JPS62267299A - Monoclonal antibody - Google Patents

Abstract

PURPOSE:The titled antibody obtained by subjecting a mammal immunocyte which is immunized against a cholestatic factor (CF) as an immune antigen and a mammalian cell of plasmacytoma to cell fusion, cloning to give a hybridoma and using the hybridoma. CONSTITUTION:An immunocyte (e.g. splenic cell, etc.) of mammal (e.g. mouse, rat, etc.) immunized against CF and a cell of plamacytoma (e.g. myeloma cell, etc.) are fused in the presence of a fusion promoter (e.g. polyetylene glycol, etc.) in a nutritive medium, then cultivated in a medium (e.g. HAT medium, etc.) for selection to give a hybridoma and reference of a strain forming the aimed antibody and monocloning are carried out by using limiting dilution method. A cloned hybridoma is cultivated by the conventional procedure and the aimed antibody is produced in the supernatant liquid of the culture mixture or the hybridoma is administered to a mammal and the aimed antibody is produced in ascites and purified by gel filtration, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to monoclonal antibodies, more specifically to cholestatic factors.
The present invention relates to a novel monoclonal antibody that specifically recognizes CFJ (hereinafter referred to as "CFJ") and is useful for the measurement of this lymphokine, CF, and for the purification of CFJ.

[Conventional technology]

CF is known as an active factor that leads to intrahepatic cholestasis [Hatahiro Mizoguchi et al., "Liver", Vol. 20, No. 7, 31.
: 673-44 : 686 (197'J); Same 2
Volume 2, No. 1, 38-44 (1981): U, A.

MAR13ET and others, European Journal
of C11nical Inve-atkgati
on, 14.346-353 (1 (J84) etc.)
.

The CF can be produced, for example, by stimulating peripheral blood lymphocytes of patients with chronic liver disease with intrahepatic cholestasis with specific antigens, or by stimulating lymph node cells of guinea pigs sensitized with killed tuberculosis bacteria using purified tuberculin (l) P. D) is detected in the culture medium by stimulation with
In addition, CF was detected in the serum of patients exhibiting symptoms of drug-allergic hepatic cholestasis, and intrahepatic cholestasis was caused by symptoms other than decreased hepatocyte function. It has been suggested that this expression occurs due to the involvement of the above-mentioned lymphokines in the immune response, and this research is attracting attention.

However, 1. 21 Self-CF is only considered to be active, and there is a need to establish separation and purification techniques and measurement techniques to study its properties and analyze its mechanism of action.

[Problem that the invention seeks to solve]

The object of the present invention is to provide a monoclonal antibody that enables the purification and measurement of CF.

[Failure to solve the problem]

Under such circumstances, the present inventor conducted intensive research and succeeded in obtaining a monoclonal antibody that specifically recognizes CF, and by using this monoclonal antibody, CF can be purified and measured immunologically. made it possible to do so. .

Hereinafter, the monoclonal antibody of the present invention will be explained.

The greatest feature of the monoclonal antibody of the present invention is that it specifically recognizes CF, that is, it exhibits specific binding to CF.

The monoclonal antibody of the present invention can be produced according to a conventional method using CF as an immunizing antigen. More specifically, for example, a hybridoma is created by fusing plasma cells (immune cells) of a sentinel mammal immunized with the above-mentioned immunizing antigen with plasmacytoma cells of a mammal; An example of a method is to select a clone that produces an antibody that recognizes ', and to obtain the desired antibody (monoclonal antibody) from the clone.

The CF of the immunizing antigen used in the above method includes, for example, a culture supernatant or a partially purified product containing CF obtained according to the method described in the above-mentioned literature, and Cl!
'Any standard product can be used.

In addition, the mammal to be immunized with the immunizing antigen in the above method is not particularly limited, but is preferably selected in consideration of compatibility with the plasmacytoma cells used for cell fusion, and is generally selected from mice, rats, etc. is used advantageously.

Immunization is carried out by a conventional method, for example, by administering the above-mentioned immunizing antigen to a mammal by intravenous, subcutaneous, subcutaneous, or intraperitoneal injection. More specifically, the immunizing antigen is administered to the animal several times every 2 to 14 days, optionally in combination with a conventional adjuvant, with a total dose of approximately
It is preferable that 1000μ be about the size of a mouse. As the immune cells, it is preferable to use pancreatic cells extracted about 3 days after the above-mentioned Injection 4.

In addition, as the mammalian plasmacytoma cell as the other parent cell to be fused with the above immune cell, various known cell lines such as p3 (p3/x63-Ag 8 ) (
Nature, 256, 495-497 (1975
)'), p3-tyt [Current Topi
cs in Microbiology and Imm
unology, 81, 1-7 (197B)
], N5-1 (Eur, J, Immunol,,
6, 511-519 (1976)), MPC-11
(Ce1l, 8.405-415 (1976)),
S P 2/0 (Nature, 276, 26
9-270 (197B)), FO (J, Immun.
ol, Meth, 35, 1-21 (1980))
X 63, 6, 5, 3, (J, Immun
ol, 12L154B-1550 (1979)),
8194 (J, Exp.

Mad,, υsu, 313-323 (1978))
etc., and R210 in rats (Nature, 2
77, 131-133 (1979)) and the like are used.

The above-mentioned fusion reaction between immune cells and plasmacytoma cells is basically carried out using known methods such as Milstein (Milate).
Method in Enzymo et al.
Logy.

Vol, 73, pI) 3 (1981) 'E et al. More specifically, the above fusion reaction is
For example, it is carried out in a conventional nutrient medium in the presence of a fusion promoter. As the fusion promoter, commonly used ones such as polyethylene glycol (PEG) and Sendai virus (HVJ)* are used, and if desired, an adjuvant such as dimethyl sulfoxide may be added to increase the fusion efficiency. You can also use The ratio of immune cells to plasmacytoma cells used is the same as in conventional methods, and for example, the ratio of immune cells to plasmacytoma cells may be about 1 to 10 times.

As the medium for the above-mentioned fusion, for example, RPMI-1640 medium used for the proliferation of the above-mentioned plasmacytoma cell line, MEM medium, and other ordinary elliptical medium used for this type of cell culture can be used. Usually fetal bovine serum (FO8)
It is best to remove serum replacement fluids such as For fusion, predetermined amounts of the above immune cells and plasmacytoma cells are thoroughly mixed in the above medium, heated to about 37°C in advance, and a 9-PEG solution, for example, one with an average molecular weight of about 1000 to 6000, is added to a normal medium. This is done by adding and mixing at a concentration of about 30 to 60 W/V%. Thereafter, the desired hybridoma is formed by repeating the steps of sequentially adding an appropriate medium, centrifuging, and removing the supernatant.

The resulting desired hybridoma is isolated by culturing it in a conventional selection medium, such as HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). 1 (Culture in AT medium is intended)・
A sufficient period of time, usually several days to several weeks, is sufficient for cells other than hybridomas (unfused cells, etc.) to die. 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 ELISA method (Eng-va
ll, E, Meth, Enzymol, 7
0419-439 (1980)), plaque method,
spot method, agglutination reaction method, octellonyi (0ucht
erlony) method, radioimmunoassay (l(IA)
) method, etc. [[) Ibridoma method and monoclonal antibody 4, @R
Published by &D Pryning, pp30-53, March 1982.
5th day of the month]. The search is performed using the immunizing antigen.

The thus obtained hybridoma that produces antibodies that recognize CF can be subcultured in Nogametsune's medium,
It can also be stored for long periods in liquid nitrogen.

The monoclonal antibody of the present invention from the hybridoma can be collected by culturing the hybridoma according to a conventional method and obtaining the culture supernatant, or by administering the hybridoma to a compatible mammal. A method is adopted in which the ascites is obtained by multiplying ascites. The former method is
Suitable for obtaining highly purified antibodies, the latter method is suitable for bulk production of antibodies. Further, the antibody obtained by the above method can be further produced in 1# by a conventional method such as salting out, gel filtration, Affinitei Clozen Raffy, etc.

The monoclonal antibody of the present invention has extremely high cFvc specificity, and can be used to purify and measure CF. Therefore, the present invention also provides a method for immunologically purifying CF' and an immunoassay method for OF of the above-mentioned specific antibody.

Purification and measurement of CF are performed according to conventional methods except for using the monoclonal antibody of the present invention. The method for measuring CF will be described in detail below.

Measurement of CF can be carried out according to conventional immunoassay methods, such as RIA method and enzyme immunoassay (EIA), and the procedures used in each of these immunoassay methods, such as hand 1 m, are different from those commonly used. For example, the known direct method, indirect method, competitive method, sandwich method, etc. can be applied.

For the purpose of diagnosis, cells, tissue pieces, and/or various body fluids are used as specimens in the above measurement method (hereinafter abbreviated as the method of the present invention). When cells and/or tissue pieces are used as specimens, conventional direct or indirect immunization methods are used. According to this method, the antibody of the present invention is applied to cells suspended in a buffer such as physiological saline or ordinary phosphate buffered saline (PBS), or to cells or tissue sections fixed on a glass slide or the like. After immunoreacting the cells or tissue fragments and thoroughly washing the cells or tissue fragments with the above buffer, the presence of the antibody of the present invention bound to the cells or tissue fragments may be directly or indirectly assayed in a conventional manner. As the labeling agent for the above assay, some of which will be described later, ordinary fluorescent light, enzymes, etc. can be used.

Conventional methods can also be followed when using body fluids as the measurement material. Here, as the body fluid, for example, blood, cell tissue fluid, lymph fluid, pleural fluid, ascites fluid, amniotic fluid, gastric fluid, urine, pancreatic fluid, cerebrospinal fluid, saliva, etc. or centrifuged supernatant after solubilizing the above-mentioned cells or tissue pieces can be used. Can be done.

When an insolubilization method (method using an insolubilized antigen or an insolubilized antibody) is employed, the above-mentioned CF or the antibody of the present invention is produced by chemically or physically reacting the insoluble antibody according to a conventional method. Examples of insoluble carriers include cellulose powder, Sephadex, Sepharose, polystyrene, P paper, carboxymethyl cellulose, ion exchange resin, text 2, glass film, glass tube, nano, glass beads, Silk, polyamine-methyl vinyl ether-maleic acid copolymer,
Amino acid copolymers, ethylene-maleic acid copolymers, etc. can be used. Insolubilization is done by the diazo method as a covalent bonding method,
Peptide methods (acid amide derivative method, carboxychloride resin method, carbodiimide resin method, maleic anhydride derivative method, isocyanate derivative method, cyanogen bromide activated polysaccharide method, cellulose carbonate derivative method, method using condensing reagents, etc.) ), chemical reactions such as alkylation method, carrier bonding method using a crosslinking reagent (geltal aldehyde, hexamethylene incyanate, etc. are used as a crosslinking reagent), carrier bonding method using Ugi reaction; or carriers such as ion exchange resins. ionic bonding method using porous glass such as glass beads; physical adsorption method using porous glass such as glass beads as a carrier.

As the labeled antigen or labeled antibody, the CF or the antibody of the present invention labeled with various labeling agents such as conventional radioactive substances, enzyme labeling substances, fluorescent substances, etc. can be used. The radioactive substances used as the labeling agent include radioactive iodine such as 12+1i, and the fluorescent substances include fluorescein isothiocyanate (fi'1Tc), tetramethylrhodamine inthiocyanate (TRITC), [transrhodamine・Inthiocyanate) (XRITC), rhodamine B isothiocyanate, cyclotriazine fluorescein (DTAF), etc., and enzyme labeling substances include peroxidase (pox), alkaline phosphatase, β-galactosidase, microperoxidase. Examples include Dase, chymotrypsinogen, procarboxypeptidase, glyceraldehyde-3-phosphate dehydrogenase, amylase, phosphorylase, D-nase, and R-nase.

These labeling methods can also follow conventional methods (
J. Biol. Chem., 4.9349-
9351 (1979); Nature, 194.49
5 (1962); Fluorescent antibody method, medical chemistry experiment course A 4
, 263-270 HActa, Endocrino
l.

5upp1.168.206 (1972) HProc
, Nat, Acad, Sct.

See USA, 713 (1967), etc.].

The method of measuring and quantifying a test substance (CF) in a specimen according to the present invention is carried out by immunoreacting using any one of the above-mentioned insolubilized antigen, insolubilized antibody, labeled antigen, and labeled antibody. At this time, the solvent used in the measurement system is a normal one that does not have a negative effect on the reaction, such as citrate buffer, phosphate buffer, tris-hydrochloric acid buffer, borate buffer, acetate buffer, etc. A preferred example is a buffer solution with a pH of about 4 to 8. Furthermore, the immunoreaction conditions during the measurement are not particularly limited, and can be the same as those used in ordinary measurement methods of this type. That is, the immune reaction generally occurs at temperatures below 45°C,
It is preferably carried out at a temperature of about 4 to 40°C for 1 to 40 hours. The separation of the conjugate and free form (B-F) after the completion of the immune reaction also follows a known method. For example, when the insolubilization method is adopted, the solid phase is separated by separation means such as centrifugation, F separation, washing, and decantation. The liquid phase can be separated. In other cases, conventional methods such as the dextran-activated charcoal method and the second antibody method may be followed.

A particularly convenient way to carry out the above assay is through the use of kits. It is important that such a kit contains the monoclonal antibody of the present invention as an antibody reagent. Stabilizers and/or preservatives such as glycerol and bovine serum proteins can be added to the antibody reagent.

Preferably, the antibody reagent is lyophilized;
The kit can contain a water-soluble or water-miscible solvent. Additionally, the antibody reagent may contain buffers to maintain a constant pH of the reconstituted reagent system and/or preservatives and/or stabilizers to prevent sample deterioration before use. can. Although a buffer solution is not considered to be an essential component of the kit reagent, it is preferable to use one that has a pH of about 4 to 8 when carrying out the measurement method of the present invention. Although the reconstitution agent preferably contains water, part or all of the water may be replaced by a water-miscible solvent. Water-miscible solvents are well known to those skilled in the art, and include, for example, glycerin, alcohols, glycols, glycol ethers, and the like.

〔Effect of the invention〕

Since the monoclonal antibody of the present invention specifically recognizes CF, the use of the antibody provides an immunological purification technique for CF by applying it to, for example, affinity chromatography. Measurement techniques are provided.

By measuring the CF of a subject, it is possible to diagnose intrahepatic cholestasis based on CF, and the above measurement technique can also be used for toxicity screening of test samples such as drugs, that is, CF.
It can be used as a screening means to determine whether or not induction of

〔Example〕

Next, reference examples and examples will be given and explained.

In the Examples, CF activity was measured as follows. That is, Wistar male rats (250-300
? ) was anesthetized with bendoparbital, and after laparotomy, a polyethylene tube (PEl 0 :Clay) was inserted into the common bile duct.
Adams) is inserted to allow bile to naturally flow out of the body. First, after collecting bile for 30 minutes, the sample was injected through the mesenteric vein, and bile was collected over time (every hour).
Measure bile flow rate, flow rate before sample injection (-7 hours)
This was confirmed by the inhibitory effect on biliary excretion in comparison with

Reference Example I Production of CF ■ Freund's Complete Adjuvant (Freund'ac) containing heat-killed Mycobacterium tuberculosis (H37Ra)
A complete adjuvant (Dlfco) was injected into each guinea pig (male: 200-3007) at a dose of 0.11 in each leg and 0.6 Inl into the posterior neck muscle. Four weeks later, lymph nodes in the axillary and inguinal regions of the sensitized guinea pigs were aseptically removed, adhered blood and connective tissue were removed, and the lymph nodes were cut into small pieces in RPMI-1640 culture medium. This piece of lymph node tissue was placed between two glass slides and gently compressed to release lymph node cells.

The cells were then agitated by pipetting and centrifuged for 7.1 minutes at 200 x 7.1 minutes using a 2000 p filter with gauze. The cell pellet was washed twice in RPMI-1640 medium and I
, a cell suspension was prepared so that the ratio was 4/4.

■ Add PPD (Puri) to the cell suspension obtained above.
fiedProtein Derivative;
Parke-Davis) at 10μ? /d and cultured for 48 hours in an a cell culture device. After incubation, low temperature centrifugation (1000X?, 10 minutes)
The cell culture supernatant was separated by

■ Transfer the culture supernatant IQmJ from ■ above to Sephadex G-7.
5 (Pharmacia) column (3,2X95tyn)
[Eluate: Q, 0.02 M Tris-HCl buffer (pH 7.4) containing 1 M Na (J),
Flow rate: 60at/hr-, 10ml/tube]. We found CF activity in Tubeya 64-77 and collected them (
partially purified CF).

Example 1 Production of antibody ■ Equal amounts of partially purified CF obtained in Reference Example and Freund's Complete Adjuvant were mixed, and 250μ4 (C
As F toOμ? ) was subcutaneously injected into the back of Ba1b/C mice (female, 8 weeks old).

The same amount of immunization was administered six times in total at two-week intervals. Three days after the final immunization, the pancreas was removed and the tile cells were incubated with RPMI-1640 medium for 3 days.
Washed twice.

Mouse myeloma cell line N5-1 (Eur, J, Im
mu-nol, 6.511-519 (1976)]
After washing in the same manner, the MS-1m cells and the top 1 cell were mixed in a 50WL centrifuge tube at a ratio of 7.5L.

After centrifugation at 200X for 4.5 minutes, the supernatant was removed with a Pasteur pipette. Polyethylene glycol 4 kept at 37℃
000 (Schiff-vaJJ) 50 W/V%cD
Add 5 ml of kLPMI-1640 solution Q dropwise for 1 minute and mix slowly over 7 minutes. RpMI-164o containing 159ftFC8 kept at 37°C
(Hereinafter, this will be referred to as “Complete RPMI J”)lQm/
was added dropwise over 1 minute, and then 35 ml of complete RPMI was added and slowly stirred. After centrifuging at 200x7 for 5 minutes,
Remove the supernatant and add cells to complete RPMI incubated at 17°C so that I x 10' ++ m/m/? M.A.
'l@t, 96-well plate (Falcon) V
100 μl of C was inoculated and cultured in a 37′” C, 5% carbon dioxide incubator. After 24 hours, 1.OX
10-'M Hyboxanthin, 4.0X 10-'
100 μp of complete 1 (PMI medium (hereinafter referred to as AT medium)) containing IVI aminopterin, 1.6 5. On the 8th and 11th day, replace with fresh HAT medium, and on the 14th day, half of the supernatant was similarly added with 1. OX 10-4 M hyboxanthin and 1.
The medium was replaced with complete RPMI medium (hereinafter referred to as FIT medium) containing 6XIQ-'M thymidine. Similarly, No. 18.20
On the 23rd day and 23rd day, half of the supernatant was replaced with HT medium, and on the 25th day, half of the supernatant was replaced with complete RPMI medium. After crucible, growth was maintained in this complete RPMI medium.

The hybridoma thus obtained was cloned by the limiting dilution method. That is, Hypuridoma 31 solids/ml
, Ba1b/c lineage mama thymocytes l×1071r
A complete RPMI medium was prepared so as to be IA/-, and this was plated in a 96-well plate at 200 μe/well and cultured. The proliferating hybridomas were further cloned in the same manner.

To search for a clone producing the desired antibody, use a 96-well plate (Dynatic Laboratory) on which the partially purified CF was immobilized, and use a goat anti-mouse immunoglobulin antibody (11 antibodies) that had been purified with Noku-oxidase. Su1
The test was carried out by the ELISA method using the laboratory).

In this way, a desired hybridoma expressed by clone ARK-1 and producing a monoclonal antibody having the specific reactivity described below was obtained.

■ Clone ASK-1 obtained in the above ■ is completely RP
37 in MI medium in a carbon dioxide incubator.
The cells were cultured at ℃ for 48 hours. Centrifuge the culture solution (3000
rpm, 10 minutes), and the monoclonal antibody of the present invention produced in the supernatant was collected.

In addition, the above antibody was tested by the Ophthalony method, and the results were as follows:
gG, belonged to Zab class.

Example 2 Antibody specificity■ CF activity inhibition test The partially purified CF obtained in Reference Example 1 was treated with the antibody of the present invention (Example 1).
- Culture supernatant obtained in ①) were mixed at a ratio of 1:2 and heated to 37°C.
and incubated for 1 hour. Centrifugal supernatant (10,000
rpm for 10 minutes) and the CF activity was measured.

The results are shown in Figure 1. In the figure, the vertical axis indicates the bile flow rate (chi) when the bile flow rate before sample injection is set to 100 chi.

In addition, l'-CFJ on the horizontal axis shows the results using partial # CF as a sample, and [CF+monoclonal antibody] shows the results using the above centrifuged supernatant as the sample.

It can be seen from FIG. 1 that CF activity was alleviated by treatment with the antibody of the present invention.

■LISA method The reaction specificity of the antibody of the present invention was evaluated using the following 4 samples.
Tested by LISA method.

Sample 1 (guinea pig CF), the partially purified CF obtained in Reference Example 1, was prepared into a 50μ juice packet with a protein concentration using an ultrafiltration device (using a MWl, 000 filter; manufactured by Amicon).

Sample 2 (human CF): Lymphocytes isolated from heparinized peripheral blood of tuberculin test positive subjects (5X 10'
Partially purified CF was prepared in the same manner as in Reference Example 1, and sample 3 (guinea pig lymphocyte culture medium) was prepared in the same manner as in Sample 1 to obtain 5o11r/kl. ): Normal guinea pig tile cells (5x IQ' cells/WLl)
was cultured for 48 hours at 37°C and s%cO, and the culture supernatant was collected. This was subjected to the same treatment as Sephadex G-75 in Reference Example 1, and the same fractions (however, , C
This sample was prepared in the same manner as Sample 1 above to a concentration of 50 pf/ml.

Sample 4 (P1)l) : 50 μt/I
1° PPD made by dKd.

The reactivity of each of the above samples with the antibody of the present invention was determined by EL and ISA methods (tV
leth, Enzymol, 70.419-43
9 (1980)). The results are shown in Table 1 below.

Table 1 - Indirect fluorescent antibody method (a) In Reference Example 1, the PPD-stimulated lymphatic S cells were washed twice with PBS, and then a smeared specimen was prepared. As a control, a smear specimen of lymph node cells that had not been stimulated with PPD was also prepared. Using this specimen, indirect fluorescent antibody method [``Sogo Clinical'', 14.458-4
64 (1965)), the reactivity with the antibody of the present invention was tested. As a result, it was confirmed that the antibody of the present invention reacts with PPD-sensitized lymphocytes [PPD-stimulated specimen: fluorescent staining positive, control specimen: fluorescent staining negative].

Note that in the above, the antibody of the present invention was pretreated with an excessive amount of CF.
No reaction was observed when used after treatment. From this, it is clear that the reaction is mediated by CF.

(b) Using frozen sections of liver tissue collected laparoscopically from patients with various liver diseases and patients with non-liver diseases as samples, the present invention was performed using indirect fluorescent antibody method in the same manner as in HQ (a) above. Antibody reactivity was tested. The results are shown in Table 2.

As shown in Table 2, the antibody of the present invention reacted with 3 out of 6 patients with intrahepatic cholestasis (diffuse fluorescence was confirmed in the liver tissue).

In addition, no fluorescence was observed in cases without intrahepatic cholestasis and cases without liver disease (patients with cholelithiasis). The above fluorescence disappeared by pretreating the antibody of the present invention with an excessive amount of CF.

In addition, in the above test, when anti-CF anti-blood # (polyclonal antibody) 't- was used as the antibody, even for samples without intrahepatic cholestasis, the entire tissue was stained non-specifically, so it was strongly stained. It could not be determined for samples other than positive.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing the CF' activity inhibiting effect of the monoclonal antibody of the present invention. Above is Figure 1

Claims (1)

[Claims] 1. Monoclonal antibody specific for cholestatic factors.