JP3232415B2 - Monoclonal antibodies, their production and use - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a monoclonal antibody against a nerve growth factor (also referred to as NGF in this specification) protein, its production and use. Further, the present invention provides a hybridoma producing the monoclonal antibody.

[0002]

2. Description of the Related Art Nerve growth factor (N)
(GF) is Levi-Monntalcini
[Annual New York Academy of Sai
Ens (Ann. N. Acad. Sci)55, 330 (195
2)] and Cohen et al.
Of the National Academy of Science
 USA (Proc. Natl. Acad. Sci. USA)4
0, 1014 (1954)].
It is a trophic factor essential for system differentiation, growth and survival. Most
Recently, NGF is a cholinergic neuron in the central nervous system.
Has been shown to have the effect of maintaining Ron's survival
Hefti, Journal of Neuro
Science (Journal of Neuroscience)6, 2155
(1986); Development by Hatanaka et al.
 Dev. Brain Res.39, 8
5 (1988)], and has some association with Alzheimer's disease.
It is attracting attention as a factor. Also, the brain of aged rats
Administration of NGF improves memory impairment
(Nature,329, 65 (1989)]
They are also expected to be used as treatments for senile dementia.

[0003] NGF (7) isolated from the submaxillary gland of male mice
SNGF) is a complex (α ₂ βγ ₂ ) composed of three types of subunits, α, β, and γ, of which NGF activity is observed only in the β subunit. β subunit
(βNGF, 2.5S NGF) is a dimer of the same polypeptide consisting of 118 amino acids, the amino acid sequence of which is that of Argeletti and Bradshaw.
(Bradshaw) [Procedings of the National Academy of Sciences USA
c. Natl. Acad. Sci. USA), 68 , 2417 (197
1)].

[0004] Scott et al. Succeeded in cloning mouse βNGF from a mouse submandibular gland cDNA library using an oligonucleotide synthesized based on the amino acid sequence of mouse βNGF as a probe [Nature, 302]. , 538 (1983)]. Furthermore, Ullrich et al. Cloned the NGF gene from a library of human genomic DNA using mouse βNGF cDNA as a probe, and found that the amino acid sequence of human NGF deduced from its nucleotide sequence showed 90% homology with that of mouse NGF. It was shown to have [Nature (Nature), 30 3, 821 (1983) ].

[0005]

SUMMARY OF THE INVENTION Such a human NGF
Although the gene has been cloned, human NG
There are few reports that F was detected or isolated. Human N
One of the reasons that GF is not detected from the living body is probably because an anti-mouse NGF antibody has been mainly used until now.

[0006]

Means for Solving the Problems In view of the above circumstances, the present inventors have conducted various studies to find a practical means for measuring NGF. As a result, the present inventors have prepared a monoclonal antibody against NGF that makes the measurement possible, and As a result of further research based on this, the present invention was completed. (1) a monoclonal antibody against an NGF protein that reacts with human NGF and does not recognize the N-terminal portion of human NGF;
(2), the hybridoma producing the monoclonal antibody according to (1), (3), wherein the hybridoma according to (2) is grown in a liquid medium or intraperitoneally in a mammal (1) And (4) a method for detecting and / or quantifying an NGF protein, which comprises performing an immunological assay using the monoclonal antibody according to (1).

As the NGF protein that immunizes mammals, any animal-derived NGF may be used, but a β subunit that exhibits biological activity is more preferable. Alternatively, the mutein may be used. Representative examples of animal-derived NGF include, for example, mouse NGF [Levi-Monntalcini R. et al., J. Exp.
6: 321 (1951); Varon S. et al., Biochemistry.
6: 2202 (1967)], NGF derived from snake venom [Angel
etti RH, Proc. Natl. Acad. Sci. USA 65: 668.
(1970)], guinea pig NGF [HarperGP.
279: 160 (1979)] and NGF derived from human placenta [Goldstein LD et al., Neurochem. Res. 3:
175 (1978)]. The NGF gene was cloned from a human genomic DNA library using mouse βNGF cDNA as a probe, and the amino acid sequence [Ulr
ich A. et al., Nature 303: 821 (1983)].

In order to obtain human NGF, for example, an expression vector containing a DNA having a nucleotide sequence encoding the above-mentioned NGF protein polypeptide is prepared and introduced into an appropriate host cell to produce NGF. Good. Examples of the expression vector include, for example, (a) transforming a host with a genomic library prepared from human leukocyte DNA, and (b) culturing the obtained transformant, and then transforming the transformant into a suitable method, such as DNA. A plasmid containing the target DNA is isolated by a colony hybridization method using a probe, and (c) the cloned DNA of interest is cut out from the plasmid, and (d) the cloned DNA is
Is linked downstream of the promoter in the vehicle.

[0009] The expression vector thus obtained is
Human NGF can be produced by incorporation into an appropriate host (eg, Escherichia coli, Bacillus subtilis, yeast, animal cells) and culturing the resulting transformant in a medium. The above human N
The GF protein also includes the mutein of human NGF. The human NGF mutein is essentially a mutation of the amino acid sequence of the original peptide or protein. Therefore, the mutation includes addition of an amino acid, deletion of a constituent amino acid, and substitution with another amino acid. Examples of the addition of the amino acid include those in which at least one amino acid is added. Examples of the deletion of the constituent amino acids include deletion of at least one NGF constituent amino acid. Examples of the substitution with another amino acid include a substitution of at least one NGF-constituting amino acid with another amino acid.

[0010] The at least one amino acid in the mutein in which at least one amino acid is added to NGF does not include methionine caused by the initiation codon used in expressing the peptide and a signal peptide. . As the number of added amino acids,
The number is at least one, but may be any number as long as the characteristics of NGF are not lost. More preferably, homology is recognized with NGF, and a part or all of the amino acid sequence of a protein having the same activity is exemplified.
The number of deficient constituent amino acids in a mutein deficient in at least one NGF constituent amino acid of NGF may be any number as long as the characteristics of NGF are not lost. The number of at least one NGF-constituting amino acid before substitution in a mutein in which at least one NGF-constituting amino acid of NGF is substituted with another amino acid may be any number as long as the characteristics of NGF are not lost.

Examples of the constituent amino acids before substitution include cysteine and those other than cysteine.
Cysteine is particularly preferred. Examples of constituent amino acids other than cysteine before substitution include aspartic acid, arginine, glycine, valine and the like. When the constituent amino acid before substitution is cysteine, the substituted 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 preferred. When the constituent amino acid before substitution is other than cysteine, another substituted amino acid is, for example, an amino acid before substitution in terms of hydrophilicity, hydrophobicity or charge of the amino acid. Choose one with different properties. As a specific example, when the amino acid before substitution is aspartic acid, examples of the amino acid after substitution include asparagine, threonine, valine, phenylalanine, and arginine. Particularly, asparagine and arginine are preferable. When the amino acid before substitution is arginine, the amino acids after substitution are glutamine, threonine,
Leucine, phenylalanine and aspartic acid are exemplified, and glutamine is particularly preferred. When the constituent amino acid before substitution is glycine, examples of the amino acid after substitution include threonine, leucine, phenylalanine, serine, glutamic acid, and arginine, with threonine being particularly preferred. When the constituent amino acid before substitution is serine, examples of the amino acid after substitution include methionine, alanine, leucine, cysteine, glutamine, arginine, aspartic acid and the like, with methionine being particularly preferred. When the constituent amino acid before substitution is valine, examples of the amino acid after substitution include serine, leucine, proline, glycine, lysine, and aspartic acid, with serine being particularly preferred. As the original constituent amino acids before substitution, aspartic acid, arginine, glycine, serine, and valine are preferable. As the amino acid after substitution, asparagine, glutamine, arginine, threonine, methionine, serine, and leucine are preferable.

In the above substitution, two or more substitutions may be made simultaneously. In particular, it is preferred that two or three constituent amino acids are substituted. The above mutein may be a combination of two or three of the above addition, deletion and substitution. In order to produce the mutein, a site-directed mutagenesis technique is employed. The technology is well known and is disclosed by R.F.Racer (Lather, RF) and J.P.
JP), Genetic E
ngineering), Academic Press (1983) 3rd
Page 1-50. Oligonucleotide directed mutagenesis was performed by M. Smith (M.) and S. Gillam (S.), Genetic Engineering: Principles and Methods, Plenum Press (1981) 3.
Volume 1-32.

In order to produce a structural gene encoding the mutein, for example, (a) one of the structural genes of NGF
The single-stranded DNA consisting of the single strand is hybridized with the mutant oligonucleotide primer (the codon for cysteine to be replaced by the single-stranded strand, or the region containing the antisense triplet which can be paired with this codon in some cases). The above primers are complementary to each other, provided that the codons do not differ from other amino acid coding codons or, in some cases, antisense triplets.) To extend the mutant heteroduplex (heteroduplex)
And (c) replicate this mutated heterodimer. Next, the phage DNA carrying the mutated gene is isolated and integrated into a plasmid.

By transforming a suitable host with the thus obtained plasmid and culturing the obtained transformant in a medium, mutein can be produced. The mutein lacking the constituent amino acids of NGF as an antigen in the present invention is preferably a mutein in which the number of amino acids in the amino acid sequence of NGF is 100 or more, more preferably 110 or more muteins. .

To produce the monoclonal antibody of the present invention, a mammal is immunized with human NGF protein, and the obtained spleen cells are fused with mammalian lymphoid cells.
It is then produced by cloning. The NGF
When immunizing a protein, the NGF protein 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 a carrier protein complex is used, the coupling ratio between the carrier protein and the NGF protein is about 0.1 to 30 times (carrier / NGF protein: weight ratio). Preferably, about 0.5 to 5 times is used.
Further, various condensing agents can be used for coupling the hapten and the carrier, and glutaraldehyde, carbodiimide and the like are conveniently used.

When immunizing with an NGF protein or a protein complex, experimental mammals such as sheep, goats, rabbits, guinea pigs, rats, and mice are used as immunizing mammals.
In order to obtain a monoclonal antibody, rats and mice are preferred. For example, when immunizing a mouse,
Injection is possible by any route such as subcutaneous, intraperitoneal, intravenous, intramuscular, subcutaneous, etc., but it is preferable to inject mainly subcutaneously, intraperitoneally, intravenously (particularly subcutaneous). The immunity interval, immunity amount, etc. are also highly variable, and various methods are possible. For example, immunization is performed about 2 to 6 times at 2 week intervals, and about 1 to 5 days, preferably about A method using spleen cells excised 4 days later is often used. It is desirable that the amount of the immunization be about 0.1 μg or more, preferably about 10 μg to 300 μg per mouse, as the amount of the peptide at one time. or,
Before removing the spleen, it is desirable to perform a fusion experiment using spleen cells after performing partial blood collection to confirm an increase in blood antibody titer.

The cell fusion between the spleen cells and the lymphoid cells is carried out by, for example, removing the spleen cells of the excised mouse from hypoxanthine-guanine-phosphoribosyltransferase deficiency.
(HGPRT ^-) or thymidine kinase deficiency (TK ^-) myeloma [Examples of suitable homologous or heterologous having such markers (preferably the same kind) of, P3-X63-Ag · 8U1 ( Ichimori other Journal of Immunological Method 8
0 55 (1985)], such as, fused with the lymphoid cell line. For example, it is produced by fusing according to the method of Keller and Millstein (Nature 256 : 495 (1975)). For example, a medium in which myeloma cells and spleen cells are mixed at a ratio of about 1: 5, for example, a 1: 1 mixture of Iscove's medium and Ham's F-12 medium
(Hereinafter referred to as IH medium), and a fusion agent such as Sendai virus and polyethylene glycol (PEG) is used. Of course, dimethyl sulfoxide (DMSO) and other fusion promoters can be added. The degree of polymerization of PEG is usually about 1000-6000, and about 0.5 for hours.
~ 30 min, concentration is about 10% ~ 80% etc.
As an example of preferred conditions, PEG 6000 is about 35
By performing the treatment at about 55% for about 4 to 10 minutes, efficient fusion can be achieved. The fused cells can be selectively grown using a hypoxanthine-aminopterin-thymidine medium [HAT medium; Nature, 256 , 495 (1975)] or the like.

The culture supernatant of the proliferating cells can be screened for the production of the desired antibody. The antibody titer can be screened as follows. That is, in this case, first, as a first step, the presence or absence of the production of an antibody against the immunized peptide can be examined by a method such as a radioimmunoassay (RIA) or an enzyme immunoassay (EIA). Various variants are possible. As an example of a preferable measurement method, one method using EIA will be described. For example, a rabbit anti-mouse immunoglobulin antibody is coupled to a carrier such as cellulose beads according to a conventional method, and a culture supernatant or mouse serum to be measured is added thereto, and a constant temperature (about 4 to 40 ° C. .
The same applies to the following. ). Thereafter, the reaction product is thoroughly washed, and a peptide labeled with an enzyme (purified after coupling the enzyme and the peptide according to a conventional method) is added, and the mixture is allowed to react at a constant temperature for a certain period of time. After thoroughly washing the reaction product, an enzyme substrate is added, and the reaction is allowed to proceed at a constant temperature for a certain period of time. Thereafter, the formed color product can be measured by absorbance or fluorescence.

Well cells showing growth on the selective medium and exhibiting antibody activity against the peptide used for immunization were:
It is desirable to carry out cloning by a limiting dilution method or the like. By screening the supernatant of the cloned cells in the same manner and increasing the number of well cells with a high antibody titer, a monoclonal antibody-producing hybridoma clone that is reactive with the immunized peptide can be obtained.

The hybridoma thus cloned is grown in a liquid medium. Specifically, for example, a liquid medium such as RPMI-1640 [Moore, G .;
E., et.al. Journal of the American Medical Association (J. Am.Med. Assoc.) 199 , 5
49 (1967)] in a medium or the like supplemented with about 0.1 to 40% bovine serum for about 2 to 10 days, preferably about 3 to 5 days to obtain the monoclonal antibody from the culture solution. it can. In addition, an antibody can be obtained by inoculating intraperitoneally into a mammal, growing cells, and collecting ascites. For this purpose, for example, in the case of mice, about 1 × 10 ⁴ to 1 × 10 ⁷ , preferably about 5 × 1 ⁷ mice, such as BALB / c, which have been inoculated with mineral oil or the like in advance.
The 0 ⁵ ~2 × 10 ⁶ hybridoma were inoculated intraperitoneally
The ascites fluid is collected after about 7-20 days, preferably about 10-14 days. The antibody produced and accumulated in the ascites can be easily isolated as a pure immunoglobulin by, for example, ammonium sulfate fractionation, DEAE-cellulose column chromatography, or the like.

Thus, a monoclonal antibody against the NGF protein is obtained. The monoclonal antibody of the present invention specifically binds to the NGF protein of the peptide of the immunogen. The monoclonal antibody of the present invention has an NGF different from that of the peptide of the immunogen used at the time of production.
In some cases, it binds to proteins. The monoclonal antibody of the present invention reacts with human NGF and does not recognize the N-terminal portion of human NGF. For example, human NGF
Does not recognize the antigen determination present in the first to fifteenth amino acid sequences from the N-terminus. Since the monoclonal antibody of the present invention binds to an NGF protein,
It is extremely useful as a reagent for measuring NGF protein. Further, facilitating the measurement of NGF protein in living organs and tissues is extremely useful from the viewpoint of obtaining basic knowledge (for example, biodistribution) of NGF protein. For the detection of NGF protein in living organs and tissues, the enzyme immunoassay (EI
A) or the like, or a fluorescent antibody method or a radioimmunoassay method (RIA method). In particular, EIA
The method is preferred. In addition, N present in these organs and tissues
To determine the size of the GF protein, a protein western blotting method is effective. In this method, a crude extract derived from an organ or tissue or a partially purified sample thereof is subjected to acrylamide electrophoresis, transferred to a membrane filter, and detected with an HRP-conjugated anti-NGF protein antibody.

Further, an antibody affinity column can be prepared by utilizing the binding ability between the antibody and the NGF protein and used as a reagent for purifying the NGF protein. NG
The antibody molecule used to detect and quantify the F protein may be IgG or 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 '. The monoclonal antibody of the present invention comprises NGF
It can be used as a reagent in a protein immunochemical assay. The amount of NGF protein in living tissues and body fluids can be measured by the immunochemical measurement method of the NGF protein. For example, by measuring NGF proteins in various tissues and body fluids, the central nervous system can be measured. It is thought to be useful for diagnosing the disease.

In the above immunological assay, when performing the assay using two kinds of antibodies, the monoclonal antibody of the present invention is used as one antibody, and the monoclonal or polyclonal antibody of the present invention is used as the other antibody. Can be used. Examples of the carrier in the antibody held on the carrier used in the method for measuring NGF protein include gel particles (eg, agarose gel [eg,
Sepharose 4B, Sepharose 6B (Pharmacia
Fine Chemical Company (Sweden)], Dextran gel [eg, Sephadex G-75, Sephadex G-
100, Sephadex G-200 (Pharmacia Fine Chemical Co., Sweden)], polyacrylamide gel [eg, Biogel P-30, Biogel P-6]
0, Biogel P-100 (manufactured by Bio-Rad Laboratories, Inc. (USA)), cellulose particles [eg, Avicel (manufactured by Asahi Kasei), ion-exchange cellulose (eg, diethylaminoethylcellulose, carboxymethylcellulose)], physical adsorbent [eg. , Glass (eg, glass sphere, glass rod, aminoalkyl glass sphere, aminoalkyl glass rod),
Silicon pieces, styrene-based resins (eg, polystyrene spheres, polystyrene particles), immunoassay plates (eg, manufactured by Nunc (Denmark)), ion exchange resins 交換 examples, weakly acidic cation exchange resins [eg, Amberlite IRC-50] (Made by Rohm and Haas (USA)), Zeocarb 226
(Manufactured by Palm Chit (West Germany)), weakly basic anion exchange resins [eg, Amberlite IR-4B, Dowex 3 (manufactured by Dow Chemical Company (USA)]}.

For retaining the antibody on the carrier, known conventional means can be applied. For example, "Metabolism", Vol.
71), p. 696, bromocyan method, glutaraldehyde method and the like. Also,
As a simpler method, it may be physically adsorbed on the carrier surface. Examples of the labeling agent in the antibody to which the labeling agent is bound include a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, and the like, and it is preferable to use an enzyme. As the enzyme, a stable and inactive large enzyme is preferable, and peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase and the like can be used, but peroxidase is preferable. As peroxidase, those of various origins can be used,
Examples include peroxidase obtained from, for example, horseradish, pineapple, fig, sweet potato, fava bean, corn, and the like, particularly horseradish peroxidase (horse) extracted from horseradish.
radish peroxidase (HRP) is preferred. Fab as an antibody molecule when binding antibody to peroxidase
In order to utilize the thiol group of ', it is convenient to use a peroxidase in which a maleimide group has been introduced in advance. As a method for introducing a maleimide group into peroxidase, a maleimide group can be introduced via an amino group of peroxidase. For that, N
-Succinimidyl-maleimide-carboxylate derivative can be used, and N- (γ-maleimidobutyloxy) succinimide (which may be abbreviated as GMBS) is preferable. Therefore, a certain group may be inserted between the maleimide group and the peroxidase. The reaction of GMBS with peroxidase is carried out by reacting both at a temperature of about 10 to 50 ° C. for about 10 minutes to 24 hours in a buffer having a pH of about 6 to 8. Examples of the buffer include p
H7.0 0.1M phosphate buffer and the like. The maleimidated peroxidase thus obtained is
For example, it can be purified by gel chromatography or the like. As the carrier used for performing the gel chromatography, for example, Sephadex G-25
[Pharmacia Fine Chemical Company (Sweden)],
Biogel P-2 [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 with each other in a buffer at a temperature of about 0 ° C. to 40 ° C. for about 1 to 48 hours. The buffer may be, for example, 0.1 mM containing 5 mM ethylenediaminetetraacetic acid sodium salt at pH 6.0.
M phosphate buffer and the like. The thus obtained peroxidase-labeled antibody can be purified by, for example, gel chromatography. As a carrier used for performing the gel chromatography, for example, Sephadex G-25 [Pharmacia.
Fine Chemicals (Sweden)], Biogel P-
2 [manufactured by Bio-Rad Laboratories (USA)].

Further, 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 carried out according to the method of peroxidase, and a per se known glutaraldehyde method, periodate method, water-soluble carbodiimide method and the like are used. The test sample in the measurement system of the present invention includes body fluids such as urine, serum, blood cells, and cerebrospinal fluid, or extracts of animal cells or bacterial cells or culture supernatants thereof. As an example of the measurement 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, the NGF to be measured is added to the antibody held on the carrier.
After an antigen-antibody reaction was performed by adding a protein-containing analyte, the peroxidase obtained above and anti-NG were added thereto.
A conjugate with the F protein antibody is added and reacted.

The test sample in this measurement system includes:
Examples include body fluids such as urine, serum, blood cells, and cerebrospinal fluid, or extracts of animal cells or bacterial cells or culture supernatants thereof. : A peroxidase substrate is added to the reaction product obtained in step (1), and the enzyme activity of the above reaction product is determined by measuring the absorbance or fluorescence intensity of the resulting substance. The above-mentioned operations are performed in advance on a known amount of a standard solution of NGF protein, and the relationship between the NGF protein and absorbance or fluorescence intensity is prepared as a standard curve. : Analyte containing unknown amount of NGF protein (test sample)
The amount of NGF protein in the analyte is measured by fitting the absorbance or fluorescence intensity obtained for the above to a standard curve.

The NGF protein can be purified using an antibody obtained using a complex of a monoclonal antibody against the NGF protein and a carrier protein as an immunogen. This can be performed by performing affinity column chromatography using the antibody. The affinity column chromatography includes, for example,
The antibody can be coupled to a suitable carrier, packed in a column, a solution containing an NGF protein can be adsorbed through the column, and then eluted.

Examples of the carrier include those similar to the carriers described above. In particular, gel particles and various synthetic resins are advantageously used. For example, CNBr
-Activated Sepharose 4B (Pharmacia Fine Chemicals), Affigel-10, Affigel15 (Bio-Rad Laboratories) and the like.

For coupling the antibody to the carrier, known conventional methods can be applied.
Vol. (1971), p. 696, bromcian method and glutaraldehyde method. Also,
A method using a water-soluble carbodiimide, an active ester method, and the like can be used, but a simpler method may be to physically adsorb to the carrier surface.

In order to carry out purification using the antibody column thus obtained, the NGF protein in a buffer near neutrality is adsorbed on an antibody column packed with a carrier to which the antibody is bound. Next, after the column is washed with the same buffer, the NGF protein specifically adsorbed is eluted. To elute specifically adsorbed NGF protein, for example,
The reaction is performed using a buffer having a high pH or a high pH, or a buffer containing a high concentration of salt.

As the low pH buffer, for example, pH
2.3 0.17 M glycine-HCl buffer, pH 1.8 0.1 M sodium citrate-HCl buffer and the like. Examples of the high pH buffer include pH 11
And a 0.2 M sodium borate buffer having a pH of 11.7. Examples of the buffer containing the salt at a high concentration include a 6M guanidine hydrochloride solution, a 7M urea solution and the like. The above-mentioned elution may be a batch method or a method using a column. The antigen eluate is purified, for example, by dialysis. For example, when eluted with a low pH buffer, for example, 0.1 M sodium carbonate buffer (pH 10.5), and when eluted with a high pH buffer, for example, 0.1 M glycine-hydrochloride buffer (pH 3.0) ) And then 0.02M containing 0.1% NaN ₃ for example.
Dialyze against phosphate buffer (pH 8.0). The antigen solution eluted with a buffer containing a high concentration of salt can be directly dialyzed and stored in the above-mentioned phosphate buffer. Further, the eluate or dialysate can be stored as a freeze-dried preparation obtained by freeze-drying. Since the NGF protein purified in this manner is of a very high unit, for example, has a nerve cell growth action,
It can be used effectively for the treatment of various neurological disorders. The human NGF protein obtained as described above is useful as a reagent used in studies on the brain and nervous system, and is also expected as a therapeutic drug for senile dementia.

To use human NGF protein for these studies, for example, human NGF protein is used in an amount of about 0.1 to 1,000 ng / ml, more preferably about 1 to 100 ng per ml of animal cell culture medium. Is preferably added. In the specification and drawings of the present invention, when bases and amino acids are indicated by abbreviations, IUPAC-IUB
It is based on the abbreviation by Commision on Biochemical Nomenclature or the abbreviation commonly used in the art,
An example is given below. In addition, when an amino acid can have an optical isomer, the L-isomer is indicated unless otherwise specified. The transformant CHO-D31- obtained in Reference Example 6 described later.
10 is the Fermentation Research Institute from November 15, 1999
(IFO) and accession number IFO 50217. The microorganism was deposited on December 7, 1999 with the Research Institute of Microorganisms and Technology (FRI) of the Ministry of International Trade and Industry under the accession number FERM BP-2674. The following hybridoma obtained in Example 1 described below has been deposited in the IFO as an accession number indicated by the next IFO number from April 5, 1990, and has been deposited in the FR since August 24, 1990.
I have been deposited with I as the accession number indicated by the following FERM BP number. NGFA-133 IFO 50239 FERM BP-3078 NGFB-260 IFO 50240 FERM BP-3079

[0034]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Reference Examples and Examples, but the present invention is not limited thereto. Reference Example 1 Construction of Human NGF Expression Vector (1) A λEMBL3 genomic library (Clontech) prepared from human leukocyte DNA was used for E. coli NM.
After infecting 538, about 3 × 1 on a soft agar plate
0 were seeded by ⁴ clone. After transferring the plaque onto a nylon membrane (Amersham Co., Ltd., Hibond-N), the plaque was added to the membrane.
After immersion in a 5N NaOH-1.5M NaCl solution for 6 minutes to denature the phage DNA, 0.5M Tris-HCl was added.
(pH 8.0)-Immersion in a 1.5 M NaCl solution for 6 minutes. This membrane is immersed in a 2 × SSC solution, air-dried, and
The DNA was immobilized on the membrane by time treatment. On the other hand, a known human NGF (Ullrich, A. et al., Nature 303 , 821 (1983))
DNA encoding human βNGF with reference to gene
(0.38 kb) was chemically synthesized, and labeled with ³² P using a DNA labeling kit (Nippon Gene) as a probe. The DNA-immobilized filter was filtered using 6 × SSC (1 × SSC = 0.15M) containing a labeled probe.
NaCl, 0.015 M sodium citrate), 5 × Den-ha
rdt's, 0.5% SDS, 20 μg / ml denatured salmon sperm DN
The solution was kept at 65 ° C. for 16 hours in 10 ml of the solution A. After the reaction, the filter was washed three times in a 2 × SSC, 0.1% SDS solution at room temperature for 5 minutes at 60 ° C. for 60 minutes in a 1 × SSC, 0.1% SDS solution. After drying the washed filter, a radioautogram was taken to find a clone that reacted with the probe. From the clone λβLN2113 obtained by this method, the method of Davis et al. (Davis et al., [Advanced Bacterial Genetics], Cold Spri
ng Harbor Laboratory 1980)
Was extracted.

Next, λβLN2113 was digested with SmaI and ApaI, a DNA (about 1 kb) containing the human NGF gene was cut out, the SmaI and ApaI sites of plasmid pBluescriptIIK (purchased from Toyo Bo) were inserted, and plasmid pNG was inserted.
FP107G was obtained. The nucleotide sequence of the inserted portion was determined using Sequenase (Toyo Bo) (sequence list: SEQ ID NO: 1) (continuous ones in FIG. 1 and FIG. 2). The determined nucleotide sequence is Nature, 30
3 , 821 (1983), in the protein coding region. Phage λβLN as described above
2113 DNA is digested with restriction enzyme BglII, and human NG
A DNA fragment containing F (1.8 kb) was isolated. On the other hand, the expression vector pKSV-10 (Pharmacia) for animal cells was digested with the restriction enzyme BglII, and ligated to the above DNA fragment (1.8 kb) containing human NGF with T4 DNA ligase. Using this reaction solution, Escherichia coli DH1 was transformed, and one of the ampicillin-resistant transformants [Escherichia coli (E.
The plasmid isolated from scherichia coli DH1 / pMNGF101 was named pMNGF101 (FIG. 3).

Reference Example 2 Construction of Human NGF Expression Vector
(2) The plasmid pNGF107G obtained in Reference Example 1 was digested with restriction enzymes BclI and ApaI, and a DNA fragment (0.8 kb) containing the human NGF gene was isolated. This 0.8kb
The BclI-ApaI fragment was mixed with the chemically synthesized adapters SN1 (SEQ ID NO: 2), SN2 (SEQ ID NO: 3) and SN3 (SEQ ID NO: 4) (see FIG. 4), and T4 DNA ligase was used. And cut with BglII to obtain 0.8 kb HindIII-BglII DN.
The A fragment was obtained. Plasmid pSV2-gpt (Science, 209 , 1422 (1980)) was digested with restriction enzymes EcoRI and HindIII, and a 2.6 kb EcoRI-HindIII DNA fragment containing the SV40 promoter was isolated. Next, the plasmid pMTVdhfr [Nature (Natur
e), 294 , 228 (1981)] to isolate a 1.6 kb BglII-EcoRI fragment containing a polyA addition region. 2.6 kb EcoRI-HindI containing the above SV40 promoter
II DNA fragment, 0.8 kb Hind containing human NGF gene
A III-BglII DNA fragment and a 1.6 kb BglII-EcoRI fragment containing a polyA addition region were ligated with T4 ligase. Using this reaction mixture, Escherichia coli (Esche
richia coli) DH1 and transformants resistant to ampicillin [Escherichia c.
oli) DH1 / pMNGF201] was designated as pMNGF201 (FIG. 4).

Reference Example 3 Construction of Human NGF Expression Vector
(3 ) Plasmid pMNGF201 obtained in Reference Example 2 was
After digestion with III, blunting by DNA polymerase Klenow fragment reaction, digestion with BglII to about 0.8
The kb DNA fragment was separated. On the other hand, plasmid pTB399
After digesting EcoRI (described in JP-A-61-63282) with EcoRI, blunting by Klenow fragment reaction,
Cleavage with BglII yielded an approximately 3.9 kb DNA fragment. These two DNA fragments were circularized by a T4 DNA ligase reaction to obtain a plasmid pTB1054. Next, the plasmid pTB34 having the hamster DHFR cDNA was used.
8 (described in JP-A-61-63282) was digested with ClaI, treated with alkaline phosphatase, and separated and purified from pTB1054 digested with ClaI. A 2.4 kb DNA fragment (MnLVLTR, human NGF (hNGF) gene, and SV40 DNA-derived DNA fragment) Splice region and poly [A] -added region) to the T4 DNA ligase reaction.
Thus, a human NGF expression vector pTB1058 was constructed (FIG. 5).

Reference Example 4 Transformation of CHO Cells Ham12 medium containing 5% fetal bovine serum was placed in a falcon dish (6 cm in diameter), and hamster DHFR ^- CHO cells were cultured at 37 ° C. overnight. After culturing, the cells (7 × 10 ⁵
Using the human NGF expression vector pTB1058 (10 μg) obtained in Reference Example 3 according to the method of Graham et al. [Virology 52 : 456-467].
(1973)]. After culturing at 37 ° C. for 4 hours, culturing was continued in place of a fresh medium. 2 days later, 5
The solution was changed in Dulbecco's modified MEM culture containing 35% dialysis fetal bovine serum and 35 µg / ml proline, and after continuing culture in this selective medium, about 2 to 3 weeks later, DMF
Cells that grew as R ⁺ formed colonies.

Reference Example 5 Cloning of transformant and
Cloning of the transformed cells obtained in Reference Example 4 and the expression of the human NGF gene was carried out according to a known method (for example, limited dilution method), and the transformant (cloned product) CHO-D
5, CHO-D42 and CHO-M36 were obtained. After completion of the cloning, each cloned cell was subjected to 5% fetal bovine serum, 35 μg / ml proline, 50 IU / ml penicillin,
The cells were cultured in Dulbecco's modified MEM medium containing 50 μg / ml streptomycin. The cells of each isolated clone are seeded on a limb dish, and when the cells are about 80% confluent, the medium is replaced with a fresh medium, and after culturing for 72 hours, NGF in the culture supernatant is subjected to EIA [Boehringer; Chemical Biophysical Research Communication (Biochem. Biophys. Res. Commun., 1
55 , 482 (1988)].

The clones producing high amounts of human NGF are shown in [Table 1]. NGF was not detected in the culture supernatant of untransformed CHO cells.

[Table 1] The above results revealed that CHO cells that permanently express the human NGF gene can produce more human NGF than COS cells that temporarily express the gene.

REFERENCE EXAMPLE 6 Transformant CHO-D obtained in the same manner as in Reference Example 5 for a human NGF-high-producing CHO cell line
31 was cultured in Dulbecco's modified MEM containing 10 nM methotrexate (MTX) (containing 35% of 5% fetal bovine serum / 35 g / ml proline). Since this clone showed normal growth at this concentration of MTX, the MTX concentration was 1
The culture was subcultured at a concentration of 00 nM. Furthermore, when the MTX concentration was 1 μM, most of the cells were killed. However, when the liquid was changed on 3 to 4 days and the culture was continued, several cells per 10 ⁵ cells began to grow in a colony. After these cells were sufficiently grown, when they were subcultured in a culture solution of 10 μM MTX, most of the cells were killed again, and several cells began to grow in a colony. The cells thus obtained show stable and normal growth in the presence of 10 μM MTX. After returning to a culture solution containing no MTX and growing for several passages, the cells are cultured in the presence of 10 μM MTX. Grew normally. CHO-D resistant to this 10 μM MTX
31-10 cells (IFO 50217, FERM BP-
2674) was cultured under the same conditions as in Reference Example 5 to find that a 4 m
g / l of human NGF was produced in the medium.
I found it in IA.

Reference Example 7 Isolation of human NGF (1) 5% of the cell line CHO-D31-10 obtained in Reference Example 6
When a large amount of cells were cultured in a Dulbecco's modified medium containing fetal bovine serum, 35 µg / ml proline, 50 IU / penicillin, 50 µg / ml streptomycin, and 10 µM methotrexate in 5% carbon dioxide gas at 37 ° C for 7 days, 2.4 mg was found in the medium. / L of human NGF is produced
I found it in IA. The culture solution was centrifuged, APMSF was added to 2.2 liters of the culture supernatant to a final concentration of 0.1 mM, and the pH was adjusted to 6.0 with 0.2N acetic acid, followed by centrifugation. The obtained supernatant was washed with 0.1 M phosphate buffer (pH 6.0).
S-Sepharose column equilibrated with -1 mM EDTA
(2.6 cm x 14 cm) and 0.1 M phosphate buffer p
H6.0-0.15M NaCl-1mM EDTA-10%
After washing with glycerol, 50 mM Tris-HCl p
H7.5-0.7M NaCl-1mM EDTA-10%
Eluted with glycerol. Fractions containing human NGF were collected and concentrated about 30-fold with a Diaflow cell (Type YM10, Amicon). The obtained concentrated solution was adjusted to 20 mM.
Tris-HCl pH 7.4-0.5M NaCl-1mM E
Sephacryl equilibrated with DTA-10% glycerol
The mixture was subjected to gel filtration with S-100HR (200 ml, 1.6 cm × 100 cm). Fractions containing human NGF were collected and concentrated about 10-fold with Centriprep 10 (Amicon).
The obtained concentrate was subjected to reverse phase HPLC to purify human NGF. That is, the concentrated solution was mixed with Asahipak ODP-50 (1
The solution was applied to a 0.0 mm ID × 250 mm L) column and subjected to a concentration gradient of 0-90% acetonitrile containing 0.1% trifluoroacetic acid to obtain 1.2 mg of purified human NGF (from amino acid analysis). As a result of SDS-polyacrylamide gel electrophoresis and reverse phase HPLC, the purity of the obtained recombinant human NGF was 94%. The purified sample thus obtained was subjected to 16% polyacrylamide gel electrophoresis containing 0.1% SDS and subjected to silver staining. As a result, a single band of 13 kilodalton (kDa) was observed. Also,
Western blotting using rabbit anti-mouse NGF antibody (Collabarative Research) and affinity-purified HRP-conjugated goat anti-rabbit IgG (Bio-Rad, USA) revealed bands at the same positions as above. The obtained purified human NGF was placed in a glass hydrolysis test tube, dried under reduced pressure, added with 5.7N hydrochloric acid containing 4% thioguticholic acid, sealed, and then hydrolyzed at 110 ° C. for 24 hours. . After the hydrolysis, the hydrochloric acid was removed and the residue was treated with 0.1 ml.
Amino acid analysis was performed after dissolving in 02N hydrochloric acid. The results are shown in [Table 2].

[0043]

[Table 2] Amino acid composition 1) Asp + Asn was calculated as 13. 2) It was calculated from the amino acid sequence deduced from the nucleotide sequence of the human NGF gene. The N-terminal amino acid sequence of purified human NGF was converted to a gas-phase protein sequencer (Applied Biosystems, Inc. model 47).
OA). The results are shown in [Table 3].

[0044]

[Table 3] N-terminal amino acid sequence ───────────────────────────── PTH-amino acid sequence amino acid sequence estimated from DNA ───────────────────────────── 1 Ser 605 Ser 2 Ser 495 Ser 3 Ser 384 Ser 4 His 785 His 5 Pro 705 Pro 6 Ile 767 Ile 7 Phe 829 Phe 8 His 341 His 9 Arg 700 Arg 10 Gly 425 Gly 11 Glu 478 Glu 12 Phe 517 Phe 13 Ser 97 Ser 14 Val 467 Val 15 - Cys 16 Asp 297 Asp 17 Ser 58 Ser 18 Val 392 Val 19 Ser 73 Ser 20 Val 476 Val 21 Trp 95 Trp 22-Val 23 Gly 166 Gly 24 Asp 179 Asp 25 Lys 245 Lys 26 Thr 74 Thr 27 Thr 102 Thr 28 Ala 179 Ala ─────────────────────────────

2.9 nmole of purified human NGF was used for the analysis. Hydrazinolysis [Natita et al., Journal of Biochemistry., 59 , 170 (196)
6)], the C-terminal amino acid of purified human NGF was alanine. As a result of a PAG isoelectric focusing method (new edition of electrophoresis experiment method, edited by the Electrophoretic Society of Japan, Spectroscopy, 1989), the isoelectric point of the obtained human NGF was pH9-10, and that of mouse NGF (Collaborative Rese-arch Incorpo
rated). Brain Research, 133 , 350 (1977), Ex
perimental Cell Research, 145 , 179 (198
3) and Journal of Neu-roscience Research, 17 ,
25 (1987).
When the activity of purified human NGF was measured using neurite outgrowth as an index, the purified human NGF showed activity comparable to that of a mouse 2.5S NGF standard (Wako Pure Chemical Industries). .

Reference Example 8 Analysis of Intramolecular Disulfide Bond Using the purified human NGF obtained in Reference Example 7, the position of the intramolecular disulfide bond was determined. Lyophilized purified human NGF (530 μg) was added to 0.2 ml of 0.9% NaCl.
The solution and 0.8 ml of 0.01 M HCl were added and dissolved. The pH of this solution was 2.2. The weight ratio is 1
1 mg / ml pepsin (Sigma)
10.6 μl of the solution was added and reacted at 37 ° C. for 22 hours. After the reaction, 950 µl was collected and 50 µl of 250 mM
The reaction was terminated by adding a phosphate buffer (pH 6.0) to obtain a pepsin digest. The pepsin digest was subjected to HPLC and peptide mapping was performed. TSK-Gel ODS
Using a -120T column (Tosoh Corporation, 0.46 x 25 cm), a mixture of 0.05% TFA (A) and 99.95% acetonitrile 0.05% TFA (B) was run according to the following elution program. Flow rate is 1 ml / min, detection wavelength is 2
20 nm and 280 nm. On the other hand, 10 μl of a 100 mM DTT solution was added to 50 μl of the pepsin digest and left at room temperature for 3 hours, and the sample in which disulfide bonds were reduced was subjected to HPLC in the same manner.
Peptide mapping was performed. By comparing these two peptide mappings, a peptide having a disulfide bond was immobilized, then the peptide was isolated, and the amino acid sequence of a gas phase protein sequencer terminal (ABI) was analyzed. As a result, the present peptide was found to be a large peptide containing the following three peptides and having three disulfide bonds.

13 15 20 SVVCDSVSV (SEQ ID NO: 5)

54 58 60 65 70 F-E-T-K-C-C-R-T-P-P-N-P-P-V-D-D-S-G-C-R-G 73 -ID-S- ( Sequence Listing: SEQ ID NO: 6) 102 105 110 115 IRIDACTVCVCLVL-S-R-K-A-VR 120-RA ( Sequence list: SEQ ID NO: 7)

Next, this peptide (2060 pmol) was concentrated to dryness under reduced pressure and dissolved in 0.3 ml of 50 mM sodium acetate (pH 6.0). 0.48 μg in this solution
After adding (14 pmol) of thermolysin (Wako Pure Chemical Industries, Ltd.) and reacting at 37 ° C. for 20 hours, the mixture was subjected to HPLC under the same conditions as described above to perform peptide mapping. However, the following elution program was used.

Further, in order to determine the position of the disulfide bond, a sample obtained by reducing the above peptide with DTT was subjected to HPLC in the same manner, and peptide mapping was performed. From the comparison of peptide mapping, three fragments containing a disulfide bond (fragment-1, fragment-2, and fragment-3) were immobilized and obtained. The amino-terminal amino acid sequences of fragment-1, fragment-2, and fragment-3 were analyzed. The results are shown in [Table 4]. On the other hand, these fragments were oxidized with performic acid to convert cysteine residues to cysteic acid, and amino acid analysis was performed. As a result, cysteic acid was detected in each of the fragments by two residues.

[0050]

[Table 4] From these results, the positions of the disulfide bonds in purified human NGF were Cys ¹⁵ -Cys ⁸⁰ , Cys ⁵⁸ -Cys ¹⁰⁸ and Cys.
It was concluded that s ⁶⁸ -Cys ¹¹⁰ . Reference Example 9 human NGF N-terminal ( 1-15) peptide H-
Ser Ser Ser His His ProIle Phe His Arg Gly G
lu Phe Ser Val Cys OH (SEQ ID NO: 8)
The N-terminal peptide of human NGF was synthesized by a solid-phase synthesis method using an automatic peptide synthesizer 430A (manufactured by Applied Biosystems). The program used was “standard”. Is like the basic synthesis process, Merrifield Arubi (Merri field, RB) (1969 ) Advance of Enzymology (Adv. Enzymol.) 32, 2
21-296. Boc-C for resin
Using ys (MeBzl) · PAM-P (0.5 mmol / g), synthesis was performed sequentially from the carboxyl terminal. Boc-Val, Boc-Ser (Bzl), Boc-Phe, Boc-Glu as Boc-amino acids
(OBzl), Boc-Gly, Boc-Arg (Tos), Boc-His
(Tos), Boc-Ile and Boc-Pro were used. Amino terminal S
After the synthesis, the peptide resin was taken out of the synthesizer and dried. 1.5 g of p-protein per gram of peptide resin
Cresol and 0.5 ml of 1,2-ethanediotyl were added, and about 8 ml of liquid hydrogen fluoride were added to give
The reaction was carried out at 2 ° C. for 2 hours. After completion of the reaction, hydrogen fluoride was removed under reduced pressure in a desiccator, and the mixture was washed with diethyl ether containing 0.1% of 2-mercaptoethanol and then with diethyl ether to remove most of the mixed reagent. The peptide was extracted with 10 ml of 3% acetic acid, and the resin mixed in the extract was removed by filtration. The filtrate was purified by gel filtration using Sephadex G-25. The gel filtration conditions were as follows: column size: 2.8 × 60 cm; detection wavelength: 230 nm; solvent: 3% acetic acid; flow rate: 40 ml / hr.

The fractions containing the peptide were collected and freeze-dried, and the obtained powder preparation was further purified by reversed-phase high performance liquid chromatography. Column YMC pack, A-324 ODS 10 × 250 nm; Column temperature 2
5 ° C; elution solvent A 0.1% -aqueous trifluoroacetic acid-9
9.9% distilled water; elution solvent B 0.1% -trifluoroacetic acid water-99.9% acetonitrile; elution program 0 min
(90% A + 10% B), 30 minutes (60% A + 40% B);
Elution rate 1.6 ml / min, detection wavelength 230 nm. Under these conditions, the main peak fraction eluted at a retention time of 29.14 minutes was collected, and used for Biorad AG 1 × 8 (AcOH type, 1.8 × 5c).
The washing solution was collected through the column of m), and acetonitrile was distilled off, followed by freeze-drying. 76 mg of a white powder was obtained. Thin layer chromatography: Rf = 0.71 (Avicel gel plate; developing solvent; n-butanol: AcOH: pyridine: water = 30: 20: 6: 4). Elman G
GL) (1959) Architectural Biochemistry and Biophysics (Arch. Biochem. Biop.
hys.) 82 , Determination of free SH groups by the 70-77 method: 9
8%. Amino acid analysis value Ser 3.99 (4); Glu 1.00 (1); Pro
0.98 (1); Gly 1.19 (1); 1 / 2Cys 0.84 (1H); Val 1.03
(1); Ile 1.01 (1); Phe 2.12 (2H); His 1.84 (2); Ar
g 0.94 (1). Recovery rate 71.1%. 1 / 2Cys was quantified by the formic acid oxidation method. The values in parentheses indicate theoretical values.

Reference Example 10 Human NGF N-terminal peptide
0 work made of bovine serum albumin of a complex with bovine serum albumin (BSA) (132mg) of 3ml.
It was dissolved in 1M phosphate buffer (pH 7.5). 1 in this solution
A dimethylamide solution containing 1.2 mg of N- (γ-maleimidobutyloxy) succinimide (GMBS) (20
0 μl) was added dropwise while stirring with a stirrer, and the mixture was reacted at 30 ° C. for 30 minutes. The reaction solution was eluted with 0.1 M phosphate buffer (pH 6.5) -0.1 M NaCl.
It was purified by xG-25 (1.5 × 30 cm) to obtain BSA (maleimidated BSA) into which a maleimide group was introduced. The above maleimidated BSA (20 mg) was dissolved in 0.1 M phosphate buffer (pH 6.5) -0.1 M NaCl. on the other hand,
Human NGFN-terminal peptide (5 mg) obtained in Reference Example 9 above
Was dissolved in 0.1 M phosphate buffer (pH 6.0) -5 mM EDTA. Mix both solutions (total volume 5 ml or less), 30 ° C
For 60 minutes, adding PBS and adding human NGF N
12 ml of a solution containing the complex of the peptide and BSA was obtained. This solution was dispensed in 1.5 ml portions and used for immunization of rabbits.

Reference Example 11 Anti-human NGF N-terminal peptide
Preparation of Antibody The complex of human NGF N-terminal peptide and BSA obtained in Reference Example 10 above was mixed well with Freund's complete adjuvant, and the mixture was injected subcutaneously into rabbits. Since then
Every two weeks the above complex and Freund's incomplete adjuvan
The mixture with t was injected into the same rabbit. Blood collected from rabbits immunized as described above was centrifuged to obtain antiserum. ELISA using the antibody titer of the obtained antiserum as a human NGF N-terminal peptide antigen
As a result, a high antibody titer was observed. The antiserum was purified by ammonium sulfate precipitation and DEAE cellulose column chromatography to obtain an anti-human NGF N-terminal peptide polyclonal antibody.

Example 1 (1) Immunization: BALB / c mice (♀8 weeks old) were challenged with 50 μg of antigen human N dissolved in 0.15 ml of physiological saline.
The same amount of GF (obtained in Reference Example 7) as Freund's complete adjuvant (Difco) was mixed and injected subcutaneously. 2
After a week, the same amount of antigen and 0.15 ml of a mixture of Freund's incomplete adjuvant were subcutaneously administered again. Further booster immunization was performed 2 weeks, 4 weeks, and 6 weeks later.
Two weeks after the next immunization, 75 μg of human N dissolved in physiological saline
GF was inoculated intravenously.

(2) Cell fusion: Three days after the final administration of the antigen, the spleen was excised from the immunized mouse obtained in the above (1) to obtain cells to be used for cell fusion. These cells were cultured in a mixture of Iscove's medium and Ham's F-12 medium at a ratio of 1: 1 (hereinafter, referred to as a medium).
IH medium). Mouse myeloma cell P
3-X63-Ag · 8UI was subcultured in RPMI 1640 medium containing 10% fetal bovine serum under the conditions of 5% carbon dioxide and 95% air. Cell fusion was performed by the method established by Kohler and Milstein et al. [Kohler, G. and Milstein, C .; Nature 256 , 495 (197).
5)]. The above myeloma cells 2.4 × 10 ⁷
And immunized lymphocytes 1.2 obtained by the method described above
× 10 ⁸ were mixed, spun down, and 45% polyethylene glycol 6000 (hereinafter P) dissolved in 0.3 ml of IH medium.
EG6000. ) Was added dropwise. The PEG6000 solution was pre-warmed to 37 ° C. and slowly added dropwise. Eight minutes later, IH medium pre-warmed to 37 ° C. was added in 0.5 ml portions per minute to make 10 ml, and then centrifuged at room temperature at 600 rpm for 15 minutes to remove the supernatant. The cell precipitate was suspended in 200 ml of IH medium containing 20% calf serum, and 100 μl of each 768 well was seeded on a 96-well microplate (Nunc). One day later, HAT (hypoxanthine 1 × 10
^-4 M, aminopterin 4 × 10 ^-7 M, thymidine 1.6 ×
100 μl of IH medium (containing 20% calf serum) (hereinafter referred to as HAT medium) containing 10 ⁻⁵ M) was added to each well, and every three days, １ ／ of the medium was added to HAT medium. Replaced. The cells grown in this way are hybrid cells.

(3) Search for antibody-producing cells: 10 μg of recombinant human NGF purified by the method described in Reference Example 7
/ 100 ml / 0.01 M carbonate buffer (pH 8.5), 100 μl of the diluted solution was added to each well of a 96-well immunoplate (manufactured by Nunc), and allowed to stand at 4 ° C. overnight. NGF was bound. After washing with 0.01M phosphate buffer (pH 7.0) containing 0.15M NaCl,
1% bovine serum albumin to block excess binding sites
200 M of 0.01 M phosphate buffer containing (BSA)
Each well was poured into wells and stored in a cool place until use.
100 μl of the hybrid cell culture supernatant was placed on the 96-well immunoplate to which human NGF was bound as described above.
The mixture was added at room temperature and incubated at room temperature for 2 hours. After removing the culture supernatant and washing, a horseradish peroxidase (HRP) -labeled anti-mouse IgG goat antibody (Kappel) as a secondary antibody
Was added and incubated at room temperature for 2 hours. After removing the secondary antibody and thoroughly washing the wells, 1 part of a peroxidase substrate solution (a sodium citrate buffer solution having a pH of 5.5 containing 0.02% H ₂ O ₂ and 0.15% o-phenylenediamine) was added.
Add 00 μl, react at 25 ° C for 10 minutes, and add 2N-sulfuric acid 1
After the enzyme reaction was stopped by adding 00 μl, the absorbance at 492 nm was measured using an automatic colorimeter for microplate (MTP-32, manufactured by Corona) (ELI).
SA method). By this method, the presence of bound antibody was observed in two wells.

(4) Cloning of hybrid cells: The cells in these wells were seeded in advance as 5 × 10 ⁴ mouse thymocytes and vegetative cells at a rate of 0.5 cells per well. The cloning was carried out on 96-well microplates. As a result, 1 representative clone cell was obtained from each well, and a total of 2 clone cells, that is, NGFA-133 (IFO 50
239, FERM BP-3078), NGFB-260
(IFO 50240, FERM BP-3079) was obtained.

(5) Production of antibody: The two hybridoma clones obtained by cloning in (4) above were intraperitoneally injected into BALB / c mice to which 0.5 ml of mineral oil had been previously administered intraperitoneally. 1 in
Ascites was inoculated by inoculating 1 × 10 ⁶ mice per animal. Ten days after the administration of the hybridoma to the abdominal cavity, ascites was collected. From about 10 ml of each ascites obtained,
Monoclonal antibodies were purified according to the method of Stalin et al. [Journal of Biological Chemistry, 256 , 9750-9754 (1981)]. First, 10,000 rotations 1 to remove fibrin-like substance from ascites
After centrifugation for 5 minutes, phosphate buffer-saline (PBS: 8.
1 mM-Na ₂ HPO ₄ , 1.5 mM KH ₂ PO ₄ , 27 mM K
Cl, 137mM NaCl, 280nm of ultraviolet absorption (A ₂₈₀₎ was diluted to a concentration showing the value of 12 to 14 at pH 7.2).
After dilution, a saturated ammonium sulfate solution was added to the sample to a concentration of 47%, salting out was performed for 60 minutes with stirring at 4 ° C, and then centrifugation (10,000 rotations, 15 minutes) was performed to obtain a precipitate. Was. The precipitate was dissolved in a 20 mM Tris buffer solution (pH 7.9) containing 50 mM NaCl, and the same solution 2 was used.
1 was dialyzed. Two hours later, 2 liters of the same dialysate was replaced with dialysis for another 15 hours. After dialysis,
The precipitate was centrifuged 10,000 rpm for 15 minutes to remove, to prepare the supernatant as the value of the A ₂₈₀ to a concentration of 20 to 30. The 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 to give 50 mM NaCl.
After washing well with Tris buffer solution containing Cl, 50 mM-5
Fractionation was carried out at a flow rate of 1.5 ml / min using a gradient salt solution of the same buffer solution containing 100 mM NaCl, and the fractions passed through were concentrated. Each purified monoclonal antibody NGF
A-133 and NGFB-260 were obtained. The purity of the antibody was confirmed by the method of Laemli et al. [Nature, 227 , 68].
0-685 (1970)], and SDS-polyacrylamide gel electrophoresis was used. That is, ammonium sulfate salting out,
The fraction passed through the DEAE cellulose column was reduced with 2-mercaptoethanol to give an acrylamide concentration of 10
The gel was run at 180 volts for 2.5 hours using a% gel. As a result, each monoclonal antibody had a molecular weight of 5
Two bands of H chain were observed around 2K, and L chain was observed around 28K.

(6) Measurement of antibody subclass: The antibody subclass was measured by the following method. That is, 100 μl of each of the cloned hybridoma culture supernatants was added to the 96-well immunoplate coated with the recombinant human NGF obtained in Reference Example 7, and incubated at room temperature for 2 hours. After removing the culture supernatant and washing, rabbit anti-mouse IgG
1. Antibodies against IgG2a, IgG2b, IgG3, K chain and λ chain
(Kappel) was added and incubated at room temperature for 2 hours. HR after removing and washing each antibody
P-labeled goat anti-rabbit IgG antibody (Kappel) was added and incubated at room temperature for 2 hours. After removing the labeled antibody and washing well, an enzymatic reaction was performed by the method described in (3) above, and the absorbance was measured. As a result, the monoclonal antibody NGF
Both A-133 and NGFB-260 were found to be subclasses of IgG1 · K type.

Example 2 (1) Enzyme labeling of antibody: Purified NGFB-260 antibody (10.
(8 mg / ml) 1.5 ml of 0.1 M NaCl
Dialyzed against acetate buffer (pH 4.2) at 4 ° C for 4 hours,
Pepsin (Sigma, USA) (0.5 mg) was added, and the mixture was added at 37 ° C.
For 20 hours. The reaction was stopped by adjusting the pH to 7 with 1 M Tris, and separated with a column of Ultrogel AcA 44 (manufactured by IBF, France) using a 0.02 M borate buffer (pH 8.0) containing 0.15 M NaCl as an eluate. F (ab ') ₂ was obtained. After concentrating the solution to 1.1 ml, the solution was adjusted to 0.1 M phosphate buffer (pH).
6.0) for 4 hours at 4 ° C, 0.1 ml of 0.2 M mercaptoethylamine, 5 mM EDTA, 0.1 M phosphate buffer (pH 6.0) was added, and the mixture was reduced at 37 ° C for 90 minutes. . The reaction solution was separated by Sephadex G-25fine (Pharmacia Fine Chemical Co., Sweden) (φ1 × 60 cm)
Then, 5 mM EDTA and 0.1 M phosphate buffer (pH 6.0) were used as an eluate to separate Fab 'fractions. On the other hand, 10 mg of horseradish peroxidase (HRP) (Boehringer Mannheim, West Germany) was dissolved in 1.4 ml of 0.1 M phosphate buffer (pH 7.0), and N- (γ-maleimidobutyloxy) succinimide (GMBS) was dissolved. 2.1 mg N, N-
Dissolved in 100 μl of dimethylformamide (DMF) and added, and stirred at 30 ° C. for 60 minutes, then Sephadex G-25fine
(φ1.2 × 60cm) 0.1M phosphate buffer (pH 7.0)
As an eluate, and the HR having a maleimide group introduced thereinto.
P was obtained (maleimidated HRP). Fab ′ and maleimidated HRP were mixed at a molar ratio of 1: 1 and mixed at 4 ° C. for 20 minutes.
Reacted for hours. After the reaction, a 0.1 M phosphate buffer (pH 7.0) was used as an eluate on a column of Ultrogel AcA44 to obtain an enzyme-labeled antibody (NGFB-260 Fab'-HRP).

(2) Preparation of antibody-bound solid phase: purified NGFA-
The 133 antibody was diluted with a 0.01 M carbonate buffer (pH 8.5) to a concentration of 10 μg / ml, and 100 μl of the diluted antibody was placed in each well of a 96-well immunoplate and allowed to stand at 4 ° C. overnight to allow the antibody to bind to the solid phase. . 0.1 containing 0.15M NaCl.
After washing with 01M phosphate buffer (pH 7.0), 1% BS
200 μl of 0.01 M phosphate buffer containing A was injected into the wells and stored in a cool place until use.

(3) Sandwich EIA method: 100 μl of NGF solutions prepared at various concentrations were placed in wells to which antibodies had been bound, and incubated at 4 ° C. overnight. After removing the NGF solution, a 100-fold diluted enzyme-labeled antibody (NGFB
-260 Fab'-HRP) was added at 100 μl per well and incubated at room temperature for 2 hours. After removing the labeled antibody, an HRP substrate solution was added, and an enzymatic reaction was carried out in the same manner as described in Example 1, and the absorbance at 492 nm was measured. As a result, the detection limit of NGF in this measurement system was 0.4.
ng / well [FIG. 6].

(4) Monoclonal Antibody Neutralizing Activity: Whether to neutralize the biological activity of the obtained monoclonal antibody-NGF was determined using rat adrenal medulla brown cell-derived PC-12 cells and chicken embryo spinal dorsal root ganglia. Examined. (a) Examination using PC-12 cells: human NGF 10 ng /
10 ⁴ PC-12 cells primed in 1 ml were seeded in a 24-well microplate and 2 ng / ml human NG
Various concentrations of the culture supernatants of the hybridoma clone cells described in Example 1 were added in the presence of F, and the effect on the projection formation of PC-12 cells was examined. As a result, NGFA-1
When 33 antibody was added, projection formation was completely suppressed [Table 5], indicating that this antibody neutralizes the biological activity of NGF.

[Table 5]

(B) Examination using ganglia: The method of Davies et al. [Journal of Neuroscience (J. Neuros
ci.) 6. 1897 (1986)], neurons obtained from chicken spinal ganglia on day 8 of fertilization were seeded in a 48-well microplate at a rate of 4 × 10 ³ cells / well. 2n
g / ml human NGF and various concentrations (0.3, 1, 3,
(10, 30, 100 ng / ml) of the purified monoclonal antibody was added to the wells, and neurite-outgrowth was added.
The effect on the was examined. As a result, NGFA-133
The antibody exhibits an inhibitory phenomenon at 3 ng / ml or more, and 30 ng / ml.
/ Ml was completely suppressed, and it was found to have strong neutralizing activity. (5) Antibody tissue site of monoclonal antibody: antibody is human N
Whether to recognize the N-terminal of GF was examined by the following method. The N-terminal peptide of human NGF obtained in Reference Example 9 was diluted to a concentration of 10 μg / ml with a 0.01 M carbonate buffer (pH 8.5), and 100 μl thereof was added to each well of a 96-well immunoplate and placed at 4 ° C. The peptide was bound to the solid phase overnight. After washing with 0.01M phosphate buffer (pH 7.0) containing 0.15M NaCl, 0.01% BSA containing 1% BSA was used to block excess binding sites.
Inject 200 μl of 1 M phosphate buffer into each well,
Stored in a cool place until use. 100 μl of the culture supernatant of the two types of clonal cells obtained in Example 1 was dispensed into the plate prepared as described above, and EIA was performed in the same manner as described in Example 1- (3). , Monoclonal antibody N
Neither GFA-133 nor NGFB-260 showed binding to the peptide. Furthermore, Example 1- (3)
The above antibody and various concentrations (0.4 to 50 μl) were added to a 96-well microplate coated with NGF in the same manner as described.
g / ml), and add the N-terminal peptide
It was examined whether or not binding to the peptide was inhibited by the peptide, but no inhibitory effect was observed at any peptide concentration. From the above results, the obtained two types of monoclonal antibodies NGFA-133 and NGFB-260 were:
It was considered that it recognized an antigenic determinant existing in addition to the N-terminal 1-15 amino acids of human NGF.

(6) Sandwich EIA method using anti-NGF N-terminal peptide antibody and monoclonal antibody: Reference Example 1
Purified rabbit polyclonal antibody and monoclonal antibody NGFA-1 against human NGF N-terminal peptide according to 1 above
It was examined by the following method whether human NGF could be quantified by 33 or NGFB-260. 100 μl of NGF solutions prepared at various concentrations were added to each well of a 96-well immunoplate in which purified human NGF N-terminal peptide antibody was bound to a concentration of 1 μg / well in the same manner as described in (2) above. Incubated at room temperature for 2 hours. After removing the NGF solution, 100 μl of a solution containing the monoclonal antibody NGFA-133 or NGFB-260 adjusted to a concentration of 1 μg / ml was added, and the solution was added at room temperature.
Incubated for hours. After removing and washing the antibody solution, 1
HRP-labeled rabbit anti-mouse IgG diluted 0000-fold
After 100 μl of an antibody (Jackson Immunoresearch Laboratory) was added and reacted at room temperature for 2 hours, the plate was washed well, and an enzymatic reaction was carried out in the same manner as described in (3) above.
The absorbance at m was measured. As a result, it was possible to detect NGF at a very low concentration of 15 pg / well in the present EIA method (FIG. 7).

Example 3 (1) Enzyme labeling of antibody: Purified NGFA-133 antibody (7 m
g / ml) and 1.5 ml of the purified rabbit anti-human NGF N-terminal peptide antibody described in Reference Example 11 (2.5 mg / ml).
ml) was obtained in the same manner as described in Example 2 (1) to obtain a Fab ′ fraction, and then reacted with maleimidated HRP to obtain an enzyme-labeled antibody NGFA-133Fab′-H.
RP and anti-human NGF (1-15) Fab'-HRP were obtained. (2) Sandwich EIA 100 μl of NGF solutions adjusted to various concentrations were placed in the wells to which the NGFA-133 antibody described in Example 2 (2) was bound, and incubated at 4 ° C. overnight. After removing the NGF solution, 100 μl of NGFA-133-HRP or anti-human NGF (1-15) Fab′-HRP diluted 100-fold was added per well and incubated at room temperature for 2 hours. After removing the labeled antibody, an HRP substrate solution was added, and an enzymatic reaction was carried out in the same manner as described in Example 1, and the absorbance at 492 nm was measured. As a result, N
When GFA-133 Fab'-HRP was used, NGF
Is 0.94 pg / well (FIG. 8), anti-human N
When GF (1-15) Fab'-HRP was used, the detection limit was 8.4 pg / well. From the above results, sandwich E using NGFA-133 as the solid phase-bound antibody and NGFA-133 as the enzyme-labeled antibody was used.
It was found that the IA system detected NGF with the highest sensitivity. In addition, the present EIA system shows high homology with human NGF, human nerve growth factor 2 (polypeptide (I))
[Human NGF-2, revised et al .: European Patent Application Publication No. 386,752, FEBS Letters, 2
66 , 189-191 (1990)], it was determined that hNGF-2 was 1 μg / ml.
No reactivity was observed even at a high concentration of (FIG. 8), and the cross-reactivity was found to be 0.01% or less. In FIG. 8, the graph of -−- shows the result of human NGF detection sensitivity, and the graph of-○-shows the result of reactivity with human NGF-2, respectively.

[0067]

EFFECT OF THE INVENTION The monoclonal antibody of the present invention comprises human N
Since it specifically binds to the GF protein and has high binding ability, it can be advantageously used as a reagent for measuring human NGF. In addition, some of the monoclonal antibodies of the present invention have a neutralizing activity, and may be used as a reagent for knowing the activity of NGF in vivo.

[0068]

[Sequence list] SEQ ID NO: 1 Sequence length: 972 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin Organism name: Human Strain name: Tissue Sequence: CCCGGGTTAC GCCTGTTGTC CCGGTATAAC CATTGCTAGC ACACCCTTTC CCTCTCAGAA 60 GTGCCCCGGT TTGAATGAAA CCTCTTCGTG ATCCCCTTGG GAGGTCAACT CTGAGGGACC 120 CAGAAACTGC CTTTTGACTG CATTTAGTAC TCCATGAAGT CACCCTCATT TCTTTTTCAT 180 TCCAGGTGCA TTC TTC ATC TTC ATC TTC ATC TTC ATC TTC ATC TTC ATC TTC ATC TTC ATC TTC ATC TTC ATC TTC ATC TTC ATC TTC TCTG ATC TTC ATC TTC ATC TTC TCTG ATC TTC ATC TTC TCTG ATC GGC ATA CAG GCG GAA CCA CAC TCA GAG AGC AAT GTC CCT 278 Phe Leu Ile Gly Ile Gln Ala Glu Pro His Ser Glu Ser Asn Val Pro 15 20 25 GCA GGA CAC ACC ATC CCC CAA GTC CAC TGG ACT AAA CTT CAG CAT TCC 326 Ala Gly His Thr Ile Pro Gln Val His Trp Thr Lys Leu Gln His Ser 30 35 40 CTT GAC ACT GCC CTT CGC AGA GCC CGC AGC GCC CCG GCA GCG GCG ATA 374 Leu Asp Thr Ala Leu Arg Arg Ala Arg Ser Ala Pro Ala Ala Ala Ile 45 50 55 GCT GCA CGC GTG GCG GGG CAG ACC CGC AAC ATT ACT GTG GAC CCC AGG 422 Ala Ala Arg Val Ala Gly Gln Thr Arg Asn Ile Thr Val Asp Pro Arg 60 65 70 75 CTG TTT AAA AAG CGG CGA CTC CGT TCA CCC CGT GTG CTG TTT AGC ACC 470 Leu Phe Lys Lys Arg Arg Leu Arg Ser Pro Arg Val Leu Phe Ser Thr 80 85 90 CAG CCT CCC CGT GAA GCT GCA GAC ACT CAG GAT CTG GAC TTC GAG GTC 518 Gln Pro Pro Arg Glu Ala Ala Asp Thr Gln Asp Leu Asp Phe Glu Val 95 100 105 GGT GGT GCT GCC CCC TTC AAC AGG ACT CAC AGG AGC AAG CGG TCA TCA 566 Gly Gly Ala Ala Pro Phe Asn Arg Thr His Arg Ser Lys Arg Ser Ser 110 115 120 TCC CAT CCC ATC TTC CAC AGG GGC GAA TTC TCG GTG TGT GAC AGT GTC 614 Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp Ser Val 125 130 135 AGC GTG TGG GTT GGG GAT AAG ACC ACC GCC ACA GAC ATC AAG GGC AAG 662 Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys Gly Lys 140 145 150 155 GAG GTG ATG GTG TTG GGA GAG GTG AAC ATT AAC AAC AGT GTA TTC AAA 710 Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val Phe Lys 160 165 170 CAG TAG TTT TTT GAG ACC AAG TGC CGG GAC CCA AAT CCC GTT GAC AGC 758 Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val Asp Ser 175 180 185 GGG TGC CGG GGC ATT GAC TCA AAG CAC TGG AAC TCA TAT TGT ACC ACG 806 Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys Thr Thr 190 195 200 ACT CAC ACC TTT GTC AAG GCG CTG ACC ATG GAT GGC AAG CAG GCT GCC 854 Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln Ala Ala 205 210 215 TGG CGG TTT ATC CGG ATA GAT ACG GCC TGT GTG TGT GTG CTC AGC AGG 902 Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu Ser Arg 220 225 230 235 AAG GCT GTG AGA AGA GCC TGACCTGCCCC ACCCGCCCCCCCCC Lys Ala Val Arg Arg Ala 240 CCTTCTACAC TCTCCTGGGC CC 972

SEQ ID NO: 2 Sequence length: 37 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA Sequence characteristics: adapter Sequence: AGCTTGCCGC CACCATGTCC ATGTTGTTCT ACACTCT 37

SEQ ID NO: 3 Sequence length: 37 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA Sequence characteristics: adapter Sequence: GATCAGAGTG TAGAACAACA TGGACATGGT GGCGGCA 37

SEQ ID NO: 4 Sequence length: 12 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA Sequence characteristics: adapter Sequence: CAGATCTGGG CC 12

SEQ ID NO: 5 Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence:

SEQ ID NO: 6 Sequence length: 20 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence: Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val Asp Ser Gly Cys 5 10 15 Arg Gly Ile Asp Ser 20

SEQ ID NO: 7 Sequence length: 19 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence: Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu Ser Arg Lys Arg 5 10 15 Val Arg Arg Ala

SEQ ID NO: 8 Sequence length: 15 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence: Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys 5 10 15

[Brief description of the drawings]

FIG. 1 shows the cloned human N obtained in Reference Example 1.
1 shows the nucleotide sequence of the GF gene and the amino acid sequence translated therefrom.

FIG. 2 shows the cloned human N obtained in Reference Example 1.
1 shows the nucleotide sequence of the GF gene and the amino acid sequence translated therefrom.

FIG. 3 shows a construction diagram of a human NGF expression vector pMNGF101 obtained in Reference Example 1.

FIG. 4 shows a construction diagram of a human NGF expression vector pMNGF201 obtained in Reference Example 2.

FIG. 5 shows a construction diagram of a human NGF expression vector pTB1058 obtained in Reference Example 3.

FIG. 6 shows the results of human NGF detection sensitivity obtained by sandwich EIA using a monoclonal antibody obtained in Example 2.

FIG. 7 shows the results of the detection sensitivity of human NGF by sandwich EIA using a monoclonal antibody and a rabbit anti-human NGFN-terminal peptide polyclonal antibody obtained in Example 2.

FIG. 8 shows anti-NGFA-133 set in Example 3.
The results of the detection sensitivity of human NGF by sandwich EIA using Fab 'of the antibody as a solid phase and a labeled antibody, and the results of reactivity with human NGF-2 are shown.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12P 21/02 G01N 33/577 B G01N 33/53 (C12P 21/02 33/577 C12R 1:91) // (C12P 21 / 02 (C12P 21/08 C12R 1:91) C12R 1:91) (C12P 21/08 C12N 5/00 B C12R 1:91) 15/00 C (72) Inventor Osamu Iwane 5--18 Tsunodai, Suita-shi, Osaka No. D73-104 (56) References JP-A-2-28199 (JP, A) JP-A-60-84299 (JP, A) JP-A-2-219593 (JP, A) (58) Fields investigated ( Int.Cl. ⁷ , DB name) C12P 21/00-21/08 C12N 5/00 C12N 15/00 C07K 16/22 BIOSIS (DIALOG) WPI (DIALOG) GenBank / EMBL / DDBJ (GENETYX)

Claims (5)

(57) [Claims] 1. A monoclonal antibody against human nerve growth factor (NGF) produced by a hybridoma having an accession number of FERM BP-3078 or FERM BP-3079. 2. The accession number for producing a monoclonal antibody against human nerve growth factor (NGF) is FERM BP-
A hybridoma that is 3078 or FERM BP-3079. 3. The method for producing a monoclonal antibody according to claim 1, wherein the hybridoma according to claim 2 is grown in a liquid medium or intraperitoneally of a non-human mammal. 4. A method for detecting and / or quantifying an NGF protein, which comprises performing an immunoassay using the monoclonal antibody according to claim 1. 5. The detection and / or quantification method according to claim 4, wherein the immunoassay is an enzyme immunoassay.