ZKX Partial Purification Reference Example 2 In the same manner as Step 1-2, Escherichia coli POP213
6/pEX-ZKX was cultured, and bacterial cells were collected from 200 ml of the culture solution. The cultured cells were suspended in 40 ml of 20 mM Tris-HCl (pH 8.0) buffer containing 4M urea, and the cells were disrupted by ultrasonication. Centrifuging the destroyed bacterial cells (
10,000×g for 10 minutes), and the precipitate was collected. This precipitate was then diluted with 20 ml of 20 mM urea containing 8M urea.
It was resuspended in Tris-HCl (pH 8.0) buffer, and the precipitate was dispersed by sonication. Centrifugation (1000
0×g for 10 min), 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 ZKX was obtained. 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. When the trypsin inhibitory activity of ZKX was measured according to the method for measuring the trypsin inhibitory activity of sPI described in the Examples of European Patent Publication No. 0304013, it was found to have a strong inhibitory activity. In addition, the anti-ZtP described in Step 6 of Example 1 of the present invention
Western blotting using I antiserum also showed that this ZKX
reacted strongly.

[Step 1-4] Preparation of anti-ZKX antiserum Reference Example 2 A rabbit was immunized with ZKX obtained in step 1-3 to obtain an antiserum. That is, the SDS-modified APP described in Step 3 of Example 1
Create SDS-denatured ZKX in the same manner as 592 creation,
200 μg of SDS-denatured ZKX in 1 ml of PBS(-)
The solution was injected subcutaneously into the backs of two domestic rabbits together with complete Freund's adjuvant, and then immunizations with incomplete Freund's adjuvant were performed at two-week intervals for a total of six times. Antibody titers in serum are non-denatured and S
This was performed by solid-phase ELISA using DS-denatured ZKX (25 ng/well) as an antigen. The final titer of the antiserum was 16,000 times that of non-denatured ZKX and 16,000 times that of SDS-denatured ZKX, as shown by the dilution of the antiserum that showed more than twice the color development of the control (well without antigen). APP667 and APP5 described in Example 1 Step 2
92, and further Zt described in Step 6-1 (vi) of this Example 1
It also reacted strongly to PI.

[0059]

[Table 1]

[0060]

[Table 2]

[0061]

Effects of the Invention: By using the measurement method, reagent, and kit of the present invention, Alzheimer's disease amyloid precursor protein APP770 and its fragments can be detected and quantified easily and with high sensitivity, and can be used to diagnose Alzheimer's disease. Useful as methods, reagents and diagnostic kits. Also,
It is also useful as a research reagent in research using Alzheimer's disease amyloid precursor protein.

[0062]

[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 Characteristic symbols: Linker 1 Sequence TGAGGCCGAT ACTGCTGTCG TCC
CCTCAA CTGGCAGAT
39

0064
] 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

0065
] Sequence number: 3 Sequence length: 50 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Characteristics of the sequence Characteristic symbols: Linker 3 Sequence CATTACGGTC AATCCGCCGT TTG
TTCCCAC GGGATCCATG AGCTCG
GTAC 50

0066
] 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 GAA
CAAACGG CGGATTGACC GT
42

0067
] 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

0068
] Sequence number: 6 Sequence length: 40 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Characteristics of the sequence Characteristic symbols: Linker 6 Sequence GTGCATCTGC CAGTTTGAGG GGA
CGACGAC AGTATCGGCC
40

[Brief explanation of the drawing]

[Figure 1] Alzheimer's disease amyloid precursor protein APP
695, APP751, APP770, APP714,
It is a schematic diagram of APP563. Blacked areas from b to f have protease inhibitor activity56
A region consisting of amino acids, the dotted portions indicate a region consisting of 19 amino acids unique to APP770, and the shaded area indicates a senile plaque amyloid portion deposited in the brain. a shows the amyloid β protein portion of APP that is deposited in the brain (shaded portion) and the transmembrane region. APP563 does not have a β protein portion.

[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] Two-antibody sandwich ELISA using a combination of anti-ZtPI antibody and 7E11 monoclonal antibody

[Figure 4] Two-antibody sandwich ELIS using a combination of 1B2 monoclonal antibody and 7E11 monoclonal antibody
A

[Figure 5] Two-antibody sandwich EL using a combination of 3B2 monoclonal antibody and 7E11 monoclonal antibody
ISA

[Figure 6] Two-antibody sandwich ELISA using a combination of 6A3 monoclonal antibody and 7E11 monoclonal antibody

[Figure 7] 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.

Claims (3)

[Claims] [Claim 1] Alzheimer's disease amyloid precursor protein APP770 or a fragment thereof obtained by a two-antibody sandwich ELISA method characterized in that an antibody that specifically recognizes Alzheimer's disease amyloid precursor protein APP770 is used for at least one of the combinations. immunological measurement method. 2. Alzheimer's disease amyloid precursor protein APP770, comprising a primary reagent in which one of the two antibodies according to claim 1 is bound to an insoluble carrier, and a secondary reagent that is the other antibody. or a reagent for measuring its fragments. 3. Alzheimer's disease amyloid precursor protein A, which comprises the measurement reagent according to claim 2 as a component.
A kit for measuring PP770 or a fragment thereof.