JPH013199A - monoclonal antibody - 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 Field of Industrial Application The present invention relates to monoclonal antibodies, specifically to screening and diagnosis of various liver diseases, and human hepatocyte growth factor (
human hepatocyte growth
The present invention relates to a novel monoclonal antibody useful for the purification of hHGF (actor: hHGF).

Conventional technology hHGF is a proteinaceous substance isolated and purified from the blood of patients with fulminant hepatitis by the present inventors, Kanayama et al.
ohda et al, Exp, Ce1l Res
,.

166.139-156 ('1986); H.

Nakayama et al., Biomed, R.
es, 6.231-237 (1985);
1-166495 etc.).

Such hHGF has the activity of significantly promoting the proliferation of hepatocytes, and its activity in the blood fluctuates in close relation to the pathology of fulminant hepatitis, especially changes in the degree of coma. It is known that it decreases during the period.

The present inventors believe that hHGF can be used as a therapeutic agent for various liver diseases such as acute hepatitis, chronic hepatitis, cirrhosis, and fulminant hepatitis, or as a therapeutic agent after hepatectomy. Furthermore, it has already been found that measurement in clinical samples is useful for screening, diagnosing, or understanding the pathology of these liver diseases.

However, as mentioned in the above report, the process for producing or purifying hHGF is extremely large and complicated, and the low yield also leaves a major problem in providing it to the above technical field.

Furthermore, hHGF can be measured using the previously reported bioassay (
The only technology that has been established is to measure the amount of activity using biological assays (biological assays), but such methods are not only inferior in operability and accuracy, but also always take into consideration the presence of components that may interfere with the measured value (activity). Due to necessity, for the above purpose,
This does not completely meet the demands of this industry, and it is desired to overcome these technical problems.

The present invention satisfactorily solves these technical problems, and its purpose is to provide a novel antibody for achieving the above-mentioned desired techniques.

Means for Solving the Problems According to the present invention, a monoclonal antibody having hHGF-specific reactivity is provided.

Utilization of the antibody of the present invention provides a means for immunologically purifying hHGF and a means for immunologically measuring hHGF.

The greatest feature of the antibodies of the present invention is that they have specific reactivity to hHGF, that is, they have specific binding properties, and such antibodies include types that inhibit or stop the hepatocyte proliferation activity of hHGF. antibodies are included.

The method for producing the antibody of the present invention will be described in detail below.

The antibody of the present invention uses IIHGF as an immunizing antigen,
It can be produced according to a conventional antibody production method.

In the above method, hHG as the immunizing antigen used
F is known as described above.

In the production of the antibody of the present invention, it is not necessarily necessary to use a purified preparation of hHGF, and it is also possible to use a crude product containing it. More specifically, in the method for producing the antibody of the present invention, for example, plasma cells (immune cells) of a mammalian mammal immunized with hHGF as the above-mentioned immunizing antigen are fused with plasma cell liver cells of a mammalian mammal to create a hybridoma (hybridoma).
A preferred example is a method in which a clone producing an antibody that recognizes hHGF as h2 is selected from the clone, and a target antibody (monoclonal antibody) is obtained from the clone.

In the above method, the mammal to be immunized with the immunizing antigen, beaten hHGF, is not particularly limited or is preferably selected in consideration of compatibility with the plasmacytoma cells used for cell fusion, and is generally a mouse. , rats etc. are advantageously used.

Immunization is carried out by a conventional method, for example, by administering the hHGF to the mammal by intravenous administration or intraperitoneal injection. More specifically, hHGF was
Dilute to an appropriate concentration with S or physiological saline, etc., and administer this to animals several times every 2 to 21 days, using a regular adjuvant if desired, so that the total dose is about 1 to 100 μg/animal. It is preferable to do so. Furthermore, a conventional carrier (Schlepper) can also be used for the above administration. As the immune cells, it is preferable to use tile cells excised about 3 days after the final administration of h l-IGF.

As the mammalian plasmacytoma cells as the other parent cells to be fused with the above immune cells, various known cell lines such as p3 (p3/X63-Aq8) (Natur
e, 256, 495-497 (1975)), p3-U
l (Current Topics in Micro
obiology and Jmmunology.

81, 1-7 (1978)), N5-1 (Eur.
.

J. Immunol, 6.511-519 (1
976) ), MPC-11 (Cell, Dan, 405-
415 (1976)), 5P210 (Nature,
276°269-270 (1978)), FO (J.

Immunol, Meth, 35.1-21 (
1980) ), X63.6.5.3. (J, Immu
nol,, 123゜1548-1550 (1979)
), 3194 (J, Exp, Med,, 148, 313
-323 (1978)) etc., and R210 in rats.
(Nature, 277.131-133 (1979
)) and other myeloma cells are used.

The above-mentioned fusion reaction between immune cells and plasma cell hepatocytes can be carried out using basically known methods such as Milstein et al.
Methods of Ein et al.
zymol, 73.3-4.6 (1981)) etc., and carried out as follows.More specifically, the above-mentioned fusion reaction [, J2, for example, in the presence of a fusion promoter in a normal nutrient medium. Commonly used fusion promoters are used, such as polyethylene glycol (PFG) and Sendai virus (HV J ), and if desired, adjuvants such as dimethyl sulfoxide are added to increase the fusion efficiency. You can also.

The ratio of immune cells to plasma hepatocytes used is the same as in the usual method, for example, about 1 to 10 times as many immune cells as plasma hepatocytes may be used. As the medium for the above-mentioned fusion, for example, RPMI-16/1○ medium used for the proliferation of the above-mentioned plasmacytoma cell line, MFM medium, and various other usual media used for this type of cell culture can be used. It is usually best to remove serum supplements such as fetal bovine serum (Fe2). Fusion is carried out by thoroughly mixing a predetermined amount of the above immune cells and plasmacytoma cells in the above medium, and adding a PEG solution preheated to about 37°C, for example, an average molecular weight of 1000 to 6.
000 to about 30-60% (W/
This is done by adding and mixing at the concentration of V).

Thereafter, desired hybridomas are formed by repeating the steps of sequentially adding an appropriate medium, centrifugation, and removing the supernatant.

The resulting desired hybridomas are isolated by culturing them in a conventional selection medium, such as HAT medium (a medium containing hypoxanthine, aminopterin, and demidine). Culture in the HAT medium may be carried out for a sufficient period of time, usually from several days to several weeks, to kill cells other than the target hybridoma (unfused cells, etc.). The hybridoma thus obtained is searched for a strain producing the antibody of interest and single cloned according to the usual limiting dilution method.

The search for the producing strain can be performed, for example, by the FLISA method ['Enc+v
all,E,,Methods Enzymol,
, 70° 419-439 (1980)), plaque method, spot method, agglutination reaction method, Ouchterlony method, radioimmunoassay (RIA) method, and various other methods commonly used for antibody detection [ [Hybridoma method and monoclonal antibodies]
, published by R&D Planning Co., Ltd., pI) 30-53,
(March 5, 1981). Note that the hHGF preparation described above can be preferably used as the antigen in the above search.

The thus obtained hybridoma producing the desired monoclonal antibody can be subcultured in a normal medium and can be stored for a long period of time in liquid nitrogen.

The monoclonal antibody of the present invention can be collected from the hybridoma by culturing the hybridoma according to a conventional method and using the culture supernatant, or by administering the hybridoma to a compatible mammal and growing it, and then using the ascites. The method of obtaining the information will be adopted.

The Usui method is suitable for obtaining highly pure antibodies, and the latter method is suitable for maximum production of antibodies.

Furthermore, the antibody obtained as described above can also be purified by conventional purification methods such as salting out method, gel filtration method, and affinity chromatography.

Utilizing the thus obtained antibody of the present invention, hHGF can be easily and specifically purified by conventional immunological purification means such as immunoprecipitation and affinity chromatography.

Furthermore, according to the use of the antibody of the present invention, radioimmunoassay (R
hHGF can be easily measured with high sensitivity, high accuracy, and high specificity by conventional immunological means such as IA>, enzyme immunoassay (E IA), and agglutination method.

Setting up a purification system and a measurement system using such an antibody of the present invention, and modification or application thereof will be obvious to those skilled in the art.

Effects of the Invention According to the present invention, a specific antibody for hHGF is provided. By using this antibody, it is possible to purify hHGF by immunological purification means, and it is extremely useful in its production. Further, the use of the antibody of the present invention provides a means for immunologically measuring hHGF, which is particularly useful for hHGF in clinical samples.
It is used to measure GF, and can be used to extremely effectively screen and diagnose various liver diseases or understand pathological conditions.

EXAMPLES Reference examples, working examples, and test examples are given below to explain the present invention in more detail.

Reference Example 1 - (Production of hHGF) hHGF was produced by the method of Kanen et al. (EXp, Ce11Res
9,166.139-150 (1986); Patent application 1986
-1664-95], it was isolated and purified from the plasma of a patient with fulminant hepatitis.

Hepatocyte proliferation activity (hereinafter abbreviated as rHGF activity) was also measured according to the method reported above.

The HGF activity of the above hHGF was determined by heat treatment (80°C11
0 min) and enzyme treatment (0,1mM+nQ trypsin,
Inactivate by 37°C for 30 minutes and 0.1mM mQ chymotrypsin at 37°C for 130 minutes).

In addition, 0.5M acetic acid, 0.1M acid buffer (pH 4,0
>, same (pH 5,0>, 0.1M phosphate buffer (pH
7,4> and 0.1 M glycine buffer (pH 9,5) (4°C, 20 hours).

According to 5DS-PAGE of the hHGF (according to the method described in the above literature, using 12% pre-separation and silver staining), under non-reducing conditions, the hHGF has a molecular weight of Two bands of 88,000 and approximately 83,000 were migrated.

Furthermore, according to 5DS-PAGE under reducing conditions, the molecule j
i56000-65000 and 32000-35000
gave both groups their main bands.

From this, it was deduced that hHGF is a proteinaceous substance in which these molecules are bonded through disulfide bonds, and that all of the above molecular types are substantially the same as hHGF.

Example 1 <Production of antibody> ■ hHGFo obtained in Reference Example 1 above, OIIIL/
+r+Q saline solution was mixed with an equal volume of Freund's complete adjuvant (Freund compl.
eteadjuvant, DIFCOLaborat
ories, DetroitMichigan US
A) and suspended. The above hH of the resulting suspension
The GF2μq-containing portion was subcutaneously administered to Balb/C mice. In the second week, the same wave containing 2 μq of hHGF was administered in the same manner, and after another 3 weeks, hHGF dissolved in physiological saline was administered.
)-IGF was administered intraperitoneally. Four days after the final administration, the spleens were removed, and the spleen cells were washed three times with RPMI-1640 medium.

Mouse myeloma cell line P3-U1 (Current
Topics in Microbiology a
nd Immunology.

1.1-7 (1978)) after washing in the same way, Part 1
×107 cells and the above tile cells 1×108 cells to 50n+C1
Mixed in a centrifuge tube. After centrifugation at 200×G for 5 minutes, the supernatant was removed with a Pasteur pipette.

Polyethylene glycol 1500 (manufactured by Boehringer Mannheim Yamanouchi) 50% (W/V) kept at 37°C
) 1 mQ of RPMI-1640 solution containing FC8 was added dropwise over 1 minute, then 1 mQ of FC8-free RPMI-1640 solution kept at 37°C was added, left for 1 minute, and then 2 mQ of the same wave was added. In addition, leave it for 2 minutes, and then apply the same wave 4
mQ was added. 15 which was kept warm at 37°C after being left for 4 minutes.
%FC3゜0.05g titer/Q
8 mQ of PMIJ) was added and centrifuged at 200XG for 5 minutes.

Remove the supernatant and add to complete RPMI kept at 37°C to give 1×10” cells/mQ!!: Turbid, 24
1 mQ each was dispensed into well-hole microplates (Costar) and cultured in a 5% carbon dioxide incubator at 37°C. After 24 hours 1.0 x 10-'M pipoxanthine, 4. OXlo-7M aminopterin and 1.6X1
Complete RPMI medium containing 0-5M thymidine (rHA
>1 mQ of "lower medium" was added to each well. Thereafter, half of the supernatant was added to fresh HAT on days 2, 3 and 4, respectively.
On the 6th day, instead of the medium, half of the supernatant was added to 1.0X
Complete RPMI medium containing 10-'M hypoxanthine and 1.6 x 10-5M thymidine (hereinafter referred to as "1 medium") was substituted. Thereafter, growth was maintained in complete RPMI medium.

The hybridoma thus obtained was cloned by the limiting dilution method. That is, using 20 mQ of RPMI-1640 medium supplemented with 10% FC3 prepared to contain 3 x 102 hybridomas and 1 x 108 Ba1b/CC mouse thymocytes, 3 hybridomas/well were plated in a 96-well plate. Cultured. Similarly, the proliferating hybridoma is divided into one hybridoma/
The cells were cloned as wells, and the hybridomas that proliferated were similarly cloned at 0.3 hybridomas/well.

The clone producing the desired antibody is the hH used for the immunogen.
The search was performed by ELISA using GF as an antigen.

Thus, a hybridoma (clone NO.
0AL-H-2-14> was obtained.

■Clone No. 0AI-11-2 obtained by the above ■
-14 was cultured in complete RPMI medium in a 5% carbon dioxide incubator at 37°C for 48 hours. The culture solution was centrifuged (3000 ppm, 10 minutes) to obtain a culture supernatant containing the monoclonal antibody of interest (hereinafter referred to as rhl-2-14J).

Note that the subclass of the above antibody was IgG+.

This is the Ophthalony Kit (manufactured by serotec)
It was confirmed using

■ Clone No. 0AL-t”-2-1 obtained in the above ■
4, 1×106 pieces, were prepared in advance using pristan (pristan).
The solution was administered intraperitoneally to 3alb/c mice that had been inoculated with A.

After 10 to 14 days, the accumulated ascites was collected to obtain ascites containing the antibody of the present invention. The concentration of this antibody is approximately 0.2-5mM
It was mG.

Purified antibody H-2-14 was obtained from the ascites using an IQG purification kit (MAPSKit; manufactured by Bio-Rad).

Test Example 1 150mM NaCQ and 0.04% Na of hHGF used as an immunogen was added to each well of a 96-well plate.
20mM Tris-HCl solution containing N3 (8.6μQ/10m
2> was added and left at 4°C for 24 hours. After washing, 2.
200 μQ of 5% BSA in PBS was added to each well and allowed to stand at 4° C. for 24 hours or more to block, thereby obtaining a plate in which the antigen was insolubilized.

Next, 100 μQ of each of the two-fold dilutions of the supernatant obtained in Example 1 (2) were added to each well, and the wells were shaken at room temperature for 1.5 hours.

After washing each well, use peroxidase-labeled goat anti-(mouse I QM+I COG>antibody (Jackson)
Jackson Immunoresearch
Add 100 μQ of X5000 (manufactured by Lab.), leave it for 1.5 hours at room temperature with shaking, wash, and then remove ortho-
Phenyldiamine (25m (1/10mQ>solution 1
Add 00 μ(!) and leave it for 20 to 30 minutes, then 2N
The reaction was stopped after one addition of 100 μQ of H2SO4.

The absorbance of the obtained reaction solution at 4.92 nm was measured using Quitartic Multiscan (manufactured by Flow Laboratories).

The results are shown in FIG.

In the figure, the horizontal axis shows the dilution factor of the culture supernatant containing the antibody of the present invention, and the vertical axis shows OD 492.

Test Example 2 (Western Plotting) This test is
According to the method of Towbin et al. (proc, Natl, ΔCad, SCi
, USA.

76.4350-4354. (1979)).

That is, after converting hHGF to 5DS-PAGF (described in Reference Example 1), the gel was combined with a nitrocellulose membrane, and a current was applied using the side with the gel as one pole and the side with the nitrocellulose membrane as a seed, and electrophoresis was performed on the gel. The resulting protein was blown onto a nitrocellulose membrane. After blocking the plotted cellulose membrane by immersing it in a PBS solution containing 2% 83Δ, the purified antibody H-2'-14 obtained in Example 1 above was reacted as the next antibody. Ta. After washing, use peroxidase-labeled goat anti (mouse I) as a secondary antibody.
QM+JΩG) antibody (manufactured by Jackson) was added,
The reaction was carried out in a PBS solution containing 2% BSΔ.

Then 50mM+-Lis-HCl (DH7,5>+0.2M
After washing the nitrocellulose membrane with NaCQ solution,
Working solution [50mM + Helis hydrochloric acid (pH 7,5
)+31+nQ of 0.2M NaCQ, 3mCJ/r
51TI of methanol solution of nQ4-chloronaphthol
25 μQ of a mixed solution of Q and 30% aqueous solution of To1202 was added to develop color.

The results of the Western plotting described above are shown in FIG.

In Fig. 2, the three lanes (1) show the results of SDS-PAGE under non-original conditions, and the three lanes (2) show the results under reducing conditions. In each result, lane Δ indicates hHGF obtained in Reference Example 1, and lanes B and C indicate hHGF from different lots obtained in the same manner as in Reference Example 1.

From the figure, the antibody H-2-1/I of the present invention has reactivity with hH'GF (both two bands) migrated by 5DS-PAGF under non-reducing conditions, regardless of the lot. However, it was also confirmed that it did not react with h l-IGF under reducing conditions.

Test Example 3 (Absorption test) Antigen (hHGF solution) and antibody (H-2 obtained in Example 1
-10-fold dilution of culture supernatant containing -14) at 37°C.
I let it react overnight. A gel containing protein Δ in this solution (manufactured by Bio-Rad)
was added to bind the monoclonal antibody to protein A, the gel was removed by centrifugation, and the supernatant was collected (J
, Biol.

Chem, 260.7219"722'5 (198'
5')).

The HGF activity of this supernatant was measured at 1251-
dUrd uptake ([3iochcm, , 23.

6295+-6299 (1984, )).

The results are shown in Figure 3.

In the figure, the horizontal axis is the supernatant (
sample) amount (μQ), the vertical axis is 125■-dUrd
The uptake amount (CI) m
Curve 1) shows the results of the above test, and curve (2) shows the results of a control using the same amount of RPMI-1640 medium instead of the antibody in the above test.

From the above test results, the antibody 1-(-2-1/l) of the present invention is
It can be seen that this antibody has specific reactivity to hHGF.

[Brief explanation of drawings]

FIG. 1 is a graph showing the results of investigating the reactivity between the antibody of the present invention and hHGF by diluting the antibody. FIG. 2 is a drawing showing the results of examining the reactivity between the antibody of the present invention and hHGF by Western blotting. FIG. 3 is a graph showing the results of an absorption test examining the reactivity between the antibody of the present invention and hHGF. (hereinafter ``2) Procedural amendment (formality) 1 Indication of the case Patent Application No. 159149 of 1988 2 Name of the invention Monoclonal antibody 3 Person making the amendment Relationship to the case Patent applicant Kyo Hara, a carpenter (two idiots) 4 Agent Sawanotsuru Building, 2-10 Hirano-cho, Higashi-ku, Osaka City June 8, 1988 (Delivery date: June 28, 1988) 6 Amendment to the column "Brief explanation of drawings" in the specification subject to amendment Contents 1 Figure 2 is shown in box 23, lines 2 to 4 of the specification. ” is corrected as follows. "Figure 2 is a photograph in place of a drawing showing the results of examining the reactivity between the antibody of the present invention and hHGF by Western blotting." (Above)

Claims (1)

[Claims] 1. A monoclonal antibody characterized by having specific reactivity to human hepatocyte growth factor.