JPH05304993A - Novel monoclonal antibody resistant to synaptophysin - Google Patents

Abstract

PURPOSE:To provide the monoclonal antibody permitting to determine synaptophysin in a small amount of the antibody on the determination of the synaptophysin contained in an small amount in a tissue by a complement- immobilizing assay. CONSTITUTION:The monoclonal antibody is a monoclonal antibody produced with the extract of a rat brain as an immunogen. The immunoglobulin class of the antibody belongs to IgM, and the antibody has a unique reactivity with the synaptophysin or P-38 of a rat.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a monoclonal antibody which specifically binds to synaptophysin.

[0002]

2. Description of the Related Art Synaptophysin is an acidic glycoprotein having a molecular weight of about 38,000, which is present in the synaptic vesicle membrane.
It was found in the bovine brain by Wiedemann et al. In 985 (Cell, 41, 1017-102).
8). On the other hand, P-38 was found in the rat brain by Joan et al. (Proc. Natl. Acad.
Sci. USA, 82, 4137-4141). However, analysis of the gene sequences of both revealed that synaptophysin and P-38 are the same protein.

Generally, these synaptophysin and P-
Since 38 is localized in synaptic vesicles, 38 is used as a detection marker for neuroendocrine tumors that retain synaptic vesicles. Neuroendocrine tumors develop in endocrine organs such as the pituitary gland and pancreas, but also metastasize to other tissues. If synaptophysin is detected in an organ that does not have synaptic vesicles, this fact indicates the presence of a neuroendocrine tumor in that organ, and the primary lesion of the tumor is the above-mentioned endocrine organ. It is shown that.

Therefore, a monoclonal antibody specific for synaptophysin is also useful as a means for locating the primary lesion of a neuroendocrine tumor.

Currently, these synaptophysin and P-3
Regarding the monoclonal antibody against 8, monoclonal antibodies belonging to the immunoglobulin class IgG have been produced.

[0006]

However, conventionally known monoclonal antibodies against synaptophysin are
Since it belongs to immunoglobulin class IgG, there are only two antigen or complement binding sites in one molecule. Therefore, there has been a problem that a large amount of antibody is required when quantifying a small amount of synaptophysin in a tissue by a complement fixation assay.

The present invention has been made in view of the above circumstances, and an object thereof is to enable quantification with a small amount of antibody when quantifying a trace amount of synaptophysin in a tissue by a complement fixation assay. More specifically, it is to provide a monoclonal antibody having specificity for synaptophysin and belonging to immunoglobulin class IgM.

[0008]

According to the present invention, a monoclonal antibody prepared by using an extract of rat brain as an immunogen, which belongs to immunoglobulin class IgM and is effective against rat synaptophysin or P-38. A monoclonal antibody characterized by having specific reactivity is provided.

The present invention also provides a monoclonal antibody having specific reactivity with a protein expressed by a gene having the following base sequence.

[0010] 10 20 30 40 50 60 AGCACCTGGC GATGCCTACG GCGATGCGGG CTACGGGCAG GGCCCCGGAG GCTATGGGCC 70 80 90 100 110 120 CCAGGACTCC TACGGGCCTC AGGGTGGTTA TCAACCCGAT TACGGGCAGC CAGCCAGCGG 130 140 150 160 170 180 TGGCGGTGGC TACGGGCCTC AGGGCGACTA TGGGCAGCAA GGCTATGGCC AACAGGGTGC 190 200 210 220 230 240 GCCCACCTCC TTCTCCAATC AGATGTAATC TGGTCAGTGA AGTCCATGAA GATCCCACGG 250 260 270 280 290 300 GTGGGCAAGA GCTCAAGAGA AGGCCTGCCC CCCTTTTCCC ATCCCCATAT CCTAGGTCTC 310 320 330 340 350 360 CACCCCTCAA CCCAGGAGAC CCTAACTGTC TTTGCTGTTT ATATATATAT ATATTATATA 370 380 390 400 410 420 TAAATATCTA TTTATCTGTC TGAGCCCTAC ATTCACCCAC TTCTCCATGC ACTAGAGGCC 430 440 450 460 470 480 CAGTCCTGAA TGGGCTCCTC CCCAACCCTG ACCTTGCATT CCTCAGCCCC TATCTGTTCC 490 500 510 520 530 540 CCAGCCCTGT CCCTTGAGGT AAGGGGCTCT AGAAAGGGGA GAGGAAGGGA ACCAGACCTT 550 560 570 580 590 600 GGCTGCATGG AGTGGGTTGG TGTGACTTTC TCTCCTTCCTTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTGCCATGCCTCTGTGCCATCAG 670 680 690 700 710 720 ACCCCAAGAG TAGAGCAGTA GACTGAGTGG AGGAGGCTTG GGTGAAACGG GCAGAGAGGA 730 740 750 GATAACCTCT GTAGAGAGAG GACTAGTCAG (where A is adenine, C is cytosine, G is guanine and T is thymine. ) Hereinafter, the present invention will be described in detail.

First, the production of the monoclonal antibody of the present invention will be described.

It can be prepared using conventional monoclonal antibody technology. That is, first, antibody-producing cells taken out from a mammal immunized with an extract of rat brain are fused with tumor cells of an appropriate animal, such as myeloma, to prepare fused cells (hybridomas) that produce the desired antibody. To do. After cloning this fused cell,
The desired monoclonal antibody can be prepared by culturing.

More specifically, the monoclonal antibody of the present invention can be produced by the following procedure.

A) Preparation of antibody-producing cells First, in order to obtain a hybridoma producing the monoclonal antibody of the present invention, an extract of rat brain is used to immunize a mammal.

As the mammal for obtaining the antibody-producing cells that produce the monoclonal antibody of the present invention, commonly used experimental animals such as mouse, rat, rabbit and guinea pig can be used, but mouse is particularly preferable. Is preferred.

As a method of immunizing a mammal, for example, a method of inoculating a mouse brain abdominal cavity or subcutaneously with an extract of rat brain as an immunogen can be used. The amount of inoculation per administration is preferably about 0.2 ml / mouse of the immunogen, and a suspension prepared by mixing this with an equal amount of adjuvant is used. This operation is repeated several times every 1 to 2 weeks. Further, 0.2 to 0.4 ml / mouse of the immunogen is directly injected into the abdominal cavity or the abdominal cavity for final immunization. The spleen is excised 3 to 4 days after the injection for the final immunization to obtain antibody-producing cells.

B) Preparation of tumor cells Tumor cells for cell fusion with the above antibody-producing cells are prepared.

Myeloma cells are usually used as the tumor cells in the present invention. The origin of the tumor cells is not particularly limited, and mammalian cell lines such as mouse, rat, rabbit and human can be used. But,
Of these, it is preferable to use a mouse cell line. Generally, a tumor cell line having an appropriate selectable marker such as hypoxanthine / guanine / phosphoribosyltransferase deficiency (HGPRT ⁻ ) or thymidine kinase deficiency (TK ⁻ ) is used. Examples of such cell lines include P3-X63-Ag8, P3-X63-Ag8-U1, SP20-Ag8-6.5.
There are three. All of these tumor cells are 8-azaguanine-resistant cell lines and cannot grow in HAT medium (containing hypoxanthine, aminopterin and thymidine).

C) Cell fusion The above antibody-producing cells and tumor cells are fused.

The medium used for cell fusion is
Commonly used Eagle's Minimal Basic Medium (MEM), Dulbecco's Modified MEM, Roswell Park Memorial
A basic medium such as Institute (RPMI) 1640 with 10% CS (calf serum), 5% FCS (fetal calf serum) + 5% CS, or 10% FCS can be used. Usually, any of the above-mentioned media may be used for maintaining the parent cells, but it is preferable to add 10% FCS when preparing the hybridoma.

The cell fusion is carried out by mixing tumor cells such as parental cells such as myeloma cells with immunized splenocytes (antibody-producing cells) in the ratio of 1: 5 to 1:20 in the presence of a fusion promoter. By doing. As a fusion accelerator,
For example, HVJ (Hennagglutinating)
Virus of Japan), polyethylene glycol (PEG) and the like can be used, but in particular,
It is preferred to use 30% to 50% PEG1500.

D) HAT selection of hybridomas Hybridomas are selected in HAT medium.

20% of the cells fused by the method of c)
It was appropriately diluted with such RPMI1640 medium containing FCS, the microculture plate (normally 96 or 24-well plates), 10 ⁴ to 10 ^6/10
Inoculate about 0 μl / well. Then add H to each well
The AT selection medium is added, and the culture is usually performed every 1-2 days while the medium is replaced.

When the 8-azaguanine resistant strain is used as the tumor cell, the unfused myeloma cell and myeloma-myeloma fused cell are about 7 in HAT medium.
Die in days. In addition, splenocytes have normal life because they are normal cells and cannot grow for more than 2 weeks in vitro. Therefore, all the cells that grow from the 7th to 10th day of culture are considered to be splenic / myeloma fused cells.

E) Screening of hybridoma The hybridoma selected by the method of d) above is screened.

The screening was carried out mainly by ELISA (Enz using the culture supernatant of the well in which the hybridoma was grown).
ime linked-immunosorbent
It can be carried out by picking up a hybridoma clone secreting the antibody of interest using a known method such as assay method or immunoblot method.

F) Cloning The desired hybridoma obtained by the screening is cloned.

There is a possibility that two or more kinds of hybridomas producing different antibodies may grow in each well. Therefore, a single hybridoma clone producing the desired monoclonal antibody may be finally obtained by cloning by the limiting dilution method or the like.

G) Obtaining Monoclonal Antibody The desired monoclonal antibody is obtained by culturing the hybridoma obtained in the above f).

The hybridomas are placed in a culture vessel (in
It can be cultivated either in vitro or in vivo. in vitr
In the case of culturing at o, the medium may be the above-mentioned normal medium supplemented with CS or FCS. After culturing in this medium for 3 to 5 days, the desired antibody can be obtained from the medium supernatant. When cultured in vivo, pristane (2,6,10,14) was added to animals syngeneic with myeloma cells.
-Tetramethylpentadecane) and the like are intraperitoneally administered, and at least one week or more after that, the hybridoma is inoculated intraperitoneally. Then, the ascites fluid stored after 7 to 14 days can be collected to obtain the target antibody.

Next, the gene expressing synaptophysin recognized by the monoclonal antibody of the present invention will be described.

The synaptophysin gene recognized by the monoclonal antibody of the present invention can be obtained by the following procedure. That is, a cDNA library prepared from rat brain using the λgt11 vector is transformed into an appropriate host cell for expression. The protein thus produced by the host cell is screened by using the above-mentioned antibody that specifically binds to rat synaptophysin to select the vector into which the gene fragment of interest is inserted. By amplifying this selected vector,
The synaptophysin gene can be obtained.

More specifically, the gene expressing synaptophysin recognized by the monoclonal antibody of the present invention can be prepared by the following procedure.

H) Screening First, host cells (for example, Escherichia coli) are cultured in an appropriate medium, and this host cells are fed with Clontech rat brain cDNA.
Introduce the A library, which has been inserted into the λgt11 vector. For example, Escherichia coli is cultured in an appropriate medium such as LBM medium, and 5 μl of this culture medium is added to
You may introduce | transduce by mixing the phage vector liquid in which the said cDNA of * 10 < ⁴ > pfu was integrated. Next, this mixed solution is spread on an agar medium and further cultured to form plaques. A nittle cellulose membrane is overlaid on the formed plaque and further incubated. This allows
The cDNA incorporated into the vector is expressed and the produced protein is transferred to the nitrocellulose membrane.

This nitrocellulose membrane is reacted with the above-mentioned anti-synaptophysin monoclonal antibody, and then with a labeled antibody that specifically binds to this anti-synaptophysin antibody. After the reaction, the treatment necessary for detecting the label of the antibody is performed, and the spot where the label is present is detected. By searching the plaque corresponding to the spot, the vector having the target rat synaptophysin gene fragment can be obtained.

I) Amplification of vector having rat synaptophysin gene The synaptophysin gene screened by the above method h) is amplified.

The vector confirmed to have the rat synaptophysin gene fragment can be amplified by culturing host cells by a method such as a plate lysate method or a liquid culture method.

J) Preparation of rat synaptophysin gene A gene of interest is prepared from the amplified vector.

First, only the phage having the rat synaptophysin gene fragment is recovered from the host cell. The recovered phages are destroyed with chloroform or the like, and the protein is removed with phenol or the like. After removing the protein, the DNA containing the rat synaptophysin gene fragment can be obtained by precipitation with ethanol.

The monoclonal antibody of the present invention specifically binds to synaptophysin as described above. Therefore,
The medicinal composition containing the monoclonal antibody of the present invention and a pharmacologically acceptable carrier or diluent is useful for diagnosing a neuroendocrine tumor using the presence of synaptophysin as an index.

When used for diagnosis, the monoclonal antibody of the present invention needs to be labeled in order to enable diagnosis. As the label, any label that can be detected after being taken into the body may be used. However, at present, image diagnosis by the scintigraphy method, which will be described later, is most commonly performed, so that a radioisotope can be preferably used as a label. As the radioisotope used as a label, any one can be selected according to the half-life, the type of radiation, the purpose of diagnosis, etc., but it is preferable to use one that is easily stored in the target organ. Specifically, ^99m Tc, ¹¹¹ In, ¹²³ I,
¹³¹ I, ²⁰¹ Tl, ⁷⁵ Se, ¹⁶⁹ Yb, ⁶⁷ Ga and the like are used.

As the carrier or diluent contained in the medicinal composition of the present invention, any of those conventionally used as pharmacologically acceptable may be used, and two or more kinds thereof may be used in combination. It is also possible to do so. A sterile and aqueous isotonic suspension or solution is preferable, and specifically, physiological saline, phosphate buffered physiological saline or the like can be preferably used.

The administration method of the medicinal composition of the present invention includes:
Parenteral administration methods such as subcutaneous administration, intramuscular administration, intravenous administration and intraperitoneal administration may be used.

The dosage of the medicinal composition according to the present invention depends on the age, body weight, administration route, etc. of the administration subject, but the daily dosage is 1-100 mg / Kg of mammal, preferably 1-10 mg / Kg. A dosage of Kg may be used.

The pharmaceutical composition of the present invention can be prepared by standard methods.

A diagnostic method using the medicinal composition thus prepared will be briefly described below.

The most commonly used diagnostic method at present is the scintigraphy method. This is the administration of an isotope that emits γ-rays into the body, and after a certain time the distribution of radioactivity in the target organ or whole body is measured using a scintillation scanner (which can be imaged by using a computer) or a scintillation camera. It is a method of displaying a diagnostic image. As a result, it becomes possible to diagnose the presence or absence of a lesion in the organ (a portion where isotopes are often taken in or a portion where isotopes are not taken in conversely).

Specifically, an effective amount of the medicinal composition of the present invention was administered, and after a certain period of time, an image of an organ having a lesion (an organ originally not having synaptic vesicles) was drawn,
It can be diagnosed whether the lesion is a neuroendocrine tumor.

[0049]

When the monoclonal antibody of the present invention is used, the protein group, ELIS, transferred to the membrane of nitrocellulose, etc.
Only the protein group adsorbed on the A plate or rat synaptophysin contained in a tissue specimen such as a tissue section can be easily detected. As the detection method, anti-mouse Ig labeled with an enzyme or a fluorescent dye is used.
The indirect enzyme antibody method and the indirect fluorescent antibody method used in combination with the M antibody can be mentioned.

The monoclonal antibody of the present invention belongs to immunoglobulin class IgM and has 10 antigen or complement binding sites in one molecule. Therefore, when a complement fixation assay is performed using the monoclonal antibody of the present invention, it is highly sensitive as compared with the case of using a conventional IgG class monoclonal antibody, and a small amount of antibody can quantify a small amount of rat synaptophysin. can do.

Further, the use of the medicinal composition containing the monoclonal antibody of the present invention makes it possible to diagnose a neuroendocrine tumor in the case of metastasis. Further, it can be detected more easily than in the case of using X-rays by a scintiscanner method using the medicinal composition and comprehensive image diagnosis using a computer.

[0052]

EXAMPLES Examples of the present invention will be described below.

Example 1 Preparation of Monoclonal Antibody Against Rat Brain Protein (1) Preparation of Immunogen The following preparation operation was carried out at 0 to 4 ° C. unless otherwise specified. In addition, 0.5 mM of protease inhibitor PMSF was added to the water and the solution as needed.

First, the rat brain was mixed with a 3-fold amount of buffer A (0.
32 mM sucrose, 20 mM Tris-citrate buffer) was added and homogenized. This homogenate may be used as it is as an immunogen. However, in this example, the homogenate was passed through a nylon mesh (mesh size: 133 μm, 77 μm), overlaid with an equal amount of buffer solution A, and centrifuged at 5000 rpm for 10 minutes to collect the supernatant. Furthermore, this supernatant is centrifuged at 15,000 rpm for 3 times.
After centrifugation for 0 minute, the obtained precipitate was collected (the extract obtained as this precipitate is referred to as P2 fraction). Next, the supernatant obtained by this centrifugation was centrifuged at 40,000 rpm for 150 minutes, and the obtained precipitate was collected (the extract obtained as this precipitate is referred to as P3 fraction). These two fractions were used alone or mixed at an appropriate ratio to prepare an immunogen.

(2) Immunization of animal: An equal amount of the immunogen prepared in (1) and Freund's complete adjuvant were placed in 1 ml syringes, respectively.
This was connected with a joint and stirred well until emulsified. This was intraperitoneally injected into a mouse for immunization. That is, basically, the second immunization was performed 2 weeks after the first immunization, and the immunization was performed as a boost 2 more weeks thereafter. Then, an experiment was conducted on a schedule in which a cell fusion operation was performed 3 days after the boost. When it was necessary to adjust the number of days, it was adjusted by delaying the boost. The antibody titer was assayed by collecting blood from the tail vein of the mouse 2 to 7 days after immunization and performing the immunoblotting method described below using the serum.

(3) Cell fusion The cervical spine of the mouse immunized by the operation of (2) was dislocated, and the spleen was taken out. This is pre-filled with 5 ml of RPMI 6
It was transferred to a cm dish to remove excess fat and the like. It was washed with two 6 cm dishes containing 5 ml RPMI. Two
The spleen was rubbed with tweezers between the 5 cm square stainless wire mesh, which was folded in two, to separate the cells. The disaggregated cells were pipetted into a centrifuge tube and washed twice with 10 ml of RPMI by centrifugation. 0.
17 M NH ₄ Cl was added, and the mixture was placed in ice for 5 minutes to cause hemolysis. After this hemolysis procedure, 5 ml of RPMI was added,
The mixture was centrifuged at 00 rpm for 5 minutes, washed with RPMI and made up to 20 ml to prepare a spleen cell suspension.

On the other hand, myelomas 2 to 4 in the logarithmic growth phase
The dish minutes were collected by centrifugation at 1200 rpm for 5 minutes. This was washed twice with serum-free RPMI and finally suspended in 10 ml of RPMI to prepare a myeloma suspension. The cell numbers of the spleen cell suspension and the myeloma suspension were counted, and the myeloma suspension was added to the spleen cell suspension so that the myeloma was 1/5 to 1/20 of the spleen cells. After centrifugation for 5 minutes, the supernatant was removed and suspended. 0.
3 ml of 50% polyethylene glycol 1500 (in
75 mM Hepes) was added to the precipitate all at once, and the mixture was immediately stirred well. RPMI was added with continued stirring. This suspension was centrifuged at 1000 rpm for 5 minutes, and HA was added to the precipitate.
T medium (1 × 10 ⁻⁷ M hypoxanthine at final concentration,
4 × 10 ^-4 M of Aminopuriten, the RPMI-FCS) containing thymidine 16 × 10 ^-4 M was added 50 ml. This was spread at 500 μl / well on two 24-well plates, and at 100 μl / well on about 3 96-well plates. After 4-5 days, about 1 ml / well for 24-well plates and about 10 for 96-well plates.
0 μl / well HAT medium was added. As a result, the hybridoma grew in most of the wells in about 1 week. This hybridoma was further cultured, and the culture supernatant was used for screening by immunoblotting.

(4) Selection and cloning of hybridomas The immunogen prepared in (1) was used in a total protein amount of several 10 mg / m 2.
It was diluted to 1 to give an antigen solution. SDS
-After PAGE, proteins were electrophoretically transferred to nitrocellulose membranes by the method of Towbin et al. (1979). The nitrocellulose membrane was blocked with an appropriate blocking agent such as Block Ace (available from Dainippon Pharmaceutical Co., Ltd.) at room temperature for 1 hour to overnight. this,
Incubated with the culture supernatant to be screened as the primary antibody. Then, this nitrocellulose membrane was washed with TBS three times for 15 minutes. Then, as a secondary antibody, a goat anti-mouse antibody labeled with horseradish peroxidase (HRP) was incubated and reacted, and then washed with TBS three times for 15 minutes. 1 in 50 mM Tris-HCl (pH 7.5) for color development
To 100 μg / ml diaminobenzidine tetrahydrochloride, 1/1
00 amount of 1% CoCl ₂ 6H ₂ O and 1% (NH ₄ ) ₂
A color developing solution containing Ni (SO ₄ ) ₂ 6H ₂₀ and 1/500 amount of hydrogen peroxide solution was used. After sufficient color development,
It was washed with distilled water and dried. As a result of this search, wells from which the culture supernatant showing strong activity was collected were selected. Hybridomas in these wells were cloned by limiting dilution using mouse thymocytes as feeder cells. This gave one clone.
The hybridoma RB2-4 thus obtained was
It has been deposited with the Institute of Microbial Science and Technology under the name of Micro Works Research Institute No. 3944. A monoclonal antibody was obtained by culturing this hybridoma. This monoclonal antibody was named RB2-4.

Sub Isotypi made by Amersham
As a result of examination using ng Kit, it was found that the subclass of this antibody was IgM.

Example 2 Investigation of reaction specificity of RB2-4 The reaction specificity of RB2-4 was examined by the same immunoblotting method as in Example 1. The results are shown in FIG.

FIG. 1 is a photograph showing the development pattern of brain homogenate by SDS-PAGE and the binding position of RB2-4 by immunoblotting. In FIG. 1, 1 is a CBB staining pattern of a molecular weight marker, and 2 is a CBB staining pattern of a rat brain homogenate. Further, 3 shows the result of the immunoblotting method using RB2-4 for rat brain homogenate. As is clear from the figure, RB2-4
Reacts with a single protein band with a molecular weight of approximately 40 kD.

Example 3 Isolation of rat synaptophysin gene (1) Screening of plaque with monoclonal antibody RB2-4 E. coli (Y1090 (Δ1acU169proA + Δ1on araD139str
AsupF [trpC22 :: Tn10] (pMC9 *), mcrA-, mcrB +)) to LB
The cells were cultured overnight in M medium (0.2% maltose, containing 50 mg / l ampicillin). On the other hand, a Clontech cDNA library prepared from rat brain using the λgt11 vector was diluted to approximately 4 × 10 ⁵ pfu. This diluted solution and 0.6 ml of the culture solution obtained by the above culture were mixed and incubated at room temperature for 20 minutes. On the other hand, top agarose (such as LBM agarose)
Was autoclaved, incubated in the molten state at 55 ° C., added to the above mixed solution, and rapidly stirred.
Bottom agar (0.8%
LBM agar etc.). After the top agarose solidified, it was incubated at 42 ° C for 3.5 hours to form plaques. A nitrocellulose membrane was overlaid on the formed plaque and incubated at 37 ° C. for 3.5 hours to express the cDNA introduced into Escherichia coli. This nitrocellulose membrane was previously dipped in 10 mM IPTG (isopropylthiogalactopyranoside) and dried. Then, the nitrocellulose membrane was washed with TBS and used for subsequent screening by antigen-antibody reaction.

Screening using RB2-4 was carried out as follows. First, the nitrocellulose membrane was blocked with TBS containing 3% gelatin at room temperature for 1 hour to overnight. The blocked nitrocellulose membrane was reacted with a monoclonal antibody (primary antibody) used for screening, and then washed with TBS for 15 minutes three times. Then, after reacting with a horseradish peroxidase (HRP) -labeled goat anti-mouse antibody (secondary antibody), washing with TBS for 15 minutes was performed for 3 minutes.
I went there. Then, in 100 μg / ml diaminobenzidine tetrahydrochloride in 50 mM Tris-HCl (pH 7.5), 1/1
00 amount of 1% CoCl ₂ 6H ₂ O and 1% (NH ₄ ) ₂
Color was developed with a color developing solution containing Ni (SO ₄ ) ₂ 6H ₂ 0 and 1/500 amount of hydrogen peroxide solution. After sufficient color development, the stained spots were searched. By searching for a plaque corresponding to the stained spot, a phage having the gene fragment of interest was obtained.

(2) Propagation of phage having rat synaptophysin gene and its complementary chain The phage having rat synaptophysin gene and its complementary chain obtained in (1) above is propagated by the following procedure using the plate lysate method. It was First, E. coli (Y1088 (ΔlacU169supEsupFhsdR-hsdM + metBtrpRt
onA21 [proc :: Tn5] (pMC9), mcra-mcrB +)) was cultured overnight in LBM medium (0.2% maltose, containing 50 mg / ml ampicillin). 10 ⁵ to 10 ^{6 in the} culture solution
Add 0.1 ml of SM buffer containing pfu phage to 1
Incubated for 0 minutes at room temperature. On the other hand, top agarose (LBM agarose etc.) was autoclaved and incubated at 55 ° C in the molten state. Add this to the above mixture, stir quickly, and prepare the bottom agar (such as LBM agar) prepared on the plate in advance.
Spread it on top. After the top agarose solidified, the plate was put in a plastic bag and incubated at 37 ° C for 6 to 7 hours. After E. coli lysis, 5 ml of SM buffer and a few drops of chloroform were added and incubated overnight at 4 ° C, keeping the SM buffer level so that it completely covered the surface. As a result, a phage suspension was obtained in which phages having the rat synaptophysin gene and its complementary chain were dispersed in SM buffer.

(3) Preparation of rat synaptophysin gene and its complementary chain The phage solution containing the rat synaptophysin gene and its complementary chain obtained in (2) above was treated with RNaseA,
Add DNase I to 5 μg / ml each, and add 3
Incubated at 7 ° C for 30 minutes. Next, an equal amount of polyethylene glycol PEG6000-2M NaCl solution was added and mixed, and then incubated at 0 ° C for 1 hour. The mixture was centrifuged at 3000 rpm for 20 minutes at 4 ° C., and then the supernatant was completely removed. The obtained precipitate was suspended in 0.5 ml of SM buffer and transferred to an Eppendorf tube. This was centrifuged at 8000 rpm for 2 minutes, and the obtained supernatant was transferred to another Eppendorf tube. To this, EDTA and SDS were added at 10 mM and 0.1%, respectively, and incubated at 65 ° C for 15 minutes. Then, phenol extraction was performed once, phenol-chloroform extraction was performed twice, and chloroform extraction was performed once. At the time of this extraction, phenol saturated with TE (Tris-EDTA buffer) was used. To the aqueous layer obtained by these extractions, 50 μl of 3M sodium acetate and 1 ml of ethanol were added, incubated at −80 ° C. for 15 minutes, and then centrifuged at 15000 rpm for 5 minutes. After centrifugation, the supernatant was discarded and the resulting residue was rinsed with cold 70% ethanol and dried. This was dissolved in 50 μl of TE to obtain a phage DNA having the rat synaptophysin gene and its complementary strand.

From the DNA thus obtained, EcoR
The rat synaptophysin gene and its complementary strand could be excised by using a restriction enzyme such as I.

Further, only the rat synaptophysin gene and its complementary strand were obtained from the phage DNA cleaved with EcoRI or the like by the following procedure. First, in a buffer solution system containing ethidium bromide and TAE (EDTA-tris acetate buffer), phage DNA cleaved with EcoRI was electrophoresed using a 1% low melting point agarose gel. After the migration, UV irradiation with a transilluminator was performed to cut out a target band of 0.8 kbp, which was transferred to an Eppendorf tube. The gel was melted at 65 ° C., distilled water was added, and the mixture was cooled slowly. After phenol extraction twice and phenol / chloroform extraction once, DNA was recovered by ethanol precipitation. TE saturated phenol was used during this extraction.

When the base sequence of this DNA was analyzed by the dideoxy method, it was found to contain the base sequence shown in SEQ ID NO: 1 in the sequence listing. SEQ ID NO: 1 shows the nucleotide sequence of rat synaptophysin gene obtained in the present invention, and the sequence is shown in the direction toward the 5'end. Here, A represents adenine, C represents cytosine, G represents guanine, and T represents thymine.

SEQ ID NO: 2 in the sequence listing shows the already known base sequence of rat synaptophysin gene. In this sequence listing, the nucleotide sequences from 721 to 1501 are shown in the direction from the 5'end to the 3'end. The base sequence of the present invention described in SEQ ID NO: 1 is the base number 7 of this known synaptophysin gene.
26 to 1486.

The homology between the nucleotide sequence of the rat synaptophysin gene obtained in the present invention and the known nucleotide sequence of the rat synaptophysin gene is shown in FIGS. The upper row shows the nucleotide sequence of the rat synaptophysin gene obtained in the present invention, and the lower row shows the nucleotide sequence of the known rat synaptophysin gene. * Indicates that both base sequences are homologous. A is adenine, C is cytosine,
G represents guanine, T represents thymine, and-indicates a portion in which the base is removed. When the two are compared, the following three bases are different, but the other three are completely the same.

Base (number) of SEQ ID NO: 1 Corresponding base (number) of SEQ ID NO: 2 G (64) A (789) C (224) T (949) T (297) C (1022) Thus, the above two DNA sequences are almost completely identical. That is, D prepared above
NA is the rat synaptophysin gene.

[0072]

INDUSTRIAL APPLICABILITY As described above, the present invention provides a monoclonal antibody that specifically binds to rat synaptophysin. By using this monoclonal antibody, it is possible to easily detect only rat synaptophysin from the protein group transferred to the nitrocellulose membrane by the indirect enzyme antibody method, the indirect fluorescent antibody, or the like.

Further, since synaptophysin serves as a good detection marker for neuroendocrine tumors, it becomes possible to detect neuroendocrine tumors. Therefore,
It is expected to be used for diagnosis in the clinical medicine field.

[Sequence Listing] SEQ ID NO: 1 Sequence length: 750 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: cDNA to mRNA Antisense: No Origin: Rattus norvegicus ( Organism Name) Method by which the characteristics were determined: S sequence 10 20 30 40 50 60 AGCACCTGGC GATGCCTACG GCGATGCGGG CTACGGGCAG GGCCCCGGAG GCTATGGGCC 70 80 90 100 110 120 CCAGGACTCC TACGGGCCTC AGGGTGGTTATCCAACCGAT TACGGGCAGC CAGCCAGCGG 130 140 150 160 170 180 TGCGGCGG. 220 230 240 GCCCACCTCC TTCTCCAATC AGATGTAATC TGGTCAGTGA AGTCCATGAA GATCCCACGG 250 260 270 280 290 300 GTGGGCAAGA GCTCAAGAGA AGGCCTGCCC CCCTTTTCCC ATCCCCATAT CCTAGGTCTC 310 320 330 340 350 360 CACCCCTCAA CCCAGGAGAC CCTAACTGTC TTTGCTGTTT ATATATATAT ATATTATATA 370 380 390 400 410 420 TAAATATCTA TTTATCTGTC TGAGCCCTAC ATTCACCCAC TTCTCCATGC ACTAGAGGCC 430 440 450 460 470 480 CAGTCCTG AA TGGGCTCCTC CCCAACCCTG ACCTTGCATT CCTCAGCCCC TATCTGTTCC 490 500 510 520 530 540 CCAGCCCTGT CCCTTGAGGT AAGGGGCTCT AGAAAGGGGA GAGGAAGGGA ACCAGACCTT 550 560 570 580 590 600 GGCTGCATGG AGTGGGTTGG TGTGACTTTC TCTCCTTCCT CCTCTCCCTC TGCCCCTCCT 610 620 630 640 650 660 AACTCTGGCC TTGGTCCTCC AGCATCACCT GAACTTCAGA AGCTCTCGAA TGGAAATCTG 670 680 690 700 710 720 ACCCCAAGAG TAGAGCAGTA GACTGAGTGG AGGAGGCTTG GGTGAAACGG GCAGAGAGGA 730 740 750 GATAACCTCT GTAGAGAGAG GACTAGTCAG SEQ ID NO: 2 Sequence length: 781 Sequence type: Nucleic acid Strand number: Single strand Topology: Linear sequence 721 726 726 736 746 756 766 776 GC786GATGCGACCACC GGCCCCGGAG GCTATGGGCC 796 806 816 826 836 846 CCAGGACTCC TACGGGCCTC AGGGTGGTTA TCAACCCGAT TACGGGCAGC CAGCCAGCGG 856 866 876 886 896 906 TGGCGGTGGC TACGGGCCTC AGGGCGACTA TGGGCAGCAA GGCTATGGCC AACAGGGTGC 916 926 936 946 956 966 GCCCACCTCC TTCTCCAATC AGATGTAATC TGGTCAGTGA AGTCCATGAA GATCCCACGG 976 986 996 1006 1016 1026 GTGGGCAAGA GCTCAAGAGA AGGCCTGCCC CCCTTTTCCC ATCCCCATAT CCTAGGTCTC 1036 1046 1056 1066 1076 1086 CACCCCTCAA CCCAGGAGAC CCTAACTGTC TTTGCTGTTT ATATATATAT ATATTATATA 1096 1106 1116 1126 1136 1146 TAAATATCTA TTTATCTGTC TGAGCCCTAC ATTCACCCAC TTCTCCATGC ACTAGAGGCC 1156 1166 1176 1186 1196 1206 CAGTCCTGAA TGGGCTCCTC CCCAACCCTG ACCTTGCATT CCTCAGCCCC TATCTGTTCC 1216 1226 1236 1246 1256 1266 CCAGCCCTGT CCCTTGAGGT AAGGGGCTCT AGAAAGGGGA GAGGAAGGGA ACCAGACCTT 1276 1286 1296 1306 1316 1326 GGCTGCATGG AGTGGGTTGG TGTGACTTTC TCTCCTTCCT CCTCTCCCTC TGCCCCTCCT 1336 1346 1356 1366 1376 1386 AACTCTGGCC TTGGTCCTCC AGCATCACCT GAACTTCAGA AGCTCTCGAA TGGAAATCTG 1396 1406 1416 1426 1436 1446 ACCCCAAGAG TAGAGCAGTA GACTGAGTGG AGGAGGCTTG GGTGAAACGG GCAGAGAGGA 1456 1466 1476 1486 1496 GATAACCTCT GTAGAGAGAG GACTAGTCAG CCAAGAGTTG AATTCCAGAC ATACT

[Brief description of drawings]

FIG. 1 SDS-PAGE development pattern of rat brain homogenate and monoclonal antibody RB2-4.
It is a photograph showing the specificity of.

FIG. 2 is a diagram showing the homology between the nucleotide sequence of the rat synaptophysin gene obtained in the present invention and the known nucleotide sequence of the rat synaptophysin gene.

FIG. 3 is a view showing the homology between the nucleotide sequence of the rat synaptophysin gene obtained in the present invention and the known nucleotide sequence of the rat synaptophysin gene.

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[Procedure amendment]

[Submission date] June 3, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Figure 1

[Correction method] Change

[Correction content]

FIG. 1 SDS-PAGE development pattern of rat brain homogenate and monoclonal antibody RB2-4.
It is a figure which shows the electrophoresis which showed the specificity of this by the immunoblotting method .

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location // A61B 10/00 T A61K 39/395 N 9284-4C G01N 33/53 D 8310-2J 33 / 577 B 9015-2J (C12P 21/08 C12R 1:91)

Claims (4)

[Claims] 1. A monoclonal antibody produced by using an extract of rat brain as an immunogen, the immunoglobulin class of which belongs to IgM, and rat synaptophysin or P.
A monoclonal antibody having a specific reactivity to -38. 2. The monoclonal antibody according to claim 1, which has specific reactivity with a protein expressed by a gene having the nucleotide sequence set forth in SEQ ID NO: 1 in the Sequence Listing. 3. A medicinal composition containing the monoclonal antibody according to claim 1 and a pharmacologically acceptable carrier or diluent. 4. A hybridoma producing a monoclonal antibody specific to synaptophysin according to claim 1, which has been deposited with the Institute of Microbial Science and Technology, under the name of Microtechnology Research Institute No. 3944.