JPH11332559A - Hybridoma - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new hybridoma free from immuno-cross-reactivity having high titer useful for diagnosis or the like of nervous tissue by producing a monoclonal antibody capable of recognizing all the isoforms of a specific clathrin assembly-protein 3 as the antigen. SOLUTION: The hybridoma produces the monoclonal antibody capable of recognizing all isoforms of the clathrin assembly-protein 3 including the amino acid sequence of formula I as the antigen, the hybridoma having the depositing number of FERMBP-6379, and the produced monoclonal antibody manifests titer of 2 to 4 times of that of the monoclonal antibody having the same antigen-recognition specificity, produced by the hybridoma R-18 cultured in the spume condition. This monoclonal antibody is capable of recognizing the 388 to 394 position of the amino acid sequence of the clathrin assembly- protein 3 described in formula II, and preferably labeled by protein, fluorescent dye or radio isotope.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a monoclonal antibody recognizing clathrin assembly protein 3 as an antigen and a hybridoma producing the monoclonal antibody.

[0002]

2. Description of the Related Art Clathrin assembly protein 3
(SEQ ID NO: 1 in the sequence listing) (hereinafter AP-3) is localized at synapses and is responsible for recycling of synaptic vesicles, thereby having a molecular weight of about 180 involved in high-speed signal transmission of nerve cells.
Kd is an acidic protein. There are at least three isoforms of AP-3 that are generated by alternative splicing during brain development. Since AP-3 is a molecule that binds to synaptic vesicles, it can be used to detect synapse formation during nerve transplantation and to detect neurosecretory cell tumors, which are tumors characterized by retaining synaptic vesicles It is. To use for these detections, AP
Monoclonal antibody recognizing -3 has already been used in a Rafer (Lafer; MabF1-20; Neuroscience 34 (2) 403-409 (199
0)), Ungewickell (MabAP180-I; Th
e EMBO Journal 5 (12) 3143-3149 (1986)), Keane
en ； CLAP3; The Journal of Cell Biology 102 1325-13
33 (1986)), Paskin (Puszkin; 8G8; The Journal o)
f Biological Chemistry 263 (15) 7418-7425 (1988)),
Sugano (Sugano; MabR-18; Neuroscience Research Comm
unications 15 (3) 177-185 (1994)).

However, a known antibody against AP-3, Ma
bF1-20, MabAP180-I, CLAP3 and 8G8 had problems with immunological cross-reactivity and the problem that only some isoforms could be recognized. In addition, the antibody MabR-18, which has no immunological cross-reactivity and recognizes all isoforms, cannot be said to have sufficient titer and cannot be used for ordinary research and clinical tests unless the monoclonal antibody is purified and concentrated. There was a problem.

[0004]

DISCLOSURE OF THE INVENTION The present invention has been made to solve the above problems, and can specifically recognize all the isoforms of AP-3 and have a high titer. An object of the present invention is to provide an antibody that can easily detect all types of AP-3 by cloning a hybridoma capable of producing an anti-AP-3 antibody having the following. It is a further object of the present invention to provide a method for simultaneously detecting and / or quantifying all isoforms of AP-3 using such an anti-AP-3 antibody.

[0005]

Means for Solving the Problems The present inventors subcloned a hybridoma R-18 producing a monoclonal antibody MabR-18 capable of recognizing all the isoforms of AP-3 as antigens, under the same conditions. Cultured R-
A sequence that is 2 to 4 times as potent as MabR-18 produced by 18 and is shared by all the isoforms of AP-3 (hereinafter, a common sequence)
A hybridoma characterized by producing a monoclonal antibody capable of recognizing "Ala Leu SerSer Val Pro Ser" (SEQ ID NO: 2 in the sequence listing) was prepared.

[0006] By using the monoclonal antibody of the present invention, AP-3 containing a consensus sequence present in a sample can be obtained.
A method for simultaneously detecting and / or quantifying all isoforms of (a), wherein (1) contacting the AP-3 present in the sample with the monoclonal antibody to thereby obtain all of the AP-3 There is provided a method comprising the steps of: forming an immune complex between an isoform and the monoclonal antibody; and (2) detecting and / or quantifying the formed immune complex.

[0007]

DETAILED DESCRIPTION OF THE INVENTION The hybridoma of the present invention is AP-3.
Was obtained by subcloning a hybridoma R-18 producing a monoclonal antibody MabR-18 capable of recognizing all the isoforms as antigens.

The hybridoma of the present invention was produced on May 25, 1998, 1-3-1 Higashi, Tsukuba, Ibaraki, Japan based on the provisions of the Budapest Treaty on the International Recognition of Deposit of Microorganisms for Patent Procedure. Deposited internationally with the Institute of Biotechnology and Industrial Technology, Faculty of Engineering, FERM B as a deposit number
P-6379 was provided.

The outline of subcloning of hybridoma R-18 is as follows. After culturing R-18 in a normal hybridoma growth medium for several days, the hybridoma is recovered from the culture solution, separated one by one, and cultured in a hybridoma selection medium. After several days, clones that have formed colonies are selected, the titer of the monoclonal antibody produced in the culture solution is measured, and the subclone that produces the antibody with the highest titer is selected.

[0010] Here, the titer is a relative index of the antibody content in the sample, and is an index expressed by the maximum dilution factor showing the minimum effective activity. Therefore, it can be assumed that the higher the antibody titer in a sample, the higher the antibody content of the sample.

The hybridoma R-18 used for subcloning was established in a research and development center of Toshiba Corporation, and then stored in liquid nitrogen for about 6 years while passing through timely. Since the monoclonality of the hybridoma has been confirmed at the time of establishment, if a hybridoma having an altered antibody-producing ability is found by subcloning, the hybridoma has been generated by mutation. Usually, since the antibody production ability is rather reduced by the mutation, it is extremely rare to find a subclone having an increased antibody production ability as described above and establish it.

According to the present invention, there is provided a reagent for simultaneously detecting and / or quantifying all isoforms of clathrin assembly protein 3 containing a consensus sequence, comprising a monoclonal antibody (MabR) produced by the hybridoma of the present invention. -18 _a12 ) is provided.

The reagent is an ELISA (enzyme-linked immuno
sorbent assay), radioimmunoassay, western blotting, immunohistochemistry, immunocytochemistry, immunofluorescence, and any immunological method known per se, including immunoelectron microscopy. it can.

When the monoclonal antibody of the present invention is used to simultaneously detect and / or quantify all isoforms of AP-3, a protein, a fluorescent dye, or a radioisotope is directly bound on the monoclonal antibody of the present invention. can do. The proteins include, but are not limited to, enzymes such as phosphatase and peroxidase, chromoproteins such as phycoerythrin, ferritin and the like. Examples of the fluorescent dye include, but are not limited to, fluorescein derivatives such as fluorescein isothiocyanate and rhodamine derivatives such as tetramethylrhodamine isothiocyanate. Radioisotopes include, but are not limited to, radioisotopes commonly used in the biological domain, such as ³ H, ¹⁴ C, ³⁵ S, ¹²⁵ I and ¹³¹ I. When administering the monoclonal antibody of the present invention to the body, it is particularly preferable to label it with ¹³¹ I.

When an enzyme such as phosphatase or peroxidase is used as a labeling substance, detection and / or quantification can be carried out using any substance for detection and / or quantification (substrate of the enzyme) which is converted into a coloring substance by the enzyme. Alternatively, it can be quantified, and such detection and / or quantification materials are well known to those skilled in the art.

[0016] The monoclonal antibody of the reagent MabR-18 _a12
In If is not directly labeled, the protein, using a labeled secondary antibody with a fluorescent dye or a radioactive isotope, for detecting and / or quantifying the immune complexes between the monoclonal antibody MABR-18 _a12 and AP-3 be able to. The proteins, fluorescent dyes and radioisotopes used to label the secondary antibody are well known to those of skill in the art, and typically include proteins, fluorescent dyes and radiolabels described herein above for labeling the monoclonal antibodies of the invention. Radioisotopes may be included.

By using the reagent of the present invention, all the isoforms of AP-3 contained in a biological sample can be detected and / or quantified according to the following method. That is, this method uses the monoclonal antibody Ma of the present invention.
A method for simultaneously detecting and / or quantifying all isoforms of AP-3 containing a consensus sequence present in a sample by bR- _18a12 , comprising: (1) presenting in the sample Contacting the AP-3 with the monoclonal antibody to form an immune complex between all the isoforms of the AP-3 and the monoclonal antibody; (2) forming the immune complex Detecting and / or quantifying.

Therefore, the method includes ELISA (enzyme-linke
d immunosorbent assay), radioimmunoassay, Western blotting, immunohistochemistry, immunocytochemistry, immunofluorescence, and immunological methods known per se, including immunoelectron microscopy.

Samples that can be used in the method of the present invention include blood, body fluids including cerebrospinal fluid, and biopsy tissues. In order to detect and / or quantify the isoform of AP-3, the monoclonal antibody Ma used in the present method is used.
bR-18 _a12 is a protein described herein above,
Labeling can be done directly with fluorescent dyes or radioisotopes.

[0020] When the monoclonal antibody MABR-18 _a12 is not labeled directly, using a secondary antibody as described above,
It can be detected and / or quantified immune complex between the monoclonal antibody MABR-18 _a12 and AP-3. In the following examples, the method for producing the hybridoma of the present invention and the AP recognized by the antibody produced by the hybridoma
The -3 epitope will be described in detail.

[0021]

EXAMPLES Example 1 Method for Producing Hybridoma of the Present Invention First, hybridoma R-18 was RPMI containing 20% fetal bovine serum.
After culturing in 1640 medium for 3 days and collecting cells by centrifugation, HAT medium (hypoxanthine, aminopterin,
(Thymidine-containing) and the number of cells was counted. The cells were then seeded by limiting dilution in a 96-well microculture plate at approximately 1 cell / well. In about 7 days, the formation of colonies was confirmed in about 50% of the wells, and 100 μL of the culture supernatant was collected from the subclones that formed the colonies. The titer of the antibody produced by the collected subclone was measured as follows by an ELISA method using a rat brain homogenate as an antigen. First, an ELISA plate was coated with a rat brain homogenate, and blocking was performed at room temperature using an appropriate blocking agent such as Block Ace. continue
The plate was washed with a PBS buffer for 10 minutes, and the culture supernatant was reacted as a primary antibody at room temperature for 1 hour. After washing four times for 10 minutes with a PBS buffer, goat anti-mouse IgG labeled with horseradish peroxidase was appropriately diluted and reacted as a secondary antibody. After four washes with PBS buffer for 10 minutes, color was developed with a color developing solution containing ABTS and hydrogen peroxide solution, and the titer of the antibody produced by the hybridoma was measured using a microplate reader (FIG. 1). ). In FIG. 1, the titer of the antibody produced by each subclone of R-18 is represented by an ELISA value.
From each subclone, the subclone producing the highest titer of the antibody was selected, cultured in large quantities, and established as hybridoma R- _18a12 . As shown in FIG. 1, the monoclonal antibody Ma produced by the hybridoma
bR-18 titer of _a12 the titer of the average value of the monoclonal antibody other subclones produced (about 0.2 in ELISA value)
It is apparent that it has a significantly higher titer, which is about 3.5 times higher.

Hybridoma R- _18a12 was obtained on May 25, 1998, 1-3-1 Higashi, Tsukuba, Ibaraki, Japan, based on the provisions of the Budapest Treaty on the International Recognition of Deposit of Microorganisms for Patent Procedure. Deposited internationally with the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (FERM BP)
-6379 was given.

Example 2 Monoclonal antibody MabR-18
Determination of AP-3 Epitope Recognized by _a12 First, a large number of peptides consisting of consecutive 10 amino acids in the amino acid sequence of the middle part of AP-3 (SEQ ID NO: 3 in the sequence listing) were synthesized by ordinary chemical synthesis. . These peptides are synthesized every three amino acids from the N-terminus of AP-3, and cover the entire intermediate region of AP-3 as a whole.
Attach the synthesized peptide to the tip of a resin pin shaped to fit a commercially available 96-well microplate,
By ELISA consisting procedure described in Example 1, it was measured the reactivity of the monoclonal antibody MabR-18 _a12 (Figure 2). The most remarkable area was AP-3
Corresponds to positions 383 to 401 (SEQ ID NO: 4 in the sequence listing),
The region includes a consensus sequence. Therefore, a peptide consisting of consecutive 8 amino acids in the region was synthesized by ordinary chemical synthesis, and the reactivity with the synthesized peptide was further measured (FIG. 3). These peptides were synthesized so as to cover the entire region of the region while being shifted by one amino acid from the N-terminus of the region. From Figure 3, epitope of AP-3, which is recognized by the monoclonal antibody MABR-18 _a12 is clear that corresponding to the consensus sequence, the monoclonal antibody MABR-18 _a12
Was shown to bind to all isoforms of AP-3 by binding to the sequence.

[0024]

As is evident from the foregoing description, according to the present invention, recognizing the AP-3 of all types as antigen, and a hybridoma which highly producing monoclonal antibodies MABR-18 _a12 and the monoclonal antibodies with high titer R-18a12 is provided. By using the antibody or a modified product of the antibody, it becomes possible to diagnose a transplanted nerve tissue or a neurosecretory cell tumor. Hybridoma R-18 _a12 of the present invention,
Since producing monoclonal antibodies MABR-18 _a12 having high titers, the use of the antibody, it becomes unnecessary concentration or purification, it can be performed such diagnostic conveniently. Further increase in detection sensitivity significantly increases the accuracy of diagnosis and reduces the false negative rate.

[0025]

[Sequence List] SEQ ID NO (SEQ ID NO): 1 Sequence length: 895 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Met Ser Gly Gln Thr Leu Thr Asp Arg Ile Ala Ala Ala Gln Tyr Ser 1 5 10 15 Val Thr Gly Ser Ala Val Ala Arg Ala Val Cys Lys Ala Thr Thr His 20 25 30 Glu Val Met Gly Pro Lys Lys Lys His Leu Asp Tyr Leu Ile Gln Ala 35 40 45 Thr Asn Glu Thr Asn Val Asn Ile Pro Gln Met Ala Asp Thr Leu Phe 50 55 60 Glu Arg Ala Thr Asn Ser Ser Trp Val Val Val Phe Lys Ala Leu Val 65 70 75 80 Thr Thr His His Leu Met Val His Gly Asn Glu Arg Phe Ile Gln Tyr 85 90 95 Leu Ala Ser Arg Asn Thr Leu Phe Asn Leu Ser Asn Phe Leu Asp Lys 100 105 110 Ser Gly Ser His Gly Tyr Asp Met Ser Thr Phe Ile Arg Arg Tyr Ser 115 120 125 Arg Tyr Leu Asn Glu Lys Ala Phe Ser Tyr Arg Gln Met Ala Phe Asp 130 135 140 Phe Ala Arg Val Lys Lys Gly Ala Asp Gly Val Met Arg Thr Met Val 145 150 155 160 Pro Glu Lys Leu Leu Lys Ser Met Pro Ile Leu Gln Gly Gln Il e Asp 165 170 175 Ala Leu Leu Glu Phe Asp Val His Pro Asn Glu Leu Thr Asn Gly Val 180 185 190 Ile Asn Ala Ala Phe Met Leu Leu Phe Lys Asp Leu Ile Lys Leu Phe 195 200 205 Ala Cys Tyr Asn Asp Gly Val Ile Asn Leu Leu Glu Lys Phe Phe Glu 210 215 220 Met Lys Lys Gly Gln Cys Lys Asp Ala Leu Glu Ile Tyr Lys Arg Phe 225 230 235 240 Leu Thr Arg Met Thr Arg Val Ser Glu Phe Leu Lys Val Ala Asp Glu 245 250 255 Val Gly Ile Asp Lys Gly Asp Ile Pro Asp Leu Thr Gln Ala Pro Ser 260 265 270 Ser Leu Met Glu Thr Leu Glu Gln His Leu Asn Thr Leu Glu Gly Lys 275 280 285 Lys Pro Gly Asn Asn Glu Gly Ser Gly Ala Pro Ser Pro Leu Ser Lys 290 295 300 Ser Ser Pro Ala Thr Thr Val Val Ser Pro Asn Ser Thr Pro Ala Lys 305 310 315 320 Thr Ile Asp Thr Ser Pro Pro Val Asp Ile Phe Ala Thr Ala Ser Ala 325 330 335 Ala Ala Pro Val Ser Ser Ala Lys Pro Ser Ser Asp Leu Leu Asp Leu 340 345 350 Gln Pro Asp Phe Ser Gly Ala Arg Ala Gly Ala Ala Ala Pro Val Pro 355 360 365 365 Pro Pro Thr Gly Gly Ala Thr Ala Trp Gly Asp Leu Leu Gly Glu A sp 370 375 380 Ser Leu Ala Ala Leu Ser Ser Val Pro Ser Glu Ala Pro Ile Ser Asp 385 390 395 400 Pro Phe Ala Pro Glu Pro Ser Pro Pro Thr Thr Thr Thr Glu Pro Ala 405 410 415 Ser Ala Ser Ala Ser Ala Thr Thr Ala Val Thr Ala Ala Thr Thr Glu 420 425 430 Val Asp Leu Phe Gly Asp Ala Phe Ala Ala Ser Pro Gly Glu Ala Pro 435 440 445 Ala Ala Serla Glu Gly Ala Thr Ala Pro Ala Thr Pro Ala Pro Val Ala 450 455 460 Ala Ala Leu Asp Ala Cys Ser Gly Asn Asp Pro Phe Ala Pro Ser Glu 465 470 475 480 Gly Ser Ala Glu Ala Ala Pro Glu Leu Asp Leu Phe Ala Met Lys Pro 485 490 495 Pro Glu Thr Ser Ala Pro Val Val Thr Pro Thr Ala Ser Thr Ala Pro 500 505 510 Pro Val Pro Ala Thr Ala Pro Ser Pro Ala Pro Thr Ala Val Ala Ala 515 520 525 Thr Ala Ala Thr Thr Thr Ala Ala Ala Ala Ala Thr Thr Thr Thr Ala Thr 530 535 540 Thr Ser Ala Ala Ala Ala Thr Thr Ala Ala Ala Pro Pro Ala Leu Asp 545 550 555 560 Ile Phe Gly Asp Leu Phe Asp Ser Ala Pro Glu Val Ala Ala Ala Ser 565 570 575 Lys Pro Asp Val Ala Pro Ser Ile Asp Leu Phe Gly Thr Asp Ala P he 580 585 590 Ser Ser Pro Pro Arg Gly Ala Ser Pro Val Pro Glu Ser Ser Leu Thr 595 600 605 Ala Asp Leu Leu Ser Val Asp Ala Phe Ala Ala Pro Ser Pro Ala Ser 610 615 620 620 Thr Ala Ser Pro Ala Lys Ala Glu Ser Ser Gly Val Ile Asp Leu Phe 625 630 635 640 640 Gly Asp Ala Phe Gly Ser Ser Ala Ser Glu Thr Gln Pro Ala Pro Gln 645 650 655 Ala Val Ser Ser Ser Ser Ala Ser Ala Asp Leu Leu Ala Gly Phe Gly 660 665 670 Gly Ser Phe Met Ala Pro Ser Thr Thr Pro Val Thr Pro Ala Gln Asn 675 680 685 Asn Leu Leu Gln Pro Asn Phe Glu Ala Ala Phe Gly Thr Thr Pro Ser 690 695 700 Thr Ser Ser Ser Ser Ser Ser Phe Asp Pro Ser Gly Asp Leu Leu Met Pro 705 710 715 715 720 Thr Met Ala Pro Ser Gly Gln Pro Ala Pro Val Ser Met Val Pro Pro 725 730 735 Ser Pro Ala Met Ser Ala Ser Lys Gly Leu Gly Ser Asp Leu Asp Ser 740 745 750 Ser Leu Ala Ser Leu Val Gly Asn Leu Gly Ile Ser Gly Thr Thr Ser 755 760 765 Lys Lys Gly Asp Leu Gln Trp Asn Ala Gly Glu Lys Lys Leu Thr Gly 770 775 780 780 Gly Ala Asn Trp Gln Pro Lys Val Thr Pro Ala Thr Trp Ser Ala Gly785 790 795 800 Val Pro Pro Gln Gly Thr Val Pro Pro Thr Ser Ser Val Pro Pro Gly 805 810 815 Ala Gly Ala Pro Ser Val Gly Gln Pro Gly Ala Gly Tyr Gly Met Pro 820 825 830 Pro Ala Gly Thr Gly Met Thr Met Met Pro Gln Gln Pro Val Met Phe 835 840 845 Ala Gln Pro Met Met Arg Pro Pro Phe Gly Ala Ala Ala Val Pro Gly 850 855 860 Thr Gln Leu Ser Pro Ser Pro Thr Pro Ala Thr Gln Ser Pro Lys Lys 865 870 875 880 Pro Pro Ala Lys Asp Pro Leu Ala Asp Leu Asn Ile Lys Asp Phe 885 890 895 Sequence number (SEQ ID NO): 2 Sequence length: 7 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type : Intermediate fragment Sequence: Ala Leu Ser Ser Val Pro Ser 15 Sequence number (SEQ ID NO): 3 Sequence length: 278 Sequence type: Amino acid Topology: Linear Sequence type: Protein Fragment type: Intermediate portion Fragment sequence: Val Gly Ile Asp Lys Gly Asp Ile Pro Asp Leu Thr Gln Ala Pro Ser 1 5 10 15 Ser Leu Met Glu Thr Leu Glu Gln His Leu Asn Thr Leu Glu Gly Lys 20 25 30 Lys Pro Gly Asn Asn Glu Gly Ser Gly Ala Pro Ser Pro Leu Ser Lys 35 40 45 Ser Ser Pro Ala Thr Thr Val Thr Ser Pro Asn Ser Thr Pro Ala Lys 50 55 60 Thr Ile Asp Thr Ser Pro Pro Val Asp Ile Phe Ala Thr Ala Ser Ala 65 70 75 80 Ala Ala Pro Val Ser Ser Ala Lys Pro Ser Ser Asp Leu Leu Asp Leu 85 90 95 Gln Pro Asp Phe Ser Gly Ala Arg Ala Gly Ala Ala Ala Pro Val Pro 100 105 110 Pro Pro Thr Gly Gly Ala Thr Ala Trp Gly Asp Leu Leu Gly Glu Asp 115 120 125 Ser Leu Ala Ala Leu Ser Ser Val Pro Ser Glu Ala Pro Ile Ser Asp 130 135 140 Pro Phe Ala Pro Glu Pro Ser Pro Pro Thr Thr Thr Thr Glu Pro Ala 145 150 155 160 Ser Ala Ser Ala Ser Ala Thr Thr Ala Val Thr Ala Ala Thr Thr Glu 165 170 175 Val Asp Leu Phe Gly Asp Ala Phe Ala Ala Ser Pro Gly Glu Ala Pro 180 185 190 Ala Ala Ser Glu Gly Ala Thr Ala Pro Ala Thr Pro Ala Pro Val Ala 195 200 205 Ala Ala Leu Asp Ala Cys Ser Gly Asn Asp Pro Phe Ala Pro Ser Glu 210 2 15 220 Gly Ser Ala Glu Ala Ala Pro Glu Leu Asp Leu Phe Ala Met Lys Pro 225 230 235 240 Pro Glu Thr Ser Ala Pro Val Val Thr Pro Thr Ala Ser Thr Ala Pro 245 250 255 Pro Val Pro Ala Thr Ala Pro Ser Pro Ala Pro Thr Ala Val Ala Ala 260 265 270 Thr Ala Ala Thr Thr Thr 275 SEQ ID NO (SEQ ID NO): 4 Sequence length 19 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate Fragment sequence: Glu Asp Ser Leu Ala Ala Leu Ser Ser Val Pro Ser Glu Ala Pro Ile 1 5 10 15 Ser Asp Pro

[Brief description of the drawings]

FIG. 1 is a diagram comparing the titers of antibodies produced by each subclone of hybridoma R-18 producing monoclonal antibody MabR-18.

FIG. 2 shows an amino acid sequence to which the monoclonal antibody of the present invention specifically binds.

FIG. 3 is a view showing an amino acid sequence to which the monoclonal antibody according to the present invention specifically binds, which sequence is commonly contained in the isoform of AP-3.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI G01N 33/577 C12N 15/00 ZNAC // (C12N 5/10 C12R 1:91) (C12P 21/08 C12R 1:91)

Claims (8)

[Claims] 1. A hybridoma having an accession number of FERM BP-6379. 2. A hybridoma producing a monoclonal antibody capable of recognizing as an antigen all isoforms of clathrin assembly protein 3 having the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing, wherein the produced monoclonal antibody The antibody titer is 2-4 of the titer of a monoclonal antibody having the same antigen recognition specificity produced by hybridoma R-18 cultured under the same conditions.
A hybridoma, characterized in that it is doubled. 3. A monoclonal antibody produced by the hybridoma according to claim 1 or 2. 4. The amino acid sequence of 388 to 3 of the amino acid sequence of clathrin assembly protein 3 shown in SEQ ID NO: 1 in the sequence listing.
A monoclonal antibody capable of recognizing position 394. 5. A monoclonal antibody capable of recognizing as an antigen all isoforms of clathrin assembly protein 3 having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. 6. The monoclonal antibody according to claim 3, which is labeled with a protein, a fluorescent dye, or a radioisotope. 7. A reagent comprising the monoclonal antibody according to any one of claims 3 to 6, wherein the clathrin assembly protein 3 comprises the amino acid sequence of SEQ ID NO: 2 in the sequence listing. For simultaneous detection and / or quantification of isoforms of 8. A clathrin assembly protein 3 containing the amino acid sequence represented by SEQ ID NO: 2 in a sample, which is present in a sample using the monoclonal antibody according to any one of claims 3 to 6. (1) contacting the clathrin assembly protein 3 present in the sample with the monoclonal antibody to thereby detect and / or quantify all the isoforms of the protein A method comprising the steps of: forming an immune complex between all the isoforms and the monoclonal antibody; and (2) detecting and / or quantifying the formed immune complex.