JPH03163095A - Partial peptide of human nerve growth factor, antibody and use thereof - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a partial peptide of human nerve growth factor (human NGF) or a partial peptide of human NGF in which some amino acids are replaced with other amino acids; The present invention relates to a complex of a peptide and a carrier protein, an antibody obtained using the complex as an immunogen, and a method for detecting and quantifying human NGF using the antibody.

Nerve growth factor
r, NGF) is Levy Montalci 12 (Le
vi-Monntalcini) [Annual New York Academy of Sciences (Ann.N; Y.A.
cad. Sci. ) 55, 330 (1952)) and Cohen et al. [Proceedings of the National Academy of Sciences USA (Pr.
oc. Natl. Acad. Sci. USA)40.
1014 (1954)] and is an essential nutritional factor for the differentiation, growth, and survival of the peripheral nervous system. Recently, it has been revealed that NGF has the effect of maintaining the survival of cholinergic neurons in the central nervous system [Journa of Neuroscience].
science) 6, 2155 (1986); Hatanaka (
Development of Brain Research (Dev.Brain Res.) 39, 8
5 (1988)), and is attracting attention as a factor associated with Alzheimer's disease. Furthermore, when NGF is administered into the brain of old rats, improvement in memory impairment is observed [Nature, 329.65 (1)
989)), it is also expected to be used as a treatment for senile dementia. NGF isolated from the submandibular gland of male mice (73NGF)
is a complex consisting of three subunits α, β, and γ (
α2βγ2), of which only the β subunit has NGF activity.

β subunit (βNGF, 2.53 NGF) is 1
It is a dimer of the same polypeptide consisting of 18 amino acids, and its amino acid sequence is
letti) and Bradshaw [
Proceedings of the National Academy of Sciences USA (Proc. Natl. Acad)
．． sci. UsA), 68. 2417 (1971)
) is determined by

Scott et al. successfully cloned mouse βNGF from a mouse submandibular gland cDNA library using an oligonucleotide synthesized based on the amino acid sequence of mouse βNOF as a probe [Nature, 302, 538 (1983)].

Furthermore, Ullrich et al.
Human genome D using F cDNA as a probe.
They cloned the NGF gene from an NA library and showed that the amino acid sequence of human NGF, deduced from its nucleotide sequence, has 90% homology with that of mouse NGF [Nature, 303,
821 (1983)).

Although the human NGF gene has been cloned in this way, there are almost no reports of the detection and isolation of human NGF from living organisms. Reasons why human NGF is not detected in living organisms
One possible reason is that until now anti-mouse NGF antibodies have been mainly used.

For the purpose of detecting and quantifying human NGF, the present inventors chemically synthesized a partial peptide of human NGF or its partially amino acid substituted product, and prepared a complex between this and a carrier protein. Then, antibodies were obtained using this complex as an immunogen. The inventors discovered that it is possible to detect and quantify human NGF by using the obtained antibody, and as a result of further research, the present invention was completed.

That is, the present invention provides (1) Ser at the 3rd position and Il at the 6th position of the human nerve exfoliation factor.
e, 9th place Arg, 37th place Net, 40th place Gly
, 50th Lys, 60th Asp, 61st Pr
o, Asp at position 65, Met at position 92, Giy at position 94, Val at position 117, and at least one amino acid at Ala at position 120 of the human NGF sequence having 10 or more consecutive amino acids. peptide, or one consecutive sequence of human nerve growth factor
(2) A partial peptide in which some constituent amino acids of a partial peptide having 0 or more amino acids are substituted with other amino acids; the partial peptide or substituted partial peptide according to (1) above, which has amino acids; (3) the partial peptide consists of 10 to 15 consecutive amino acids of the 1st to 15th sequences of human nerve growth factor; the partial peptide or substituted partial peptide according to (1) above, which consists of (4) the partial peptide consisting of human nerve growth factor Nos. 88-11;
The partial peptide or substituted partial peptide according to (1) above, which is composed of 10 to 3 consecutive amino acids of the 8th sequence, (5) the 108th position and/or the substituted partial peptide of human nerve growth factor. The substituted partial peptide according to (4) above, having a sequence in which Cys at position 110 is replaced with another amino acid, (6) above (1), (2), (3), (4) or (
5) a complex of the partial peptide and carrier protein described above; (7) an antibody obtained using the complex described in (6) above as an immunogen; (8) inoculating an animal with the complex described in (6) above. and (9) a method for detecting and quantifying human nerve growth factor, which uses the antibody described in (7) above. Hereinafter, the above partial peptide and the substituted partial peptide are collectively referred to as the human NGF partial peptide of the present invention.

The human NGF partial peptide of the present invention is based on the amino acid sequence of human NGF [Nature, 303, 8
21 (1983), see also FIG. 1 of the present invention], especially consecutive 10 to 4 of them.
Peptides containing 0 amino acids, especially those having 10 to 20 consecutive amino acids, are preferred. Partial peptides are preferred that allow antibodies obtained using a complex of these and a carrier protein as an immunogen to recognize human NGF. The partial peptide of the present invention is particularly preferably SerSer Ser His Pro.
Ile Phe His ArgGly Glu P
he Ser Val C! /! lya, Lys Ala
Leu Thr Met Asp GlyLys Gl
n Ala Ala Trp Arg Phe IIs
Arg Ile AspThr Ala Cys V
al Cys Val Leu Ser Arg Ly
Examples include sAlaValArg.

Among the human NGF partial peptides of the present invention, those that are not substituted include Set at the 3rd position, 11e at the 6th position, Arg at the 9th position, Met at the 37th position, and Met at the 40th position of human NGF.
Gly + 50th ys, 60th Asp, 61st
1st place Pro, 65th place Asp, 92nd place Net, 94th place Giy, 117th place Val and 120th place A
It has at least one amino acid of la.

Among the human NGF partial peptides of the present invention, substituted partial peptides do not necessarily need to contain amino acids at the positions described above.

Furthermore, it is preferable that the partial peptide of the present invention contains at least one or more Cys residues.

This is because a complex of a peptide and a carrier protein can be easily obtained by using the SH group of this Cys residue. Fragments containing Cys forming an S-S bond in the NGF amino acid sequence are
By linking with -S bonds, a molecule structurally closer to natural NGF can be obtained, which is a better immunogen and can also be expected to have NGF activity and NGF inhibitory activity.

The partial peptide of the present invention can be produced by known conventional methods of peptide synthesis. This may be achieved by either solid phase synthesis or liquid phase synthesis. Examples of such peptide combination methods include, for example, "The Peptides".
ides) J. Volume 1 (1966) + Schro
by der and Lubke, Acade++ic
Press, New York, U. S. A. , “Peptide Combination”, Izumiya et al., Maruzen Co., Ltd. (1975)
Alternatively, the method described in "Basics and Experiments of Peptide Coupling" by Izumiya et al., Maruzen Co., Ltd. (1985) may be mentioned. Further, the partial peptide of the present invention may be produced by cleaving human NGF with an appropriate enzyme. As the cutting method, for example, "Biochemistry Experiment Course 1 Chemistry of Proteins", edited by the Japanese Biochemical Society, Tokyo Kagaku Doujin (197
6), pages 255 to 332.

At least l before substitution in a peptide in which some of the constituent amino acids of the partial peptide are substituted with other amino acids
The number of constituent amino acids may be any number as long as the characteristics of the partial peptide are not lost.

Examples of the constituent amino acids before substitution include cysteine and amino acids other than cysteine.

Cysteine is particularly preferred. Constituent amino acids other than cysteine before substitution include aspartic acid, arginine, glycine, and valine.

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 acids include glycine, parine, alanine, leucine, inleucine, tyrosine, phenylalanine, histidine, tryptophan, serine, threonine, and methinine. Particularly preferred are serine and threonine.

If the constituent amino acid before substitution is other than cysteine, the substituted amino acid may be, for example, in terms of hydrophilicity, hydrophobicity, or charge of the amino acid.
Choose an amino acid that has properties different from those of the amino acid before being substituted. Specifically, when the amino acid before substitution is aspartic acid, examples of the amino acid after substitution include asparagine, threonine, palin, phenylalanine, and arginine, with asparagine and arginine being particularly preferred.

When the amino acid before substitution is arginine, examples of the amino acid after substitution include glutamine, threonine, leucine, phenylalanine, and aspartic acid, with glutamine being 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.

If the constituent amino acid before substitution is serine,
Examples of amino acids after substitution include methionine, alanine, leucine, cysteine, glutamine, arginine, and aspartic acid, with methionine being particularly preferred.

If 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.

The original amino acids before substitution are preferably aspartic acid, arginine, glycine, serine, and valine.

Preferred amino acids after substitution are asparagine, glutamine, arginine, threonine, methionine, serine, and leucine.

The most preferred substituted muteins include those in which the constituent amino acid cysteine is replaced with alanine or serine.

In the above substitutions, two or more substitutions may be performed simultaneously. In particular, it is preferred that two or three constituent amino acids are substituted.

The substituted partial peptide can be produced by, for example, replacing the corresponding amino acid residue with another desired amino acid residue in peptide synthesis.

When producing the complex of the present invention, the partial peptide of the present invention is combined with a carrier protein. Examples of the carrier protein include bovine serum albumin, bovine thyroglobulin, bovine gammaglopurin, and hemocyanin.

The binding of the partial peptide to the carrier protein can be carried out using known conventional methods. Examples of reagents used for binding include glutaraldehyde and water-soluble carbodiimide.

The appropriate ratio of the peptide to the carrier protein is about 1:E to about 1:4, and the pH of the reaction is around neutral,
In particular, values around 7.3 often give good results. The time required for the reaction is often about 2 to 6 hours, but about 3 hours is particularly appropriate. The composite thus prepared may be stored by dialysis against water at about 4° C. in a conventional manner, frozen, or freeze-dried.

To produce polyclonal antibodies, the immunogen produced as described above is inoculated into warm-blooded animals. Examples of warm-blooded animals used in the production of the above-mentioned antibodies include clear-breasted warm-blooded animals (e.g., rabbits, sheep, cows, rats, mice, guinea pigs, horses, pigs), birds (e.g., chickens, pigeons, ducks, Goose, quail), etc. As a method for inoculating an immunogen into a warm-blooded animal, the amount of immunogen inoculated into the animal may be effective for producing antibodies. For example, a rabbit may be inoculated with LM in 1 ml of physiological saline and Freund's complete Antibodies are often produced when emulsified with an adjuvant and inoculated subcutaneously on the back and palms of the hind limbs 5 times every 4 weeks. As a method for collecting antibodies that have been expressed in warm-blooded animals in this way, for example, in rabbits,
It is usually collected from the ear vein between 7 and 12 days after the final inoculation and centrifuged to obtain serum. Polyclonal antibodies can be purified from the obtained antiserum by recovering both adsorbed components using affinity chromatography, which uses a carrier holding each antigenic peptide. Also, the method of Milstein et al. [Nature, Vol. 256 (1975)]
Monoclonal antibodies obtained by a method similar to that described in , p. 495] can also be used.

That is, individuals with a recognized antibody titer are selected from warm-blooded animals such as mice that have been immunized in the same manner as in the polyclonal antibody preparation method described above, and their spleens or lymph nodes are collected 2 to 5 days after the final immunization. An anti-human NGF antibody-producing hybridoma can be prepared by fusing the antibody-producing cells contained in the above with myeloma cells. The fusion operation can be performed using known methods, such as the Köhler and Milstein method [
Nature, 256,495 (1
975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used. Examples of myeloma cells include MS-1, P3U1, SP
Examples include 2/O, and P3Ul is particularly preferably used. The preferred ratio of the number of antibody-producing cells (tile cells) to the number of bone marrow cells used is about 1:1 to 20:1, and PEG (preferably PEG 1000 to P
E G 6000) is added at a concentration of about 10 to 80%, and the temperature is 1 to 10 at 20 to 40°C, preferably 30 to 37°C.
Cell fusion can be efficiently carried out by incubating for minutes.

Various methods can be used to screen hybridomas producing anti-human NGF antibodies. For example, a hybridoma culture supernatant is added to a solid phase (e.g., a microplate) on which human NGF has been adsorbed, and then horseradish peroxidase (H R Anti-immunoglobulin antibody labeled with P ) (
When the cells used for cell fusion are mice, an anti-mouse immunoglobulin antibody is used) or an enzyme-linked immunosorbent assay (EIA method) that detects an anti-human NGF monoclonal antibody bound to a solid phase by adding protein A; Human NG labeled with HRP by adding hybridoma culture supernatant to a solid phase adsorbed with globulin antibody or protein A.
Examples include the EIA method in which hybridomas producing anti-human NGF monoclonal antibodies bound to a solid phase are detected by adding F. Selection and breeding of anti-human NGF monoclonal antibodies are usually carried out in an animal cell medium (eg RPMI 1640) containing 10-20% fetal bovine serum with the addition of HAT (hyboxanthin, aminopterin, thymidine). The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the above-mentioned measurement of the anti-human NGF antibody titer in the antiserum. The hybridoma obtained here is inoculated into mammalian animals or cultured in an animal cell culture medium to produce and accumulate monoclonal antibodies, which are then collected and manufactured.

Anti-human NGF monoclonal antibody portion i! Purification is carried out in accordance with the method for separating and purifying immunoglobulins, similar to the separation and purification of polyclonal antibodies described above.

The antibody molecule may be an IgG or a fraction C thereof, eg, F(ab')z, Fab' or Fa
It may be b. Among these, the antibody molecule to which the labeling agent is directly bound is preferably Fab'.

The antibody thus obtained can be used as a reagent for immunochemical assay of human NGF or Western blotting6.In addition, this antibody can also be used as a material for a diagnostic agent.

Detection and quantification of human NGF using this antibody can be carried out by conventional methods, but can also be carried out by immunochemical assay. The method includes, for example, human N N
GF can be measured.

The antibodies retained on the carrier include anti-human NGF antibody and anti-mouse NGF antibody.
Either a GF antibody or an anti-human NGF partial peptide antibody may be used.

The labeling agent-conjugated antibody used in the assay method of the present invention is an anti-human NGF partial peptide antibody that has a different antigen-determining site from that of the antibody held on the carrier, and the labeling agent is directly bound to the antibody. It is preferable that Examples of carriers for antibodies supported on carriers used in the method for measuring human NGF include gel particles (
Examples, agarose gel [example, Sepharose 4B, Sepharose 6B (manufactured by Pharmacia Fine Chemicals (Sweden)], dextran gels [examples, Sephadex G-75, Sephadex G-100, Sephadex G-200 (Pharmacia Fine Chemicals) Polyacrylamide gel [eg, Biogel P-30, Biogel P-60. Biogel P-1
00 (manufactured by Bioland Laboratories (USA))
], Cellulose particle c example, Avicel (manufactured by M Kasei), ion-exchanged cellulose (e.g., diethylaminoethynoresenolose, canoleboxymethylcellulose)], physical adsorbent [e.g., glass (e.g., glass bulb, glass rod, aminoalkyl glass bulb, aminoalkyl glass rod),
Silicon pieces, styrene resins (e.g. polystyrene spheres, polystyrene particles), immunoassay plates (e.g. manufactured by Nunc (Denmark)), ion exchange resins (e.g. weakly acidic cation exchange resins [e.g. Amberlite IRC- 5
0 (manufactured by Rohm and Haas (USA)), Zeocarb 226 (manufactured by Palmchit (West Germany))], weakly basic anion exchange resins (e.g., Amberlite IR-4B)
, DOWEX 3 (manufactured by Dow Chemical Company (USA))]6.To retain antibodies on a carrier, known conventional methods can be applied, but for example, ``Metabolism'', Vol. (1971),
Examples include the Promcien method and the glutaraldehyde method described on page 696. Alternatively, as a simpler method, it may be physically adsorbed onto the surface of the carrier. Examples of the labeling agent in an antibody bound to a labeling agent include a radioactive isotope, an enzyme, a fluorescent substance, a luminescent substance, and the like, and it is preferable to use an enzyme.

The enzyme is preferably one that is stable and has a high specific activity.
peroxidase, alkaline phosphatase, β-D-
Galactosidase, glucose oxidase, etc. can be used, but peroxidase is preferred. As peroxidase, those of various origins can be used, examples of which include peroxidase obtained from horseradish, pineapple, figs, sweet potato, fava beans, corn, etc. In particular, peroxidase extracted from horseradish horseradish peroxidase
(HRP) is preferred.

When binding peroxidase to an antibody, it is convenient to use 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 this purpose, N-succinimidyl-maleimidolupoxylate derivatives can be used, preferably N-(γ-maleimidobutyloxy)succinimide (sometimes abbreviated as GMBS). Therefore, a certain group may be present between the maleimide group and the peroxidase.

The reaction of GMBS with peroxidase is carried out by reacting the two in a buffer solution with a pH of about 6 to 8 at a temperature of about IO to 50° C. for about 10 minutes to 24 hours. As the buffer solution, for example, 0 with a pH of 7.0
．． Examples include 1M phosphate buffer.

The maleimidated peroxidase thus obtained can be purified by, for example, gel chromatography. Examples of carriers used in gel chromatography include Sephadex G.
-25 (manufactured by Pharmacia Fine Chemicals (Sweden))], Biogel P-2 (manufactured by Bioland Laboratories (USA)), and the like.

The reaction between maleimidated peroxidase and antibody molecules is
This can be carried out by reacting both in a buffer solution at a temperature of about 0° C. to 40° C. for about 5 to 48 hours.

Examples of the buffer include a 0.1M phosphate buffer containing 5mM ethylenediaminetetraacetic acid sodium salt having a pH of 6.0.

4ml of peroxidase obtained in this way! Antibodies can be purified, for example, by gel chromatography. Examples of carriers used in gel chromatography include Sephadex G-2.
5 (Pharmacia Fine Chemicals (Sweden) 1!) ). Biogel P-2 [Bio-Rad・
Laboratories, Inc. (USA)]. Furthermore, a thiol group may be introduced into peroxidase and reacted with a maleimidated antibody molecule.

Direct binding of enzymes other than peroxidase to antibodies can be carried out in the same manner as for peroxidase.
Additionally, the glutaraldehyde method, periodic acid method, water-soluble carbodiimide method, etc., which are known per se, are used. Test samples in the measurement system of the present invention include urine, serum,
Examples include body fluids such as plasma and cerebrospinal fluid, extracts of animal cells and bacterial cells, and culture supernatants thereof. As an example of the measurement method of the present invention, a case where the labeling agent is peroxidase will be specifically explained below, but it is not limited to peroxidase.

First, ■: human NG to be measured on the antibody retained on the carrier.
After adding the F-containing analyte and performing an antigen-antibody reaction,
This was combined with peroxidase obtained above and anti-human NGF.
A conjugate with the partial peptide antibody is added and reacted.

(2): Add a peroxidase substrate to the reaction product obtained above, and measure the absorbance or fluorescence intensity of the resulting substance to determine the enzymatic activity of the reaction product.

(2) The operations (1) to (2) above are performed in advance on a known amount of a standard solution of human NGF, and the relationship with the absorbance or fluorescence intensity of human NGF is prepared as a standard curve.

■: Analyte containing unknown amount of human NGF (test sample)
The amount of human NGF in the analyte is measured by applying the absorbance or fluorescence intensity obtained for the sample to a standard curve. In addition, the anti-human NGF partial peptide antibody is Western
Blotting (W.N. Burnstte, Analytical Biochemistry (Analytical B)
iochemistry) e month. 2,195 (198
1)) can be used for the detection and quantification of human NGF.

A specific example of Ilestern blotting is shown below. A sample containing human NGF, for example, sample buf
fer (U.K., Laemmli, Nature (Na
ture), 227, 680 (1970)). In this case, 2-mercaptoethanol may be added as a reducing agent (under reducing conditions) or not (under non-reducing conditions), either of which may be used. Approximately 10% of this solution
After heating at 0°C for 5 minutes, it is subjected to electrophoresis. As for electrophoresis, any method that can separate proteins is suitable.
Specific examples include polyacrylamide gel electrophoresis containing SDS. After electrophoresis, proteins are transferred from the gel to a nitrocellulose membrane. This method itself is known; for example, Burnette's method [Analytical Biochemistry]
emistry), 112, 195 (1981)). Next, human NGF on the nitrocellulose membrane
is detected using immunological methods. That is, after blocking the nitrocellulose membrane with, for example, a 3% gelatin solution, the first antibody reaction is performed. The human NGF partial peptide antibody used as the first antibody may be an antiserum or a purified one, but a purified one is preferable. The conditions for the first antibody reaction after blocking may be any conditions as long as the human NGF on the membrane can bind to the first antibody, for example, the reaction is carried out at room temperature for about 4 to 16 hours. After the first antibody reaction described above, a second antibody reaction is performed. The second antibody used is
Anything can be used as long as it can bind to the antibody and can be detected, such as IgG bound to a labeled enzyme. Specific examples of the 6-label rar# element include horseradish peroxidase (HRP), alkaline phosphatase, etc. Examples include.

The conditions for the second antibody reaction may be any conditions as long as the second antibody can bind to the first antibody, and for example, the reaction can be carried out at room temperature for about 1 hour. After the above second antibody reaction, color development is performed to detect the human NGF band on the nitrocellulose membrane. In the above υstern blocking, human NGF of approximately 50 mg or more can be detected, and by comparing the band density of various amounts of human NGF or mouse NGF with the band density of the sample, it is possible to detect human NGF of approximately 50 mg or more. Human N in specimen
GF can also be quantified. Note that instead of the above first antibody, for example, anti-human NGF partial peptide antibody and H
A complex with RP may also be used.

The partial peptide of human NGF obtained in the present invention is novel. In addition, in the present invention, antibodies are obtained using this partial peptide of human NGF as part of the antigen, but since it is a partial peptide, raw materials can be procured by simple methods such as chemical synthesis, and the isolation and purification itself is complicated. Compared to the case where human NGF whole protein is used, human NGF antibodies can be produced more easily, and this can be said to be a simpler method for detecting and quantifying human NGF.

In addition. In the specification and drawings of the present invention, when bases, amino acids, etc. are indicated by abbreviations, IUPAC-IU
BCommission on Biochemi
It is based on the abbreviation according to the cal nomenclature or the abbreviation commonly used in the field, examples of which are given below.

Furthermore, if an amino acid has optical isomers, the L-isomer is indicated unless otherwise specified.

DNA Deoxyribonucleic acid A Adenine C Cytonine G Guanine T Thymine Ala: Alanine Arg: Arginine Asn: Asparagine Asp: Aspartic acid Cys: Cysteine Gln: Glutamine Glu: Glutamate G1y: Glycine His: Histidine IIs: Isoleucine Leu: Leucine Lys: Lysine Me t: Methionine Phe: Phenylalanine Pro: Proline Ser: Serine Thr. : Threonine Trp : Tryptophan Tyr : Tyrosine Val : Valine Boc : t-butyloxycarbonyl MeBzl:
p-Methylbenzyl Bzl: Benzyl P: Polystyrene resin PAM for solid-phase peptide synthesis
: p-oxymethylphenylacetamidomethyl resin AaOH: e. Acid OBzl: Benzyl ester Tos: h silo Jl The present invention will be explained in more detail with reference to Examples and Reference Examples below, but the present invention is not limited thereto. Transformant Escherichia coli obtained in Reference Example 1 described below
/ p M N GF101 (IF○ 14869,
FERM BP-2385) has been transferred to the Fermentation Research Institute (IFO) since April 6, 2001, with accession number IF0 1.
It has been deposited as No. 4869. In addition, the microorganism
It has been deposited with the Ministry of International Trade and Industry, Agency of Industrial Science and Technology, Microbial Research Institute (FRI), under accession number FERM BP-2385, on April 15, 2013.

Synthesis of the N-terminal peptide of human NGF was performed using a solid-phase synthesis method using an automatic peptide synthesis machine 430A (manufactured by Applied Biosystems). The "Standard" program was used.The basic synthesis process etc. was explained by Merrifield, R.B.
(1969) Advance of Enzymology (Adv
．． Enzymol. ) 32, 221-296. The resin is Boc-Cys (MeB
z1) - Using PAM-P (0.5 mmol/g), synthesis was carried out sequentially from the carboxyl end. Boa-Val, Boa-Ser (Bz1) as Boc-amino acids
, Boc-Phe, Boc-Glu (OBz1), B
oa-Gly, Boa-Arg (Tos), Boa-
His (Tos), Boc-Ile, Boa-Pro
was used. After synthesis up to the amino terminal Ser, the peptide resin was taken out of the synthesizer and dried. Add 1.5 m Q of p-cresol and 0.5 m Q of 1.2-ethanediothyl to 1 g of peptide resin,
Add approximately 8 ml of liquid hydrogen fluoride and heat at 0°C for 2 hours.
A time reaction was performed. After the reaction was completed, hydrogen fluoride was removed under reduced pressure in a desiccator, and most of the mixed reagents were removed by washing with diethyl ether containing 0.1% 2-mercabutoethanol and then with diethyl ether. 10 peptides
The mixture was extracted with 3% mQ acetic acid, and the resin mixed in the extract was removed by filtration. The filtrate was treated with Sephadex (Se
phadex) G-25. Gel filtration conditions are column size 2.8X60
cn; detection wavelength 230 nm; solvent 3% acetic acid; flow rate 40 m
It was Q/hr. Fractions containing the peptide were collected and lyophilized, and the resulting powder preparation was further purified by reversed-phase high performance liquid chromatography. Column MMC pack, A-324 0DS 10X250m
m: column temperature 25°C; elution solvent AO. 1% monotrifluoroacetic acid water-99.9% distilled water; Elution solvent BO. 1% monotrifluoroacetic acid water - 99.9% acetonitrile; Elution program O minutes (90% A + 10% B), 30 minutes (6
0% A + 40% B); elution rate 1.6 m Q /min, detection wavelength 230 nm. The main peak fraction eluted at a retention time of 29.14 minutes under these conditions was collected and
Pass through a column of X8 (AcOH type, 1.8X5■),
The washing liquid was also collected, and after distilling off the acetonitrile, it was freeze-dried. 76 ■ of white powder were obtained. Thin layer chromatography:
Rf: 0.71 (Avicel gel plate; developing solvent; n
-Butanol: AcOH: Pyridine: Water = 30:20:
6:4). Elman, G.L.
(1959) Architectural Biochemistry and Biophysics (Arch. Biochem.
Biophys. ) Determination of free SH groups by method 82.70-77: 97%.

Amino acid analysis value Set 3.99 (4); Glu
1.00(1): Pro 0.98(1); Gly
1.19(1): 1/2Cys O. 84(1);
Val 1.03(1): Ile 1.01(1);
Phe 2.12(2);}1is 1.84(2)
, Arg O. 94(1) - Recovery rate 71.1%.

1/2 C y s was determined by performic acid oxidation method. Values inside brackets indicate theoretical values.

Bovine serum albumin (BSA) (132■) was dissolved in 3 mQ of 0.1M phosphate buffer (PH7.5). A 2-dimethylamide solution (200μQ) containing 11.2μ of N-(γ-maleimidobutyloxy)succinimide (GMBS) was added dropwise to this solution while stirring with a stirrer, and the reaction was carried out at 30°C for 30 minutes. 0.1M liquid
Sephadex G-25 (1.5X30a
n) I was purified to obtain BSA into which a maleimide group had been introduced (malimidized BSA).

The above maleimidated BSA (20 µ) was dissolved in 0.1 M phosphate buffer (pH 6.5) - 0.1 M NaCQ, while the human NGF N-terminal peptide obtained in Example 1 (5 µ ) in 0.1M phosphate buffer (pH 6.0) -
Dissolved in 5 nM EDTA. Both solutions were mixed (total volume less than 5 m breath), reacted at 30°C for 60 minutes, and P1S
was added to obtain a solution 12mQ containing a complex of human NGF N-terminal peptide and BSA. This solution was dispensed into 1.5 mQ portions and used for rabbit immunization.

3 Hairmaking of human NGF N-terminal peptide The complex of the human NGF N-terminal peptide obtained in Example 2 and BSA was prepared using Freund's complete adju
vat and the mixture was injected subcutaneously into rabbits. Thereafter, a mixture of the above complex and Freund's complete adjuvant was injected into the same rabbit every two weeks.

The blood collected from the immunized rabbit as described above was centrifuged to obtain antiserum. As a result of measuring by ELISA using the complex as an antigen, a high antibody titer was observed.

The above antiserum was purified by ammonium sulfate precipitation and DEAE cellulose column chromatography, and anti-human NGF N
A terminal peptide antibody was obtained.

1. Structure of human NGF vector 2. λEMB L3 genomic library prepared from human leukocyte DNA [Clontech]
After infecting Escherichia coli NM538, approximately 3×10′ clones were plated on a soft agar plate. After transferring the plaque onto a nylon membrane (Amersham, Hypondo N), 0.5N NaOH-1.5M N
a After denaturing phage DNA by soaking in C1 solution for 6 minutes, 0.5M Tris-HCI (pH 8.0
) - 1.5M N a C l 71! Immersed it in the liquid for 6 minutes. This membrane plan was immersed in a 2xssc solution, air-dried, and then treated at 80°C for 2 hours to fix DNA to the membrane plan.

On the other hand, the known [Ullrich, A.
) et al., Nature 303,821
(1983)) based on the human NGF gene.
DNA (0.38 kb) encoding NGF was chemically synthesized and labeled with 32p using a DNA labeling kit (Nitspon Gene), which was used as a probe. A filter with immobilized DNA containing a labeled probe,
6xSSC (1xSSC=0.15M NaC 1
．． 0.015M sodium citrate), 5XDe
nhardt's, 0.5%SDS, 20ttg/m
Q-denatured salmon sperm DNA solution in 10 mQ at 65°C, 16
It was kept warm for hours.

After the reaction, the filter was soaked in 2xSSC, 0.1% SDS solution at room temperature for 3 times for 5 minutes each.
Washed in solution at 60°C for 60 minutes. After drying the washed filter, a radioautogram was taken to search for clones that reacted with the probe. From the clone λβLN2113 obtained by this method, Dav
is) et al.'s method (Davis et al., "Advanced Bacterial Genetics")
terial Genetics)”, Cold Sp
ring Harbor Laboratory 19
Phage DNA was extracted using 80).

Next, λβLN2113 was cut with Sma I and Apal, and the DNA (approximately 1 kb) containing the human NGF gene was excised, and the plasmid pB bluescript II was cut out.
SmaI, Apa of K (purchased from Toyobo)
One site was inserted to obtain plasmid pNGFP107G.

The nucleotide sequence of the inserted portion was determined using Sequenase (Toyobo) (Fig. 1). The determined base sequence is published in Nature, 303, 821 (19
The protein coding region was completely identical to the sequence described in 83).

The above phage λβL N 2113 DNA was cut with restriction enzyme BgQU to obtain a DNA fragment containing human NGF (
1.8 kb) was isolated. On the other hand, the expression vector PKSV-10 (Pharmacia) for animal cells was used with the restriction enzyme BgQ.
This was cut with U and ligated with the above DNA fragment containing the human NGF gene using T4 DNA ligase.

Using this reaction solution, Escherichia coli (Escher
One of the ampicillin-resistant transformants [Escherichia coli (Escherichia coli) DH 1] was transformed.
/ p MNG F 101 (IFO 1486
9, FERM BP-2385) was named pMNGF101 (Fig. 2).

Monkey COS-7 cells in DMEM containing 10% fetal bovine serum
Media Virology 8:396
(1959), and then the medium was replaced with the same medium. After 4 hours of exchange, the known method [Graha
M) et al., Virology 52:4
56 (1973)), human N obtained in Reference Example 1
GF expression vector pMN GF 101 (10 μg)
Alternatively, a calcium phosphate gel containing pKSV-10 DNA (10 μg) was prepared and added to the cells to obtain transfected COS-7 cells. After 4 hours, the cells were treated with glycerol (Gorman et al., Science, 221, 551 (1983).
)) L. 1 in DMEM medium containing 10% fetal bovine serum.
Cultured for 6 hours. The medium was DM containing 0.5% fetal bovine serum.
The medium was replaced with EM medium, and the NJ cells were further cultured for 2 days. The obtained culture solution was centrifuged, and the supernatant (0.5 m Q
) was added with trichloroacetic acid to precipitate the protein, and the precipitate was added to Sallple Buffer (Laemm11
, Naturs, 227, 68
0 (1970)), heated at 100°C for 5 minutes, and subjected to electrophoresis on a 17% polyacrylamide gel containing 0.5% SDS.

Burnette's method [Analytical Biochemist]
ry), 112, 195 (1981)], and the protein was transferred from the gel to a nitrocellulose membrane using the anti-human NGFN-terminal peptide antibody obtained in Example 3 and a Wester protein test using Bechstein's ABC kit (Funakoshi Pharmaceutical Co., Ltd.).
By n blotting, p M N G F 10
Approximately 13 kilodaltons of human NGF was detected in the culture supernatant of COS-7 cells transfected with 1. On the other hand, COS-transfected with pKSV-10
No human NGF was detected in the culture supernatant of 7 cells.

The nerve fiber elongation activity in PC12 cells in the above culture supernatant was determined by Brain Research.
ch),. Li. 3,350 (1977). Human NGF expression vector pM
In the culture supernatant of COS-7N cells transfected with 101 NGF, nerve fiber elongation activity was observed as in the case of mouse NGF. On the other hand, no activity was observed in the culture supernatant of cos-tm cells transfected with pKSV-10.

From the above results, it became clear that the anti-human NGF N-terminal peptide antibody is useful for detecting human NGF.

4 Human NGF C Peptide 88 Alai1' 88-118 Human NGF or H-L s-Ala-Leu-Thr-Met-As
-Gl -L s-Gin-Ala-Ala - Same as Example 1, the resin contains Boa-^rg (
Tos) -PAM-P (0.5m mol/g
) was used to sequentially synthesize from the carboxyl terminus. Boc
- BoaLys (Cl-Z), Boc as amino acids
-Ala, Boc-Leu, Boa-Thr(Bz
l), Boc-Met. Boc-Asp(OBzl)
, Roe-Gly, Boc-Gin, Boc-Tr
p(CHO), Boc-Arg(Tos), Boc
-Phe, Boa-Ile, Boa-Cys (MeB
zl), Boa-Val. Boa-Ser (Bzl
) was used. After repeating condensation to the amino terminal, the obtained peptide resin was treated with hydrogen fluoride in the same manner as in Example 1 (however, 8 m of hydrogen fluoride was added to 1 g of peptide resin).
(using 1 ml of l+P-cresol and 1 ml of 1,2-ethenedithiol), and then purified by gel filtration and reversed-phase high performance liquid chromatography, and the obtained target product was freeze-dried. 10.1 square meters of white powder was obtained. This peptide was tested under the high performance liquid chromatography conditions shown in Example 1, with only the elution program set to O minutes (70% A + 30% B).
Under conditions changed to 20 minutes (60% A + 40% B), it eluted at a retention time of 11.43. Amino acid analysis value Asp
1.92(2). Thr1.83(2), SarO
．． 96(1), Glu 1.00(1). Gly O
．． 91(1), Ala 6.36(6), 1/2 Cy
sO. 71(1), Val 3.17(3). Ne
t 0.88(1), He 1.76(2). Leu
1.81(2), Phe 1.02(1), Lys
2.56(3), Arg 3,84(3), TrpO
．． 43(1). Recovery rate 77%. Hydrolysis was performed with 1,6N hydrochloric acid in the presence of 4% thioglycolic acid. 1/2 Cys
was determined by performic acid oxidation method. The inside of the cutout indicates theoretical profits. As shown in Reference Example 2, this peptide inhibits the nerve fiber elongation activity of PC-1 cells by human NGF expressed in monkey COS-7 cells by approximately 80 μg/ml at a concentration of 100 μg/ml.
% inhibition. Human NGF C-terminal peptide analog obtained in Example 4 [
Ala"') (88-118) using human NGF,
In a manner similar to that shown in Example 1, the peptide and BSA
A solution containing the complex was obtained.

6 AIa11@88-118 Human NGF Anti-(Ala11') (88-1
18) Human NGF antibody was generated. Similar to Example 1,
Boc-Ser (Bzl)-PAM-P for resin
(0.5 m@ol/g) as a Boc-amino acid condensed sequentially from the carboxyl terminal.
-Gly, Boc-Asp(OBzl), Boa-
Lys (CI-Z), Boc-Thr (Bzl),
Boa-Ala, Boa-Ile, Boa-Glu
(OBzl)+Boc-Val, Boc-Mat,
Boa-Leu, Boc-Asn, etc. were used. After repeating condensation up to the amino terminal, the resulting peptide resin was treated with hydrogen fluoride in the same manner as in Example 1, followed by gel filtration.
It was purified by reverse-phase high performance liquid chromatography. 10 cm of white powder was obtained.

Of the high performance liquid chromatography conditions shown in Example 1, this peptide was tested using only the elution program for O minutes (80
%A+20%B) and 40 minutes (50%A+50%B), it was eluted at a retention time of 34.3 minutes. Amino acid analysis value Asp 5.00 (5), Thr 2
．． 7g (3), Set 0.86 (1), Glu 2
．． 04(2), Gly 2.99(3). Ala0
．． 98(1), Val 2.88(3), Ile
1.97(2), Leu 1.07(1), Net
0.95(1), Lys 2.78(3). Same as Example 1. The resin is Boa-Ser (Bz
l)-PAM-P (0.5 mmol/g) as a Boc-amino acid sequentially condensed from the carboxyl terminal. Boc●Arg(Tos). Boc-A
sp(OBzl), Boc-Pro, Boc-Asn.
Boa-Val, Boa-Ssr (Bzl), 8oc
-Gly, Boc-Cys (MeBzl). Boc
-I1e. Boc-Lys(Cl-Z), Boa-H
is(Tos) and Hoe-Trp(CHO) were used.

After repeating condensation to the amino terminal, the resulting peptide resin was purified by hydrogen fluoride treatment in the same manner as in Example 4, followed by gel filtration and reversed-phase high performance liquid chromatography. 13μ of white powder was obtained. This peptide was tested under the high performance liquid chromatography conditions shown in Example 1, with elution programs of 0 minutes (85% A + 15% B) and 40 minutes (70%
A + 30% B) and the flow rate was changed to 1.20 ll/win, and it was eluted at a retention time of 20.2 minutes. Amino acid analysis value Asp 4,80(5), Set 3
．． 12(3), Pro 1.93(2), Gly 2
．． 38(2), Cys O. 91(1), Val1
．． 11(1), Ile 1.02(1), Lys
1.00 (1). }Iis 1.00(1), Ar
g 1.92(2), Trp O. 88(1). The conditions for hydrolysis are the same as in Example 4. The recovery rate for amino acid analysis was 58%. As in Example 1, the resin contained Boa-Gly-PAM.
-P (0.5 mmol/g) was used to sequentially condense the carboxyl terminal. As a Boa-amino acid, B
oa-Val, Boa-Phe, Boc-Lys (C
l-Z) + Boc-Gin, Boc-Tyr(Br
-Z), Boa-Glu (OBzl), Boc-
Thr(Bzl), Boc-Cys(MeBzl),
Boc-Arg (Tos), Boc-Asp (OB
zl), Boc-Pro, Boa-Asn, Bo
c-Ser (Bzl) etc. were used. After repeating the condensation up to the amino terminal, the resulting peptide resin was treated with hydrogen fluoride in the same manner as in Example 1, and then purified by gel filtration and reversed-phase high performance liquid chromatography. 28 cm of white powder was obtained. Of the high performance liquid chromatography conditions shown in Example 1, only the elution program for this peptide was changed to O min (aO%A+20%B). 30 minutes (60% A + 40% B
), it was eluted at a retention time of 26.0 minutes.

Amino acid analysis value A. sp 3.00(3), Thr
O. 97(1), Set 0.92(1), Glu
2.14(2), Gly 1.25(1), Val
2.15(2), Tyr O. 98(1), Phe
3.19(3), Lys 2.02(2), Ar
g 1.01(1), Pro 1.91(2), C
ys O. 74(1). Recovery rate 71%. Cys is over!
It was quantified as cysteic acid by the ifi acid oxidation method.

10 Human NGF peptide positions 70-100, H
-Gl -I1e-As -Ser-L s-}
1is-Tr-Asn-Ser-Tr-As in Example 1, Boc-Arg (Tos
Boc-Gly. Boc-I1e. Boa-
Asp(OBzl). Boc-Ser (Bzl).
Boc-Lys (Cl-Z), Boc-His (Tos
), Boc-Trp (CHO), Boa-Asn. B
oc-Tyr (Br-Z), Boc-Cys (MeBz
l). Boc-Thr (Bzl), Boc-Phe,
Boc-Val, Boc-Ala. Boa-L
eu. Boc-Net. Boa-Gln. was used. After repeating the condensation up to the amino terminal, the resulting peptide resin was treated with hydrogen fluoride in the same manner as in Example 4, and then purified by gel filtration and reversed-phase high performance liquid chromatography. 9.2 cm of white powder was obtained. This peptide is Example 1
Of the high performance liquid chromatography conditions shown in , only the elution program was 0 minutes (80% A + 20% B), 40%
It was eluted at a retention time of 37.0 minutes under conditions changed to (50% A + 50% B). Amino acid analysis value Asp 3
．． 00(3), Thr 5, 27(5), Ser 1
．． 71(2), Glu 1.12(1), Gly
1.97(2). Ala3.41(3), Cys
1.20(1). Val 1.09(1). Net
1.01(1), Ile 1.00(1),
Leu 1.03(1), Tyr O. 99
(1), Phe 1.04 (1), Lys 3.0
9(3), }Iis 1.97(2), Arg1.
09(1), Trp 1.76(2). Recovery rate 74%
．． The conditions for acid hydrolysis are the same as in Example 4.

As in Example 1, the resin contained Boc-Val-PAM.
-P (0.5 mmol/g) was used to sequentially condense from the carboxyl end. As a Boa-amino acid, Boa
c-Arg(Tos), Boc-Gly, Glu(
OBzl), Boc-Phe, Boc-Ser(B
zl), Boc-Val, BoC-Cys (MeB
zl), Boa-Asp(OBzl), Boa-T
rp(CHO), Boa-Lys(Cl-Z), B
oa-Thr(Bzl), Boc-Ala, Boa
-I1e etc. were used. After repeating condensation up to the amino terminal, the resulting peptide resin was treated with hydrogen fluoride in the same manner as in Example 4, and then purified by gel filtration and reversed-phase high performance liquid chromatography. 4.0 cm of white powder was obtained. Of the high performance liquid chromatography conditions shown in Example 1, only the elution program was changed for this peptide (85% A
+15%B) and 30 minutes (60%A+40%B), it was eluted at a retention time of 28.7 minutes.

Amino acid analysis value Asp 3.00 (3), Thr 2
．． 85(3), Set 2.36(3), Glu 2
．． 12(2), Gly 3.12(3). CysO
．． 81(1), Val 5.11(5), Ile
1.20(1), Phe O. 84(1), Lys
3.01(3), Trp 1.00(1), Ar
gO. 93(1). Recovery rate 7-S%. The conditions for acid hydrolysis are the same as in Example 4. As in Example 1, the resin contained Boc-^sn-PAM.
-P (0.5 mm+ol/g) was used to sequentially condense from the carboxyl end. As a Boa-amino acid, B
oc-Val, Boc-Leu, Boc-Gly,
Boa-Glu(OBzl), Boc-Asn,B
oc-Ile, Boc-Ser(Bzl), Bo
c-Phe, Boc-Lys(Cl-Z), Bo
a-Gin, 13oc-Tyr(Br-Z), Bo
c-Thr, Boa-Cy (MeBzl), Bo
a-Arg(Tos), Boc-Asp(OBzl
), Boc-Pro, etc. were used. After repeating the condensation to the amino terminal, the resulting peptide resin was treated with hydrogen fluoride in the same manner as in the examples, and then purified by gel filtration and reversed-phase high-performance organic chromatography. A white powder] O■ was obtained. This peptide was tested under the conditions of column speed liquid chromatography shown in Example 1, with only the elution program being O minutes (85% A + 15% B) and 40 minutes (4% A + 60% B).
It was eluted at a retention time of 30.2 minutes under conditions changed to .

Amino acid analysis value Asp 4.95 (5), Thr 1
．． 02(1). Ser O. 83(1), Glu 3
．． 17(3), Pro 0.91(1), Gly1
．． 11(1), Cys O, 89(1), Val
2.93(3), Ile 0.98(1), Leu
O. 94(1), Tyr 1.06(1), Ph
e 3.01(3), Lys 200(2), Arg
1.06(1). Recovery rate is 80%. The acid hydrolysis conditions were the same as in Example 4.

Appearance: Anti-human NGF antibodies can be obtained by using the human NGF partial peptide of the present invention as part of the antigen, but since it is a partial peptide, raw materials cannot be procured by simple methods such as chemical synthesis. This makes it possible to produce anti-human NGF antibodies more easily than in the case of using whole human NGF protein, which itself is complicated to isolate and purify, and thus to provide a simpler method for detecting and quantifying human NGF.

[Brief explanation of the drawing]

Figure 1 shows the cloned human NG obtained in Reference Example 1.
The base sequence of the F gene and the amino acid sequence translated from this are shown. FIG. 2 shows a construction diagram of the human NGF expression vector pMNGF101 obtained in the reference example.

Claims (1)

[Claims] (1) Ser at the 3rd position and Ile at the 6th position of human nerve growth factor
, 9th Arg, 37th Met, 40th Gly, 50th Lys, 60th Asp, 61st Pro, 6th
5th place Asp, 92nd place Met, 94th place Gly, 11th place
10 consecutive sequences of human NGF having at least one amino acid at position 7 Val and position 120 Ala
A partial peptide having 10 or more amino acids, or a partial peptide in which some constituent amino acids of a partial peptide having 10 or more consecutive amino acids in the sequence of human nerve growth factor are substituted with other amino acids. (2) The partial peptide or substituted partial peptide according to claim 1, wherein the partial peptide has 10 to 40 consecutive amino acids in the sequence of human nerve growth factor. (3) The partial peptide or substituted partial peptide according to claim 1, wherein the partial peptide consists of 10 to 15 consecutive amino acids of the 1st to 15th sequences of human nerve growth factor. (4) The partial peptide is human nerve growth factor Nos. 88 to 11
The partial peptide or substituted partial peptide according to claim 1, which consists of 10 to 31 consecutive amino acids in the 8th sequence. (5) The substituted partial peptide according to claim 4, which has a sequence in which Cys at position 108 and/or position 110 of human nerve growth factor is replaced with another amino acid. (6) A complex of the partial peptide according to claim 1, 2, 3, 4 or 5 and a carrier protein. (7) An antibody obtained using the complex according to claim 6 as an immunogen. (8) The method for producing the antibody according to claim 7, which comprises inoculating an animal with the complex according to claim 6. (9) A method for detecting and quantifying human nerve growth factor, which comprises using the antibody according to claim 7.