JP3005284B2 - Antibodies to smooth muscle myosin heavy chain - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an antibody or an active fragment thereof that discriminates a single isoform of smooth muscle myosin heavy chain, a labeled product thereof, and a method for producing them. It is.

Background Art Myosin, a contractile protein that forms the thick filament of muscle cells, reacts with actin filaments to generate AT.
It has the function of converting chemical energy into mechanical work by decomposing P (ATPase activity). The myosin molecule has a subunit structure consisting of a heavy chain and a light chain. It is known that the myosin heavy chain is not a single molecule but exists in the form of several isoforms in the skeletal muscle and cardiac muscle heavy chains. These isoforms have different ATPase activities, and their phenotypes are changed by stresses such as developmental processes, hormone administration, and pressure overload.

On the other hand, it is known that smooth muscle has physiological characteristics completely different from skeletal muscle and cardiac muscle not only in myofibrils but also in calcium regulation mechanism and continuous contraction mode. However, little research has been done on the molecular structure and molecular diversity of smooth muscle myosin.

Heretofore, it has been reported that smooth muscle myosin heavy chain is separated into two bands by gel electrophoresis, and only one of them is phosphorylated (Eur. J. Biochem., 152 , 207-211 (1).
985), Circulation Res., 59 , 115-123 (1986), Am. J.
Physiol., 19 , C861-C870 (1986), J. Biol. Chem., 262 , 7
282-7288 (1987), Biochemistry, 27 , 3807-3811 (198
8) etc.). Nagai et al. Prepared c from rabbit smooth muscle.
Myosin heavy chain cDNA was cloned from a DNA library and its nucleotide sequence was determined (Proc. Natl. Ac
ad. Sci. USA, 85 , 1047-1051 (1988)).

In general, as one method for elucidating the physiological significance of various myosin isoforms or the function of regulating the expression of myosin isoforms in ontogeny, an immunological method using an antibody against myosin isoform has been adopted. I have.

However, the conventional antibody against smooth muscle myosin heavy chain has a problem that it is not possible to specifically distinguish and recognize the isoform of smooth muscle myosin heavy chain that has been recently revealed (J. Biol. Chem. , 262 , 7282-7288 (1
987)).

DISCLOSURE OF THE INVENTION The present inventors have continued research to obtain an antibody that has a completely different specificity from previously reported antibodies, that is, an antibody that recognizes a single isoform of myosin heavy chain in smooth muscle. As a result, the inventors succeeded in elucidating the difference in the amino acid sequence of the isoform of the smooth muscle myosin heavy chain, and succeeded in obtaining an antibody specific to the different amino acid sequence. The antibodies obtained are those which recognize and specifically recognize a single isoform of the smooth muscle myosin heavy chain.

Accordingly, the present invention provides an antibody or an active fragment thereof that recognizes a single isoform of smooth muscle myosin heavy chain; an antibody or an active fragment thereof labeled with a labeling agent; and an amino acid sequence of the isoform. A method for producing the above antibody or an active fragment thereof, comprising immunizing an animal with the oligopeptide or a complex thereof to obtain an antiserum thereof; and immunizing an animal with the oligopeptide or the complex thereof containing the amino acid sequence of the above isoform. Fusing the antibody-producing cells with myeloma cells to obtain a hybridoma that produces a monoclonal antibody recognizing the isoform of the smooth muscle myosin heavy chain; and culturing the hybridoma to obtain the monoclonal antibody. Obtaining an antibody, including The present invention relates to a method for producing the above antibody or an active fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows SDS-P of rabbit aortic smooth muscle myosin heavy chain
The results of AGE and immunoblotting are shown. Line 1 is the result of immunoblotting using anti-C1 antiserum after SDS-PAGE, and line 2 is the result of anti-C2 after SDS-PAGE.
The results when immunoblotting using antiserum and the results when line 3 was treated with amide black after SDS-PAGE are shown.

Fig. 2 shows SDS-PAG of smooth muscle myosin heavy chain in rabbit uterus.
E and the results of immunoblotting are shown. In the figure, lines 1 to 3 show the results when the same processing as in FIG. 1 is performed.

FIG. 3 shows a fluorescence micrograph of a rabbit arterial duct (Botaro duct) on the first day of life stained with anti-C1 antiserum or anti-C2 antiserum. Photo A is stained with anti-C1 antiserum, and Photo B is stained with anti-C2 antiserum.

FIG. 4 shows a fluorescence micrograph of a growing rabbit aorta stained with anti-C1 antiserum or anti-C2 antiserum. Photo C is stained with anti-C1 antiserum, and Photo D is stained with anti-C2 antiserum.

FIG. 5 shows fluorescence micrographs of the aorta of rabbits in fetal, neonatal (10 days after birth) and growing (30 days after birth) using anti-C1 or anti-C2 antiserum. Photos E, G and I are anti-C1 antiserum, photos F, H and J are anti-serum.
Stained with C2 antiserum. Photos E and F show the fetal period, photos G and H show the neonatal period, and photos I and J show the growing period.

FIG. 6 shows a fluorescence micrograph of a growing rabbit uterus stained with an anti-C1 antiserum or an anti-C2 antiserum. Photo K is stained with anti-C1 antiserum, and Photo L is stained with anti-C2 antiserum.

FIG. 7 shows a fluorescence micrograph of normal blood vessels of a human adult stained with anti-C2 antiserum.

FIG. 8 shows a fluorescence micrograph of a rabbit aorta stained with an anti-C3 antiserum. A indicates the fetal stage, and B indicates the anagen stage.

FIG. 9 shows a fluorescence micrograph of the umbilical artery stained with anti-C3 antiserum.

FIG. 10 shows the N of isoform of smooth muscle myosin heavy chain.
The amino acid sequence excluding the terminal portion is shown. Amino acid sequence of isoform SM-3 in upper row, isoform SM in middle row
-1 and the amino acid sequence of isoform SM-2 are shown below. As for the amino acid sequences of SM-1 and SM-2, only the amino acids different from the amino acid sequence of SM-3 are shown. In FIG. 10, (-) indicates that there is no corresponding amino acid, and * indicates the C terminal of each isoform. Each amino acid has a single letter symbol [Lubert
Stryer, Biochemistry, 2nd ed., P. 16 (1981), WHFree
man and Company, San Francisco], and the L-amino acids represented by each symbol are as follows.

A: alanine, R: arginine, N: asparagine, D: aspartic acid, C: cysteine, Q: glutamine, E: glutamic acid, G: glycine, H: histidine, I: isoleucine, L: leucine, K: lysine, M : Methionine, F: phenylalanine, P: proline, S: serine, T: threonine, W: tryptophan, Y: tyrosine, V: valine.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.

(1) Features of the Antibody of the Present Invention The antibody of the present invention is characterized in that it recognizes and recognizes a single isoform of smooth muscle myosin heavy chain, and further, does not show cross-reactivity with myosin heavy chain of cardiac muscle and skeletal muscle. Any of polyclonal antibodies (antisera) and monoclonal antibodies may be used as long as they have this characteristic. Here, “discriminating and recognizing” means that there is substantially no cross-reactivity between the isoforms of the smooth muscle myosin heavy chain.

In addition, “single isoform” means a single type of isoform, and the same type of isoform is similarly recognized as being derived from two or more species of animals as described later. It may be something.

The isoform of the smooth muscle myosin heavy chain recognized by the antibody of the present invention includes human, horse, cow, goat, sheep, pig, monkey, rabbit, guinea pig, rat, mouse, hamster, dog, cat and other mammal-derived isoforms. It may be of any origin, such as those derived from birds such as chickens, chickens, pigeons, and ducks, and is not particularly limited. The antibody of the present invention may be one that recognizes the corresponding isoforms of the smooth muscle myosin heavy chain derived from two or more animals redundantly. For example, the antibodies prepared in the examples described below were those that repeatedly recognized the corresponding isoforms of rabbit and human smooth muscle myosin heavy chains.
Among them, those that recognize human isoforms of smooth muscle myosin heavy chain can be examined histologically for the expression state of isoforms of smooth muscle myosin heavy chain in various human diseases. It is extremely meaningful for objectively diagnosing and studying the progress after the onset.

The present inventors have recognized to date that there are three isoforms in the smooth muscle myosin heavy chain, determined their amino acid sequences, and published SM-1 and SM-2 [ J. Biol. Chem., 264 , No. 17, 9734-9737 (1
Published June 15, 989)]. Three types of isoform N
The amino acid sequence excluding the terminal portion is shown in FIG. The three isoforms are defined as follows.

(1) Smooth muscle myosin heavy chain isoform 1 (hereinafter referred to as "SM-1"): an isoform that is expressed and phosphorylated in all phases of fetal, neonatal, and anagen during the ontogeny process; (2) Smooth muscle myosin heavy chain isoform 2 (hereinafter referred to as “SM-2”): an isoform that is not expressed in the fetal period, but is expressed in the neonatal and anagen stages of ontogenesis; Smooth muscle myosin heavy chain isoform 3 (hereinafter referred to as "SM-3"): an isoform that is expressed in the fetal and neonatal stages of ontogenesis but not expressed in the anagen stage [Fetal isoform] .

The recognition site of the antibody of the present invention is an amino acid sequence portion whose amino acid sequence differs depending on the isoform of the smooth muscle myosin heavy chain. That is, an antibody capable of recognizing a portion that is specifically present in each isoform in the amino acid sequence shown in FIG. 10 is an example of the “antibody that recognizes (discriminates) a single isoform” of the present invention. . Examples of the amino acid sequence portion specifically present in each isoform that can be used to prepare the antibody of the present invention include, for example, SM-1
Is any sequence portion present 50 to 60 amino acid residues from the C-terminal. For SM-2 from C terminal
Any sequence present in 10-20 amino acid residues.

As for SM-3, SM-1 and SM- in FIG.
2 is an arbitrary sequence selected from portions different from the amino acid sequence of 2.

For example, SM-1 and SM of rabbit smooth muscle myosin heavy chain
The amino acid sequence of -2 is the same except for the vicinity of the C-terminal. However, the amino acid sequence around the respective C terminals site of SM-1 and SM-2 has the formula [I] and [II] (SM-1) H 2 N- ...... -SKLRRGNETSFVPTRRSGGRRVIENADGSEEEVDARD
ADFNGTKSSE-COOH [I] (SM-2) H ₂ N... -SKLRGPPPQETSQ-COOH [II], and the amino acid sequence and length after “SKL” are greatly different.

SM-3 differs from SM-1 and SM-2 in many parts of the amino acid sequence. In particular, the amino acid sequence near the C-terminal of SM-3 of rabbit smooth muscle myosin heavy chain has the formula [II
I] (SM-3) H ₂ N-----NRLRRGGPISFSSSRSGRPQLHIEGASLELSDDDTES
KTSDVNETQPPQSE-COOH [III], which differs from the amino acid sequence near the C-terminal of SM-1 and SM-2. The antibody of the present invention recognizes, for example, the difference between the isoforms on the amino acid sequence near the C-terminal portion of the smooth muscle myosin heavy chain as described above. It became possible to distinguish and recognize a single isoform of muscle myosin heavy chain.

(2) Immunization antigen The antigen (immunization antigen) used for producing the antibody of the present invention is an amino acid sequence having the same or extremely high homology to the amino acid sequence of a portion different between each isoform of the smooth muscle myosin heavy chain. Oligopeptide having

Specific examples of such oligopeptides include, for example, an oligopeptide represented by the following formula [IV] and SM-2 suitable for producing an antibody that specifically recognizes SM-1.
Oligopeptide represented by the following formula [V] suitable for preparing an antibody specifically recognizing SM-3, and formula [V] suitable for preparing an antibody specifically recognizing SM-3
The oligopeptide represented by I] can be exemplified.

H ₂ N-RRGNETSFVPTRRSGGRRVIENADGSEEEVDARDADFNGTKSS
E-COOH [IV] H ₂ N-RGPPPQETSQ-COOH [V] H ₂ N-NRLRRGGPISFSSSRSGRPQLHIEGASLELSDDDTESKTSDVN
ETQPPQSE-COOH [VI] The oligopeptides represented by the above formulas [IV], [V] and [VI] may use the whole of the oligopeptide itself as an immunizing antigen. Preferably, six or more peptide fragments may be used as the immunizing antigen.

The oligopeptide may be prepared by any method including a method of chemically synthesizing, a method of synthesizing by genetic engineering using a corresponding DNA sequence, and a method of enzymatically preparing a smooth muscle myosin heavy chain. When the number of amino acid residues of the oligopeptide to be prepared is short (for example, around 20 residues), a method of chemically synthesizing is particularly convenient and preferable. In the case of chemically synthesizing an oligopeptide, if the amino acid sequence of the isoform of the smooth muscle myosin heavy chain is known, three or more consecutive portions of the amino acid sequence that differ between the isoforms Preferably, an arbitrary amino acid sequence consisting of six or more amino acids is selected and chemically synthesized. Further, even when the amino acid sequence of each isoform is unknown, for example, hydrazinolysis method,
Tritium labeling, carboxypeptidase, dinitrofluorobenzene (DNP), phenylisothiocyanate (PTC), dansyl chloride (DNS)
Method, cyanic acid method, direct Edman method, Dansyl-Edman method [for the above, protein chemistry II;
Biochemistry Experiment Course 1, edited by The Chemical Society of Japan, pp. 118-211 (1976
FD) and FAB-Mass Spectrometry [Protein Chemistry (above): Seminar on Seismic Chemistry 2, edited by The Biochemical Society of Japan, p. 375 (19
1987)], and a partial amino acid sequence of each isoform may be determined, and then chemically synthesized in the same manner as when the amino acid sequence is known.

The method for chemically synthesizing an oligopeptide is not particularly limited, and may include an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride method, a DCC (N, N'-dicyclohexylcarbodiiminod) method, and an active ester. Method (p-nitrophenyl ester method, N-hydroxysuccinimide ester method, cyanomethyl ester method, etc.), method using Woodward reagent K, carbonyldiimidazole method, redox method, DC
A commonly used method such as the C / additive (eg, HONB, HOBT, HOSu) method may be appropriately selected and performed. These chemical synthesis methods may be either a liquid phase method or a solid phase method. As a synthesis procedure, a stepwise method in which amino acids are condensed one by one, and a block consisting of several amino acids are condensed. Any of the block condensation methods can be applied. For the specific procedures and techniques of these synthetic methods, reference can be made to books [[The Pept
ides "Vo1.1, Academic Press, New York, USA (1966)
"Peptide synthesis" (Maruzen Co., Ltd., 1975)].

Oligopeptides prepared in this manner may be used directly as immunizing antigens, but when antigenicity is low,
It is preferred to bind or adsorb to a suitable carrier to form a complex, which is used as an immunizing antigen.

As a carrier for binding the oligopeptide, a natural or synthetic polymer carrier commonly used for preparing an antibody against a hapten antigen can be used. Natural polymer carriers include, for example, animal serum albumins such as bovine serum albumin, rabbit serum albumin, human serum albumin; bovine serum globulin;
Animal serum globulins such as rabbit serum globulin, human serum globulin, sheep serum globulin; animal thyroglobulins such as bovine thyroglobulin and rabbit thyroglobulin; animal hemoglobins such as bovine hemoglobin, sheep hemoglobin and human hemoglobin; keyhole limpet Hemocyanins such as hemocyanin can be exemplified. Examples of the synthetic polymer carrier include a polymer or copolymer of an amino acid such as polylysine, polyglitamate, and lysine-glutamic acid copolymer;
Styrene, chlorostyrene, α-methylstyrene, divinylbenzene, sodium styrenesulfonate, (meth) acrylic acid, ethyl (meth) acrylate, (meth)
Acrylonitrile, (meth) acrolein, (meth) acrylamide, butadiene, isoprene, vinyl acetate,
Vinylpyridine, N-vinylpyrrolidone, vinyl chloride,
Aromatic vinyl compounds such as vinylidene chloride, α, β-unsaturated carboxylic acids or their salts; polymers or copolymers prepared from α, β-unsaturated nitrile compounds, vinyl halide compounds, conjugated diene compounds, etc. Various latexes can be exemplified.

The binding between such a carrier and the oligopeptide may employ any binding method such as a physical adsorption method, a covalent bonding method, and an ionic bonding method.

As a specific example of the physical adsorption method, for example, the physical adsorption method can be carried out by slowly stirring latex particles and an oligopeptide at room temperature for 1 to 3 hours (for details, see Endoor
inology, 93 , 1092 (1973), JP-A-61-43124, etc.). Covalent bonding method, amino group present in the carrier, carboxyl group, in the presence of an excess amount of a binding agent capable of binding the oligopeptide and the carrier by activating various reactive residues such as a sulfhydryl group, In various buffers such as a borate buffer and a phosphate buffer, the oligopeptide is used in an amount of 1 to 50 times mol per mol of the polymer carrier, and the reaction temperature is 0
The reaction can be carried out at 5050 ° C. for 30 minutes to 30 hours.

As the binding agent used for binding the oligopeptide and the carrier, those commonly used for binding the hapten or peptide to the carrier or the enzyme can be used. Examples of such a binder include bisdiazonated compounds such as bisdiazotized benzidine, bisdiazotized 3,3'-dianisidine, which crosslink tyrosine, histidine, and tryptophan; glyoxal, malondialdehyde, glutar, which crosslink amino groups. Dialdehyde compounds such as aldehyde, succinaldehyde and adipaldehyde; diisocyanate compounds such as toluene-2,4-diisocyanate and xylene diisocyanate; 2,4-dinitro-1,5-difluorobenzene, p,
halonitrobenzene compounds such as p'-difluoro-m, m'-dinitrophenylsulfone; imide ester compounds such as diethylmalonimidate; N, N'-O-phenylenedimaleimide which crosslinks thiol groups, N, N ' −
dimaleimide compounds such as m-phenylenedimaleimide; N- (m-maleimidobenzoyloxy) -succinimide, 4- (maleimidomethyl) benzoic acid-N-hydroxysuccinimide ester, m-maleimidobenzoyl-, which crosslinks an amino group and a thiol group. Maleimide carboxy-N-hydroxysuccinimide compounds such as N-hydroxysuccinimide ester and 4- (maleimidomethyl) -cyclohexane-1-carboxyl-N'-hydroxysuccinimide ester; N, N-dicyclohexylcarbodiimide which crosslinks amino and carboxy groups , N-ethyl-N'-dimethylaminocarbodiimide, 1-ethyl-3-diisopropylaminocarbodiimide, 1-cyclohexyl-3- (2-morpholinyl-4-ethyl) cal Carbodiimide compounds such as diimidomethyl-P-toluenesulfonate; isoxazolium salt compounds such as N-ethyl-5-phenylisoxazolium-3'-sulfonate; alkylchloroforms such as ethyl chloroformate and isobutyl chloroformate Formate compounds can be exemplified. What is necessary is just to select and use suitably from these binding agents according to the kind of the binding site of the oligopeptide used.

The immunizing antigen (complex of the oligopeptide and the carrier) thus prepared is isolated and purified according to a conventional method such as a dialysis method or a gel filtration method, and used for the production of the next antibody.

(3) Production of Antibody As the immunizing antigen, the oligopeptide itself or a complex of the oligopeptide prepared as described above and a carrier is used. The complex of the oligopeptide and the carrier is the carrier 1
It is preferable to use, as the immunizing antigen, one to which 2 to 50 times mol of the oligopeptide is bound per mole, particularly one to which 5 to 20 times mol of the oligopeptide is bound per 1 mol of the carrier.

When an antiserum is produced using such an immunizing antigen, the immunizing antigen is administered to a different animal different from the animal from which the immunizing antigen is derived, and a single isoform of the smooth muscle myosin heavy chain is administered in vivo. Can be produced according to a conventional method of producing an antibody that recognizes and distinguishing the antibody.

That is, the heterologous animal to which the immunizing antigen is administered may be any of bovine, horse, sheep, goat, rat, mouse, guinea pig, hamster, dog, cat, pig, rabbit, monkey, pigeon, chicken, duck, etc. Particularly preferred are mice, rats, guinea pigs, rabbits.

The administration of the antigen to such a heterologous animal may be performed according to a conventional method, for example, various adjuvants such as complete Freund's adjuvant, incomplete Freund's adjuvant, alum, adjuvant aluminum hydroxide, and B. pertussis adjuvant. A suspension of a band, preferably complete Freund's adjuvant, and the immunizing antigen described above may be prepared and administered to the animal intravenously, intraperitoneally, subcutaneously, or intradermally.

The dose is 0.01 to 10 mg / animal as an immunizing antigen (oligopeptide or a complex of an oligopeptide and a carrier) when using rabbits and guinea pigs as animals, and 0.001 when using mice and rats. ~ 1 mg / animal is preferred.

By performing the same additional immunization as described above about 1 to 5 times every 1 to 4 weeks after the initial administration, it is possible to obtain an antibody that can distinguish and recognize a single isoform of the smooth muscle myosin heavy chain.

The antibody thus obtained can be obtained as an antiserum by collecting blood 1-2 weeks after the final immunization and centrifuging the blood. When antibody purification is required, further, a selective fractionation method using a difference in solubility (eg, salting out, alcohol precipitation, etc.), a fractionation method using a difference in electric charge (eg, ion exchange chromatography,
Electrophoresis), fractionation using molecular weight differences (eg, ultracentrifugation, gel filtration, etc.), fractionation using specific binding to ligands (eg, affinity chromatography (eg, using a protein A column)) And the like. Antibodies present in the antiserum may be fractionated and purified for each antibody class by appropriately combining conventional methods, and smooth muscle myosin weight may be determined using the immobilized antigen obtained by immobilizing the immunizing antigen. Only those antibodies that recognize the single isoform of the chain in a discriminating manner may be fractionated and purified.

Next, preparation of a monoclonal antibody will be described.
The monoclonal antibody can be prepared by appropriately applying a known cell fusion method, a transformation method using an EB virus, or the like.

The cell fusion method suitable for mass production of monoclonal antibodies will be described as an example. For example, a large amount of a monoclonal antibody that recognizes and recognizes a single isoform of smooth muscle myosin heavy chain can be obtained in the following procedure.

a) Preparation of antibody-producing cells: A spleen cell from an animal obtained by immunizing a heterologous animal, preferably a mouse, rat, rabbit, hamster, chicken or the like, with the above-mentioned immunizing antigen in the same manner as in the preparation of antiserum, and acquiring antibody-producing ability And antibody-producing cells such as lymph node cells and peripheral blood cells are obtained by a conventional method.

b) Preparation of myeloma cells As myeloma cells, cell lines derived from various animals such as mice, rats, chickens, and humans and available to those skilled in the art are used. Cell lines used include:
Those which have drug resistance and cannot survive in a selection medium in an unfused state, but can survive only in a state fused to an antibody-producing cell are preferable. Usually, an 8-azaguanine resistant strain is used, and this cell line is hypoxanthine-phosphoribosyltransferase (Hypoxanthine phosphoritransferase).
bosyl transferase) and cannot grow on hypoxanthine / aminopterin / thymidine (HAT) medium. In addition, a so-called non-secretory cell line that does not secrete immunoglobulin as a cell property is preferable.

Specific examples of myeloma cell lines include P3X63Ag8 (ATCC
TIB-9) (Nature, 256 , 495-497 (1975)), P3X63A
_{g8U.1 (P 3 U 1) (} ATCC CRL-1597) (Current Topics i
n Microbiology and Immunology, 81 , 1-7 (1978))
P3X63Ag8.653 (ATCC CRL-1580) (J. Immunology, 123 ,
1548-1550 (1979)), P2 / NSI / 1-Ag4-1 (ATCC TIB
-18) (European J. Immunology, 6 , 511-519 (197
6)), Sp2 / O-Ag14 (ATCC CRL-1581) (Nature, 276 ,
269-270 (1978)) and mouse myeloma cell lines; 21
0.RCY.Ag1.2.3 (Y3-Ag1.2.3) (ATCC CRL-1631) (N
ature, 277, 131-133 (1979) ) a rat myeloma cell lines such _{as; U-266-AR 1 (} Proc.Natl.Acad.Sci.USA, 77, 5
429 (1980)), GM1500 (Nature, 288 , 488 (1980), KR
-4 (Proc. Natl. Acad. Sci. USA, 79 , 6651 (1982))
And other human myeloma cell lines.

c) Cell fusion For cell fusion, select myeloma cells suitable for the antibody-producing cells. Cell fusion was performed using Eagle's minimal basic medium (MEM), Dulbecco's modified Eagle's medium (DMEM), R
10 ⁷ to 10 ⁸ / in animal cell culture medium such as PEI 1640 medium
Efficient fusion can be achieved by mixing ml myeloma cells and antibody-producing cells at a mixing ratio of 1: 4 to 10 and contacting the cells at 37 ° C. for 1 to 10 minutes. In order to promote cell fusion, a fusion promoter such as polyethylene glycol (PEG) having an average molecular weight of 1000 to 6000, polyvinyl alcohol, and Sendai virus can be used.

Antibody-producing cells and myeloma cells can also be fused using a commercially available cell fusion device utilizing electric pulses.

d) Selection of hybridoma in selective medium As a means for selecting a target hybridoma from cells after the cell fusion treatment, a method utilizing selective growth of cells in a selective medium can be used. For example, the cell suspension is converted to RPMI containing 10-15% fetal calf serum (FCS)
After appropriately diluting with I1640 medium or the like, spread about 10 ⁵ to 10 ⁶ / well on a microplate, add a selection medium (for example, HAT medium) to each well, and replace the selection medium as appropriate. Do. When an 8-azaguanine-resistant strain is used as a myeloma cell and an HAT medium is used as a selection medium, unfused myeloma cells are killed by about 10 days of culture, and antibody-producing cells that are normal cells are in vitro (in vitro).
In vitro), cells that grow from day 10 to 14 of culture can be obtained as hybridomas because they cannot grow for a long time.

e) Search for hybridomas producing monoclonal antibodies that recognize and discriminate a single isoform of smooth muscle myosin heavy chain Search for hybridomas that produce a monoclonal antibody that recognizes a single isoform of smooth muscle myosin heavy chain Can be performed by enzyme-labeled immunoassay (ELISA), radioisotope immunoassay (RIA), or the like. For example, a 96-well EL adsorbed with the aforementioned immunizing antigen
The culture supernatant containing the monoclonal antibody is added to the ISA microplate to react with the immunizing antigen, and then the bound specific antibody is reacted with the enzyme-labeled anti-immunoglobulin antibody, or the biotin-labeled anti-immunoglobulin antibody is reacted. The avidin D-enzyme label is reacted, and an enzyme substrate is added to each well to develop color. Based on the presence or absence of color development, a well to which a culture supernatant containing a hybridoma that produces an antibody capable of binding to the isoform of smooth muscle myosin heavy chain can be discriminated, and the desired hybridoma can be searched.

f) Cloning Cloning of the hybridoma can be performed by a limiting dilution method, a soft agar method, a fibrin gel method, a fluorescence excitation cell sorter method, or the like.

g) Production of monoclonal antibody As a method for producing a monoclonal antibody from the thus obtained hybridoma, a usual cell culture method, ascites formation method and the like can be adopted.

In cell culture, hybridomas are
The cells can be cultured in an animal cell culture medium such as an S-containing RPMI 1640 medium or a serum-free medium, and an antibody can be obtained from the culture supernatant.

In the ascites formation method, animals whose hybridomas and major histocompatibility are matched are given intraperitoneal injection of mineral oil such as pristane (2,6,10,14-tetramethylpentadecane).
For example, in the case of a mouse, about 10 ⁶ to 10 ⁷
Per animal intraperitoneally. Hybridomas form ascites tumors in about 10 to 18 days and produce antibodies at high concentrations in serum and ascites, so that antibodies can be obtained from the ascites obtained.

If antibody purification is required, ammonium sulfate precipitation, DE
Ion exchange chromatography using an anion exchanger such as AE cellulose; known methods such as affinity chromatography using protein A-Sepharose and molecular sieve chromatography (gel filtration) are appropriately selected and combined. It can be purified.

(4) Antibody fragment The active fragment of the antibody may be any as long as it retains the characteristics of the antibody of the present invention (for example, various fragments such as F (ab ') ₂ , Fab', and Fab). . Preparation of these active fragments can be carried out by applying a known method such as a method of degrading the purified antibody with a protease such as papain, pepsin, and trypsin. 5, edited by the Biochemical Society of Japan, p. 89 (1986)].

(5) Labeled antibody or active fragment As a labeling agent for labeling (labeling) the antibody or active fragment, an enzyme (for example, β-galactosidase, peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, etc.) ;
Coenzyme / prosthetic group (eg, FAD, FMN, ATP, heme, etc.); in vivo ligand / receptor (eg, biotin, avidin, streptavidin, etc.); fluorescein derivatives [eg, fluorescein isothiocyanate (FITC), fluorescein thiol Fluvamil), rhodamine derivatives (eg, tetramethylrhodamine B isothiocyanate), umbelliferone, 1-anilino-8-naphthalenesulfonic acid, and other fluorescent substances; luminol derivatives [eg, luminol, isoluminol, N- ( 6-aminohexyl) N-ethylisoluminol and the like; and radioisotopes ( ¹²⁵ I, ¹³¹ I, ³ H, ¹¹¹ In, and ^99m Tc). These may be bound directly to the antibody or active fragment or indirectly via other substances (anti-immunoglobulin antibodies, biotin-avidin system, etc.). The binding between the antibody or the active fragment thereof and the labeling agent can be performed, for example, by the method described in “Seizoku Chemistry Laboratory Course 5 Immunobiochemical Research Method” (Tokyo Kagaku Dojin, 1986) No. 102
Pp. 112; "Sequence radioimmunoassay" (supra), EP, B
The method may be appropriately selected from known methods described in 1,0163041 and the like.

(6) Utility of the Antibody of the Present Invention The antibody of the present invention distinguishes and recognizes a single isoform of the smooth muscle myosin heavy chain, and thus elucidates, for example, the mechanism of regulating the expression of the smooth muscle myosin heavy chain isoform in ontogeny. Useful as a biochemical reagent or diagnostic reagent, specifically, a tissue staining reagent or the like.

Among them, those that recognize human isoforms of smooth muscle myosin heavy chain can be examined histologically for the expression state of isoforms of smooth muscle myosin heavy chain in various human diseases. It is extremely useful for objectively diagnosing and studying the progress after the onset.

In addition, those antibodies or active fragments thereof labeled (labeled) with a labeling agent such as a radioisotope can be administered to the body of an animal, including a human, and subjected to diagnostic imaging to determine the site and degree of vascular damage. It is useful as a diagnostic agent in the body for diagnosis.

Further, the antibody of the present invention or an active fragment thereof, or a product obtained by binding a labeling agent to the antibody or the active fragment thereof may be used for a body fluid (blood,
It is also useful as an immunoassay reagent for quantitatively or qualitatively measuring the smooth muscle myosin heavy chain in urine and the like. The immunoassay is performed by a known method [Hiroshi Irie, edited by Radio Immunoassay, Kodansha Co., Ltd., May 1, 1979]
Ed. Eiji Ishikawa et al., Enzyme Immunoassay, Second Edition,
Published by Medical Shoin, December 15, 1982; Methods in ENZY
MOLOGY, Vol.92, Immunochemical techniques, Part E: Mon
oclonal Antibodies and General Immunoassay Method
s, Academic Press, Inc.], for example, by a sandwich method. Such an immunoassay makes it possible to diagnose various diseases such as vascular disorders, arteriosclerosis, and cancer.

EXAMPLES Hereinafter, the present invention will be described specifically with reference to Examples.

Example 1 Rat Antiserum Against Smooth Muscle Myosin Heavy Chain Isoform Rabbit Smooth Muscle Myosin Heavy Chain Isoform 1 (SM-
Synthetic peptide C1 (H ₂ N-ARDADFNGTKSSE-COOH) corresponding to 13 amino acid residues from the C-terminal of 1) and 10 amino acids remaining from the C-terminal of isoform 2 (SM-2) of rabbit smooth muscle myosin heavy chain Synthetic peptide C2 (H ₂ N-RGP
Synthetic peptide C3 (H ₂ N-TSDVNETQPPQSE) corresponding to 13 amino acid residues from the C-terminal part of PPQETSQ-COOH) and isoform 3 of rabbit smooth muscle myosin heavy chain (embryo type: SM-3)
-COOH) as an antigen, and these antigens and bovine serum albumin (BSA) were combined with the normal maleimide method [Immunological Experiment Procedure XI, (Immunological Society of Japan, published in 1982) Nos. 3529-3534.
Page]] and a complex of synthetic peptide and BSA (C
1-BSA, C2-BSA, C3-BSA) were prepared. The complex thus obtained has an average of 10 synthetic peptides per BSA molecule.
It was a molecular bond.

Next, C1-BSA was dissolved in physiological saline to a concentration of 1 mg / ml, and this was mixed with complete Freund's adjuvant at a ratio of 1: 1 to prepare a suspension. Rats (female, 6 weeks old) were administered intradermally on the back.

Further, the same amount of the suspension was administered four times every two weeks for booster immunization.

Blood was collected 10 days after the final immunization, left at 4 ° C. for 16 hours, and centrifuged at 3000 rpm for 10 minutes to obtain a supernatant, which was used as rat anti-C1 antiserum.

C2-BSA was treated in the same manner as C1-BSA to obtain rat anti-C2 antiserum.

Further, C3-BSA was dissolved in physiological saline to a concentration of 1 mg / ml, and this was mixed with complete Freund's adjuvant:
A suspension was prepared by mixing at a ratio of 1
Was administered intradermally to the back of BALB / c mice (female, 6 weeks old).
Hereinafter, an antiserum was obtained in the same manner as C1 and C2, and this was
Antiserum was used.

Example 2 Specificity of at-anti-C1 and anti-C2 antisera The specificity of rat anti-C1 antiserum and rat anti-C2 antiserum was confirmed by immunoblotting.

Preparation of Smooth Muscle Myosin Heavy Chain Rabbit aorta and rabbit uterine tissues were extracted 10 times in volume from an extraction solution (50 mM sodium dihydrogen phosphate, 1 mM ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (EG
TA), 0.125 mM phenylmethanesulfonyl fluoride (P
MSF), pH 7.0) and homogenized at 4 ° C at 1000 rp
The precipitate was obtained by centrifugation at m for 10 minutes. Gub the total amount of the resulting precipitate
a-Straub solution (150 mM sodium dihydrogen phosphate, 300 mM
Sodium chloride, 10mM ATP, 1mM EGTA 0.125mM PMS
F, 1 mM 2-mercaptoethanol, pH 6.7), and gently stirred at 4 ° C for 1 hour.
After centrifugation for minutes, a supernatant containing the smooth muscle myosin heavy chain was obtained. After adding an equal volume of glycerin, the obtained supernatant was stored at −20 ° C. until use.

Smooth muscle myosin heavy chain contained in the extract obtained by electrophoresis
Each isoform was separated by performing SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

SDS-PAGE was carried out at 60 V for 12 hours using a polyacrylamide gel on which a 3% concentrated gel and a 4% separation gel were layered.

As a sample for electrophoresis, the same amount of a reducing solution (2% SDS, 5% 2-mercaptoethanol, 20% glycerol, 0.01% bromophenol blue- (BPB) in 100 mM Tris -Hydrochloric acid buffer (pH 6.
8)) was added, and the mixture was heated and reacted at 100 ° C. for 2 minutes.

A nitrocellulose membrane was placed on the separation gel obtained by SDS-PAGE of immunoblotting, and the protein was transferred to the nitrocellulose membrane by applying a current of 60 V for 12 hours. The nitrocellulose membrane thus obtained was cut into strips along the electrophoresis line of the sample, and a part thereof was stained for protein with amide black. The remaining membrane was immersed in a 3% gelatin solution, reacted at 37 ° C. for 1 hour to perform a blocking treatment, and then diluted to 1/50 concentration with phosphate buffered saline (PBS) to obtain rat anti-C1 Antiserum and rat anti-C2 antiserum
The reaction was performed at 37 ° C. for 1 hour. After the reaction, the nitrocellulose membrane was washed by immersing it in PBS (containing 0.05% Tween 20) for 5 minutes, and the same washing was repeated three more times.
Was reacted with a peroxidase-labeled anti-rat IgG antibody (manufactured by EY Laboratories) diluted to a concentration of 1/200 using the above method at 37 ° C. for 1 hour. After the reaction, the nitrocellulose membrane was washed with PBS (0.05
% Tween 20), and then washed with a substrate solution (30 mg of color developer (manufactured by Biorat), 10 m of methanol)
(1) containing 50 ml of PBS and 30 μm of hydrogen peroxide solution) to form a color, and when the color was appropriately developed, the reaction was stopped by washing with water.

First SDS-PAGE and immunoblotting results
This is shown in FIG. 2 and FIG.

Fig. 1 shows SDS-P of smooth muscle myosin heavy chain in rabbit aorta.
The results of AGE and immunoblotting are shown. Line 1 shows the result of immunoblotting using anti-C1 antiserum after SDS-PAGE, and line 2 shows the result of SDS-PAGE.
-Results after immunoblotting using anti-C2 antiserum after -PAGE, and line 3 shows the results when treated with amide black after SDS-PAGE.

Fig. 2 shows SDS-PAG of smooth muscle myosin heavy chain in rabbit uterus.
It shows the results of E and immunoblotting. Lines 1 to 3 in the figure show the results when the same processing as in FIG. 1 is performed.

From FIGS. 1 and 2, the anti-C1 antiserum was SM-1 only,
It was confirmed that the anti-C2 antiserum specifically reacted with only SM-2.

Example 3 Immunofluorescent tissue staining Rabbit tissue (rabbit fetal, neonatal and growing aorta, rabbit botarotube, growing rabbit uterus)
And human tissue (human adult normal blood vessels, human umbilical artery) were cryostated using a cryostat, washed by immersion in PBS for 5 minutes, and this washing was repeated three times. Tissue sections were reacted with rat antiserum (anti-C1 antiserum, anti-C2 antiserum) or mouse antiserum (anti-C3 antiserum) diluted with PBS at a concentration of 1/10 to 1/50 for 1 hour at 37 ° C. Thereafter, the plate was washed by immersion in PBS for 5 minutes, and this washing was repeated three times. Washed tissue sections 1/1 in PBS
Fluorescein isothiocyanate (FITC) -labeled anti-rat IgG antibody or FITC-labeled anti-mouse IgG antibody (manufactured by EY Laboratories) diluted to a concentration of 0 to 1/50 at 37 ° C.
After reacting for 1 hour, washing with PBS and glycerol treatment were performed. As a result of observing the tissue section thus obtained under a fluorescence microscope, the following items were confirmed.

The rabbit arterial tract (Botaro tract) on the first day of life was confirmed (stained) with anti-C1 and anti-C2 antisera. Although SM-2 was scarcely present in general vascular smooth muscle on the first day of life, it was revealed that SM-2 was abundantly present in the Botaro tube, which is a blood vessel having the characteristic of closing immediately after birth (No. 3). See figure).

Both the anti-C1 antiserum and the anti-C2 antiserum recognized (stained) rabbit vascular smooth muscle during growth (see FIG. 4).

The anti-C1 antiserum recognized (stained) rabbit vascular smooth muscle in the fetal period, neonatal period (10 days after birth) and growing period (30 days after birth). The anti-C2 antiserum recognized neonatal and growing vascular smooth muscle, but did not recognize (stain) fetal vascular smooth muscle. That is, tissue staining using both anti-C1 and anti-C2 antisera could reveal changes in the expression of smooth muscle myosin heavy chain isoform in blood vessels (see FIG. 5).

Both the anti-C1 antiserum and the anti-C2 antiserum recognized (stained) rabbit uterine smooth muscle during growth. In consideration of the above results, it was shown that SM-1 and SM-2 coexist in the smooth muscle cells during the growth phase (see FIG. 6).

Normal human normal vascular smooth muscle cells were recognized by the anti-C2 antiserum (see FIG. 7).

The anti-C3 antiserum recognized (stained) fetal rabbit vascular smooth muscle, but did not recognize (stain) vascular smooth muscle during growth (see FIG. 8).

The human umbilical artery was recognized (stained) by anti-C3 antiserum, and it was confirmed that the anti-C3 antiserum recognized not only rabbits but also human fetal smooth muscle myosin heavy chain isoform (SM-3). (See FIG. 9).

From the above results, the antibody of the present invention is useful as a biochemical or diagnostic reagent used for tissue staining, and the antiserum prepared in Example 1 can be used to distinguish human smooth muscle myosin heavy chain isoforms. It turned out to be recognizable.

Tissue samples of rabbit myocardium and skeletal muscle were prepared in the same manner as described above, and anti-C1 antiserum, anti-C2 antiserum and anti-C3
Staining was performed in the same manner as above using antiserum, but these tissues were anti-C1 antiserum, anti-C2 antiserum or antisera.
No staining was observed with the C3 antiserum.

Example 4 Monoclonal Antibody Against Smooth Muscle Myosin Heavy Chain Isoform Preparation of Monoclonal Antibody C1-BSA prepared in Example 1 was dissolved in physiological saline to a concentration of 1 mg / ml, and this solution and complete Freund's adjuvant were used. Were mixed in a 1: 1 volume to prepare a suspension. This suspension was intraperitoneally administered to BALB / c mice (female, 6 weeks old) at 50 μl, and the same amount of the suspension was administered three times every two weeks in the same manner to carry out booster immunization. C1-BSA (100μ
(g / ml) 100 μ was injected intravenously for final immunization.

Three days after the final immunization, spleen cells of the mouse were excised and washed with Eagle's minimal basic medium (MEM). The mouse myeloma P3 × 63Ag8U.1 (P ₃ U ₁ ) (ATCC CRL-1597) was washed with MEM, and the spleen cells and P ₃ U ₁ were mixed at a ratio of 10: 1, followed by centrifugation. % Polyethylene glycol (PEG) 100
1 ml of a MEM solution containing 0 was gradually added to perform cell fusion.
Additionally, suspensions such as 10ml by adding MEM solution, a 3 × 10 ⁴ cells /0.1ml pellet obtained by centrifugation in 10% fetal calf serum (FCS) RPMI 1640 medium containing as P ₃ U ₁ Then, 0.1 ml of each well was dispensed into a 96-well microplate. One day later, 0.1 ml of HAT medium was added, and then 3 to 4
Every day, half of the medium was replaced with fresh HAT medium.

On day 10 after the fusion, the synthetic peptide C1 (1 μg /
ml), and 50 μl of the culture supernatant was added to a 96-well polyvinyl chloride (PVC) plate that had been blocked with 3% gelatin, followed by biotinylated horse anti-mouse Ig.
G (Vector) solution (50 μ) was added and reacted at room temperature for 1 hour.

After the reaction, each well was thoroughly washed with PBS, and avidin D-
After adding 50 μ of a peroxidase (manufactured by Vector) and reacting at room temperature for 30 minutes, the plate was washed three times with PBS, and a substrate solution (4-aminoantipyrine (0.25 mg / ml), phenol (0.25 mg / ml), 0.425 After adding 200μ (M hydrogen peroxide) and reacting at room temperature, the absorbance at 550nm of each well is measured using a 96-well microplate photometer to produce a monoclonal antibody that specifically reacts with the synthetic peptide C1. A hybridoma was obtained (No. 1
table).

Hybridomas producing monoclonal antibodies specifically reacting with synthetic peptide C2 or synthetic peptide C3 were obtained in the same manner as described above (Table 1).

Each of the hybridomas thus obtained was cloned by the limiting dilution method to produce 10 hybridoma strains (1B4, 1C8, 1C10, 1H9, 2H) that produce an antibody against the synthetic peptide C1.
G10, 3C8, 4C6, 4E10, 5C4, 5D5, 5D10) and two hybridoma strains producing antibodies against the synthetic peptide C2 (1A1
0, 2B8) and 9 hybridomas producing antibodies to the synthetic peptide C3 (1G5, 2A6, 2C5, 2D7, 2G11, 3A
4, 3H2, 4H3, 5H6) were established.

Next, the established hybridoma 1B4 was cultured to increase the number, and 3 × 10 ⁶ cells / mouse were administered to the abdominal cavity of a mouse about one month after pristane was administered intraperitoneally in advance.

 Two weeks later, about 10 ml of ascites was collected per mouse.

After adding and diluting the same amount of PBS as about 40 ml of ascites (for 2 animals), 80 ml of a saturated ammonium sulfate solution was added, and the fraction precipitated under 50% ammonium sulfate saturated conditions was collected by centrifugation. About 10 ml of 0.1 M Tris-
Hydrochloric acid buffer (pH 7.2) was added for dissolution, and this was dialyzed against the same buffer for 2 days.

Next, the antibody solution was added to a column (22 mm × 65 cm) packed with DEAE-cellulose DE52 (manufactured by Whatman), and the flow-through fraction was collected.
It was added to a column (22 mm x 65 cm) packed with (LKB) and subjected to gel filtration to obtain a purified antibody that specifically reacts with the synthetic peptide C1.

Using hybridoma 1A10, a purified antibody specifically reacting with synthetic peptide C2 was obtained in the same manner as described above.

Using hybridoma 2D7, a purified antibody specifically reacting with synthetic peptide C3 was obtained in the same manner as described above.

Properties of monoclonal antibodies against smooth muscle myosin heavy chain isoforms 1) Class, type Synthetic peptide C1, synthetic peptide C2 or synthetic peptide
After adding the culture supernatant of each of the above hybridomas to a 96-well PVC rate coated with C3 and subjected to a blocking treatment with 3% gelatin, MonoAb-ID EIA kit (manufactured by Zymed)
Was used to search for the class and type of antibody.
Tables, Tables 3 and 4).

2) Specificity The specificity of the monoclonal antibody for producing the hybridoma was confirmed by immunoblotting and immunofluorescent tissue staining.

Immunoblotting was performed on a monoclonal antibody that reacts with the synthetic peptides C1 and C2. The culture supernatant of the hybridoma was used instead of the antiserum of Example 2, and a peroxidase-labeled anti-mouse IgG antibody (E
Example 2 was performed except that Y Laboratory was used.

For immunofluorescent tissue staining, the culture supernatant of the hybridoma was used in place of the antiserum of Example 3, and a second antibody was used.
The procedure was performed in the same manner as in Example 3 except that a FITC-labeled anti-mouse IgG antibody was used. The sample subjected to tissue staining is a tissue section of a rabbit aorta in the fetal period and the growing period.

As a result, exactly the same results as those obtained with the antiserum (see Examples 2 and 3) were obtained, and the monoclonal antibody of the present invention discriminately recognizes a single isoform of the smooth muscle myosin heavy chain. Was confirmed.

Example 5 Preparation of ¹³¹ I-Labeled Anti-Smooth Muscle Myosin Heavy Chain SM-1-Specific Monoclonal Antibody Hybridoma 1C10 strain was administered at a dose of 5 × 10 ⁶ cells / animal to a mouse previously administered with pristane, and ascites tumor did. The ascites obtained 10 to 20 days after the mouse was pooled, and a fraction precipitated from the ascites under 50% ammonium sulfate saturation conditions was obtained. The precipitated fraction was purified by DEAE-cellulose DE52 column chromatography to obtain a purified monoclonal antibody (MHM-1C10).

To ¹³¹ I 3 mCi, the purified antibody (8.7mg / ml) 200μ,
Gloramine T (1mg / ml) 150μ, sodium metabisulfate (1mg / ml) 600μ, potassium iodide (50mg / ml) 150
μ and 150 μ of 0.5 M phosphate buffer (pH 7.5)
After reacting for 1 minute at room temperature, Sephadex G-preliminarily equilibrated with a phosphate buffer containing 0.5% bovine serum albumin.
The column is subjected to 50 (manufactured by Fumarasia) column chromatography to separate free ¹³¹ I to obtain a ¹³¹ I-monoclonal antibody (MHM-1C10).

Example 6 Preparation of Fab Fragment of Monoclonal Antibody Specific to ¹¹¹ In-Labeled Anti-Smooth Muscle Myosin Heavy Chain SM-1 A monoclonal antibody (MHM-1C10) was prepared in the same manner as in Example 5 and lyophilized. 30 mg of this purified antibody is added to 2.5 ml of phosphate buffer (pH 7.0), 0.3 mg of pepsin (manufactured by Sigma) is added, and the mixture is reacted at 37 ° C. for 2 hours.

The reaction solution was subjected to gel filtration column chromatography using a protein A-Sepharose (manufactured by Fumarumasia) column which had been equilibrated with a phosphate buffer (pH 7.4) beforehand.
Adsorb fragments and still undegraded antibodies. Collect the non-adsorbed fraction, and add 5 mg with Amicon B15 (Amicon).
/ ml and the method of Krejcarek et al. (Biochem. Biophys. Res. Commum. 77 , 581-587 (197
7)) by diethylenetriaminepentaacetic acid (DTPA)
And mixed gradually with carboxycarbonic anhydride, and reacted at 4 ° C. overnight.

Next, the reaction solution was dialyzed against a 0.1 M acetate buffer (pH 5.0), and a Fab-DTPA fraction was collected using Sephadex G-25, and the fraction was collected against a 0.1 M glycine hydrochloride buffer (pH 3.5). Dialysis. The thus obtained MHM-1C10 Fab-DTPA is mixed with indium chloride ¹¹¹ In in the same buffer and reacted for 30 minutes. As a ^{result, MHM-1C10 Fab-DTPA- 111} In is obtained.

INDUSTRIAL APPLICABILITY The antibody of the present invention is used for distinguishing and recognizing a single isoform of smooth muscle myosin heavy chain, for example, for elucidating the mechanism of regulating the expression of smooth muscle myosin heavy chain isoform in ontogeny. It is useful as a biochemical or diagnostic reagent, specifically a tissue staining reagent. In particular, an antibody or an active fragment thereof that recognizes the isoform of human smooth muscle myosin heavy chain can be histologically examined for the isoform of smooth muscle myosin heavy chain in various human diseases. It is useful for diagnosing progress after onset.

Those in which these antibodies or active fragments thereof are labeled with a radioisotope or the like are useful as in-vivo diagnostic agents, and are also useful as immunoassay reagents.

Continuation of the front page (56) References JP-A-57-6363 (JP, A) Eur. J. Biochem. , Vol. 179, no. 1, (1989), p. 79-85 (58) Fields surveyed (Int. Cl. ⁷ , DB name) G01N 33/53, 33/563, 33/577

Claims (7)

(57) [Claims] An antibody or an active fragment thereof that recognizes the amino acid sequence of the C-terminal portion of a single isoform of smooth muscle myosin heavy chain. 2. The antibody or the active fragment thereof according to claim 1, which recognizes a single isoform of smooth muscle myosin heavy chain and does not show cross-reactivity with myosin heavy chain of cardiac muscle and skeletal muscle. 3. The isoform SM- of the smooth muscle myosin heavy chain.
1. The antibody or the active fragment thereof according to claim 1, which recognizes SM-2 or SM-3. 4. The antibody or the active fragment thereof according to claim 1, which recognizes a single isoform of human smooth muscle myosin heavy chain. 5. The antibody or the active fragment thereof according to claim 1, which recognizes an oligopeptide containing all or at least three consecutive amino acid residues of any of the following amino acid sequences: H ₂ N-RRGNETSFVPTRRSGGRRVIENADGSEEEVDARDADFNGTKSSE
−COOH; H ₂ N−RGPPPQETSQ−COOH; H ₂ N−NRLRRGGPISFSSSRSGRPQLHIEGASLELSDDDTESKTSDVNET
QPPQSE-COOH. 6. The antibody or the active fragment thereof according to claim 1, which is labeled with a labeling agent. 7. The antibody according to any one of claims 1 to 6, wherein the antibody is a monoclonal antibody, or an active fragment thereof.