JPH07163393A - Anti-nitrogen monoxide synthase monoclonal antibody, its production and use - Google Patents

Abstract

PURPOSE:To obtain the subject new monoclonal antibody showing integrity to an NO synthase, useful for measuring the NO synthase simply and in high sensitivity and purifying the NO synthase, comprising an induction type nitrogen monoxide (NO) synthase as an immunogen. CONSTITUTION:An NO synthase derived from macrophage or a brain such as nerve cell or glia cell is administered to a BALB/c mouse, which is immunized. Three days after the final administration of the antigen, a spleen is extracted to collect a cell to be used for cell fusion. Then the cell is subjected to cell fusion with a myeloma cell of mouse and selectively cultured in a HAT medium to give a hybridoma cell. Then the hybridoma cell is cultured, a clone capable of producing an antigen to be reacted with an NO synthase is selected and the positive clone is cloned. The prepared monocloned hybridoma is cultured to give a monoclonal antibody showing integrity to an NO synthase, useful for measuring the NO synthase simply and in high sensitivity and purifying the NO synthase.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a monoclonal antibody capable of binding to nitric oxide synthase, a method for producing the same, and its use. Further, the present invention provides a hybridoma producing the monoclonal antibody.

[0002]

BACKGROUND OF THE INVENTION Nitric oxide (NO) is a physiological activity in the body of mammals, for example, as a vasodilator in the vascular system [Pharmacol.
v.) Vol. 43, pp. 109-142 (1991)], in the leukocyte system [Cu]
rr. Opin. Immunol. Volume 3, pp. 65-70 (199)
1)], it is believed that the nervous system plays various roles as a neurotransmitter [Neuron, Vol. 8, page 3-11 (1992)]. NO is synthesized from arginine by NO synthase. The gene for NO synthase has been found in the brain [Nature No. 35].
1, Vol. 714-718 (1991)], Macrophages [Journal of Biological Chemistry] Vol. 267, Vol. 6370-637.
4 (1992)], vascular endothelial cells [Proceeding Natl. Acad. Sci.] 89, 6348-6.
352 (1992)] and the like. The amino acid sequences deduced from these nucleic acid sequences are
Although they show 50-60% homology with each other, they are all recognized as different molecular species. Of these NO synthases, the macrophage type is calcium / calmodulin-independent and immunologically induced (in
ducible enzyme, and those of the brain and vascular endothelial cell types are calcium / calmodulin-dependent and constitutive (consti
It is said that it is a tutive) enzyme [Cell (Vol. 70), pp. 705-707 (1992)]. Although several kinds of antibodies have been obtained against NO synthase, most of them are polyclonal antibodies, and bovine endothelial cell-derived NO synthase is recognized as a monoclonal antibody capable of separating NO synthase. Antibodies are known [JS Pollock et al., Journal Vascular Research (J. Vasc. Res.), Vol. 29, p. 184 (1992)].

[0003]

Therefore, various N
If a monoclonal antibody capable of highly sensitively binding to O synthase can be prepared, it is possible to know the distribution of the expression of each NO synthase in cells and tissues and its expression amount, and at the same time, in vivo, etc. It becomes possible to further elucidate the role of NO in the NO synthase.

[0004]

[Means for Solving the Problems] In view of the above circumstances, the present inventors have conducted extensive studies and as a result have found that macrophage-derived NO synthase, which is one of the inducible NO synthases, is used as an immunogen to produce various NO synthases. We succeeded in producing a monoclonal antibody with high binding ability. When the monoclonal antibody is subjected to immunohistochemical or immunochemical assay, various cell-derived NO synthases can be detected or quantified with high sensitivity. By using the monoclonal antibody, cell-derived N such as macrophages, brain cells (nerve cells, glial cells, etc.) or vascular endothelial cells, etc.
O synthase can be efficiently purified. As a result of further research based on these findings, the present invention has been completed. That is, the present invention relates to (1) an IgG-type monoclonal antibody that uses inducible nitric oxide synthase as an immunogen and exhibits binding properties to nitric oxide synthase, and (2) binds to brain-derived nitric oxide synthase. (3) a monoclonal antibody which exhibits binding properties to nitric oxide synthase derived from macrophages and which does not bind to nitric oxide synthase derived from vascular endothelial cells, (3) 4) A cloned hybridoma consisting of spleen cells of a mammal immunized with inducible nitric oxide synthase and allogeneic or heterologous lymphoid cells, and (5) a mammal immunized with nitric oxide synthase derived from macrophages. The above-mentioned (4), characterized in that the spleen cells of the animal and the same or different lymphoid cells are fused and cloned.
(6) The method for producing the hybridoma described above, (6) The monoclonal antibody according to (1), (2) or (3) above, which comprises growing the hybridoma described in (4) above in a liquid medium or in the abdominal cavity of a mammal. A method for producing an antibody, (7) a method for detecting or quantifying nitric oxide synthase, which comprises using the monoclonal antibody according to (1), (2) or (3), and (8) an enzyme immunoassay method. (9) A method for purifying nitric oxide synthase, which comprises using the monoclonal antibody according to (1), (2) or (3) above.

N used for immunization of mammals in the present invention
The O synthase may be any animal-derived inducible NO synthase, and the molecule may be not only a natural type but also a partial peptide and a mutein. Representative examples of animal-derived NO synthases include, for example, rat macrophage-derived NO synthase [Yui, Y.
Et al., Journal of Biological Chemistry (J. Biol. Chem.) 266, 12544-1254.
7 (1991)], but macrophage-derived ones are preferably used. As the immunogen, the above-mentioned NO synthase may be isolated from each tissue, and D having a nucleotide sequence encoding NO synthase
An expression vector containing NA may be prepared and introduced into an appropriate host cell to produce a large amount of NO synthase. The expression vector includes, for example, (a) RNA encoding a NO synthase, and (b) a single-stranded complementary DNA from the RNA.
(CDNA) and then double-stranded DNA are synthesized, (c) the complementary DNA is incorporated into a plasmid, (d) the host is transformed with the obtained recombinant plasmid, and (e) the obtained transformant is After culturing, the transformant can be transformed by a suitable method such as D
A plasmid containing the target DNA was isolated by a colony hybridization method using an NA probe, and (f) the cloned DNA of interest from the plasmid.
Can be prepared by excising each of the clones and (g) ligating the cloned DNA downstream of the promoter of the vector.

The expression vector thus obtained is
Suitable host (eg E. coli, Bacillus subtilis, yeast, animal cells, etc.)
The NO synthase can be produced by culturing the transformant thus obtained in a medium and culturing the obtained transformant in a medium. The NO synthase protein also includes the NO synthase mutein. As the NO synthase mutein, originally,
It is a mutation in the amino acid sequence of the original peptide or protein, and includes addition of amino acids, deletion of constituent amino acids, and substitution with other amino acids. The addition of the amino acid includes an addition of at least one amino acid. Examples of the deletion of the constituent amino acids include those lacking at least one NO synthase constituent amino acid. Examples of the substitution with the other amino acid include a substitution of at least one NO synthase-constituting amino acid with another amino acid.

[0007] The addition of an amino acid in a mutein in which an amino acid is added to NO synthase does not include the addition of methionine due to the initiation codon used for expressing a peptide and the addition of a signal peptide. . The number of amino acids added should be at least 1.
However, any number may be used as long as the characteristics of the inducible NO synthase are not lost. More preferably, a part or all of the amino acid sequence in which high homology (homology) is recognized among NO synthases can be mentioned. The NO synthase in which the constituent amino acids are deficient in the mutein has at least one defective amino acid, but may be any number as long as the characteristics of the inducible NO synthase are not lost. N in which a constituent amino acid is replaced with another amino acid
The number of constituent amino acids (replacement amino acids) of NO synthase before substitution in the mutein of O synthase is
At least one, but any number may be used as long as the characteristics of the inducible NO synthase are not lost.

Examples of constituent amino acids to be substituted (amino acids to be replaced) include cysteine and any amino acid other than cysteine, but cysteine is particularly preferably substituted. Substituted amino acids other than cysteine include aspartic acid, arginine, glycine,
Valine etc. are mentioned. When the substituting amino acid is cysteine, the substituting amino acid (substitution amino acid) is preferably, for example, a neutral amino acid. Specific examples of the neutral amino acid include glycine, valine, alanine, leucine, isoleucine, tyrosine, phenylalanine, histidine, tryptophan, serine, threonine and methionine. Particularly, serine and threonine are preferable. When the substituting amino acid is other than cysteine, the substituting amino acid having a property different from that of the substituting amino acid is selected, for example, in terms of hydrophilicity, hydrophobicity or charge of the amino acid. Specifically, when the substituting amino acid is aspartic acid, examples of the substituting amino acid include asparagine, threonine, valine, phenylalanine, and arginine, with asparagine and arginine being particularly preferable. When the substituting amino acid is arginine, examples of the substituting amino acid include glutamine, threonine, leucine, phenylalanine and aspartic acid, with glutamine being particularly preferred. When the substituting amino acid is glycine, examples of the substituting amino acid include threonine, leucine, phenylalanine, serine, glutamic acid, and arginine, and threonine is particularly preferable. When the substituting amino acid is serine, examples of the substituting amino acid include methionine, alanine, leucine, cysteine, glutamine, arginine and aspartic acid, and methionine is particularly preferable. When the substituting amino acid is valine, examples of the substituting amino acid include serine, leucine, proline, glycine, lysine, and aspartic acid.
Serine is particularly preferable. The substituting amino acid is preferably aspartic acid, arginine, glycine, serine, valine, and the substituting amino acid is preferably asparagine, glutamine, arginine, threonine, methionine, serine, leucine.

In the above substitution, two or more substitutions may be carried out simultaneously. In particular, it is preferred that 2 or 3 constituent amino acids be substituted. The above mutein may be a combination of two or three of the above addition, deletion and substitution. To produce the mutein, a site-directed mutagenesis technique is adopted. The technique is well known and is known to Lf Racer (Lather, RF) and Jay P. Lecoq (Lecoq,
JP), Genetic Engineering (Genetic E
ngineering), Academic Press, Inc. (1983) 3rd
See pages 1-50. Oligonucleotide-directed mutagenesis is described by Smith, M. and Gillam, S., Genetic Engineering: Principles and Methods, Plenum Press, Inc. (1981) Vol. It is shown on page 32.

In order to produce the structural gene encoding the mutein, for example, (a) single-stranded DNA consisting of a single strand of the structural gene of NO synthase is hybridized with a mutant oligonucleotide primer ( The primer is complementary to the codon for cysteine to be replaced by this single strand, or optionally to the region containing the antisense triplet pairing with this codon, provided that (This does not apply to the mismatch with the amino acid coding codon, or in some cases the antisense triplet), (b) extending the primer with DNA polymerase,
Form a mutant heterodimer,
And (c) replicate this mutant heterodimer. Next, a phage DN carrying the mutated gene
A is isolated and integrated into a plasmid.

A mutein can be produced by transforming an appropriate host with the thus obtained plasmid and culturing the obtained transformant in a medium. As the mutein lacking the constituent amino acids of NO synthase as an antigen in the present invention, a mutein having 100 or more amino acids in the amino acid sequence of NO synthase is preferable, and more preferably 110 or more. Mutein is given. In order to produce the monoclonal antibody of the present invention, a NO synthase, its mutein or its partial peptide is immunized into a mammal, the resulting spleen cells are fused with mammalian lymphoid cells, and then cloned. It is manufactured by When immunizing the NO synthase, the mutein thereof or a partial peptide thereof, the NO synthase may be used as a complex with a carrier protein and then used for immunization. Examples of the carrier protein include bovine serum albumin, bovine thyroglobulin, hemocyanin and the like. When the carrier protein complex is used, the coupling ratio between the carrier protein and NO synthase is about 0.1 to 30 times (carrier / NO synthase: 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.

[0012] When immunizing with NO synthase or a complex, laboratory animals such as sheep, goats, rabbits, guinea pigs, rats and mice are used as the mammals to be immunized. Rats and mice are preferred. Immunization can be performed by any route such as intraperitoneal, intravenous, intramuscular, intracutaneous, and subcutaneous when immunizing a mouse, but mainly subcutaneously, intraperitoneally, and intravenously.
Injection (especially subcutaneously) is preferred. In addition, the immunity interval, immunity, etc. are highly variable, and various methods are possible, but for example, immunization is performed about 2 to 6 times at 2-week intervals, and about 1 to 5 days, preferably about 2 after the final immunization. A method using spleen cells extracted after 4 days is often used. It is desirable to use an immunization amount of about 0.1 μg or more per mouse, preferably about 10 μg to 300 μg, as the amount of peptide per dose. or,
Before extracting the spleen, it is desirable to perform partial blood sampling, confirm the increase in antibody titer in blood, and then perform fusion using spleen cells.

[0013] Spleen cells of the mice above cell fusion is for example removed, hypoxanthine - guanine - phosphoribosyl transferase deficiency (HGPRT ^-) or thymidine kinase deficiency (TK ^-) suitable homologous or with such markers Different (preferably same) myeloma [eg P3
-X63-Ag / 8U1 (Ichimori et al. Journal of Immunological Method, Vol. 80, p. 55 (198)
5)] and the like. For example, the method of Keller and Milstein [Nature
(Nature) Vol. 256, p. 495 (1975)]. For example, myeloma cells and spleen cells are suspended in a ratio of about 1: 5, for example, in a medium in which Iscove's medium and Ham's F-12 medium are mixed in a ratio of 1: 1 (hereinafter referred to as IH medium), and the cells are suspended in Sendai virus.
Fusing can be performed using a fusing agent such as polyethylene glycol (PEG). Of course dimethyl sulfoxide
(DMSO) and other fusion promoters can be added instead of these. The average molecular weight of PEG is usually about 1
000 to 9000, the reaction time is about 0.5 to 30 minutes, and the concentration is about 10% to 80%. As an example of preferable conditions, PEG 6000 is about 35 to 55% and about 4 to 4%.
By treating for 10 minutes, the fusion can be efficiently performed. The fused cells were hypoxanthine-aminopterin-thymidine medium [HAT medium: Nature, vol. 256,
P. 495 (1975)] and the like.

The culture supernatant of the grown cells is screened for the production of the desired antibody.
The antibody titer can be screened as follows.
That is, first, as a first step, the presence or absence of production of antibodies against the antigenic peptide used for immunization is checked by radioimmunoassay (RI
The method A) or the enzyme immunoassay (EIA) method can be used for the examination, but various modifications of these methods are possible. As one example of a preferable measurement method, one method using EIA will be described. To a carrier such as cellulose beads, for example, rabbit anti-mouse immunoglobulin antibody is coupled according to a conventional method,
The culture supernatant to be measured and mouse serum are added to this, and the mixture is allowed to react for a certain period of time at a constant temperature (about 4 to 40 ° C .; the same applies below). After this, after washing the reaction product well,
An enzyme-labeled antigen peptide (coupling the enzyme and the antigen peptide according to a conventional method and then purifying) is added, and the mixture is allowed to react 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, after which the formed color product can be measured by absorbance or fluorescence intensity.

It is desirable that cells in the wells that show growth in the selective medium and that have antibody activity against the antigen peptide used for immunization be cloned by the limiting dilution method or the like. By similarly screening the supernatant of cloned cells and increasing the number of cells in wells having high antibody titers, monoclonal antibody-producing hybridoma clones showing reactivity with the antigen peptide used for immunization can be obtained. The hybridoma thus cloned is grown in liquid medium. Specifically, for example, a liquid medium such as RPMI-1640 [Moore, GE, et al.
Journal of American Medical Association (J. Am. Med. Assoc.) 199,549 (196)
7)] with about 0.1-40% bovine serum added to the medium, etc.
The monoclonal antibody can be obtained from the culture medium by culturing for 10 days, preferably for 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, when a mouse is used, about 1 × 10 ⁴ to 1 × 10 ⁷ cells, preferably about 5 × 10 ⁷ cells, are preliminarily inoculated with mineral oil or the like in a BALB / c mouse or the like.
^{5 to} 2 × 10 ⁶ hybridomas are intraperitoneally inoculated, and ascites fluid is collected after about 7 to 20 days, preferably about 10 to 14 days. The antibody produced and accumulated in the ascites can be easily isolated as a pure immunoglobulin from the monoclonal antibody by separation and purification methods such as ammonium sulfate fractionation and DEAE-cellulose column chromatography.

In this way, a monoclonal antibody against inducible NO synthase can be obtained. The monoclonal antibody of the present invention refers to an antibody that binds to various NO synthases (preferably derived from macrophages or brain) with inducible NO synthase as an immunogen with high sensitivity. Not only N at the same time but different from this
An antibody that also binds to O synthase (for example, a constitutive NO synthase such as brain-derived NO synthase) is also included in the antibody of the present invention. Since the monoclonal antibody of the present invention binds to NO synthase with high sensitivity, it is extremely useful as a reagent for measuring NO synthase. Furthermore, facilitating the measurement of NO synthase in living organs and tissues is a basic finding regarding NO synthase (for example, biodistribution).
It is also extremely useful in obtaining Enzyme-linked immunosorbent assay (EIA method) is usually used to detect NO synthase in living organs and tissues.
The quantification by the method such as, or the fluorescent antibody method or the RIA method is used, and the EIA method is particularly preferable. Further, Western blotting of proteins is effective for determining the size of NO synthase existing in these organs and tissues using the antibody of the present invention. In this method, a crude extract derived from an organ or a tissue or a partially purified sample thereof is subjected to acrylamide electrophoresis, and then transferred to a membrane filter to obtain H
Detect with RP-labeled anti-NO synthase antibody.

Furthermore, by utilizing the binding ability between the antibody of the present invention and NO synthase, an antibody affinity column is prepared to produce NO.
It can also be used for the purification of synthetic enzymes. The antibody molecule used for detecting and quantifying NO synthase may be a fraction thereof (eg, F (ab ') ₂ , Fab' or Fab}. Among them, the antibody molecule to which the labeling agent is directly bound is preferably Fab '. Thus, the monoclonal antibody of the present invention can be used as a reagent in the immunochemical assay for NO synthase. NO in living tissues and cells by immunochemical assay of NO synthase
It is possible to measure the amount of synthase, and by measuring NO synthase of various tissues or cells, for example, central nervous system disease, vascular system disease, leukocyte-related disease such as inflammatory disease, etc. It is thought to be useful for diagnosis of.

When carrying out the assay method using two kinds of antibodies in the above immunological assay method, the monoclonal antibody of the present invention is used as one antibody and the monoclonal antibody or polyclonal antibody of the present invention is used as the other antibody. Can be used. Further, three or more kinds of antibodies can be appropriately combined and used for the purpose of increasing the measurement sensitivity. Examples of the carrier for the antibody (solid phase antibody) retained on the carrier used in the method for measuring NO synthase include gel particles (eg, agarose gel [eg, Sepharose 4B, Sepharose 6B (trade name, Pharmacia Fine Chemicals)). (Manufactured by Sweden)), dextran gel [eg, Sephadex G-75, Sephadex G-100, Sephadex G-200 (registered trademark,
Pharmacia Fine Chemicals (Sweden)
Manufactured)], polyacrylamide gel [eg, Biogel P-
30, Biogel P-60, Biogel P-100 (registered trademark, Bio-Rad Laboratories (USA)
Manufactured)), cellulose particles [eg, Avicel (registered trademark, manufactured by Asahi Kasei), ion exchange cellulose (eg, diethylaminoethyl cellulose, carboxymethyl cellulose)], physical adsorbent [eg, glass (eg, glass spheres, glass rod,
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 I
RC-50 (registered trademark, manufactured by Rohm and Haas Company (USA)), Zeocurve 226 (trade name, manufactured by Palmchid Company (West Germany)), weakly basic anion exchange resin [eg, Amberlite IR-4B, Dowex 3 (registered trademark, manufactured by Dow Chemical Company (USA)]} and the like.

For holding the antibody on the carrier, known conventional means can be applied. For example, “Metabolism”, Vol. 8, No. 6
The bromocyan method and the glutaraldehyde method described on page 96 (1971) can be mentioned. Also,
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 (labeled antibody) include a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, and the like, and an enzyme is preferably used.
As the enzyme, a stable and highly inactive one is preferable,
Peroxidase, alkaline phosphatase, β-D-
Galactosidase, glucose oxidase and the like 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 (HRP) is preferred. In binding the peroxidase and the antibody, it is convenient to use a peroxidase into which a maleimide group has been introduced in advance in order to utilize the thiol group of Fab ′ as an antibody molecule. 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, N-succinimidyl-maleimide-carboxylate derivatives can be used, preferably N-
(γ-maleimidobutyloxy) succinimide (GMBS
It may be abbreviated as "). 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 0.1 M phosphate buffer having a pH of 7.0. The maleimidated peroxidase thus obtained can be purified by, for example, gel chromatography. Examples of the carrier used when performing the gel chromatography include Sephadex G-25 [registered trademark, manufactured by Pharmacia Fine Chemicals (Sweden)], Biogel P-2 [registered trademark,
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. The buffer solution may be, for example, 0.1 mM containing 5 mM ethylenediaminetetraacetic acid sodium salt having a pH of 6.0.
M phosphate buffer etc. are mentioned. The peroxidase-labeled antibody thus obtained can be purified by, for example, gel chromatography. Examples of the carrier used for performing the gel chromatography include Sephadex G-25 [Pharmacia.
Fine Chemical Company (Sweden)], Biogel P-
2 (manufactured by Bio-Rad Laboratories (USA)) and the like. 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. The test sample in the measurement system of the present invention includes tissues, cells,
Examples include cell lines and cell extracts, 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, the analyte retained by the NO synthase to be measured is added to the antibody retained on the carrier to carry out an antigen-antibody reaction, and then the peroxidase and anti-NO obtained above are added thereto.
A product (enzyme-labeled antibody) bound to the synthetic enzyme 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-mentioned operations (1) to (3) are performed in advance on a standard solution of a known amount of NO synthase, and the relationship between the NO synthase and the absorbance or fluorescence intensity is prepared as a standard curve. : Analyte containing unknown amount of NO synthase (test sample)
The absorbance or fluorescence intensity obtained in (1) is applied to a standard curve to measure the amount of NO synthase in the analyte.

NO synthase can be purified using the antibody obtained in the present invention. This can be done by performing affinity column chromatography using the antibody. The affinity column chromatography can be carried out, for example, by coupling the antibody to a suitable carrier, packing this in a column, adsorbing a solution containing NO synthase through the column, adsorbing it, and then eluting it. 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-activated Sepharose 4B (manufactured by Pharmacia Fine Chemicals), Affigel-1
0, Affi-Gel 15 (trade name, 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 bromocyan method and glutaraldehyde method described in "Metabolism", Vol. 8, page 696 (1971). To be Also, a method using a water-soluble carbodiimide,
Although the active ester method or the like can be used, it may be physically adsorbed on the surface of the carrier as a simpler method.

In order to carry out purification using the antibody-bound carrier thus obtained, the NO synthase solution prepared in a buffer solution near neutral is applied to the antibody column packed with the antibody-bound carrier. , NO synthase is adsorbed. Next, after washing the column with the same buffer, the N
Elute O synthase. To elute the specifically adsorbed NO synthase, for example, a low pH or high pH buffer solution or a buffer solution containing a high concentration of salt is used. The buffer solution having a low pH is, for example, 0.3 at pH 2.3.
17M glycine-hydrochloric acid buffer solution, pH 1.8 0.1M secondary sodium citrate-hydrochloric acid buffer solution and the like can be mentioned. Examples of the high pH buffer include pH 11 ammonia water and pH 11.7 0.2M sodium borate buffer. The buffer containing the high concentration of salt,
For example, 6M guanidine hydrochloric acid solution, 7M urea solution and the like can be mentioned. The elution may be performed by a batch method or a column method. The eluate of the antigen is dialyzed and purified. For example, when eluted with a low pH buffer solution, for example, 0.1M sodium carbonate buffer solution (pH 10.
When eluted with a high pH buffer such as 5) or a high pH buffer, for example, 0.1 M glycine-hydrochloric acid buffer (pH 3.
0) or the like and then neutralized with a low pH buffer such as 0.02M phosphate buffer (pH) containing 0.1% NaN _3.
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 NO synthase thus purified using the antibody of the present invention is of extremely high purity and high unit, and is a reagent in studies such as physiology of the cranial nervous system, vascular system, blood cell system or elucidation of diseases. It is considered to be useful as

In the specification and drawings of the present invention, 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 Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: Threonine Cys: Cysteine Met: Methionine Glu: Glutamic acid Asp: Aspartic acid Lys: Lysine Arg: Arginine His: Histidine Phe: Phenylalanine Tyr: Tyrosine Trp: Tryptophan Pro: Proline Asn: Asparagine Gln: Glutamine Clz: 2 Chlorobenzyloxycarbonyl BrZ: 2-Bromo-benzyloxycarbonyl Bzl: Benzyl Boc: t-butoxycarbonyl

[0024]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. The antibody-producing hybridomas of the present invention obtained in the examples described below have been deposited as follows. (IFO) (NIBH) Animal cell IFO No. FERM No. ────────────────────────────────── Mouse hybridoma N2 50398 BP-4258 (1993. 4.2) (1993. 4.14) Mouse hybridoma N5 50399 BP-4259 (1993. 4.2) (1993. 4.14) Mouse hybridoma N21 50400 BP-4260 (1993. 4.2) (April 14, 1993) Mouse hybridoma N11 50440 (March 7, 1994) ─────────────────────────────────── IFO: Fermentation Research Institute (Osaka) NIBH: Ministry of International Trade and Industry, Institute of Industrial Science and Technology, Institute of Life and Industrial Technology In the table, the number in parentheses () indicates the contract date. Example 1 Preparation of NO synthase Male Kb1 Wistar rats (body weight: 200 to 230 g) were intraperitoneally injected with 40 mg of B dissolved in 10 ml of physiological saline per mouse.
Inoculated with CG. Two weeks after the inoculation, 50 ml of phosphate buffered saline (PBS) was inoculated into the abdominal cavity of the rat, and peritoneal exudate cells were collected. After centrifugation (300 × g, 10 minutes), the supernatant was removed, and 25 ml of hypotonic NaCl (0.2%) solution was added to lyse the mixed red blood cells, and then 1.6% Na was added.
25 ml of Cl was added to return to an isotonic solution. Centrifuge again (300 x
cells, 10% fetal bovine serum, 10 ng
/ Ml lipopolysaccharide containing DMEM (Dulbec
Suspend in a co's modified Eagle's medium) and inoculate it in a 10 cm culture dish at 37 ° C and 5% carbon dioxide gas (C
Culturing was performed under O ₂ ) for 24 hours. After culturing, non-adherent cells were removed by washing 3 times with PBS, and adherent cells were detached and collected with a rubber policeman. The macrophages thus obtained were suspended in 50 mM Tris-hydrochloric acid buffer (pH 7.4) containing 1 mM DTT (dithiothreitol) at a concentration of 1 × 10 ⁸ cells / ml to give 0.1 mg / ml. ml phenylmethylsulfonyl fluoride, 0.01 mg / ml trypsin inhibitor, 0.01 mg / ml leupeptin, 0.01 mg / ml antipain, 0.01 mg / ml pepstatin, etc. were added.
Ultrasonic disruption of macrophage suspension
ptor, output 6,3 seconds, Tommy Seiko), 100,00
The supernatant was obtained by centrifugation at 0 × g for 60 minutes. Purification was performed as follows. 7.5 ml of the supernatant was applied to a 2 ', 5'-ADP-agarose column equilibrated with 50 mM Tris-HCl buffer (pH 7.4) containing 1 mM DTT. The column was washed successively with 10 ml of 50 mM Tris-hydrochloric acid containing 1 mM DTT and 750 mM NaCl and 10 ml of 50 mM Tris-hydrochloric acid containing 1 mM DTT, and 1 mM.
50 mM Tris-containing DTT and 1 mM NADPH
Elution was performed with 2 ml of hydrochloric acid. The eluted NO synthase sample was concentrated to 50 μl using Centricon 10 (Amicon, Danvers, MA), and 50 m containing 1 mM DTT was added.
Gel filtration column Superose equilibrated with M Tris-HCl
12HR10 / 30 (300 × 10mm, Pharmacia
LKB Biotechnology Uppsala (Sweden) was used to obtain the NO synthase fraction.

Example 2 Acquisition of Anti-NO Synthase Monoclonal Antibody (1) Immunization For BALB / c mice (8 weeks old), 10 μg of NO synthase obtained in Example 1 and Freund's complete adjuvant (Difco Laboratories, (US) was inoculated subcutaneously. Two months later, a mixture of 10 to 15 μg of NO synthase and Freund's incomplete adjuvant (Difco) was subcutaneously inoculated in the same mouse, and the same immunization was carried out at intervals of about 1 month for 4 more times.
1 month after the final immunization, 50 mM Tris-HCl (pH
The mouse was intravenously inoculated with 15 μg of NO synthase dissolved in 7.4).

(2) Cell fusion From the immunized mouse obtained in (1) above, 3 days after the final administration of the antigen, the spleen was extracted to obtain cells used for cell fusion.
Suspended in MEM medium. Mouse myeloma cell P3-X
63-Ag.8U1 (P3U1) was 5% carbon dioxide in a GIT medium (Nippon Pharmaceutical Co., Ltd.) containing 5% fetal bovine serum, 95%.
Subculture was performed under the condition of% air. Cell fusion is a division method established by Köhler and Milstein [Nature No. 2
56, p. 495 (1975)]. P3
U1 cells (6.9 × 10 ⁷ cells) and the immunized lymphocytes (3.2 × 10 ⁸ cells) obtained by the above method were mixed and centrifuged, and the cells were preheated to 37 ° C. with 0.5 ml of MEM. 45% PEG6000 dissolved in the medium was slowly added dropwise. After 7 minutes, the MEM medium heated to 37 ° C. was adjusted to 0.
After adding 5 ml each to make 15 ml, the supernatant was removed by centrifugation at room temperature for 600 rpm for 15 minutes. This cell precipitate was suspended in 210 ml of GIT medium containing 5% fetal bovine serum, and 10 cells were suspended in a 96-well microplate (Nunc, Naperville, IL).
20 μl of each of 0 μl was seeded. 1 day later, HAT
(Hypoxanthine 1 × 10 ^-4 M, aminopterin 4 × 1
GIT medium (containing 5% fetal bovine serum) containing 0 ^-7 M, thymidine 1.6 × 10 ^-5 M) (hereinafter referred to as HAT medium)
Was added to each well in an amount of 150 μl, and half of the medium was replaced with fresh HAT medium every 3 days. The cells thus grown are hybrid cells.

(3) Search for antibody-producing cells NO synthase purified by the method described in Example (1) was diluted to 0.5 μg / ml with PBS (pH 7.4) and 100 μl thereof was added to 96 wells. It was put in each well of an immunoplate (Nunc) and left overnight at 4 ° C. to bind NO synthase to the solid phase. After washing with PBS, 250 μl of PBS containing 20% of Block Ace (Snow Brand Milk Products Co., Ltd.) was injected into each well to block excess binding sites, and stored in a cool place until use. NO obtained as described above
100 μl of each hybrid cell supernatant was added to a 96-well immunoplate to which synthase was bound, and the mixture was incubated at room temperature for 2 hours. After removing the culture supernatant and washing, HRP as a secondary antibody
Labeled goat anti-mouse IgG antibody (Cappel, USA) was added and incubated at room temperature for 2 hours. Remove the secondary antibody,
After well washing well, HRP substrate solution (0.02
% H ₂ O ₂ and 0.15% o-phenylenediamine, pH 5.5 sodium citrate buffer) 100 μl
In addition, after reacting at 25 ° C. for 10 minutes to stop the enzymatic reaction by adding 100 μl of 2N sulfuric acid, the absorbance at 492 nm was measured using an automatic colorimeter for microplates (MTP-32, Corona). As a result 14
The presence of an antibody capable of binding to NO synthase was confirmed in the wells.

(4) Cloning of hybrid cells 5 × 10 ⁴ cells / well of mouse thymocytes were pre-seeded as vegetative cells so that the number of cells in the well in which the presence of the antibody was recognized was 0.5 cells per well. The seeds were seeded on a 96-well microplate that had been set aside and cloned. As a result, one representative clone cell was obtained from each well, and a total of 14 clone cells N1, N2, N3, N were obtained.
4, N5, N7, N8, N9, N11, N12, N1
4, N16, N18 and N21 were obtained.

(5) Production of antibody 14 obtained by cloning in the above (4)
Hybridoma clones of each species were previously
BA that had been intraperitoneally administered 5 ml of mineral oil
LB / c mice were intraperitoneally inoculated with 1 × 10 ⁶ cells per mouse to perform ascites. Ascites was collected 10 days after the hybridoma was intraperitoneally administered. From about 10 ml of each ascites obtained, Stalin et al. [Journal of Biological Chemistry, Volume 256, 97
The monoclonal antibody was purified according to the method of P. 50-9754 (1981)]. First, after centrifugation at 10,000 rpm for 15 minutes to remove fibrin-like substances from ascites,
PBS (8.1 mM disodium phosphate, 1.5 mM potassium phosphate, 27 mM KCl, 137 mM NaCl, pH)
In 7.2), it was diluted to a concentration at which the ultraviolet absorption (A ₂₈₀ ) at 280 nm showed a value of 12-14. After dilution, add saturated ammonium sulfate solution to the sample to a concentration of 47%, and add 4 ° C.
Salting out for 60 minutes with stirring, and then centrifuging (10,
(000 rotations, 15 minutes) to obtain a precipitate. The precipitate was added to a 20 mM Tris buffer solution containing 50 mM NaCl (pH 7.
It melted in 9) and dialyzed against 2 liters of the same solution.
After 2 hours, the same dialysate solution was replaced with 2 liters of the same and dialyzed for another 15 hours. After dialysis, centrifugation was performed at 10,000 rpm for 15 minutes to remove the precipitate, and the supernatant was prepared 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 the same buffer solution containing 50 mM-500 mM NaCl. Fractionation was performed at a flow rate of 1.5 ml / min using the gradient salt solution of No. 3, and the flow-through fraction was concentrated to obtain purified monoclonal antibodies N1, N2, N3, N4.
N5, N7, N8, N9, N11, N12, N14, N
16, N18 and N21 were obtained. The method of Laemli et al. [Nature, Vol. 227, 680-
SDS-polyacrylamide gel electrophoresis was used according to pp. 685 (1970)]. That is, salting out with ammonium sulfate, D
The fraction passed through the EAE cellulose column was reduced with 2-mercaptoethanol, and the acrylamide concentration was 10%.
Gel electrophoresis was performed at 180 V for 2.5 hours. As a result, the molecular weight of all monoclonal antibodies
Two bands, an H chain around 52 KDa and an L chain around 28 KDa, were observed.

(6) Measurement of Subclass of Antibody Subclass of antibody was measured by the following method. That is, Example 1
100 .mu.l of each cloned hybridoma culture supernatant was placed in the 96-well immunoplate coated with NO synthase obtained in 1. above and incubated at room temperature for 2 hours. After removing the culture supernatant and washing, rabbit anti-mouse γ ₁ , γ _2a ,
100 μl of antibodies against γ _2b , γ ₃ , K chain and λ chain (Cappel) were added and incubated at room temperature for 2 hours.
After removing each antibody and washing, HRP-labeled goat anti-rabbit IgG antibody (Cappel) was added and incubated at room temperature for 2 hours. After removing the labeled antibody and washing well, (3) above
The enzyme reaction was performed by the method described and the absorbance was measured. As a result, N8, N9, N11, N12, N14, N16,
It was revealed that the N18 and N21 antibodies are γ ₁ , κ, N1 and N7 antibodies, and γ _2a , κ, N2, N3, N4 and N5 are antibodies belonging to the γ _2b , κ subclass.

Example 3 Characteristics of antibody (1) Western blotting method The NO synthase obtained in Example 1 was electrophoresed on a 10 to 15% gradient gel using the Phast-system (Pharmacia). After migration, transfer to a membrane filter to obtain each anti-N obtained in Example 2.
The reaction was carried out with a monoclonal antibody of O-synthesizing enzyme, and with alkaline phosphatase-labeled goat anti-mouse IgG antibody,
The binding properties of anti-NO synthase antibody were examined. As a result, 1
It was found that all of the four types of antibodies showed binding properties to 150 kDa NO synthase. (2) Histochemical Staining Wister rats aged 5 weeks were inoculated into the abdominal cavity with 20 mg of BCG dissolved in physiological saline, and 2 weeks later, various organs were excised.
It was immediately frozen and sliced to a thickness of 5 μm with cryostat. After fixing with cold acetone, endogenous peroxidase was blocked, and N4, N5, N16 and N21 monoclonal antibodies were reacted for 1 hour. After washing well with PBS, HRP-labeled goat anti-mouse IgG antibody was reacted for 1 hour. After the reaction, the plate was washed with PBS, and histochemical staining was performed using 3-3 'diaminobenzidine as a substrate. As a result, all four antibodies reacted with macrophages of various organ tissues including lung / spleen macrophages and liver Kupffer cells. N5 antibody is nerve cell, N21 antibody is
It also showed reactivity with astrocytes. The immunoelectron microscopic examination was performed as follows. The rat peritoneal or alveolar macrophages were cultured for 24 hours in the presence of 1 ng / ml of lipopolysaccharide (LPS). The macrophages activated in this way were fixed with periodate-lysine-paraformaldehyde, then stained in the same manner, and post-fixed with osmic acid. After fixing, Epon resin [E
pok 812, Dodecenyl succinic anhydride, Methyl n
A mixture of adic anhydride (above Oken Shoji Co., Ltd.) and DMP-30 (Polyscience Co., Ltd.)], and an ultrathin section was prepared and observed with an H300 electron microscope (Hitachi). As a result, antibody positive reaction was observed in the synthetic system such as nuclear membrane of macrophage, rough endoplasmic reticulum, and Golgi apparatus. (3) Fluorescent antibody staining Mouse peritoneal macrophages induced with thioglycolate were adhered on a cover glass, stimulated with LPS and interferon (IFN) -γ, fixed with 4% paraformaldehyde, and adjusted to 5 μg / ml. Each monoclonal antibody was reacted overnight at 4 ° C. After the reaction, the plate was washed with PBS and stained with a fluorescent antibody-labeled goat anti-mouse IgG antibody at room temperature for 2 hours. After washing well with PBS, observation with a fluorescence microscope was performed. As a result, N1, N2, N3, N
5, N8, N9, N11, N12, N14, N16, N
Twelve monoclonal antibodies of 18, N21 showed reactivity to macrophages.

Example 4 Effect of Antibody on Enzymatic Activity The macrophage suspension described in Example 1 was ultrasonically disrupted and centrifuged (100,000 × G, 60 minutes) to obtain a supernatant (cytoso).
l) To 200 μl, 100 μl of each monoclonal antibody described in Example 2 prepared at 1 mg / ml was added, and reacted at room temperature for 60 minutes while gently stirring. An anti-endothelin monoclonal antibody prepared in the same manner as a control (Suzuki, N.
Et al., Journal of Immunological Methods (J.
Immunol. Methods) 118, 245-250 (1989)] and 50 mM Tris buffer solution (pH
7.4) was used. The reaction mixture was divided into two equal volumes, one of which was mixed with 30 μl of 50 mM Tris buffer suspension in which 3.3 mg of Protein A Sepharose CL-4B (Pharmacia) was suspended, and the other was mixed with 30 μl of 50 mM Tris buffer solution at room temperature. The reaction was carried out for 30 minutes with gentle stirring. After the reaction, the mixture was centrifuged at 1,000 × G for 2 minutes and the supernatant was collected. The enzyme activity of NO synthase in the supernatant is
Quantification was carried out by measuring nitrite and nitrate, which are degradation products of [Yui, Y., et al., Journal of Biological Chemistry (J. Biol. Chem.) Vol. 266, 12544-12547 (1991)]. Also, above
NO synthase (10μ
g / ml) was changed to 200 μl (900 nmol / min / mg protein) and the same operation was performed. The obtained results are shown in [FIG. 1] and [FIG. 2]. In the figure, B indicates Tris buffer, C indicates anti-endothelin antibody, and each numeral indicates the number (N number) in the antibody name. When the Cytosol fraction was used as a zymogen, none of the anti-NO synthase monoclonal antibodies showed enzyme activity inhibitory activity, but rather higher enzyme activity was observed as compared to 50 mM Tris buffer alone (Fig. 1 shaded column). ). The N4, N5, N7 and N21 antibodies showed about twice the enzyme activity as in the case of using Tris buffer alone. The anti-endothelin antibody used as a control also showed a slight increase in enzyme activity, but the enzyme activity was not removed by the coexistence of protein A sepharose. In contrast, when the anti-NO synthase monoclonal antibody was used, removal of the enzyme activity was confirmed by the coexistence of protein A sepharose (Fig. 1, black column). When purified NO synthase was used, the enzyme activity was almost inactivated by Tris buffer alone (Fig. 2, B). On the other hand, when the antibody was reacted with the anti-NO synthase antibody, the enzyme activity remained 5 to 17% (lower diagonal line column in FIG. 1), and the enzyme activity was removed by protein A sepharose (black column in FIG. 2). Although some enzyme activity remained with the anti-endothelin antibody, it was not removed with protein A sepharose (Fig. 2, C). The above results are anti-N
It is presumed that this is because the O synthase monoclonal antibody stabilized the enzyme activity by binding to the NO synthase. It is more unstable than the Cytosol fraction (Yui, Y.
Et al., Supra), the purified NO synthase is used,
The result that the stabilization of the enzyme activity was more clearly recognized by the addition of the antibody also strongly supports this estimation.

Example 5 (1) Enzyme labeling of antibody 1.5 ml of purified N11 antibody (6.5 mg / ml) was added to 0.1M.
It was dialyzed against 0.1 M acetate buffer (pH 4.2) containing NaCl at 4 ° C. for 6 hours, pepsin (Sigma, USA) (0.25 mg) was added, and the mixture was digested at 37 ° C. for 20 hours. Stop the reaction by adjusting the pH to 7 with 1M Tris buffer and
A trogel AcA 44 (IBF, France) column was used as a eluent to separate 0.02M borate buffer (pH 8.0) containing 0.15M NaCl to obtain F (ab ') ₂ . After concentrating this to 0.9 ml, it was dialyzed against 0.1 M phosphate buffer (pH 6.0) at 4 ° C. for 20 hours to give 0.2
M mercaptoethylamine, 5 mM EDTA, 0.1
Add 0.1 ml of M phosphate buffer (pH 6.0) to 37
Reduced at 90 ° C for 90 minutes. The reaction solution is Sephadex G-25fi
Ne (Pharmacia Fine Chemical Co., Sweden) (φ1 × 60 cm) was used to separate 5 mM EDTA and 0.1 M phosphate buffer (pH 6.0) as eluents and Fa
A b'fraction was obtained. On the other hand, horseradish peroxidase (HRP) (Boehringer Mannheim, West Germany)
10 mg of 0.9 ml of 0.1 M phosphate buffer (pH 7.
0), 2.1 mg of N- (γ-maleimidobutyloxy) succinimide (GMBS) was dissolved in 100 μl of N, N-dimethylformamide (DMF) and added, and the mixture was stirred at room temperature for 45 minutes, and then Sephadex G-25fine (φ1). ．
HRP with maleimide group introduced was separated with 0.1M phosphate buffer (pH 6.0) as the eluent at 2 x 60 cm).
Was obtained (maleimidized HRP). Fab ′ and maleimidated HRP were mixed at a molar ratio of 1: 1 at 20 ° C. at 4 ° C.
Reacted for hours. After the reaction, 0.1 M phosphate buffer (pH 6.5) was separated as an eluent by a column of Ultrogel AcA 44 to obtain an enzyme-labeled antibody (N11Fab'-HRP).

(2) Preparation of antibody-bound solid phase Purified N5 antibody was diluted with 0.1 M Tris-HCl buffer (pH 8) to 10 μg / ml, and 100 μl thereof was added to 96
Each well of the well immunoplate was placed in each well and left overnight at 4 ° C. to bind the antibody to the solid phase. After washing with 0.01 M phosphate buffer (pH 7.0) containing 0.15 M NaCl, 25% Block Ace (Snow Brand Milk Products Co., Ltd.), 0.15
0.01 M phosphate buffer containing M NaCl (pH
7.0) 250 μl of the solution was injected into each well and stored in a cool place until use. (3) Sandwich EIA method 100 μl of NO synthase prepared at various concentrations was put in the antibody-bonded wells and incubated overnight at 4 ° C. After removing the NO synthase solution, 100-fold diluted enzyme-labeled antibody (N11Fab'-HRP) was added to each well at 1
00 μl was added and incubated at room temperature for 2 hours. After removing the labeled antibody, the HRP substrate solution was added, and the following enzymatic reaction was carried out in the same manner as in Example 2 to measure the absorbance at 492 nm. As a result, the detection limit of NO synthase in this assay system was 2.5 to 5 ng / well [Fig. 3].

[0035]

INDUSTRIAL APPLICABILITY Since the monoclonal antibody of the present invention binds to NO synthase with high sensitivity and its binding ability is high,
It can be advantageously used as a reagent for detecting or measuring NO synthase derived from various cells. Therefore, NO
It is possible to know the distribution and expression of synthase expression in cells and tissues, and to further elucidate the roles of NO synthase itself and NO.

[Brief description of drawings]

FIG. 1 shows the stabilization of the activity of NO synthase in the Cytosol fraction by the anti-NO synthase antibody obtained in Example 4.

FIG. 2 shows the stabilization of the activity of purified NO synthase by the anti-NO synthase antibody obtained in Example 4.

FIG. 3 shows a calibration curve of NO synthase obtained in Example 5 by sandwich EIA using an anti-NO synthase antibody.

[Explanation of symbols]

 B indicates Tris buffer. C indicates an anti-endothelin antibody.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] March 25, 1994

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0024

[Correction method] Change

[Correction content]

[0024]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. The antibody-producing hybridomas of the present invention obtained in the examples described below have been deposited as follows. (IFO) (NIBH) Animal cell IFO No. FERM No. ────────────────────────────────── Mouse hybridoma N2 50398 BP-4258 (1993. 4.2) (1993. 4.14) Mouse hybridoma N5 50399 BP-4259 (1993. 4.2) (1993. 4.14) Mouse hybridoma N21 50400 BP-4260 (1993. 4.2) (14 Apr 1993) Mouse hybridoma N11 50440 P-1 4234 (7 Mar 1994) 3.16 Mar 1994 ────────────────────────── ───────── IFO: Fermentation Research Institute (Osaka) NIBH: Ministry of International Trade and Industry, Institute of Industrial Science and Technology, Institute of Life and Industrial Technology In the table () indicates the date of entrustment. Example 1 Preparation of NO synthase Male Kb1 Wistar rats (body weight: 200 to 230 g) were intraperitoneally injected with 40 mg of B dissolved in 10 ml of physiological saline per mouse.
Inoculated with CG. Two weeks after the inoculation, 50 ml of phosphate buffered saline (PBS) was inoculated into the abdominal cavity of the rat, and peritoneal exudate cells were collected. After centrifugation (300 × g, 10 minutes), the supernatant was removed, and 25 ml of hypotonic NaCl (0.2%) solution was added to lyse the mixed red blood cells, and then 1.6% Na was added.
25 ml of Cl was added to return to an isotonic solution. Centrifuge again (300 x
cells, 10% fetal bovine serum, 10 ng
/ Ml lipopolysaccharide containing DMEM (Dulbec
Suspend in a co's modified Eagle's medium) and inoculate it in a 10 cm culture dish at 37 ° C and 5% carbon dioxide gas (C
Culturing was performed under O ₂ ) for 24 hours. After culturing, non-adherent cells were removed by washing 3 times with PBS, and adherent cells were detached and collected with a rubber policeman. The macrophages thus obtained were suspended in 50 mM Tris-hydrochloric acid buffer (pH 7.4) containing 1 mM DTT (dithiothreitol) at a concentration of 1 × 10 ⁸ cells / ml to give 0.1 mg / ml. ml phenylmethylsulfonyl fluoride, 0.01 mg / ml trypsin inhibitor, 0.01 mg / ml leupeptin, 0.01 mg / ml antipain, 0.01 mg / ml pepstatin, etc. were added.
Ultrasonic disruption of macrophage suspension
ptor, output 6,3 seconds, Tommy Seiko), 100,00
The supernatant was obtained by centrifugation at 0 × g for 60 minutes. Purification was performed as follows. 7.5 ml of the supernatant was applied to a 2 ', 5'-ADP-agarose column equilibrated with 50 mM Tris-HCl buffer (pH 7.4) containing 1 mM DTT. The column was washed successively with 10 ml of 50 mM Tris-hydrochloric acid containing 1 mM DTT and 750 mM NaCl and 10 ml of 50 mM Tris-hydrochloric acid containing 1 mM DTT, and 1 mM.
50 mM Tris-containing DTT and 1 mM NADPH
Elution was performed with 2 ml of hydrochloric acid. The eluted NO synthase sample was concentrated to 50 μl using Centricon 10 (Amicon, Danvers, MA), and 50 m containing 1 mM DTT was added.
Gel filtration column Superose equilibrated with M Tris-HCl
12HR10 / 30 (300 × 10mm, Pharmacia
LKB Biotechnology Uppsala (Sweden) was used to obtain the NO synthase fraction.

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Claims (9)

[Claims] 1. An inducible nitric oxide synthase as an immunogen,
An IgG type monoclonal antibody that exhibits binding properties to nitric oxide synthase. 2. The monoclonal antibody according to claim 1, which exhibits binding to nitric oxide synthase derived from brain. 3. A monoclonal antibody which shows binding to macrophage-derived nitric oxide synthase and does not show binding to vascular endothelial cell-derived nitric oxide synthase. 4. A cloned hybridoma comprising mammalian spleen cells immunized with inducible nitric oxide synthase and allogeneic or xenogeneic lymphoid cells. 5. The hybridoma according to claim 4, wherein the spleen cells of a mammal immunized with macrophage-derived nitric oxide synthase and the same or different lymphoid cells are cell-fused and cloned. Manufacturing method. 6. The method for producing a monoclonal antibody according to claim 1, 2 or 3, wherein the hybridoma according to claim 4 is grown in a liquid medium or intraperitoneally of a mammal. 7. A method for detecting or quantifying nitric oxide synthase, which comprises using the monoclonal antibody according to claim 1, 2 or 3. 8. The detection or quantification method according to claim 7, which is an enzyme immunoassay method. 9. A method for purifying nitric oxide synthase, which comprises using the monoclonal antibody according to claim 1, 2 or 3.