JPH06125784A - Monoclonal antibody, hybridoma, its production and use - Google Patents

Abstract

PURPOSE:To obtain a monoclonal antibody capable of detecting and determining various non-ceptor type tyrosine kinases. CONSTITUTION:A monoclonal antibody shows affinity to a specific region of non-receptor type tyrosine kinase. Since the monoclonal antibody is specifically bonded to non-receptor type tyrosine kinase and has high bonding ability, the non-receptor type tyrosine kinase is advantageously useful as a reagent for detecting and measuring various non-receptor type tyrosine kinases. Further, by measuring the non-receptor type tyrosine kinase in an organism organ, tissue or cell, the monoclonal antibody is expected to be useful for diagnosing diseases caused by incontinence of information transmission mechanism in cell resulting from the protein such as cancer.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a monoclonal antibody capable of binding to a specific region of a non-receptor tyrosine kinase, a method for producing the same and use thereof. Further, the present invention provides a hybridoma producing the monoclonal antibody.

[0002]

2. Description of the Related Art Information for a physiologically active substance to exert its functions such as proliferation, differentiation and maintenance of homeostasis of individual cells constituting a living body is supplied to specific receptors on the cell membrane. When the ligand corresponding to the receptor binds, the first signal is transmitted to the cell. As one of the transfer systems, a reaction system involving a tyrosine residue-specific protein kinase (hereinafter sometimes referred to as tyrosine kinase) is known. The gene encoding tyrosine kinase is epidermal growth factor (Epid
ermal growth factor (EGF) receptor gene typified by a receptor gene and non-receptor type gene typified by an oncogene src. Non-receptor tyrosine kinase genes are src, yes, lyn, fgr, f
src genes such as yn, lck, hck, blk and other abs
It is divided into l gene group and fps / fes gene group, and their gene products all have tyrosine kinase activity. Receptor-type tyrosine kinase exists across the cell membrane, and it is understood that when a ligand binds to a domain outside the cell membrane, tyrosine kinase activity in the domain inside the cell membrane is enhanced, and cell proliferation information is transmitted inside the cell. There is. However, the non-receptor tyrosine kinase does not have the extracellular domain and transmembrane domain, but has only the intracellular domain, and this portion exists by being bound to the cell membrane by myristic acid. Therefore, it was unclear what kind of information was specifically transmitted. Recently, a non-receptor tyrosine kinase, p56 ^lck, is present on CD4 or CD8 present on T lymphocytes [Cell, 55, 301 (198
8)], and also p53 ^lyn and p56 ^lyn with IgM present on B lymphocytes [Science, 251, 192 (199
1)] was shown to form a complex, CD4 / CD
8. It has been assumed that the non-receptor tyrosine kinase plays an important role in signal transduction through cell surface IgM. The non-receptor tyrosine kinase is divided into a specific region, a regulatory region, a kinase region and a regulatory region from its amino terminus. Of these, the specific region is a sequence unique to each product, and therefore it is presumed that it may be necessary for interaction with a molecule unique to each product.
In fact, p56 ^lck is said to bind to the CD4 molecule through this region.

[0003]

Therefore, if a monoclonal antibody capable of binding to a specific region of a non-receptor tyrosine kinase can be produced, the distribution of expression of various tyrosine kinases in cells and tissues and the expression amount thereof can be known. At the same time, it is expected to further clarify the roles of various tyrosine kinases.

[0004]

[Means for Solving the Problems] In view of the above circumstances, the present inventors have conducted extensive studies and as a result, as a result of using a partial peptide of a non-receptor tyrosine kinase or a specific region thereof as an immunogen,
We have succeeded in producing a monoclonal antibody having a high binding ability to the tyrosine kinase. When the monoclonal antibody is subjected to immunohistochemical or immunochemical assay, the tyrosine kinase can be detected and quantified with high sensitivity, and the tyrosine kinase can be efficiently purified by using the monoclonal antibody. As a result of further research based on these findings, the present invention has been completed.
That is, the present invention provides: (1) a monoclonal antibody whose immunoglobulin class belongs to IgG and exhibits binding to a specific region of a non-receptor tyrosine kinase; (2) immunization with a non-receptor tyrosine kinase or a partial peptide of the specific region. A cloned hybridoma consisting of a spleen cell of the mammal and a lymphoid cell of the same or different species; (3) a spleen cell of a mammal immunized with a non-receptor tyrosine kinase or a partial peptide of its specific region; A method for producing the hybridoma according to (2) above, which comprises cell fusion with allogeneic or xenogeneic lymphoid cells and cloning; (4) the hybridoma according to (2) above in a liquid medium or in a mammal. Propagated in the abdominal cavity to produce and accumulate a monoclonal antibody, which is then collected, and described in (1) above. (5) A method for detecting and quantifying a non-receptor tyrosine kinase, which comprises using the monoclonal antibody according to (1) above, and (6) using the monoclonal antibody according to (1) above. Is a method for purifying a non-receptor type tyrosine kinase.

The non-receptor tyrosine kinase may be any of warm-blooded animals (eg, mammals, birds), and their partial peptides and muteins (mutes).
ein). Non-receptor tyrosine kinase gene clusters include src, yes, lyn, fgr, fyn, lck, hck,
src genes such as blk and other abl genes, fps
They are divided into the / fes gene group, and their gene products all have tyrosine kinase activity. As a specific example thereof, the src gene product (sometimes referred to as Src) is avian Src [T. Takeya et al., Cell 32: 881 (1
The amino acid sequence is shown in Fig. 3 of 983)], but as a yes gene product (sometimes referred to as Yes), human Y
es [J. Sukegawa et al., Mol. Cell. Biol. 7:41 (198
The amino acid sequence is shown in Fig. 2 of 7)], but human Ly is used as the lyn gene product (sometimes referred to as Lyn).
n [Y. Yamanashi et al., Mol. Cell. Biol. 7: 237 (198).
7)] is the fgr gene product (sometimes called Fgr)
As human Fgr [K. Inoue et al., Oncogene 1: 301
(1987)], but as a fyn gene product (sometimes referred to as Fyn), human Fyn [K. Semba et al., Proc. Natl. Aca
d. Sci. 83: 5459 (1986)] is a mouse Lck [J.
D. Marth et al., Cell 43: 393 lsk ^T in FIG. 6 (1985)
The amino acid sequence is shown as the gene product], but h
Human Hck [SF Ziegler et al., Mol. Cell. Biol. 7: 2] is used as the ck gene product (sometimes referred to as Hck).
The amino acid sequence is shown in Figure 1 of 276 (1987)].
However, as a blk gene product (sometimes referred to as Blk), mouse Blk [SM Dymecki et al., Science 24
7: 332 (1990), the amino acid sequence is shown in FIG. 1]. To obtain a non-receptor type tyrosine kinase, an expression type vector containing a DNA having a nucleotide sequence encoding a polypeptide of the tyrosine kinase is prepared and introduced into an appropriate host cell to produce the tyrosine kinase. Good. For example, as an expression vector for obtaining human Lyn, Fgr, Fyn, (a) RNA encoding Lyn, Fgr or Fyn is isolated, and (b) a single-stranded complementary DNA (cDNA) from the RNA.
Then, double-stranded DNA is synthesized, (c) the complementary DNA is incorporated into a plasmid, (d) the host is transformed with the obtained recombinant plasmid, and (e) after culturing the obtained transformant, A plasmid containing the target DNA is isolated from the transformant by a suitable method, for example, a colony hybridization method using a DNA probe, and (f) the target cloned DNA is excised from the plasmid,
(G) The cloned DNA can be produced by ligating the cloned DNA downstream of the promoter in the vehicle.

Lyn-encoding RNA is B lymphocyte,
RNA that encodes macrophages and Fgr is RNA that encodes macrophages, granulocyte leukocytes, and Fyn.
It can be obtained from T lymphocytes, platelets, etc., respectively.
The expression vector thus obtained is incorporated into an appropriate host (eg, Escherichia coli, Bacillus subtilis, yeast, animal cell),
Human Lyn, Fgr, and Fyn can be produced by culturing the obtained transformant in a medium. The human Lyn, Fgr, and Fyn proteins also include muteins in which some of them are mutated or deleted. The mutein may be one in which a part of the specific region is mutated or deleted. Specific regions of non-receptor tyrosine kinases include amino acid sequences 1 to 84 in the case of human Src, amino acid sequences 1 to 92 in the case of human Yes, and amino acid sequences 1 to 67 in the case of human Lyn. The amino acid sequence of is 1 to 77 in the case of human Fgr, the amino acid sequence of 1 to 82 in the case of human Fyn, and the amino acid sequence 1 to 65 in the case of mouse Lck, In the case of human Hck, the 1st to 61st amino acid sequences are included, and in the case of mouse Blk, the 1st to 56th amino acid sequences are included. As the partial peptide of the specific region of the non-receptor tyrosine kinase as an immunogen, any peptide consisting of any amino acid sequence can be used as long as a monoclonal antibody that specifically binds to the non-receptor tyrosine kinase can be obtained. Although it is good, a peptide consisting of 10 to 100 is preferable, and a peptide consisting of 10 to 25 is more preferable. The partial peptide used in the present invention can be produced by a known conventional method for peptide synthesis. And it may be either a solid phase method or a liquid phase method. Examples of such peptide synthesis methods include, for example, "The Peptides (Th
e Peptides) ”, Volume 1 (1966), Schroder and Lubke
Authors, Academic Press, New York, USA and “Peptide Synthesis”, Izumiya et al., Maruzen Co., Ltd. (1975). Further, the partial peptide may be produced by cleaving the non-receptor type tyrosine kinase with an appropriate enzyme. Examples of the method include the method described on pages 255 to 332 of “Biochemistry Experiment Course 1 Protein Chemistry II”, edited by The Biochemical Society of Japan, Tokyo Kagaku Dojin (1976).

To produce the monoclonal antibody of the present invention, a mammal is immunized with a non-receptor tyrosine kinase or a partial peptide of its specific region, and the resulting spleen cells are fused with mammalian lymphoid cells. , Then cloned. When immunizing a non-receptor tyrosine kinase or a peptide in its specific region, the peptide may be complexed with a carrier protein and then used for immunization. In particular, peptides that are considered to be weakly immunogenic are preferably immunized with a complex with a carrier protein. As the carrier protein,
For example, bovine serum albumin, bovine thyroglobulin,
Examples include hemocyanin. When the carrier protein complex is used, the coupling ratio between the carrier protein and the non-receptor tyrosine kinase or the peptide in its specific region is about 0.1 to 30 times (carrier / the peptide: weight ratio). Desirably, about 0.5 to 5 times is used. Various condensing agents can be used for coupling the hapten and the carrier, and glutaraldehyde, carbodiimide and the like are conveniently used. When immunizing with a non-receptor tyrosine kinase or protein complex, mammals to be immunized are experimental animals such as sheep, goats, rabbits, guinea pigs, rats and mice. Rats and mice are preferred. Immunization can be performed by any route such as subcutaneous, intraperitoneal, intravenous, intramuscular, and subcutaneous when immunizing a mouse, but mainly subcutaneous,
It is preferably injected intraperitoneally or intravenously (especially subcutaneously). Further, the immunity interval, the immunization amount, etc. are highly variable, and various methods are possible. A method using spleen cells extracted after 4 days is often used.
The immunization amount is about 0.1 per mouse as the peptide amount.
It is desirable to use more than or equal to μg, preferably about 10 to 300 μg. In addition, it is desirable to perform partial blood collection before removing the spleen, confirm the increase in antibody titer in the blood, and then perform a fusion experiment using spleen cells.

[0008] The spleen cells of mice cell fusion is, for example removal of the spleen cells and lymphoid cells, hypoxanthine - guanine - phosphoribosyl transferase deficiency (HGPRT ^-) - such as and, thymidine kinase deficiency ^(TK) Suitable homologous or heterologous (preferably homologous) myeloma carrying a marker [eg P3-X63-Ag.8U
1 (Ichimori et al. Journal of Immunological Methods (J. Immunol. Methods) 80:55 (1985))]
Etc. and a lymphoid cell line. For example, the method of Kahler and Milstein [Natur (Natu
re) 256: 495 (1975)]. For example, myeloma cells and spleen cells are mixed at a ratio of about 1: 5, for example, Iscove's medium and Ham's F.
A -12 medium is suspended in a 1: 1 mixed medium (hereinafter referred to as IH medium), and a fusion agent such as Sendai virus and polyethylene glycol (PEG) is used. Of course, it is also possible to add dimethyl sulfoxide (DMSO) and other fusion promoters. When PEG is used as the fusing agent, it usually has a degree of polymerization of about 1000 to 6000.
Is used at a concentration of about 10% to 80% for about 0.5 to 30 minutes. As an example of preferable conditions, PEG
By treating 6000 with about 35 to 55% for about 4 to 10 minutes, the fusion can be efficiently performed. The fused cells are hypoxanthine-aminopterin-thymidine medium [HAT medium; Nature, 256, 495 (1975)].
Etc. can be used to selectively proliferate. The culture supernatant of the grown cells can be screened for the production of the desired antibody, and the antibody titer can be screened as follows. That is, in this case, first, as a first step, the presence or absence of antibody production against the immunized peptide is checked by a radioimmunoassay (RIA) method or an enzyme immunoassay (EI).
It can be examined by methods such as the method A), but various modifications of these methods are possible. As one example of a preferable measurement method, one method using EIA will be described. For example, a rabbit anti-mouse immunoglobulin antibody is coupled to a carrier such as a cellulose bead according to a conventional method, and a culture supernatant to be measured and mouse serum are added to the carrier, and a constant temperature (about 4 to 40 ° C) The same applies to the following). After this, the reaction product is thoroughly washed, and then an enzyme-labeled peptide (purification after coupling the enzyme and the peptide by a conventional method) is added, and the reaction is carried out at a constant temperature for a fixed time. After thoroughly washing the reaction product, an enzyme substrate is added and the reaction is carried out at a constant temperature for a certain period of time, and then the produced color product can be measured by absorbance or fluorescence.

The cells in the wells showing the growth in the selective medium and having the antibody activity against the peptide used for immunization are
It is desirable to carry out cloning by the limiting dilution method or the like. By similarly screening the supernatant of the cloned cells and increasing the number of cells in wells having high antibody titers, monoclonal antibody-producing hybridoma clones showing reactivity with the immunized peptide can be obtained. The hybridoma thus cloned is grown in liquid medium. Specifically, for example, about 0 in a liquid medium such as RPMI-1640 [Moore, GE, et. Al. Journal of American Medical Association (J. Am. Med. Assoc.) 199: 549 (1967)]. The monoclonal antibody can be obtained from the culture solution by culturing in a medium or the like supplemented with 1 to 40% bovine serum for about 2 to 10 days, preferably about 3 to 5 days. Alternatively, the antibody can be obtained by inoculating the mammal intraperitoneally, growing the cells, and collecting ascites. For this purpose, for example, in the case of a mouse, about 1 x 10 is applied to a mouse such as BALB / c previously inoculated with mineral oil or the like.
⁴ to 1 × 10 ⁷ , preferably about 5 × 10 ⁵ to 2 × 10 ⁶ hybridomas were intraperitoneally inoculated, and about 7 to 20 days later,
Ascites fluid is preferably collected after about 10 to 14 days. The antibody produced and accumulated in ascites can be easily isolated as a pure immunoglobulin from the monoclonal antibody by, for example, ammonium sulfate fractionation, DEAE-cellulose column chromatography and the like. In this way, a monoclonal antibody against the non-receptor tyrosine kinase is obtained.

The monoclonal antibody of the present invention specifically binds to a non-receptor tyrosine kinase and does not cross-react with non-receptor tyrosine kinases other than the tyrosine kinase. Since the monoclonal antibody of the present invention specifically binds to non-receptor tyrosine kinase, it is extremely useful as a reagent for measuring various non-receptor tyrosine kinases. Furthermore, facilitating the measurement of non-receptor tyrosine kinases in living organs, tissues, and cells is extremely useful in obtaining basic knowledge (for example, biodistribution) regarding non-receptor tyrosine kinases. For detection of non-receptor tyrosine kinases in living organs, tissues and cells, quantification by enzyme immunoassay (EIA method) or the like, or fluorescent antibody method or radioimmunoassay method (RIA method) is usually used. Especially, the EIA method is preferable. In addition, western blotting of proteins is effective for determining the size of non-receptor tyrosine kinases present in these organs, tissues and cells. In this method, crude extracts derived from organs, tissues, cells or partially purified samples thereof are subjected to acrylamide electrophoresis and then transferred to a membrane filter to obtain horseradish peroxidase (hors).
Eradish peroxidase (HRP) -conjugated anti-non-receptor tyrosine kinase antibody is used for detection.

Further, by utilizing the binding ability between the antibody and the non-receptor tyrosine kinase, an antibody affinity column can be prepared and used as a reagent for purifying the non-receptor tyrosine kinase. The antibody molecule used to detect and quantify the non-receptor tyrosine kinase may be IgG, or a fraction thereof (eg, F (ab ') ₂ , F).
It may be ab 'or Fab}. Among them, the antibody molecule to which the labeling agent is directly bound is preferably Fab '. The monoclonal antibody of the present invention can be used as a reagent in an immunochemical assay for non-receptor tyrosine kinase. The amount of non-receptor tyrosine kinase in living tissue or body fluid can be measured by an immunochemical assay method for non-receptor tyrosine kinase. Therefore, measuring non-receptor tyrosine kinases in various tissues and body fluids is expected to be useful in diagnosing diseases caused by abnormalities in cell proliferation and differentiation signal transduction mechanisms, such as cancer. In the above immunological assay method, when performing the assay method using two kinds of antibodies, it is preferable to use the monoclonal antibody of the present invention as one antibody and the monoclonal antibody or polyclonal antibody of the present invention as the other antibody. it can.

[0012] Examples of the carrier of the antibody retained on the carrier used in the method for measuring non-receptor tyrosine kinase include gel particles (eg, agarose gel [eg, Sepharose 4B, Sepharose 6B (Pharmacia Fine Chemical Co., Sweden ))], Dextran gel [eg, Sephadex G-75, Sephadex G-100, Sephadex G-200 (Pharmacia Fine Chemical Co. (Sweden))], polyacrylamide gel [eg, Biogel P-30, Biogel P-60, Biogel P-100 (manufactured by Bio-Rad Laboratories (USA))], cellulose particles [eg, Avicel (manufactured by Asahi Kasei), ion exchange cellulose (eg, diethylaminoethyl cellulose, carboxymethyl cellulose)], physics Adsorbent Example, glass (e.g.,
Glass spheres, glass rods, aminoalkyl glass spheres, aminoalkyl glass rods), silicon pieces, styrene resins (eg polystyrene spheres, polystyrene particles), immunoassay plates (eg Nunc (Denmark))
Ion exchange resin {eg, weakly acidic cation exchange resin [eg, Amberlite IRC-50 (Rohm and
Haas Company (USA)), Zeocurve 226 (Palmtit Company (West Germany))], weakly basic anion exchange resin [eg, Amberlite IR-4B, Dowex 3 (Dow Chemical Company (USA)]]} And so on.

For holding the antibody on the carrier, known conventional means can be applied. For example, “Metabolism”, Vol. 8 (1971).
Year), the Bromcian method and the glutaraldehyde method described on page 696. Further, as a simpler method, it may be physically adsorbed on the surface of the carrier.
Examples of the labeling agent in the antibody to which the labeling agent is bound include a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like, and it is preferable to use an enzyme. As the enzyme, stable and large inactive ones are preferable, and peroxidase,
Alkaline phosphatase, β-D-galactosidase,
Glucose oxidase can be used, but peroxidase is preferred. As the peroxidase, those of various origins can be used, and examples thereof include peroxidase obtained from horseradish, pineapple, fig, sweet potato, broad bean, corn and the like, and in particular, horseradish extracted from horseradish. Radish peroxidase (horserad
ish peroxidase) (HRP) is preferred. Fa as an antibody molecule for binding peroxidase and antibody
In order to utilize the thiol group of b ′, it is convenient to use a peroxidase into which a maleimide group has been introduced in advance.

As a method for introducing a maleimide group into peroxidase, a maleimide group can be introduced via an amino group of peroxidase. for that purpose,
An N-succinimidyl-maleimide-carboxylate derivative can be used, preferably N- (γ-maleimidobutyloxy) succinimide (sometimes abbreviated as GMBS). Therefore, a certain group may be included between the maleimide group and peroxidase. The reaction of GMBS with peroxidase is carried out by reacting both in a buffer solution having a pH of about 6 to 8 at a temperature of about 10 to 50 ° C. for about 10 minutes to 24 hours. Examples of the buffer include p
Examples include H7.0 0.1M phosphate buffer. The maleimidated peroxidase thus obtained is
For example, it can be purified by gel chromatography or the like. Examples of the carrier used when performing the gel chromatography include Sephadex G-25.
[Pharmacia Fine Chemicals (Sweden)
Manufactured by Bio-Rad Laboratories (USA), and the like. The reaction between the maleimidated peroxidase and the antibody molecule can be carried out by reacting both in a buffer solution at a temperature of about 0 ° C. to 40 ° C. for about 1 to 48 hours. Examples of the buffer include 0.1 M phosphate buffer containing 5 mM ethylenediaminetetraacetic acid sodium salt having a pH of 6.0. The peroxidase-labeled antibody thus obtained can be purified by, for example, gel chromatography. Examples of the carrier used when performing the gel chromatography include Sephadex G-25 [manufactured by Pharmacia Fine Chemicals (Sweden)], Biogel P-2 [manufactured by Bio-Rad Laboratories (USA)] and the like. To be Furthermore, a thiol group may be introduced into peroxidase and reacted with a maleimidated antibody molecule. Direct binding of an enzyme other than peroxidase to the antibody can be performed according to the case of peroxidase, and the glutaraldehyde method, periodate method, water-soluble carbodiimide method and the like known per se can be used.

Examples of test samples in the assay system of the present invention include body fluids such as urine, serum, plasma, and cerebrospinal fluid, or extracts of animal cells or fungi or culture supernatants thereof. As an example of the measuring method of the present invention, the case where the labeling agent is peroxidase will be specifically described below, but is not limited to peroxidase. First of all, an antigen-antibody reaction is carried out by adding an analyte containing a non-receptor tyrosine kinase to be measured to the antibody retained on a carrier, and then the peroxidase and the anti-non-receptor tyrosine kinase obtained above are added thereto. A bound substance with the antibody is added and reacted. A substrate of peroxidase is added to the reaction product obtained in step (1), and the enzyme activity of the reaction product is determined by measuring the absorbance or fluorescence intensity of the resulting substance. : The above steps (1) to (3) are performed in advance on a standard solution of a known amount of non-receptor tyrosine kinase, and the relationship between the amount of non-receptor tyrosine kinase and the absorbance or fluorescence intensity is prepared as a standard curve. : The absorbance or fluorescence intensity obtained for an analyte (test sample) containing an unknown amount of non-receptor tyrosine kinase is applied to a standard curve to measure the amount of non-receptor tyrosine kinase in the analyte.

The non-receptor tyrosine kinase can be purified using an antibody obtained by using a complex of the non-receptor tyrosine kinase or a partial peptide of its specific region and a carrier protein as an immunogen. This can be done by performing affinity column chromatography using the antibody. The affinity column chromatography can be performed, for example, by coupling the antibody to a suitable carrier, packing the column in a column, adsorbing a solution containing a non-receptor tyrosine kinase through the column, and then adsorbing the solution. it can. Examples of the carrier include those similar to the carriers described above. In particular, gel particles and various synthetic resins are conveniently used. For example, CNBr-activat
ed Sepharose 4B (manufactured by Pharmacia Fine Chemicals Co., Ltd.), Affigel-10, Affigel-15 (manufactured by Bio-Rad Laboratories) and the like. In order to couple the antibody to the carrier, known conventional means can be applied, and examples thereof include the Bromcian method and the glutaraldehyde method described in “Metabolism”, Volume 8 (1971), page 696. To be Further, a method using a water-soluble carbodiimide, an active ester method and the like can be used, but as a simpler method, they may be physically adsorbed on the surface of the carrier.

To perform purification using the antibody column thus obtained, the non-receptor tyrosine kinase in a buffer solution near neutral is adsorbed to the antibody column packed with the carrier to which the antibody is bound. Next, the column is washed with the same buffer, and the specifically adsorbed non-receptor tyrosine kinase is eluted. To elute the specifically adsorbed non-receptor tyrosine kinase, for example, a low pH or high pH buffer solution or a buffer solution containing a high concentration of salt is used. Examples of the low pH buffer solution include p
H2.3 0.17 M glycine-HCl buffer, pH 1.8
0.1 M secondary sodium citrate-hydrochloric acid buffer solution and the like. Examples of the high pH buffer solution include pH
11 aqueous ammonia, 0.2M sodium borate buffer solution having a pH of 11.7 and the like can be mentioned. As the buffer solution containing the high-concentration salt, for example, a 6M guanidine hydrochloric acid solution,
7M urea solution etc. are mentioned. The elution may be performed by a batch method or a column method.

The eluate of the antigen is purified by, for example, dialysis. For example, when eluted with a low pH buffer, for example, 0.1M sodium carbonate buffer (pH 10.5), high pH
When eluted with the buffer solution of 0.1 M glycine-
After neutralizing with a hydrochloric acid buffer (pH 3.0), for example, a 0.02 M phosphate buffer containing 0.1% NaN ₃ (pH)
Dialyse against 8.0). Alternatively, the antigen solution eluted with a buffer solution containing a high concentration of salt can be directly dialyzed against the above-mentioned phosphate buffer solution and stored. Further, it can be stored as a freeze-dried preparation obtained by freeze-drying the above-mentioned eluate or dialysate. The non-receptor tyrosine kinase purified in this manner is of extremely high unit, and may be utilized for elucidation of the structure of the protein by, for example, X-ray analysis, and substances that interact with these proteins are clarified. It is thought that this will be useful for analysis of signal transduction system.

In the present specification and drawings, when abbreviations are used for bases, amino acids, etc., IUPAC-IU
The abbreviations according to B Commision on Biochemical Nomenclature or abbreviations commonly used in this field are used, and examples thereof are shown below. When amino acids may have optical isomers, the L-form is shown unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine triphosphate dCTP: deoxy Cytidine triphosphate ATP: Adenosine triphosphate Tdr: Thymidine EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate G, Gly: Glycine A, Ala: Alanine V, Val: Valine L, Leu: Leucine I, Ile: Isoleucine S, Ser: Serine T, Thr: Threonine C, Cys: Cysteine M, Met: Methionine E, Glu: Glutamic acid D, Asp: Aspartic acid K, Lys: Lysine R, Arg: Arginine H, His: Histidine F, Phe: Fe Lualanine Y, Tyr: Tyrosine W, Trp: Tryptophan P, Pro: Proline N, Asn: Asparagine Q, Gln: Glutamine Clz: 2-Chlorobenzyloxycarbonyl Brz: 2-Bromobenzyloxycarbonyl Bzl: Benzyl Boc: t-butoxy. Carbonyl Hybridomas LY8 and LY obtained in Example 1 described later
9 and the hybridoma FGA-1 obtained in Example 2.
2 has been deposited with the Institute for Fermentation (IFO) and the Institute for Microbial Technology (FRI), Ministry of International Trade and Industry. The acceptance date and acceptance number are shown in [Table 1] below.
Shown in.

[Table 1] Hybridoma IFO FRI LY8 IFO 50346 FERM BP-3711 (August 7, 1991) (January 17, 1992) LY9 IFO 50347 FERM BP-3712 (August 7, 1991) (January 17, 1992) FGA-12 IFO 50348 FERM BP-3713 (August 12, 1991) (January 17, 1992)

[0020]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. Example 1 (1) Expression of Lyn-β-gal fusion protein: Lyn protein (amino acid sequence is Mol. Cell. Biol, 7, 237-
243 (1987) (shown in FIG. 2), a cDNA encoding the 95 amino acid residues Arg25 to Ala119.
The A fragment (285 bp) was inserted into the SmaI site of the pIC-III vector to construct pIC-LYN [Proc. Natl. A.
cad. Sci. USA 86, 6538-6542 (198
9)]. pIC-LYN was introduced into Escherichia coli UT481 strain,
100 mM I in medium (L-broth) during logarithmic growth phase
1 PTG (isopropyl-β-D-galactopyranoside)
/ 50 volume was added and the mixture was cultured for 12 hours to produce a fusion protein of Lyn protein and β-galactosidase (Lyn-β-gal). (2) Purification of Lyn-β-gal fusion protein: Lyn-β-
Purification of the gal fusion protein is performed by Alman [Gene (Ge
ne) 29, 27-31 (1984)]. E. coli UT481 / pIC-LY collected from 2 liter culture
11.45 g of N cells was suspended in 46 ml of 20 mM Tris-HCl (pH 7.4) containing 10 mM MgCl ₂ on ice, and the suspension was sonicated under ice cooling and centrifuged. To the supernatant, NaCl and dithiothreitol (DTT) were respectively added to 1.6M,
10 mM MgCl ₂ , 1.6 was added to 10 mM.
20 mM Tris-HCl containing 10 mM DTT and M NaCl
P-aminophenyl β-D-t equilibrated with (pH 7.4)
hiogalactopyranoside-agarose (TPEG-agarose)
It was applied to a column (manufactured by Sigma) (diameter 1.0 cm × 15.3 cm). After washing the column with the buffer used for equilibration,
100 mM sodium borate (pH 10.0) containing 10 mM 2-mercaptoethanol was flowed at a flow rate of 30 ml / hr, and fractionation was performed every 5 ml. 37 ml of the eluted fraction was added to 2 liters of 40 mM Tris- containing 1 mM MgCl ₂ , 0.1 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM DTT.
It was dialyzed overnight against HCl (pH 7.5). After concentrating to 7.4 ml with a PM10 membrane (Diaflow Amicon), equilibrated with the same solution as the dialysis buffer to a Sephacryl-S-200HR column (Pharmacia) (diameter 2.6 cm x 68 cm). Gel filtration,
6.5 mg of Lyn-β-gal fusion protein was obtained.

(3) Immunization: 140 μg of antigen Lyn-β-gal fusion protein (obtained with (2)) dissolved in 0.2 ml of physiological saline in BALB / c mice (8 weeks old). The same amount of Freund's complete adjuvant (manufactured by Difco) was mixed and injected subcutaneously. Two weeks later, a mixture of the same amount of antigen and 0.2 ml of Freund's incomplete adjuvant was re-administered subcutaneously. The same booster was given 2 weeks later, and 10 days later, 200 μg was dissolved in physiological saline.
Lyn-β-gal fusion protein was inoculated intravenously. (4) Cell fusion: From the immunized mouse obtained in (3) above, 3 days after the final administration of the antigen, the spleen was extracted to obtain cells used for cell fusion. The cells are from Iscove's medium and Ham's F-
Twelve mediums were suspended in a medium (hereinafter abbreviated as IH medium) mixed at a ratio of 1: 1. Mouse myeloma cell P3-X6
3-Ag / 8U1 is RPMI containing 10% fetal calf serum
The cells were subcultured in 1640 medium under the conditions of 5% carbon dioxide and 95% air. Cell fusion is a method established by Köhler and Milstein et al. [Kohler, G. and Milstein, C .; Nature 256: 495 (197).
5)]. Myeloma cells above 4.0 × 10
⁷ and 2.0 immunized lymphocytes obtained by the method described above
45% polyethylene glycol 6000 (hereinafter PE) was mixed and spun down to 10 × 10 ⁸ and dissolved in 0.5 ml of IH medium.
(Sometimes referred to as G6000). PEG6
The 000 solution was previously warmed to 37 ° C. and slowly added dropwise. After 7 minutes, preheat IH medium to 37 ° C for 0.5 minutes per minute.
After adding l to make 10 ml, the mixture was centrifuged at room temperature at 700 rpm for 15 minutes to remove the supernatant. This cell precipitate was suspended in 130 ml of IH medium containing 20% fetal bovine serum, and 100 μl of each was seeded in a 96-well microplate (Nunc). One day later, 20% fetal bovine serum-containing IH medium containing hypoxanthine 1 × 10 ⁻⁴ M, aminopterin 4 × 10 ⁻⁷ M and thymidine 1.6 × 10 ⁻⁵ M (hereinafter referred to as HAT).
100 μl) was added to each well, and half of the medium was replaced with HAT medium every 3 days. The cells thus grown are hybrid cells.

(5) Screening for antibody-producing cells: Lyn-β-gal fusion protein or β-galactosidase purified in (2) above was adjusted to 2.5 μg / ml in 0.01 M carbonate buffer (pH 8.5). ), And 100 μl of which is diluted with 96
Each well of a well immunoplate (manufactured by Nunc) was placed at 4 ° C. overnight, and Lyn-β-gal fusion protein or β-galactosidase was bound to the solid phase. 0.15
0.01 M phosphate buffer containing M NaCl (pH 7.
After washing with 0), 1% to block excess binding sites
200 μl of 0.01 M phosphate buffer containing bovine serum albumin (BSA) was injected into each well and stored in a cool place until use. Lyn− obtained as described above
100 μl of the hybrid cell culture supernatant was added to each of the 96-well immunoplates to which β-gal fusion protein or β-galactosidase was bound, and the mixture was incubated at room temperature for 2 hours. After removing the culture supernatant and washing, a horseradish peroxidase (HRP) -labeled goat anti-mouse IgG antibody (manufactured by Kappel) was added as a secondary antibody and incubated at room temperature for 2 hours. After removing the secondary antibody and thoroughly washing the wells, 100 μl of a peroxidase substrate solution (sodium citrate buffer solution containing 0.02% H ₂ O ₂ and 0.15% o-phenylenediamine at pH 5.5) was added. After reacting at 25 ° C. for 10 minutes to stop the enzymatic reaction by adding 100 μl of 2N-sulfuric acid, an automatic colorimeter for microplates (MTP-32, manufactured by Corona) was used to obtain 492 nm.
The absorbance at was measured (ELISA method). By this method, Lyn- was not shown to bind to β-galactosidase.
The presence of an antibody that specifically reacts with the β-gal fusion protein was observed in 7 wells. (6) Cloning of hybrid cells: The number of cells in these wells was previously 6 × so that the number of cells in each well was 0.5.
10 ⁵ cells / well of mouse thymocytes are seeded on a 96-well microplate, which has been seeded as feeder cells,
Cloning was performed. As a result, 1 type of each representative clone cell was obtained from each well, and a total of 7 types of clone cells, that is, LY1, LY3, LY4, LY6.
LY7, LY8 (IFO 50346, FERM BP
-3711), LY9 (IFO 50347, FERM
BP-3712) was obtained.

(7) Production of antibody: Hybridoma clone L obtained by cloning in (6) above
Each of Y3, LY6, LY8, and LY9 was intraperitoneally administered with 0.5 ml of mineral oil B
Ascites was performed by inoculating 1 × 10 ⁶ cells per mouse into ALB / c mouse intraperitoneally. Ascites was collected 10 days after the hybridoma was intraperitoneally administered. From about 10 ml of each of the obtained ascites, Stalin et al. [Journal of Biological Chemistry, 256: 9750-
9754 (1981)] to purify the monoclonal antibody. First, after centrifugation at 10,000 rpm for 15 minutes to remove fibrin-like substances from ascites, phosphate buffer-saline (PBS: 8.1 mM-Na ₂ HPO ₄ , 1.5
mM KH ₂ PO ₄ , 27 mM KCl, 137 mM NaCl,
It has an ultraviolet absorption (A ₂₈₀ ) of ₂₈₀ nm of 12 to 12 at pH 7.2).
It was diluted to a concentration showing a value of 14. After the dilution, a saturated ammonium sulfate solution was added to the sample to a concentration of 47%, salting out was performed for 60 minutes while stirring at 4 ° C., and then centrifugation (10,000 rpm, 15 minutes) was performed to obtain a precipitate. The precipitate was melted in a 20 mM Tris buffer solution (pH 7.9) containing 50 mM NaCl, and dialyzed against 2 liters of the same solution. After 2 hours, it was replaced with 2 liters of the same dialyzing solution and dialyzed for another 15 hours. After dialysis, the precipitate was removed by centrifugation at 10,000 rpm for 15 minutes, and the supernatant was adjusted so that the A ₂₈₀ value was 20 to 30. A sufficient amount of this sample is 50 mM-
20 ml of DEA equilibrated with Tris buffer containing NaCl
50 on E-cellulose column (Whatman DE ₅₂ )
After washing well with Tris buffer containing mM NaCl, 50m
Fractionation was carried out using a gradient salt solution of the same buffer containing M-500 mM NaCl at an outflow rate of 1.5 ml / min to concentrate the flow-through fraction.
AbLY3, MAbLY6, MAbLY8 and MAb
LY9 was obtained. To confirm the purity of the antibody, SDS-polyacrylamide gel electrophoresis was used according to the method of Laemli et al. [Nature, 227, 680-685 (1970)]. That is, a fraction obtained by salting out with ammonium sulfate and passing through a DEAE cellulose column was reduced with 2-mercaptoethanol, and electrophoresed at 180 volts for 2.5 hours using a gel having an acrylamide concentration of 10%. As a result, all the monoclonal antibodies had H chains of around 52 kDa and 28 kDa.
Two L chain bands were observed around a. (8) Measurement of antibody subclass: The antibody subclass was measured by the following method. That is, Lyn-β obtained in the above (2)
100 μl of each cloned hybridoma culture supernatant was added to a 96-well immunoplate coated with -gal fusion protein and incubated at room temperature for 2 hours. After removing the culture supernatant and washing, rabbit anti-mouse IgG1,
100 μl each of antibodies against IgG2a, IgG2b, IgG3, κ chain and λ chain (manufactured by Kappel) were added and incubated at room temperature for 2 hours. After removing each antibody and washing, HRP-labeled goat anti-rabbit IgG antibody (manufactured by Kappel) was added and incubated at room temperature for 2 hours. After removing the labeled antibody and washing it well, the enzyme reaction was carried out by the method described in (5) above and the absorbance was measured. As a result, MAbL
Y3 and MAbLY6 are IgG1κ type, MAbLY7
And MAbLY9 were found to be subclasses of IgG2aκ type, and MAbLY1 and MAbLY8 were subclasses of IgG2bκ type.

(9) Examination of Antigen-Binding Property of Antibody Whether the antibody recognizes the Lyn protein was determined by immunoprecipitation / kinase assay or Western blotting. Human B cell line Daudi cell or mouse B cell line WE
1 × 10 ⁷ HI-231 cells with 1 ml of cell lysis buffer (1% digitonin, 10 mM triethanolamine, 150 mM NaCl, 10 mM iodoacetamide,
1 mM EDTA, aprotinin 10 μg / ml, pH 7.
8) was added and treated for 30 minutes at 0 ° C. 15,000 rpm
The supernatant was collected by centrifugation at 4 ° C for 20 minutes. Each antibody was added to the obtained supernatant and reacted at 4 ° C. for 1 hour. After completion of the reaction, protein G sepharose was added and shaken at 4 ° C. for 1 hour. The reaction mixture was washed thoroughly with a cell lysis buffer, and a sample buffer (20% glycerol, 10% 2ME) was added.
(Mercaptoethanol), 6% SDS, 130 mM T
ris pH6.8 BPB (Bromophenol Blue) ~ 2
mg) and subjected to immunoprecipitation / kinase assay. For Western blotting, 1 × 10 ⁷ cells of each of the above two types of cells were treated with 1 ml of a cell lysis buffer, boiled for 5 minutes and subjected to 10% polyacrylamide gel electrophoresis. After electrophoresis, the protein was transferred to a membrane and treated with various anti-Lyn antibodies, and the binding property of the anti-Lyn antibody to the Lyn protein was examined with an alkaline phosphatase-labeled antibody. The results are shown in Table 2 below.
bLY3, MAbLY6, MAbLY8, MAbLY9
Was found to bind to the Lyn protein.

[Table 2] Binding of anti-Lyn monoclonal antibody to Lyn protein ─────────────────────────────────── ─ Monoclonal antibody ───────────────── Cell line assay LY3 LY6 LY8 LY9 ────────────────────── ───────────── Human B cell line Immunoprecipitation / Kinase method ＋＋＋＋ Daudi Western blotting ＋ Mouse B cell line Immunoprecipitation / Kinase method ＋ WEHI-231 Western blotting ± ── ────────────────────────────────── + indicates reactivity.

Example 2 (1) H-Cys-Ala-His-Ile-Pro-Asn-Tyr
-Ser-Asn-Phe-Ser-Ser-Gln-Ala-Ile-
Asn-Pro-Gly-Phe-Leu-Asp-OH (Cys-F
Production of gr [48-67] peptide Fgr protein (amino acid sequence 1 to 143 is O
A peptide corresponding to Ala48 to Asp67 of ncogene 1,301-304 (1987, shown in FIG. 2) (hereinafter sometimes referred to as Fgr [48-67]) was added with Cys at the N-terminal portion as a linker. The peptide added with was synthesized by a solid phase synthesis method using an automatic peptide synthesizer 430A (manufactured by Applied Biosystems). The program used "standard 1". The basic synthesis process is based on Merrifield,
R. B. Advance of Enzymology (Ad
v. Enzymol.) 32: 221-296 (1969). Boc-Asp (OBzl)-for the resin (solid phase)
PAM-P (phenylacetamidomethyl polymer)
(0.5 mmol, corresponding to 700 mg of resin) was used to sequentially synthesize the following amino acids from the carboxyl terminal. Boc-
As amino acids, Boc-Leu-OH, Boc-Phe-O
H, Boc-Gly-OH, Boc-Pro-OH, Boc-Asn
-OH, Boc-Ile-OH, Boc-Ala-OH, Boc-
Gln (OBzl) -OH, Boc-Ser (Bzl) -OH, B
oc-Ser (Bzl) -OH, Boc-Phe-OH, Boc-A
sn-OH, Boc-Ser (Bzl) -OH, Boc-Tyr (B
rz) -OH, Boc-Asn-OH, Boc-Pro-OH, B
oc-Ile-OH, Boc-His (Tos) -OH, Boc-A
la-OH, Boc-Cys (MeBzl) -OH was used. After synthesizing to the amino terminal Cys, the peptide resin was taken out from the synthesizer and dried. To 1 g of the peptide resin, 1.5 ml of p-cresol and 0.5 ml of 1,2-ethanedithiol were added, and further about 8 ml of liquid hydrogen fluoride was added thereto, and the mixture was added at 0 ° C.
And reacted for 2 hours. After completion of the reaction, hydrogen fluoride was removed under reduced pressure in a desiccator, and washed with diethyl ether containing 0.1% 2-mercaptoethanol and then with diethyl ether to remove most of the mixed reagents. The peptide was extracted with 10 ml of 3% acetic acid, and the resin mixed in the extract was removed by filtration. The filtrate is Sephadex
adex) G-25 and purified by gel filtration. Gel filtration conditions are column size φ3 × 70 cm; detection wavelength 23
0 nm; solvent 3% acetic acid; flow rate 30 ml / hr. The peptide-containing fractions were collected, freeze-dried, and the resulting powder preparation was further purified by reverse phase high performance liquid chromatography [column: YMC-pack, D-ODS.
-7 φ20 × 250 mm (YMC), column temperature:
25 ° C., elution conditions: elution solvent A 0.1% trifluoroacetic acid-99.9% distilled water; elution solvent B 0.1% trifluoroacetic acid-99.9% acetonitrile; elution program 0
Minute (75% A + 25% B), 2 minutes (75% A + 25%
B), 32 minutes (45% A + 55% B); efflux rate 5 ml /
min, detection wavelength 230 nm]. Retention time of about 23 under these conditions
Bio-Rad AG
It was passed through a 1 × 8 (AcOH type 1.8 × 5 cm) column, a washing solution was collected, and acetonitrile was distilled off, followed by freeze-drying to obtain 46 mg of a white powder. The obtained peptide, when analyzed by high performance liquid chromatography under the same conditions as above, gave a sharp single peak at a retention time of 23 minutes. Ellman G. El (Elman, GL) method [architecture Le Biochemistry and Biotechnology Physics box (Arch. Biochem. Biophys.) 8 2, 70-77 (195
9)] was used to quantify the free SH groups.
It was 0%. The results of amino acid analysis were as follows. Asp 3.95 (4); Ser 2.72 (3); Gln 1.04 (1); Pro 1.7
9 (2); Gly 1.00 (1); Ala 1.92 (2); Cys 0.93 (1); Il
e 1.86 (2); Leu 1.00 (1); Tyr 0.94 (1); Phe 2.01 (2);
His 0.83 (1).

(2) Preparation of immunogen Cys-Fgr [48-67] peptide (2.0 mg) and bovine thyroglobulin (BTG)
4.0 mg was dissolved in 2 ml of 0.1 M phosphate buffer (pH 7.0), 0.5 ml of 1% glutaraldehyde aqueous solution was added, and the mixture was stirred under ice cooling for 30 minutes and then at room temperature for 3 hours. . After stirring, 2 liters of phosphate saline (PB
S) is dialyzed twice and Cys-Fgr [48-67] -BTG
got the conjugate. (3) Immunization 0.2 ml of PBS for BALB / c mice (8-week old)
Dissolved in Cys-Fgr [48-67] -BTG conjugate
Freund's complete adjuvant (Difco)
(Manufactured by the company) was mixed and injected subcutaneously. Two weeks later, a mixture of the same amount of antigen and 0.2 ml of Freund's incomplete adjuvant was re-administered subcutaneously. In addition, the same booster immunization was performed four times every two weeks, and two weeks later, 282 μ was dissolved in PBS.
g of Cys-Fgr [48-67] -BTG conjugate was inoculated intravenously. (4) Cell fusion From the immunized mouse obtained in (3) above, 3 days after the final administration of the antigen, the spleen was excised to obtain cells used for cell fusion. The cells were suspended in a medium (hereinafter, abbreviated as IH medium) in which Iscove's medium and Ham's F-12 medium were mixed at a ratio of 1: 1. Mouse myeloma cell P3-X63-Ag.8U1
Was subcultured in RPMI 1640 medium containing 10% fetal bovine serum under the conditions of 5% carbon dioxide and 95% air. The cell fusion was performed according to the method established by Koehler and Milstein [Kohler, G. and Milstein, C .; Nature 256, 495 (1975)].
The myeloma cells (4.3 × 10 ⁷ ) and the immunized lymphocytes (2.0 × 10 ⁸ ) obtained by the above method were mixed, spun down, and dissolved in 0.4 ml of IH medium to prepare 45% polyethylene. Glycol 6000 (hereinafter sometimes referred to as PEG6000) was added dropwise. PEG6000 solution is 37 ° C in advance
It was warmed to and slowly added dropwise. After 7 minutes, 0.5 ml of IH medium preheated to 37 ° C. was added to each well for 10 ml.
Then, the mixture was centrifuged at room temperature at 700 rpm for 15 minutes to remove the supernatant. This cell precipitate was suspended in 100 ml of IH medium containing 20% fetal bovine serum, and 100 μl of each was seeded in a 96-well microplate (Nunc). One day later, hypoxanthine 1 × 10 ⁻⁴ M, aminopterin 4 × 10
100 μl of IH medium containing 20% fetal bovine serum (hereinafter referred to as HAT medium) containing ⁻⁷ M and thymidine 1.6 × 10 ⁻⁵ M was added to each well, and 1/3 of the medium was added every 3 days. Two volumes were replaced with HAT medium. The cells thus grown are hybrid cells.

(5) Search for antibody-producing cells Cys-Fgr [48-67] peptide was diluted with 0.01 M carbonate buffer (pH 8.5) to 2.5 μg / ml, and 100 μl thereof was added to 96 wells. Immuno plate (made by Nunc)
In each well and left overnight at 4 ° C to allow Cys-Fgr on the solid phase.
The [48-67] peptide was conjugated. After washing with 0.01 M phosphate buffer (pH 7.0) containing 0.15 M NaCl, 0.01 M phosphate buffer containing 1% bovine serum albumin (BSA) was added to block excess binding sites. 200 μl was injected into each well and stored in a cool place until use. Cys-Fgr [48-6 obtained as described above
7] To the 96-well immunoplate with the peptide bound,
100 μl each of hybrid cell culture supernatant was added and incubated at room temperature for 2 hours. After removing the culture supernatant and washing, a horseradish peroxidase (HRP) -labeled goat anti-mouse IgG antibody (manufactured by Kappel) was added as a secondary antibody and incubated at room temperature for 2 hours. After removing the secondary antibody and thoroughly washing the wells, a peroxidase substrate solution (0.02% H ₂
O ₂ and pH5 containing 0.15% o-phenylenediamine.
100 μl of the sodium citrate buffer solution of 5) was added, and 2
After reacting at 5 ° C. for 10 minutes and stopping the enzymatic reaction by adding 100 μl of 2N-sulfuric acid, an automatic colorimeter for a microplate (MTP-32, manufactured by Corona Co.) was used.
The absorbance at 92 nm was measured (ELISA method). The presence of bound antibody was observed in 4 wells by this method. (6) Cloning of hybrid cells: The number of cells in these wells should be 5 × in advance so that the number of cells in each well is 0.5.
10 ⁵ cells / well of mouse thymocytes are seeded on a 96-well microplate, which has been seeded as feeder cells,
Cloning was performed. As a result, 1 type of each representative clone cell was obtained from each well, and a total of 4 types of clone cells, FGA-12 (IFO 50348, FERM.
BP-3713), FGA-19, FGA-22, F
GA-53 was obtained.

(7) Production of antibody The hybridoma clone FGA-12 obtained by the cloning in (6) above was intraperitoneally administered to BALB / c mice to which 0.5 ml of mineral oil had been intraperitoneally administered. Ascites was performed by inoculating 1 × 10 ⁶ animals per animal. Ascites was collected 10 days after the hybridoma was intraperitoneally administered. From about 10 ml of the obtained ascites, Stalin et al. [Journal of Biological Chemistry, 256, 9750-9754 (1981)]
The monoclonal antibody was purified according to the method described in 1. First, 10,000r to remove fibrin-like substances from ascites
After centrifugation for 15 minutes at pm, phosphate buffer-saline (PB
S: 8.1 mM-Na ₂ HPO ₄ , 1.5 mM KH ₂ PO ₄ , 2
28 with 7 mM KCl, 137 mM NaCl, pH 7.2)
It was diluted to a concentration showing an ultraviolet absorption (A ₂₈₀ ) at 0 nm of 12 to 14. After dilution, add saturated ammonium sulfate solution to the sample to a concentration of 47%, perform salting out for 60 minutes while stirring at 4 ° C, and then centrifuge (10,000 times).
(0 rpm, 15 minutes) was performed to obtain a precipitate. 20 mM Tris buffer solution containing 50 mM NaCl (pH)
7.9), and dialyzed against 2 liters of the same solution. After 2 hours, it was replaced with 2 liters of the same dialyzing solution and dialyzed for another 15 hours. After dialysis, centrifuge at 10,000 rpm for 15 minutes to remove the precipitate,
The supernatant was adjusted so that the A ₂₈₀ value was 20 to 30. This sample was applied to a 20 ml DEAE cellulose column (Whatman DE ₅₂ ) equilibrated with a sufficient amount of Tris buffer solution containing 50 mM-NaCl, washed well with Tris buffer solution containing 50 mM NaCl, and then washed with 50 mM-500 mM.
1.5 using a gradient salt solution of the same buffer containing NaCl.
Fractionation was performed at an outflow rate of ml / min to concentrate the flow-through fraction to obtain a purified monoclonal antibody MAbFGA-12. To confirm the purity of the antibody, SDS is performed according to the method of Laemli et al. [Nature, 227, 680-685 (1970)].
-Polyacrylamide gel electrophoresis was used. That is, the fraction which was subjected to salting out with ammonium sulfate and passed through a DEAE cellulose column was reduced with 2-mercaptoethanol, and a gel having an acrylamide concentration of 10% was used to obtain a voltage of 180 V at 2.
The electrophoresis was performed for 5 hours. As a result, H around 52kDa
Two bands of the L chain were observed around the chain, 28 kDa. (8) Measurement of antibody subclass: The antibody subclass was measured by the following method. That is, the Cys-F obtained in (1) above
100 μl of each cloned hybridoma culture supernatant was added to a 96-well immunoplate coated with gr [48-67] peptide and incubated at room temperature for 2 hours. After removing the culture supernatant and washing, rabbit anti-mouse IgG1,
100 μl each of antibodies against IgG2a, IgG2b, IgG3, κ chain and λ chain (manufactured by Kappel) were added and incubated at room temperature for 2 hours. After removing each antibody and washing, HRP-labeled goat anti-rabbit IgG antibody (manufactured by Kappel) was added and incubated at room temperature for 2 hours. After removing the labeled antibody and washing it well, the enzyme reaction was carried out by the method described in (5) above and the absorbance was measured. As a result, MAbF
GA-12 is an IgG2bκ type, and the other three monoclonal antibodies MAbFGA-19, MAbFGA-22, and MAb.
All bFGA-53 were found to be IgG1κ type subclasses.

(9) Examination of Antigen-Binding Property of Antibody Whether the antibody recognizes Fgr protein or not was determined by immunoprecipitation method or Western blotting method. Immunoprecipitation method: Human acute pre-leukemia cell line HL-60 cell 4
× 10 ⁶ suspended in 1.5 ml of P ^- medium, 200 μC
i / ml [32 _P ] -orchophosphate was added, and the mixture was cultured in a 6 cm Petri dish at 37 ° C for 4 hours. After culturing, the supernatant is discarded and 1 ml of RIPA (radio immunoprecipitation) is added to the cell pellet.
assay) buffer (50 mM HEPES (N- [2-Hydroxy
ethyl] piperazine-N '-[2-ethanesulfonic acid]) pH
7.5, 150 mM NaCl, 1% Triton X-100,1
mM EDTA, 100 kIU (kiro international unit) / m
l Aprotinin, 5μg / ml PMSF (phenylmethylsulf)
onyl fluoride), 10 μg / ml leupeptin, 0.1% S
DS, 0.5% DOC (sodium deoxycholate), 10 mM
NaF, 2 mM Na ₃ VO ₄ , 10 mM Na pyrophosphat
e) was added and stirred well to lyse the cells. 15,000
The supernatant was collected by centrifugation at 4 ° C for 20 minutes at rpm. The FGA-12 antibody was added to the obtained supernatant and reacted at 4 ° C for 1 hour. After the reaction, add Protein G Sepharose, 4 ℃, 1
Shake for time. After thoroughly washing with RIPA buffer, the bound product was eluted by treating in sample buffer at 100 ° C. for 5 minutes. The supernatant was electrophoresed on a 10% SDS-polyacrylamide gel and then a radioautogram was taken. As a result, MAb FGA-12 was
HL cultured in retinoic acid for 5 days to differentiate into granulocytes
It was revealed that the protein exhibited binding properties to the Fgr protein expressed at −60. Western blotting: 2 × 10 ⁶ HL-60 cells were dissolved in a sample buffer, shaken, boiled for 5 minutes, and subjected to 10% SDS-polyacrylamide gel electrophoresis. After transferring to the membrane, MAbFGA-12 antibody was reacted for 1 hour at room temperature, treated with a rabbit anti-mouse immunoglobulin antibody labeled with ¹²⁵ I, and then autoradiogram was taken. As a result, re of 1 μM
When lysate of HL-60 cells differentiated with tinoic acid was used, a single band was observed near 58 kDa,
MAbFGA-12 was found to show binding properties to the Fgr protein.

Example 3 (1) Fyn-PEA (Pseudomonas aeruginosa exotoxi
n A) Expression of fusion protein (PEFN) Fyn protein (amino acid sequence is Proc. Natl. Acad. Sci.
USA 83, 5459-5463 (1986) (shown in FIG. 2), a cDNA fragment (165 bp) encoding 55 amino acid residues from Asp19 to Thr73 was added to pVC4D vector [Biotechniques 10 (2): 202-4, 20
6,208-9 (1991)] to construct pVC-FN by incorporating it into the PpuMI / EcoRI site of E. coli BL21 (λD
E3) strain was introduced. E. coli BL21 (λDE3) / p
When the OD _{600 of} VC-FN is 0.45, the final concentration is 1 mM.
IPTG was added so that the amount became 3 times, and the cells were collected 3 hours after the addition.
20 ml of ice-cold TEN buffer (50 m
M Tris-HCl pH 8.0, 1 mM EDTA, 100 m
After suspending in MNaCl), 20 mg of lysozyme (manufactured by Wako Pure Chemical Industries, Ltd.) was added and gently mixed at room temperature for 30 minutes.
After centrifugation at 800 xg for 30 minutes at 4 ° C, the precipitate was reduced to 0.
Ice-cold TEN buffer 20 containing 1% deoxycholic acid
suspended in ml. 160 μl 1M MgCl ₂ and 200 μl 1
mg / ml DNase (manufactured by Sigma) was added and gently mixed at room temperature for 30 minutes. 4 ℃, 10 minutes, 12000 × g
After centrifugation at 20, the precipitate was suspended in 20 ml of ice-cold TEN buffer containing 0.5% Triton X-100, sonicated under ice-cooling, and centrifuged again under the same conditions. Ice-cold PBS for precipitation
20 ml of buffer was added, and the suspension was sonicated and centrifuged. 2 ml of ice-cold PBS was added to the precipitate, and the suspension was sonicated. As a result, 37.5 mg of PEFN was obtained. (2) Solubilization of antigen The PEFN antigen suspension obtained in (1) above had a final concentration of 2%.
10% (w / w) sodium dodecyl sulphate solution was added and the mixture was allowed to stand at room temperature for 5 minutes, then 5 to 5 in boiling water.
A solubilized PEFN antigen was obtained by leaving it for 10 minutes. (3) Immunization BALB / c mouse (♀ 8 weeks old) with physiological saline 0.1
Freund's complete adjuvant (Dif) in the same amount as 150 μg PEFN antigen (obtained in (2)) suspended in ml.
Co.) was mixed and injected subcutaneously. Two weeks later, a mixture of the same amount of the antigen and 0.1 ml of Freund's incomplete adjuvant was re-administered subcutaneously. Further, two weeks and four weeks later, the same booster immunization was performed, and two weeks after the fourth immunization, 200 μl of the supernatant of the PEFN antigen suspension was inoculated intravenously.

(4) Cell fusion: From the immunized mouse obtained in (3) above, 3 days after the final administration of the antigen, the spleen was excised to obtain cells used for cell fusion. The cells were suspended in a medium (hereinafter, abbreviated as IH medium) in which Iscove's medium and Ham's F-12 medium were mixed at a ratio of 1: 1. Mouse myeloma cells P3-X63-Ag.8U1 were subcultured in RPMI 1640 medium containing 10% fetal bovine serum under the conditions of 5% carbon dioxide and 95% air. Cell fusion is the method established by Koehler and Milstein et al. [Koehler, G. and Milstein, C .; Nature 256: 49.
5 (1975)]. Myeloma cells above 6.
45 × polyethylene glycol 6000 dissolved in 0.3 ml of IH medium was prepared by mixing 8 × 10 ⁷ cells and 3.5 × 10 ^{8 of} immunized lymphocytes obtained by the method described above, centrifuging.
(Hereinafter referred to as PEG6000) was dropped. PEG6
The 000 solution was previously warmed to 37 ° C. and slowly added dropwise. After 7 minutes, preheat IH medium to 37 ° C for 0.5 minutes per minute.
After adding l to make 10 ml, the mixture was centrifuged at room temperature at 700 rpm for 15 minutes to remove the supernatant. This cell precipitate was suspended in 200 ml of IH medium containing 20% fetal bovine serum, and 100 μl of the suspension was seeded in a 96-well microplate (Nunc). One day later, an IH medium containing 20% fetal bovine serum (hereinafter referred to as HAT medium) containing hypoxanthine 1 × 10 ⁻⁴ M, aminopterin 4 × 10 ⁻⁷ M, and thymidine 1.6 × 10 ⁻⁵ M was added. 100 μl was added to each well, and half of the medium was replaced with HAT medium every 3 days.
The cells thus grown are hybrid cells.

(5) Search for antibody-producing cells Solubilized PEFN antigen or similarly solubilized PEA was adjusted to 10 μg / ml in 0.01 M carbonate buffer (pH).
It was diluted with 8.5) and 100 μl thereof was put into each well of a 96-well immunoplate (manufactured by Nunc) and left overnight at 4 ° C. to bind PEFN or PE to the solid phase. 0.1
0.01M phosphate buffer containing 5M NaCl (pH
After washing with 7.0), to block excess binding sites,
0.01% containing 1% bovine serum albumin (BSA)
200 μl of M phosphate buffer was injected into each well and stored in a cool place until use. P obtained as described above
100 μl each of the hybrid cell culture supernatant was added to a 96-well immunoplate to which EFN or PE was bound, and 2 at room temperature.
Incubated for hours. After removing the culture supernatant and washing, a horseradish peroxidase (HRP) -labeled goat anti-mouse IgG antibody (manufactured by Kappel) was added as a secondary antibody and incubated at room temperature for 2 hours. After removing the secondary antibody and thoroughly washing the wells, a peroxidase substrate solution (0.0
100 μl of 2% H ₂ O ₂ and 0.15% o-phenylenediamine in pH 5.5 sodium citrate buffer was added, and the mixture was reacted at 25 ° C. for 10 minutes, and 2N-sulfuric acid 100 μl.
After the enzymatic reaction was stopped by adding, the absorbance at 492 nm was measured using an automatic colorimeter for microplates (MTP-32, manufactured by Corona) (ELISA).
Law). By this method, PEF has no binding property and PEF
An antibody-producing hybridoma that specifically reacts with N was obtained.

Example 4 (1) Fyn-GST (Glutathione S-Toausferase)
Expression of fusion protein (GSFN) A cDNA fragment (219 bp) encoding the amino acids from Met1 to Thr73 of Fyn protein was amplified by the PCR method to introduce NcoI and EcoRI sites. Nco
After enzymatic treatment with I and EcoRI, the digested fragment was cut out from an agarose gel and collected. This fragment was inserted into the NcoI / EcoRI sites of the plasmid pGEX · 2TN and introduced into E. coli DH5α. E. coli DH5α /
When OD ₅₉₅ of pGEX · 2TN-Fyn is 1, 0.
IPTG was added to 1 mM, and 20 hours later, the cells were collected. 50 ml of L buffer (50
mMTris-HCl pH 7.5, 25% sucrose, 0.5
% NP40, 5 mM MgCl ₂ ), ultrasonically disrupted and centrifuged, 3 ml of glutathione agarose beads was added to the supernatant, and the mixture was stirred overnight. Place the beads in the column,
WE buffer (20 mM Tris-HCl pH 7.5,
After washing with ₂ mM MgCl ₂ , 1 mM DTT), 5 mM
Glutathione, 50 mM Tris-HCl (pH 9.6)
The bound protein was eluted with, and this was dialyzed against WE buffer overnight. About 15 mg of fusion protein GSFN was obtained from 500 ml of culture. (2) Immunization BALB / c mice (♀ 8 weeks old) were supplemented with physiological saline.
50 μg of GSFN suspended in 1 ml and the same amount of Freund's complete adjuvant were mixed and injected intraperitoneally. Two
After a week, the mixture of the same amount of the antigen and 0.1 ml of Freund's incomplete adjuvant was re-administered intraperitoneally. Two weeks later, 20 μg of GSFN dissolved in physiological saline was inoculated intravenously.

(3) Cell fusion From the immunized mouse obtained in (2) above, 3 days after the final administration of the antigen, the spleen was excised and collected spleen cells (1.5 × 1).
0 ⁸⁾ and mouse myeloma cells P3-X63-Ag ·
8 UI (3.0 × 10 ⁷ pieces) were mixed and centrifuged. To the obtained precipitate, 50% polyethylene glycol 1500 dissolved in 1 ml of T medium (manufactured by Nippon Pharmaceutical Co., Ltd.) was added in advance to 37%.
Warm to ℃, add gradually for 1 minute to allow cell fusion, add 7 ml of T medium at 37 ℃ for 5 minutes, then 1500 rp
It was centrifuged for 5 minutes at m. Add 3 HAT medium to the cell pellet.
Add 0 ml and add 100 μl to a 96-well microplate.
The seeds were inoculated with each other, and the culture was continued while exchanging half of the HAT medium at 2-3 day intervals. In this way, proliferation of hybridomas was observed in 280 wells. (4) Search for antibody-producing cells GSFN antigen or GST was diluted with 0.01 M carbonate buffer (pH 8.5) to 10 μg / ml, and 100 μl thereof was placed in each well of a 96-well immunoplate at 4 ° C. Let stand overnight and solid phase GSFN or GST
Combined. 0.0 containing 0.15 M NaCl
After washing with 1 M phosphate buffer (pH 7.0), 200 μl of 0.01 M phosphate buffer containing 1% bovine serum albumin was injected into each well to block excess binding sites, and used until use. Stored in a cold place. 100 ml of the hybridoma culture supernatant was added to the 96-well immunoplate bound with GSFN or GST obtained as described above.
μl was added, and the mixture was reacted at room temperature for 1 hour. Remove the culture supernatant,
After washing goat anti-mouse I labeled with horseradish peroxidase
gG antibody (manufactured by Cappel) was added, and the mixture was reacted at room temperature for 1 hour. After removing the secondary antibody and washing the wells, a peroxidase substrate solution (0.02% H ₂ O ₂ and 0.15% o) was added.
-Phenylenediamine-containing pH 5.5 sodium citrate buffer) (100 μl) was added and reacted at room temperature.
After 30 minutes, the reaction was stopped with 100 μl of 1N-sulfuric acid, and the absorbance at 492 nm was measured using an automatic colorimeter for microplates. By this method, a monoclonal antibody-producing hybridoma that specifically binds to Fyn protein was obtained.

INDUSTRIAL APPLICABILITY Since the monoclonal antibody of the present invention specifically binds to non-receptor tyrosine kinase and has high binding ability, it can be advantageously used as a reagent for detecting / measuring various non-receptor tyrosine kinases. You can

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12N 15/06 G01N 33/577 B 9015-2J // A61K 39/395 P 9284-4C B 9284- 4C G01N 33/573 A 9015-2J (C12P 21/08 C12R 1:91)

Claims (16)

[Claims] 1. A monoclonal antibody whose immunoglobulin class belongs to IgG and exhibits binding to a specific region of a non-receptor tyrosine kinase. 2. The monoclonal antibody according to claim 1, wherein the non-receptor tyrosine kinase is a lyn gene product and does not cross-react with non-receptor tyrosine kinases other than the lyn gene product. 3. The monoclonal antibody according to claim 1, wherein the non-receptor tyrosine kinase is an fgr gene product and does not cross-react with non-receptor tyrosine kinases other than the fgr gene product. 4. The monoclonal antibody according to claim 1, wherein the non-receptor tyrosine kinase is a fyn gene product and does not cross-react with non-receptor tyrosine kinases other than the fyn gene product. 5. A cloned hybridoma consisting of a spleen cell of a mammal immunized with a non-receptor tyrosine kinase or a partial peptide of its specific region and a lymphoid cell of the same or different species. 6. The hybridoma according to claim 5, wherein the mammal is a mouse. 7. The hybridoma according to claim 5, wherein the lymphoid cells are myeloma. 8. A cell fusion of a mammalian spleen cell immunized with a non-receptor tyrosine kinase or a partial peptide of a specific region thereof and a homologous or heterologous lymphoid cell,
The method for producing a hybridoma according to claim 5, which comprises cloning. 9. The method according to claim 8, wherein the mammal is a mouse. 10. The method according to claim 8, wherein the lymphoid cells are myeloma. 11. The monoclonal antibody according to claim 1, wherein the hybridoma according to claim 5 is grown in a liquid medium or in the abdominal cavity of a mammal to produce and accumulate a monoclonal antibody, which is then collected. Manufacturing method. 12. The method according to claim 11, wherein the mammal is a mouse. 13. The method according to claim 11, wherein the lymphoid cells are myeloma. 14. A method for detecting and quantifying a non-receptor type tyrosine kinase, which comprises using the monoclonal antibody according to claim 1. 15. The detection / quantification method according to claim 14, wherein an enzyme immunoassay method is performed. 16. A method for purifying a non-receptor tyrosine kinase, which comprises using the monoclonal antibody according to claim 1.