JPS61294365A - Antiserum for antihuman epithelia cell growing factor - Google Patents

Abstract

PURPOSE:To obtain an antiserum which has extremely high specificness and is usable for a quantitative determination method with high accuracy by using refined h-EGF as an antigen and sensitizing the same to a warm-blooded animal then preparing the antiserum in accordance with the conventional practice. CONSTITUTION:The h-EGF which is obtainable by a genetic engineering technique and is isolated and refined to high purity (99%) is used as the antigen. Such h-EGF is dissolved in a physiological salt soln. and an equal amt. of Freund's complete adjuvant is added thereto to obtain an emulsion. The emulsion is sensitized several times at weekly intervals with a domestic rabbit (female) and thereafter the blood is drawn therefrom. The hemofibrin thereof is centrifugally separated after a tube contg., for example, a blood separating agent is treated according to the conventional practice and is thereby removed, by which the antiserum is prepd.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an antiserum that specifically reacts with human epidermal growth factor, ie, an anti-human epidermal growth factor antiserum.

Prior Art Human Epidermal Growth Factor
alGrOWth Factor (hereinafter referred to as h-EGF)
was introduced as a human-derived factor that promotes the proliferation and keratinization of epithelial tissue isolated from human urine in 1975 by Cohen et al. [Reference 1], and in the same year by Gregory ( Human urogastrone (htJlan U rOQastro), which has the effect of suppressing gastric acid secretion, was isolated from human urine by H.
It is the same substance as the polypeptide introduced as ne) [Reference 2], is composed of 53 amino acids with a molecular weight of about 6000, and has 3 disulfide bonds in its molecule [Metabolism, 17,51 ( 1980)).

By the way, there are reports on antiserum against h-EGF [Reference 3], but conventional anti-h-EGF
The serum is prepared using a known method in which h-EGF isolated from human urine is used as an antigen, and a bloodletting animal (rat, mouse, rabbit, etc.) is sensitized with the antigen, and then blood cells and fibrin are removed from the collected blood. It was prepared according to [Reference 4].

The h-EGF used here as an antigen is obtained from urine, and the h-EGF contained in urine is
EGF is very small (25 to 25 Or+g/jd), and there are so many impurities in urine that it is difficult to isolate.
Isolation and purification costs are high.

Therefore, it goes without saying that the antiserum prepared using h-EGF as an antigen is octavalent, and if the antigen (h-EGF extracted from urine) is not highly purified. It is also difficult to obtain an antiserum with very high specificity that reacts only with h-EGF. Furthermore, various methods for obtaining h-EGF have been proposed at present, including methods for extracting h-EGF from biological components (usually urine) [JP-A No. 58-99418, No. 58-219124, Japanese Patent Publication No. 59-Sho. 42650, etc.], chemical synthesis methods [JP-A-59-27858, etc.] and genetic engineering methods [JP-A-57-122096].
No. 58-216697, No. 59-132892, etc.] have also been proposed. Here, h-EGF is chemically synthesized by linking one to several amino acids according to a known peptide synthesis method (in-phase method or liquid phase method).
The higher-order structure (S
-8 bond), but the synthesis operation is complicated because the steps of protection of a functional group that is not subjected to reaction → condensation reaction → deprotection are repeated. Since it also involves a rough oxidation operation of stin, the synthesis efficiency is low, only a trace amount of h-EGF can be obtained, and the production cost is high.

On the other hand, the method for preparing h-EGF using genetic engineering methods is
After transforming a host bacterium according to a given genetic recombination procedure, a transformant is obtained, and by culturing it, h-E
Is there a method in which h-EGF is recovered after expressing GF as a fusion protein with another protein in the host bacterial cell (Japanese Patent Laid-Open Nos. 57-122096 and 58-21669)?
7), or a method in which h-EGF is secreted outside the bacterial cell by utilizing a signal sequence in the above gene recombination operation, and then h-EGF is recovered (see JP-A-59-132892). However, in order to recover and produce h-EGF prepared in this way, the former method requires destroying the host cell and obtaining the fusion protein from the miscellaneous cell components. Later, it is necessary to separate h-EGF from this fusion protein and purify it, and if the latter method is used, the h-EGF secreted outside the bacterial cell
EGF has an extra peptide or amino acid attached to it (i.e., a linker is usually used to connect the signal sequence and the structural gene of h-EGF, and a corresponding base sequence is interposed). , this part is also expressed with the expression of the protein, and as a result, it is bound to the N-terminal side of h-EGF as an excess). Therefore, after specifically removing this part, h- EG
Since it is necessary to purify F, even if h-EGF is produced in large quantities, there are various problems in obtaining a highly purified purified product.

There are also reports that h-EGF that has been prepared chemically or genetically and purified to a high degree of purity has been obtained, and that anti-h-EGF antiserum was prepared using this as an antigen. Not obtained.

Therefore, there is a strong desire to provide an antiserum with high specificity and low cost by using highly purified h-EGF.

The present invention aims to solve the above-mentioned problems by providing an antiserum prepared using purified h-EGF as an antigen, which specifically reacts with h-EGF. This is what we are trying to achieve. The present invention was achieved by confirming that this antiserum does not react with other in-vivo peptides or hormones, but specifically reacts with h-EGF.

Therefore, the anti-h-EGF antiserum according to the present invention is an antiserum obtained from blood collected after sensitizing a warm-blooded animal with purified human epidermal growth factor as an antigen. , which is characterized by specifically reacting with human epidermal growth factor.

肱-1 Thus, the antiserum of the present invention uses purified h as an antigen.
- EGF was used to sensitize a warm-blooded animal, then blood was collected and prepared according to a conventional method. Therefore, the difference from conventional anti-h-EGF antiserum is that the antigen used to prepare the antiserum is extracted from human urine (conventional), whereas it is purified (in the present invention). That is the point.

Since the antiserum of the present invention is prepared using the above-mentioned highly purified h-EGF as an antigen, it solves the above-mentioned problems and has the following advantages.

b) Very specific.

The antiserum of the present invention specifically reacts only with h-EGF without reacting with other Babtides (insulin, human gonadotropin, secretin, etc.) present in the human body. This is shown in Reference Example 2 below,
This is supported by the fact that when cross-reactivity with other peptides and hormones was examined by radioimmunoassay using the antiserum of the present invention, most of them showed cross-reactivity of 0.01% or less. It can be said that the antiserum has extremely high specificity. Therefore, the antiserum of the present invention can be used for immunohistochemistry, and can be used, for example, to investigate the distribution of h-EGF in the human body, and for various pathological analyzes.

口) Can be used for high-sensitivity constant m method.

The conventionally known anti-1f-EGF antiserum is prepared using h-EGF extracted from human urine as an antigen. It is difficult to isolate h-EGF from human urine, and there are problems with the specificity of the anti-EGF anti-autoclear prepared using h-EGF obtained from urine as an antigen. (This is because if h-EGF is not isolated, antibodies are produced by recognizing impurities bound to h-EGF as antigens, and antibodies containing such antibodies are produced. (This is because such antisera have low specificity.)

On the other hand, the anti-h-EGF antiserum of the present invention uses purified h-EGF as an antigen, so the above-mentioned problems are solved, and the anti-h-EGF antiserum of the present invention is highly specific.
It is an antiserum. Therefore, it can be used in immunoassay methods (radioimmunoassay, enzyme immunoassay, etc.) and can perform highly sensitive measurements. For example, in the radioimmunoassay system, the measured value is compared with a standard curve and the measured value is usually expressed by the grade of the hormone used as the standard. Where it is desirable to use
In the present invention, h-E used as an antigen in one example
GF is prepared by genetic engineering and has high purity (99%
It goes without saying that the antiserum prepared using this h-EGF has high specificity, and since this highly purified h-EGF is used as a standard product, it is easy to measure. The error is small (and the measurement sensitivity is also high (see application example below). In addition, in preparing h-EGF, as shown in one embodiment of the present invention, the method previously proposed by the present inventors h-EGF produced in this manner can be obtained efficiently (and in large quantities) by using h-EGF purified in this way. The production cost of anti-h-EGF antiserum prepared as 6 anti-h-EGF antiserum will also be lower than in the past.

Antigen is a general term for substances that can induce antigen-antibody reactions and immune responses, and in nature, it consists of proteins with a molecular weight of about 1000 or more, polysaccharides, complexes thereof, or complexes with lipids. It is said to consist of the body (from ``Biology Dictionary'' published by Iwanami Shoten).

In the present invention, h-EGF that has been isolated and purified to a high degree of purity can be used as the antigen. As mentioned above, there are various methods for preparing h-EGF (e.g., extraction from urine, chemical synthesis, genetic engineering techniques, etc.). It is preferable to use h-EGF purified as follows. In the present invention, ``ryigJed'' means ``substantially single'', and the term h-EGF used as an antigen in the present invention has a purity of 99%.
% or more is preferable.

Various methods for preparing h-EGF using such genetic engineering methods have been proposed (Japanese Patent Application Laid-Open No. 57-122096,
No. 58-216697, No. 59-132892, etc.], but the method previously proposed by the present inventors [
It is most preferable to carry out according to the specification of Japanese Patent Application No. 60-22630]. This is because h-EGF can be efficiently prepared and a purified sample with high purity (99% or more) can be easily obtained.

The method for preparing h-EGF using such genetic engineering techniques consists of the following steps.

A. (a) A vector containing a gene encoding a signal peptide to which the structural gene of a desired exogenous protein can be ligated immediately after the downstream end of the gene and capable of proliferating within the intended host cell; A gene encoding a desired foreign protein is introduced, (b) a Durham-negative microorganism is transformed with this recombinant, and (c) the resulting transformed microorganism is transformed into a late logarithmic growth phase during the growth process of the microorganism. From ll to early arrest phase, protein synthesis ability increases! (d) The microorganisms are then treated by the osmotic shock method to obtain a fraction containing the desired foreign protein. to collect the amount.

B. After subjecting both fractions containing the recovered desired protein to ion exchange chromatography, recovering the desired exogenous protein fraction.

C9: After further subjecting the fraction containing the desired exogenous protein recovered above to high performance liquid chromatography, the desired exogenous protein fraction is recovered.

Details of this method can be found in the reference examples and patent application filed in 1986-226.
Please refer to the specification of No. 30.

As mentioned above, h-EGF is a peptide with a molecular weight of about 6,000, so for example, purified h-EGF is dissolved in physiological saline and then exsanguinated from animals (rats, mice, etc.).
Needless to say, it acts as an antigen in vivo just by injecting it (rabbits, etc.).

Furthermore, when administering the antigen (h-EGF), it is common to mix it well with Freund's complete adjuvant [Reference 5] to prepare an emulsion, which is then administered.

Antiserum and Part-1 Antiserum refers to serum containing antibodies, and its preparation is as follows:
This can be carried out according to a known conventional method (for example, Reference 6) in which an animal is injected with an antigen to produce antibodies against the antigen in vivo, blood is collected, and blood cells and fibrin are removed from the blood.

The antiserum according to the present invention is a serum containing an antibody against h-EGF, and an example of its preparation method is as follows. That is, after dissolving h-EGF produced by genetic engineering methods and purified to a purity of 99% or more in physiological saline,
Equal amounts of Freund's complete adjuvant (Freund's complete adjuvant)
's Complete A adjuvant) (Reference 6) was added to obtain an emulsion, which was then injected into a rabbit (♀
) is sensitized several times at intervals of 1311, then blood is collected, and blood cell fibrin is removed according to a conventional method (in the case of the present invention, as an example, the blood is treated with a tube containing a serum separating agent (commercially available product), and then centrifuged. ) to prepare antiserum (see Examples below for details).

W double 1 The specificity of the antiserum of the present invention can be confirmed by qualitative precipitation reaction, in-gel diffusion reaction, radioimmunoassay method, or the like.

In the present invention, as an example, the in-gel diffusion method (the book "Useful Immunology Experimental Methods" p64, (1984) published by Kodansha) and the radioimmunoassay method [Seishan [Biochemistry Experiment Course - Hormones - J pl 73 (1977)], Specificity was confirmed according to [Tokyo Kagaku Doujin Publishing].

In the present invention, among the in-gel diffusion methods, the offtelony method, which is easy to operate and allows visual confirmation of the specificity of the antiserum, is used as a method for confirming the specificity of the obtained antiserum [the above-mentioned "Useful Immunology Experiment Method"]. ] is preferred.

This method consists of the following steps. That is, using a heated solution of 1-2% purified agar or agarose in a neutral buffer solution, it is placed on a glass plate or a glass Petri dish for 2-3 minutes.
! A layer of IIR thickness is applied and cooled to form a gel plate. Then, on this gel plate at an appropriate interval (6 to 8 It
I11 is normal) and drill a hole with a diameter of 2 to 5jIl. Then, a sample (antigen solution or antibody solution) is placed in this hole, and the sample is transferred to a wet chamber or placed in a cold room (4° C.) overnight to allow a reaction to occur, and the resulting sedimentation line is determined. Therefore, when examining the specificity of the antiserum of the present invention using this method, prepare a gel plate with a hole in the center and radially spaced holes around the gel plate. Serum is added to the surrounding hole, and antigen solution is added to the surrounding hole, respectively, and specificity is confirmed according to the above method.

For details on confirming the specificity of the antiserum of the present invention according to the above method, please refer to Examples below.

On the other hand, the characteristics of the antiserum of the present invention can be investigated by radioimmunoassay as follows. That is, first, h-EGF was labeled with I-h-E
Prepare GF. Then, the antiserum of the present invention and unlabeled h
- Add the above to the system previously incubated with EGF.
After adding I-h-EGF, the mixture is left at 4°C overnight. Goat anti-rabbit antiserum was added to this, and h-
Separation of EGF (B) and unbound h-EGF (F) (two-antibody method, adsorption method, and filter paper chromatography are used as typical methods with good reproducibility [see "Biochemistry Experiments" above) [Refer to lecture]) After that, measure the radioactivity of the precipitate (B). Then, use the above method to create a standard curve by varying the concentration of unlabeled h-EGF, and use this standard curve and In the above method, unlabeled h-EG
Calculate the cross-reactivity (%) by comparing the dose-response line results obtained by adding various peptides and hormones at various concentrations in place of F to the above system [see above [Biochemistry Experiment Course Jp162]] The specificity of the antiserum of the present invention is confirmed by (for details, see Reference Examples below).

In preparing the anti-h-EGF antiserum of the present invention, h-EGF used as an antigen was prepared as follows and further purified to obtain a specimen with islet purity (99% or higher purity).

Step A: Preparation of osmotic supernatant (1) Creation of transformants Transformants E and coliK1 were prepared according to the following method.
2 YK 537 (pTA1522) was constructed.

pTA1529 (details of construction are in patent application No. 59-15970)
5 μ9 (see specification of No. 3) in a 5 μA buffer solution (10 m
M Tris-1 acid MI solution (hereinafter referred to as Tris-HCI) (pH
7,5), 10mM fvlocI, 50mM N
aC1) with 4 units of restriction enzyme Hindll (Takara)
(hereinafter referred to as Hindll) at 37° C. for 1 hour. Next, ethanol precipitation was performed, and the resulting precipitate was diluted with a 30 μm reaction solution <67 mM Tris −
HCl, (pH 8,8), 16.6mM ammonium sulfate [hereinafter (NH4) 2804: +, a, 7mM ethylenediaminetetraacetic acid (hereinafter referred to as EDTA), 0.66mM each of dAT'P, dCTP, and dGTP.

The cells were treated with 1 unit of T4-DNA polymerase in TTP) for 15 minutes at 37°C. Next, the precipitate obtained by ethanol precipitation was diluted with a 50 μm reaction solution (6 mM
Tris-HCI (pH 8,0), 6mM
Hydrolysis was carried out at 37°C for 1 hour using 4 units of restriction enzyme 5alI (Takara) (hereinafter referred to as 5alI) in MIIIC+, 150mM NaCI).
By agarol gel electrophoresis, a 3900 bp DNA
Fragment A (■ in Figure 1) was obtained.

Plasmid pBR322-hUG (1) BR322 (
E. coli K12 C600 (pBR322
) has been deposited as EC.
A fragment of the artificially synthesized h-EGF structural gene digested with ECOR and 5alI was added to the fragment digested with 0RI and 5alI.
, 50mM NaCl゜50mM MoCl2)
3 using 4 units of restriction enzyme EcoRI (Takara) in
After hydrolysis at 7°C for 1 hour, T4
After DNA polymerase treatment and further 5alI treatment, 160b was isolated by agarose gel electrophoresis.
A DNA fragment of p (■ in Figure 1) was obtained.

The two DNA fragments prepared above (■ and ■ in Figure 1)
) was added to a 30 μm reaction solution (20 mM Tris-HCl
(pH 7,5), 10mM MaCI2.10mM
The mixture was reacted for 16 hours at 14°C using 300 units of T4 ligase [Takara] in DTT, 0.5mM ATP). After the reaction is completed, E. coli is transformed with 12 YK537 (Kushner's method (Reference 7)) to create a transformed strain (E. coli) containing the target plasmid (hereinafter referred to as pTA1522) (■ in Figure 1). Kl 2YK
537 (pTAL 522)) was obtained.

(2) Cultivation of the transformant The transformed microorganism E, coli K12YK
537 (E)TAL 522) was cultured as follows. Single cell pure isolated E. coli K12
YK537 (pTA1522)-Platinum loop per liter of tryptone 10g, yeast extract 5g, NaCl 5g,
Inoculated into 100I11 medium consisting of 20Mg ampicillin, 500a! ! Shaking culture was performed overnight at 37°C in Yonosaka Rokolben.

Next, take the entire ff1 (100d) of this culture solution and inoculate it into 20 liters of a medium consisting of 30g of glucose per liter, 2(1) tryptone, 1.0g of yeast extract (LF, Mg5O.7H201,0g), and ampicillin 20#19. , cultured in a 30 liter jar fermenter.

The culture temperature was 37°C, and the aeration rate was 0,5 vva (1
vv+a is 1 volume-volume-1inu
te means that 1 liter of air is introduced per 1 liter of culture solution per minute. ), pH is 4N NaOH and 4N HCI
The dissolved oxygen (Do) is adjusted to 7.2, and the dissolved oxygen (Do) is 11 degrees.
By changing the stirring speed using an O-control device 41)
It was kept near 0m. The culture was carried out until the amount of h-EGF produced reached the maximum by quantitating glucose and inorganic phosphorus, measuring the number of viable bacteria, and measuring the amount of h-EGF produced over time.

(3) Osmotic supernatant made by gJ First, transfer the above culture solution (20 liters) to a 5CR continuous centrifuge.
Centrifugation (14,600 g) by 20B (Hitachi) L,
, to obtain bacterial cells (wet weight: 672.1 g). Next, in order to subject this bacterial cell to the osmotic shock method, 10 liters of a high concentration sucrose solution (20% sucrose, 30mM Tris-HCI <pH 8,0), 1
(mMEDTA). After being left at room temperature for 10 minutes, the cells were collected by centrifugation in the same manner as above.

Next, the bacterial cells were soaked in 4°C water (8°C) containing 2 kg of ice.
After standing for 10 minutes, centrifugation is performed in the same manner as above, and the supernatant is collected. The sample is subjected to ion exchange chromatography (osmotic supernatant).
And so.

Step B: Ion exchange chromatography The above osmotic supernatant was subjected to the same continuous centrifugation operation as above to obtain supernatant 93.
40d was obtained, and using this as a sample, ion exchange chromatography was performed as follows.

(1) Preparation of ion exchange column Add resin (D
EAE-TOYOPEARl 650M) was filled and equilibrated with 20mM ammonium acetate buffer (pH 6.0).

(2) Ion Exchange Column Chroma 1 - Graphie The above sample was applied to an ion exchange column (column temperature 4°C) to treat h-EGF to the column, and then 20mM ammonium acetate! 1ili solution (p l-16.0) 25
00! The column was washed with d. Elution of adsorbed h-EGF was performed using 20mM ammonium acetate buffer (pH 6,0
) 1500Id and 250mM ammonium acetate buffer (
pH6,0) 1500jd! Which linear gradient (
The flow rate was 2 d/min). The ion exchange column chromatogram at that time is as shown in FIG. ○ in the same figure
indicates the absorbance (280 nm) of each fraction, and . indicates the strength of activity of each fraction by radioreceptor assay (RRA) [expressed as binding inhibition rate (%)].

As h-EGF fraction, absorbance peak and RR of each fraction
Two portions where the activity peaks measured by method A overlapped were collected. One is for both the 51st to 60th fractions (h-EGF-10 and below, rh-EGF-I in Figure 2).
The other one is the 97th to 105th fractions (h-EGF-Il in Figure 2, hereinafter referred to as rh-
It is called EGF-nJ).

Here, the RRA method is performed as follows.

That is, RRA of h-EGF was performed using KB cells (ATCCNαCCL17) derived from human nasopharyngeal epithelial carcinoma cells;
, King et al. [Reference 8]. That is, first, monolayer culture was performed in a DME medium using an 800 m flask.

Next, the medium was removed and cells were detached using phosphate balanced salt solution (PBS) containing 0.05% EDTA to prepare a cell suspension. Then, 20mM Hep
es) (pH 7,4).
) Cells were washed twice with balanced salt solution (HBSS). Binding trusion ([3inding
5 solution) (DME medium/20mM Heibes (Hel) eS) (pH 7,4) ・0.35g
/liter NaHCO-100μq・d streptomycin), then calculate the number of cells to 300,000 to 400,001.
It was prepared to give a 0.2 d binding trusion and dispensed into tubes in 0.2 d portions. 1 liter of various concentrations
-EGF and I-mEGF ('?us EGF)
Add 0.27 of sample solution containing
Incubated for hours. The cells were then cooled on ice.
After washing twice with IBSS, the radioactivity of I-mEGF bound to the cells was measured using a gamma counter (Aroki ARC300 (Aloka Co., Ltd.)). This was converted into a binding inhibition rate (%) as shown in FIG.

In addition, h-EGF-1f obtained here is h-EGF
-I consists of 53 amino acids that constitute epidermal growth factor (described later), but two amino acids (leucine and arginine) were missing from the C-terminus.

Step C: High Performance Liquid Chromatography The fraction h-EGF-I obtained in the above step B was subjected to the following operations to purify these fractions. That is, after freeze-drying the fraction h-EGF-I, it was
60 t of solution containing % acetonitrile and 2% acetic acid
It was dissolved in d. This solution was then centrifuged and the supernatant was
d was obtained and subjected to high performance liquid chromatography. The conditions for high performance liquid chromatography are as follows.

High performance liquid chromatography m: HCl803D system (Toyo Soda) Column temperature = 45°C Flow rate: 2d1 min Eluent and elution conditions: Solution A (10% acetonitrile, 2% acetic acid) Solution B (80%
Elution (acetonitrile, 2% acetic acid) was performed using a gradient based on the concentrations of liquids A and B as shown in the table below.

Detection wavelength: absorbance 280 nm The elution pattern of h-EGF-I at that time is as shown in FIG. In addition, in the figure, the storage time of 17.23 minutes is the peak of purified h-EGF-I.

The specific activity and yield of h-EGFffi purified by the above steps are as shown in Table 1.

Table 1 Calculated by.

**Performed by dye-binding method (performed using BIO-RAD protein assay kit).

In addition, the h-EGF-II fraction could also be purified using the same method.

The purity of the shoe last J and the purified h-EGF was tested using conventional methods such as polyacrylamide gel electrophoresis and reversed phase high performance liquid chromatography. From these results, the purity is 99%
It turns out that's all.

The results of electrophoresis at that time are shown in FIG. 4, and the results of high-performance liquid O-mography are shown in FIG. In addition, (a) in FIG. 4 is a reproduction of an electrophoresis gel, the arrow (0) indicates the band of h-EGF, and (opening) is a densitometry.

The amino acid composition of h-EGF in the amino acid phase was analyzed by taking a part of the fraction obtained in the above example, treating this fraction according to a known conventional method, and then using an amino acid analyzer (HLC-825A).
A (Toyo Soda)].

The results obtained are shown in Table 2. This result is in complete agreement with literature values.

Table 2 ☆　I had a foot as Cystine.

− and two The primary structure was determined according to (Reference 9).

That is, using a gas phase protein sequencer (Applied Biosystems), a sample (h-EGF-I
60μg (10nn+ol) dissolved in water (100μm)] was subjected to Edman degradation to release amino acid residues from the amino terminal side as anilinothiazolinone derivatives, which were then converted into phenylthiohydantoin derivatives (PTH amino acids). Converted to . and,
This PTH amino acid was identified by reverse phase high performance liquid chromatography (HPLC). By performing the steps up to this point, except for the 6 sites corresponding to hand cystine (if you run bevtide containing cystine as it is on a sequencer, the half cystine site will be blank; see Figure 6).
Fixed up to the third.

Next, in order to identify half-cystine, the sample (above) was treated with bromo cyanide and then subjected to reductive carboxymethylation (CM conversion) to obtain two fragments (CMI and CMII) [see the above-mentioned biochemistry experiment course].

When the amino acid sequences of these two 7-ragment sequences were then analyzed by HPLC, all half-cystines were detected as S-ruboxymethylcystines. The above results are as shown in FIG.

In the figure, ASn%Ser, etc. are abbreviations commonly used in the art to indicate amino acids such as asparagine and serine, respectively. Numbers such as 1.5.10 on the abbreviated amino acid sequence indicate the sequence number of the amino acids from the N-terminus to the C-terminus of h-EGF. Also, the right arrow (−
1) shows directly analyzed PTH amino acids, and the other arrow (-7) shows the identification results of PTH amino acids after CM conversion treatment. Furthermore, in the figure, CMI and CMn indicate fractions obtained by converting to 0M after treatment with bromo cyanide. In addition, the leftward arrows (-) from the C-terminus to the 6th position in the figure indicate the results of hydrolyzing the sample using carboxypeptidase W (CPW) and analyzing the released amino acids over time. There is (C
Terminal analysis: CPWl is reacted with h-EGF20 at a ratio, amino acid analysis is repeated over time, and released amino acids are identified).

From the above, the primary structure of h-EGF was confirmed to be that shown in FIG. 6.

Next, the secondary structure was investigated by the method described below.

First, a sample (h-EGFl 74 μ9 dissolved in 100 μm pyridine/acetate buffer (pH 6,5)) was digested with thermolysin [-Reference 10], and then
Fragments were separated by reverse phase HPLC. Then, as a result of amino acid analysis using a conventional method, three fragments containing cystine were obtained. The S-8 bridge was then cleaved by performic acid oxidation of each of these fragments. Then, cystic acid-containing fragments were fractionated by reverse-phase HPLC, and each fragment was subjected to amino acid analysis and amino acid sequence analysis using conventional methods (see FIG. 7 for the results). As a result, the position of the S-8 bridge is number 6 from the N-terminal side [
1 and 20 (CI in Figure 7)), 14th and 31st (([)) in Figure 7, and 33rd and 42nd ([■] in Figure 7). . The identification results of the above primary and secondary structures were published by Gregory in 1975.
This structure corresponds to the structure of h-EGF proposed by (Reference 2).

In addition, when we investigated h-EGF-I in the same way, we found that
h-EG except that two amino acids on the C-terminal side are missing.
It was the same as F-I.

1 Tanseihuang 11 h-EG purified to the above high purity (99% or higher purity)
Antiserum was prepared using F as an antigen according to the following procedure.

h-EGF (0,2rRg) was added to physiological saline (0,5rRg).
d), and then added Freund's complete adjuvant of the highest quality to create an emulsion [Seijai, Biochemistry Experimental Course Hormone JP175 (1977), published by Tokyo Kagaku Doujin].Next, 1mf! of this emulsion was prepared at home. Rabbit (♀)
The patient was subcutaneously injected into the armpit and back of the patient at several locations (approximately 0.2 d each), and this procedure was repeated six times at one-week intervals. One week after the last sensitization, blood was collected from the ear vein.

Then, this blood was placed in a Heparavit tube (tube containing a serum separating agent: Miki Chemical Industry ■), left for 20 to 30 minutes, and then centrifuged (3000 rpm).

30 minutes) to remove blood cells and fibrin to obtain the desired antiserum.

The specificity of the antiserum of the present invention was confirmed by examining cross-reactivity between the antiserum and other substances according to the following offtelony method.

After putting 7 m of 1.2% purified agar solution (added with 0.1% sodium azide (Na N3)) into a glass petri dish and gelling the agar, holes with a diameter of about 5J111 were made in the gel. . As shown in Figure 8, six such holes are made (A to A), 20μρ of antiserum is poured into the center hole, and h-EGF and 21-Leu-h are poured into the surrounding holes, respectively.
-EGF (h-jGF in which the 21st amino acid is substituted from methionine to leucine. For details on construction, see JP-A-60-28994), urogastrone, normal rabbit serum, and anti-heron gG anti-blood serum. 120
After each addition of μρ, incubation was performed overnight at a low temperature (4° C.). Figure 8 shows the sedimentation line that occurred at that time. In the figure, Ab indicates a hole containing the antiserum solution of the present invention, and A to A indicate holes containing the following solutions.

(b) h-EGF (b) h-EGF-II (The C-terminal amino acids of h-EGF are two what is missing) (c) 2l-Leu-EGF (d) Ulogastrone (e) Normal rabbit serum (f) Family rabbit I (IG antiserum Moreover, the solid line around the Ab hole indicates a sedimentation line.

Therefore, a clear sedimentation line was confirmed between the anti-h-EGF antiserum of the present invention and A, H, H, and II (Fig. 8), and the inventive Mouse-EGF (m-EGF
), secretin, insulin, human sex-stimulating hormone, etc., and no sedimentation lines were generated, which confirmed that the antiserum of the present invention has high specificity for h-EGF. .

Reference Example 2 Radioimmunoassay was performed using the anti-h-EGF antiserum prepared according to the present invention to examine cross-reactivity with other peptides and hormones.

l! Gooza 11 The h-EGF obtained above was processed using the chloramine T method [Reference 11
]. That is, h-EGF 1
After dissolving the microorganism in 10 μm of 0.5 M phosphate buffer (pH 7,5), Na I (manufactured by NEN, USA; purchased through the Japan Isotope Association) was added to the mixture.
j! Stirred. Next, 710 μp of 3M8/Id chloramine was added to this and the reaction was carried out for 1 minute at room temperature.
Rg/l+11! The reaction was stopped with sodium metabisulfite.

To this solution, 1/10 volume of 2% bovine serum albumin (BSA) solution was added to the reaction solution, and Sephadex G-25 (Pharmacia') (0.15M sodium chloride (NaCl) was added.
125I-labeled antigen (125I-labeled antigen (1
-h-EGF) was obtained.

Radioimmunoassay (RIA) Test sample, anti-h-EGF antiserum and 125■-11-
EGF was diluted with each dilution buffer (0.01M phosphate buffer,
0.15M NaCl, 0.1% gelatin, 0.01
% NaN5) and mixed with 100 μp of each of these, and then left at 4° C. overnight. Next, goat anti-rabbit IQG serum (second antibody) diluted 20 times with a dilution buffer and 100 μp of normal rabbit serum diluted 100 times were added to this.
After adding and incubating at 4℃ overnight, centrifuge (300 rpm).
30 minutes) to obtain a precipitate, and after washing this precipitate,
The radioactivity (B) of the precipitate was measured using a γ-Autowell scintillation counter (manufactured by Aloka). On the other hand, excluding the test sample, anti-h-EGF antiserum and I
, -h -E The same operation as above was performed using only G, the radioactivity (Bo) was measured, and B, /Bo was calculated [the above-mentioned "Biochemistry Experimental Course - Hormones - Jp162". The above method was performed in the presence of various h-EGF concentrations to obtain a standard curve (Figure 9).

Then, in order to examine cross-reactivity with other peptides and hormones, various concentrations of other peptides or hormones were added to the system instead of h-EGF in the above RIA procedure to create a standard curve as shown in Figure 9. did. The concentration of various peptides or hormones such that B/Bo = 50% is adjusted to h-EG.
B/Bo= calculated when performing the above RIA method on F
The concentration of 50% is taken as 100%, and it is expressed as a ratio [cross-reactivity (%)] to the above concentration of h-EGF (
Table 3).

From this result, the antiserum of the present invention was found to contain h-EGF, h-E
GF-11r and 2l-Leu-h-EGF and 100
% cross-reactivity, 0.1% cross-reactivity with m-EGF, and less than 0.01% cross-reactivity with other peptides or hormones, so specificity is very high. It is suggested that it is something.

Table 3 Reference Example 3 h-EGF in human bodies (blood plasma, serum, urine, etc.) was measured by the RIA method described above. The measurement results and literature values at that time are shown in Table 4.

Table 4 * Book “Cell Growth Factors-Growth Fact”
ors-Jp251 edited by the Japanese Society of Tissue Culture, published by 11 Suijinten.As described above, the antiserum of the present invention can also be used in immunoassay methods.

The mycological properties and accession numbers of the microorganisms disclosed in the present invention are as follows.

Contract date (1) June 9, 1981 (2) April 30, 1988 (3) June 9, 1981 (4) November 14, 1980 * Ministry of International Trade and Industry Industrial Technology Accession number of the National Institute of Microbiology and Technology: Mycological properties (1) E. coli K12C600 This bacterium is
It is a Durham-negative bacillus that does not produce spores and has the general attributes of the genus Escherichia coli, such as being facultatively anaerobic. It also does not contain the F factor, lacks the function of suppressor gene E, and the recBC gene encodes a nuclease involved in genetic recombination. It has a defect. As an auxotroph, it requires threonine and leucine for growth on its minimal medium. Furthermore, taxonomically, it belongs to the family Enterobacteriaceae and the genus Escherichia coli. The literature regarding this bacterium is as follows.

(b) Genetics (Q genetics),
1, 440 (1954) (b) Nature, 217
．． 1110 (1,968) (2) E. coli K12C600 (pYK
283) This bacterium is a Durham-negative bacillus, does not produce spores, has general attributes of the genus Escherichia coli such as facultative anaerobic properties, does not contain F factor, lacks the function of suppressor gene E, and is involved in genetic recombination. It has a defect in the recBC gene, which encodes a nuclease. As an auxotroph, it requires threonine and leucine for growth on its minimal medium. Promoter-operator region of pho A gene Replication initiation region from pACYC177 and pBR32
Plasmid pYK28 composed of two bla genes
3 and is resistant to ampicillin. In addition, taxonomically, it belongs to the Endobacteriaceae family and the genus Escherichia coli.

In addition, except for the traits derived from plasmid pYK283, the mycological properties of this strain are those of its parent strain 1:, coltK12C.
It is the same as that of 600.

(3) E, coliKl 2C600 (pBR3
22) This bacterium is a Durham-negative bacillus, does not produce spores, has general attributes of the genus Escherichia coli such as facultative anaerobic properties, does not contain E factor, lacks the function of suppressor gene E, and is involved in genetic recombination. It has a defect in the recBC gene, which encodes a nuclease. As an auxotroph, it requires threonine and leucine for growth on its minimal medium. It also contains drug resistance plasmid pBR322. In addition, for plasmid pBR332, Gene
>, 2.95 (1977), 12C600 to E. coli
Regarding this, the above-mentioned Nature can be referred to. pBR3
Except for the traits derived from E. coli K 12
The mycological properties of C600(+)BR322) are the same as those of the parent strain.

(4) E. coli K12 YK537 E. coli 12 YK 537 is a known strain of E. coli [Microbiological ψ Reviews
12 RR1 (Gene, 21.9
5 (1977) Biochemical and Biophysics Acta (Biochem, B1ophys)
, A cta,.

655.243 (1981)), it exhibits the following properties, and other properties are described in 12
This strain is no different from that of RR1.

(rec Al, Pho A8, pro)
” [Erin 1] Proceedings of the National Academy of Sciences of the United States of America (Proc., Nat.
l, Acad, Sci.

tJsA), 7U, 1317 (1975) 2) Nature t'-(Nature), 257.3253)
Journal of Clinical Endocrinology
and Metabolism (J, Clnic, Endocli, Me
tab, ), ± support, 1144 (1977) 4) Same as above (J, Clnic, Endocli
oMetab, ), Tuberc, 988 (1971) 5) Advances in label crosis research (Advances Tuberc, Res,
)ヱ、6) Endoc IJ / Logy (EndOc
IinoIoOy>, 90.1261 (1972) 7) Genetics Engineering (Genet
ic Engineering), 1978.8)
Journal of Biological Chemistry M)-L
l, B111, ChQm, ) 257.9)
Ibid. (J, Biol, Chew, ), L.
l ball, 7°990 (1981) 10) Journal of Biochemistry (J
, Biochem, ), 74.697'1
1) Nature t-(Nature), 194.

[Brief explanation of drawings]

FIG. 1 is a flowchart for the construction of plasmid pTA15223B. FIG. 2 is a graph showing a chromatogram of an ion exchange column and the results of RRA method. FIG. 3 is a reproduction of a chromatogram obtained by high performance liquid chromatography. Figure 4 shows the results of polyacrylamide gel disk electrophoresis, Figure 4 (a) is an explanatory diagram replicating the electrophoresis gel, and Figure 4 (b) is a diagram of densimetry. It is. FIG. 5 is a reproduction of a chromatogram obtained by high-performance liquid chromatography for analysis. FIG. 6 is an explanatory diagram showing the procedure for determining the amino acid sequence and the determined amino acid sequence when determining the primary structure of h-EGF. Figure 7 shows the S-
FIG. 8 is an explanatory diagram showing the position of 8 bonds and the amino acid sequence in the vicinity thereof. FIG. 8 is a reproduction of a gel when the specificity of anti-h-EGF antiserum was investigated by the offtelony method. FIG. 9 is a standard curve obtained when RIA was performed using anti-h-EGF antiserum. Applicant's representative Inomata Seiyu Retention time (minutes) Child 3 Zu Ma 8 Figure h-EGl' 1 degree (n9/Nayu Ichiriki Sei 9 Procedural Amendment (Method) % formula % 1, Indication of the case 1985 Patent Application No. 135460 2, Title of the invention: Anti-human epidermal growth factor antiserum 3, Relationship with the person making the amendment Patent applicant Yusui Pharmaceutical Co., Ltd. 4, Agent (zip code 100) ( Shipping date September 24, 1985) 6. Drawing subject to amendment 7. Contents of amendment =: l, 0.10.23'.

Claims (1)

[Scope of Claims] 1. An antiserum obtained from blood collected after sensitizing a warm-blooded animal with purified human epidermal growth factor as an antigen, which suppresses human epithelial cell growth. An anti-human epidermal growth factor antiserum characterized by specifically reacting with the factor. 2. The anti-human epidermal growth factor antiserum according to claim 1, wherein the purified human epidermal growth factor is obtained from one created by genetic engineering techniques.