JPH04267888A - Analysis of endoserine - Google Patents

Abstract

PURPOSE:To provide the subject sandwich analystic technique for endoserine, free from quantitative scattering by using a monoclonal antibody. CONSTITUTION:An endoserine antigen in a sample is bonded to an anti- endoserine monoclonal antibody immobilized into a solid phase and a non-labeled anti-endoserine monoclonal antibody or a labeled anti-endoserine monoclonal antibody marked with a labeling substance is then bonded to the above-bonded endoserine antigen. In the case of using the non-labeled anti-endoserine monoclonal antibody, an anti-immunoglobulin antibody marked with a labeling substance is further bonded to the above-bonded non-labeled anti-endoserine monoclonal antigen. By measuring the quantity of the labeling substance, the quantity of endoserine antigen in the sample is quantitatively analyzed.

Description

[Detailed description of the invention]

0001

TECHNICAL FIELD The present invention relates to a method for quantifying endothelin that can be applied to clinical diagnosis of various human diseases, and a monoclonal antibody that can be used in the method.

0002

[Prior Art] Endothelin is a physiologically active peptide consisting of 21 amino acid residues, is produced from vascular endothelial cells, and is known to have a strong vasoconstrictive effect [Nature, 332, 411- 41
5 (1988)]. Endothelin is quantitatively determined by radioimmunoassay using one type of polyclonal antibody [Biochemical and Biophysical Research Communication (Bioch.
em. Biophys. Res. Commun. )
, 162, 340-346 (1989)] and a sandwich quantitative method using a mouse monoclonal antibody as the first antibody and a rabbit polyclonal antibody as the second antibody [J. Immunol. Methods, 11
8, 245-250 (1989)].

[0003] Anti-endothelin monoclonal antibodies include Takeda Pharmaceutical Co., Ltd. (EP-31100A1);
Yamasa Soy Sauce Co., Ltd. (WO 8911099, abbreviation: M
Antibodies such as CA ET-01, MCA ET-02) are already known. Furthermore, the mouse anti-endothelin monoclonal antibody KM565 was announced by the applicant at the 63rd General Meeting of the Japanese Pharmacological Society (March 25-28, 1990) [Japanese Journal of Pharmacology (Japanese J
our own of Pharmacology),
vol 52, suppl 1, p202 (19
90)]. However, there is no report on the use of these antibodies as the first and second antibodies in a sandwich assay for endothelin.

0004

[Problems to be Solved by the Invention] In the conventional radioimmunoassay quantitative method using one type of polyclonal antibody and the sandwich quantitative method using a polyclonal antibody as the second antibody, quantitative performance is affected due to differences in lots of polyclonal antibodies. There is a problem with variations. An object of the present invention is to provide a sandwich quantitative method for endothelin using a monoclonal antibody with consistent quantitative performance.

[0005]

[Means for Solving the Problems] According to the present invention, an endothelin antigen in a specimen is bound to a solid-phase anti-endothelin monoclonal antibody (first antibody), and then an anti-endothelin antigen that is unlabeled or labeled with a labeling substance is produced. If a monoclonal antibody (second antibody) is combined with the bound endothelin antigen and an unlabeled anti-endothelin monoclonal antibody is used, an anti-immunoglobulin antibody (third antibody) labeled with a labeling substance is further added to the bound endothelin antigen. A method can be provided for quantifying the amount of endothelin antigen in a specimen by binding it to a labeled anti-endothelin monoclonal antibody and measuring the amount of bound labeled substance.

In the quantitative method of the present invention, either an unlabeled or labeled anti-endothelin monoclonal antibody can be used as the second antibody, but if endothelin is to be quantified with higher sensitivity, an unlabeled anti-endothelin antibody can be used. It is preferred to use endothelin monoclonal antibodies. The present invention will be explained in detail below.

First, a hybridoma is produced by fusing a mouse myeloma cell line with the splenocytes of a mouse immunized with a complex of endothelin and a carrier protein or a complex of a partial peptide of endothelin and a carrier protein. Hybridomas that produce monoclonal antibodies that react with the carrier protein and do not react with the carrier protein are selected. The hybridoma is cultured in a medium or administered to a mouse to cause ascites cancer in the mouse, and several mouse monoclonal antibodies are collected from the culture fluid or ascites. Then,
Hybridomas are produced by fusing rat splenocytes immunized with a complex of endothelin and a carrier protein or a complex of a partial peptide of endothelin and a carrier protein with a mouse myeloma cell line, and the mouse obtained previously A hybridoma that produces a monoclonal antibody that can bind the monoclonal antibody and endothelin by sandwiching them together is selected. The hybridoma is cultured in a medium or administered to a mouse to induce ascites cancer, and some rat monoclonal antibodies are collected from the culture fluid or ascites. Several mouse monoclonal antibodies and several rat monoclonal antibodies thus obtained are used as first and second antibodies, respectively, and a sandwich quantitative method is examined to select a combination of the first and second antibodies.

[0008] The method for producing the monoclonal antibody used in the present invention will be explained below. (1) Immunization of animals and preparation of antibody-producing cells: immunize mice or rats aged 3 to 20 weeks with an antigen (a complex of endothelin and a carrier protein or a complex of a partial peptide of endothelin and a carrier protein). , prepare antibody-producing cells in the spleen, lymph nodes, and peripheral blood of the animal. The method of immunization is to inject the animal subcutaneously, intravenously, or intraperitoneally with an appropriate adjuvant [for example, Complete Freund's Adjuvant (Complete Freund's Adjuvant)].
or aluminum hydroxide gel and Bordetella pertussis vaccine]. These administrations are performed 5 to 10 times every 1 to 2 weeks after the first administration. Blood is collected from the fundus venous plexus 3 to 7 days after each administration, and the reaction of the serum with the antigen is examined by enzyme immunoassay [ELISA method: Igaku Shoin, 1976]. A mouse or rat whose serum shows a sufficient antibody titer against the antigen used for immunization is provided as a source of splenocytes. For fusion of spleen cells and myeloma cells, spleen cells are extracted from immunized mice or rats 3 to 7 days after the final administration of the antigenic substance, and spleen cells are prepared. The spleen was shredded in MEM medium (Nissui Pharmaceutical Co., Ltd.), loosened with tweezers, and centrifuged (1200 rpm).
, 5 minutes), discard the supernatant, treat with Tris-ammonium chloride buffer (pH 7.65) for 1 to 2 minutes to remove red blood cells, wash three times with MEM medium, and provide as splenocytes for fusion. .

(2) Preparation of myeloma cells As myeloma cells, established cell lines obtained from mice are used. For example, 8-azaguanine resistant mice (derived from Balb/c)
Myeloma cell line P3-X63Ag8-U1 (P3-U1)
[Current Topics in Microbiology]
And Immunology 1 (Current Topi
cs in Microbiology and Im
munology-1), European Journal of Immunology (European J.I.
mmunology) 6, 511-519 (19
76) ], SP2 /0 -Ag14 (SP-2) [
Nature 276, 269-
270 (1978)], P3-X63-Ag865
3 (653) [J. Immunology 123, 1548
-1550 (1979)], P3-X63 -Ag8 (
X63) [Nature 256,
495-497 (1975)] etc. are used. These cell lines were grown in 8-azaguanine medium [RPMI-1
640 medium containing glutamine (1.5mM), 2-mercaptoethanol (5x10-5M), gentamicin (10 μg/ml) and fetal calf serum (FCS).
(manufactured by CSL, 10%) (hereinafter referred to as normal medium) was further supplemented with 8-azaguanine (15 μg/
3 to 4 days after cell fusion.
Passage the cells into a normal medium one day in advance to ensure a cell count of 2 x 107 cells or more on the day of fusion.

(3) Cell fusion: Antibody-producing cells immunized in (1) and myeloma cells obtained in (2) were mixed in MEM medium or PBS (1.83 g of disodium phosphate, 0.21 g of monopotassium phosphate, Salt 7
．． 65 g, 1 liter of distilled water, pH 7.2), and the number of cells was determined to be: antibody-producing cells: myeloma cells = 5-10:
1, and centrifuged (1,200 rpm, 5
After discarding the supernatant and thoroughly loosening the precipitated cell group, 2 g of polyethylene glycol-1,000 (PEG-1,000), M
Add 0.2 to 1 ml of a mixture of 2 ml of EM and 0.7 ml of dimethyl sulfoxide/103 antibody-producing cells, and add 1 to 2
After adding 1-2 ml of MEM medium several times every minute, MEM
Add medium to make a total volume of 50 ml. After centrifugation (900 rpm, 5 minutes), discard the supernatant, gently loosen the cells, and gently transfer the cells to HAT medium [normal medium with hypoxanthine (10-4M), thymidine ( 1.5×10-5M
) and aminopterin (4 x 10-7M)]. Dispense 100 μl/well of this suspension into a 96-well culture plate, and add 5% CO2
Culture in an incubator at 37°C for 7-14 days. After culturing, a portion of the culture supernatant is taken and the antibody titer against the antigen is measured by enzyme immunoassay or the like, and those that specifically react with endothelin and do not react with carrier protein alone are selected. Next, cloning was repeated twice by the limiting dilution method [the first time was using HT medium (HAT medium with aminopterin removed), and the second time was using normal medium], and a monoclonal antibody that strongly reacts with endothelin was used. A hybridoma strain that stably produces an anti-endothelin monoclonal antibody is selected as an anti-endothelin monoclonal antibody-producing hybridoma strain.

(4) Preparation of monoclonal antibodies Mice aged 8 to 10 weeks were treated with pristane [0.5 ml of 2,6,10,14-tetramethylpentadecane (Pristane) was administered intraperitoneally and kept for 2 weeks]. 2 x 106 to 5 x 107 anti-endothelin monoclonal antibody-producing hybridoma cells obtained in (3)
Cells/mouse are injected intraperitoneally. The hybridoma turns into ascites cancer in 10 to 21 days. Ascitic fluid was collected from this mouse, centrifuged (3,000 rpm, 5 minutes) to remove solids, salted out with 40-50% ammonium sulfate, dialyzed against a solution of PBS and sodium chloride, and DEAE- The column is passed through a Sepharose column, a Protein A column, or a Cellulofine GSL2000 (Seikagaku Corporation) column, and the IgG or IgM fraction is collected and used as a purified monoclonal antibody. The antibody subclass is determined using a mouse monoclonal antibody typing kit (manufactured by Zymet) or a rat monoclonal antibody typing antibody (manufactured by Nordic Immunology). The amount of protein is quantified using the Lowry method and absorbance at 280 nm.

(5) Selection of a monoclonal antibody that can be used in the sandwich quantitative method (4) Add 1 to 50 μg/ml of the purified mouse anti-endothelin monoclonal antibody or the purified rat anti-endothelin monoclonal antibody to a 96-well plate at 10 to 100 μl/well. The first antibody is then coated on a plate after being left overnight at 4°C. Next, blocking is performed with a bovine serum albumin (BSA) solution or the like. 50 to 100 μl of an endothelin solution with a concentration of 10 pg to 100 ng/ml is added thereto, and the mixture is allowed to react at room temperature for 2 hours or at 4° C. overnight. PBS or 0.05% Tween
After thorough washing with a PBS solution containing PBS-20 (PBS-Tween), culture supernatant of a hybridoma producing a mouse anti-endothelin monoclonal antibody or a hybridoma producing a rat anti-endothelin monoclonal antibody obtained by the method described in (3). culture supernatant (
If the first antibody is a mouse antibody, it is of rat origin; if the first antibody is a rat antibody, it is culture supernatant of a mouse-derived hybridoma) as the second antibody. Dispense 50 to 100 μl/well and leave it at room temperature for 2 hours. Alternatively, react overnight at 4°C. If the second antibody is unlabeled, add PBS or PBS-T.
After washing thoroughly with 30% immunoglobulin, use enzyme-labeled anti-mouse immunoglobulin antibody as the third antibody if the second antibody is mouse-derived, or enzyme-labeled anti-rat immunoglobulin antibody if the second antibody is rat-derived at room temperature. Allow time to react. After thorough washing with PBS or PBS-Tween, color is developed using an appropriate substrate solution and the absorbance is measured. Based on this result, a combination of a mouse anti-endothelin monoclonal antibody and a rat anti-endothelin monoclonal antibody that can detect endothelin with high sensitivity is selected.

[0013] Specific examples of endothelin monoclonal antibodies selected by the method described above include mouse anti-endothelin monoclonal antibody KM565 produced by hybridoma cell line KM565 and rat anti-endothelin monoclonal antibody KM714 produced by hybridoma cell line KM714. I can give it to you. Hybridoma cell line KM565 and hybridoma cell line KM714 were each submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology under the Budapest Treaty on February 5, 1991, under FAIKEN Article No. 3264 (FERM BP-3264).
and No. 3265 (FERM BP-3265)
It has been deposited as.

[0014] Labeling substances for antibodies include enzymes, biotin, chemiluminescent substances, and radioluminescent substances. For example, the label may be an enzyme such as peroxidase,
Periodate crosslinking method using urease, alkaline phosphatase, β-galactosidase, etc.
of Histochemistry and Cytochemistry (J. Histochem. Cytochem.
) 22, 1084-1091 (1971)],
Method using biotin [Journal of Histochemistry and Cytochemistry (J. His
tochem. Cytochem. ) 27, 1
131-1139 (1979)].

For example, the biotin labeling method is as follows. 1ml purified monoclonal antibody 1mg/m
l to 0.1M NaHCO3 -0.5M NaCl (
After dialysis in 1 liter of pH 8.0 at 4°C overnight, N
-100 μl of N-hydroxysuccinimide biotin (manufactured by Sigma) dissolved in N'-dimethylformamide
Add 1 mg/ml and react at room temperature for 4 hours. Then, the reaction solution is dialyzed overnight at 4°C in 1 liter of PBS-0.5M NaCl. At this time, PBS-0.5M
Replace 1 liter of NaCl with fresh water 3 times. The amount of protein in the reaction solution after dialysis is measured and used as a biotin-labeled monoclonal antibody.

Next, a method for quantifying endothelin using the selected first and second antibodies will be explained. Purified mouse anti-endothelin monoclonal antibody 1-50μg/
ml or purified rat anti-endothelin monoclonal antibody 1 to 50 μg/ml is dispensed into 10 to 100 μl/well, left overnight at 4° C., and coated on a plate as the first antibody. Then, blocking is performed with a BSA solution or the like. There, add 50 to 100 μl of the specimen solution containing endothelin.
and react at room temperature for 2 hours or at 4°C overnight. After washing thoroughly with PBS or PBS-Tween, add 1 to 50 μg/ml of unlabeled or labeled mouse anti-endothelin monoclonal antibody or rat anti-endothelin monoclonal antibody (if the first antibody is a mouse antibody, use rat-derived, When the first antibody is a rat antibody, a mouse-derived anti-endothelin monoclonal antibody (mouse-derived anti-endothelin monoclonal antibody) is dispensed as a second antibody at 50 to 100 μl/well, and allowed to react at room temperature for 2 hours or at 4° C. overnight. When using an unlabeled antibody as the second antibody, additional P
After washing well with BS or PBS-Tween, use an anti-mouse immunoglobulin antibody labeled with a labeling substance as the third antibody when the second antibody is mouse, or an anti-mouse immunoglobulin antibody labeled in the same way when the second antibody is rat. Incubate rat immunoglobulin antibody at room temperature for 2 hours and add PBS or PBS-T.
Wash thoroughly with ween.

As described above, when the labeling substance bound to the hole is an enzyme, a chemiluminescent substance, a radioactive compound, etc., its activity is measured. When the labeling substance is biotin, avidin-
The enzyme complex or avidin-biotin-enzyme complex is further reacted, and the enzyme activity is measured. By quantifying an appropriately diluted standard solution of endothelin at the same time as the sample, a standard straight line is drawn, and the amount of endothelin in the sample is calculated based on it.

Examples, comparative examples, and reference examples of the present invention are shown below.

[0019]

[Examples] Example 1 (1) Animal immunization and preparation of antibody-producing cells Rat immunization Endothelin C-terminal 8-residue peptide (hereinafter abbreviated as RET8) was injected into hemocyanin keyhole limpet (Hem).
ocyanin Keyhole Linpet
; Hereinafter abbreviated as KLH) [Calbiochem
chem)] as a carrier protein and m-maleimido-benzoyl-N-hydroxysuccil (m-ma
lenimido-benzoyl-N-hydro
xy succil (hereinafter abbreviated as MBS) (manufactured by Nacalai Tesque), and mixed with KLH-MBS purified using a Sephadex G25 column at a ratio of 1:5, and then mixed with KLH-MBS purified by a Sephadex G25 column using a known method [Cell, 28, 477- 487
, (1982)] and dialyzed against PBS to use as an antigen. 50 μg RET8-KLH in rats
The complex (hereinafter abbreviated as RET8-KLH) was administered together with 2 mg of aluminum gel and 1 x 109 cells/rat of pertussis vaccine (manufactured by Chiba Prefecture Serum Research Institute), and after 2 weeks, 50 μg of RET8-KLH was administered once a week. Total 6
Administered twice. Blood was collected from the fundus venous plexus, and the antibody titer of the serum was examined by the enzyme immunoassay method described below, and the spleen was removed from rats that showed a sufficient antibody titer 3 days after the final immunization. The spleen was cut into pieces in MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.), loosened with tweezers, and centrifuged (1200 rpm, 5 minutes).
(pH 7.65) for 1 to 2 minutes to remove red blood cells.
The cells were washed three times with MEM medium and used for cell fusion.

Enzyme immunoassay method 96-well EIA plate [Greine
r) Add 1 to 50 μg/ml of antigen to 10 to 10
0 μl/well was dispensed and left overnight at 4° C. to coat the plate with the antigen. Next, 100 to 200 μl/well of PBS solution containing 1% BSA (BSA-PBS) was dispensed and left to stand at room temperature for 1 to 2 hours or at 4°C for 1 to 2 nights to separate the remaining proteins on the plate. The binding residues were blocked. After that, discard the BSA-PBS and
After washing well with S, use BSA-PBS as the first antibody.
Samples (mouse serum, rat serum, hybridoma culture supernatant, purified monoclonal antibody) diluted with
0 μl/well was dispensed and left at room temperature for 2 to 3 hours or at 4° C. overnight. PBS or PBS-Tween,
After washing thoroughly, add 50 to 100 μl of peroxidase-labeled anti-mouse immunoglobulin antibody [manufactured by DAKO Co., Ltd.] or peroxidase-labeled anti-rat immunoglobulin antibody [manufactured by DAKO Co., Ltd.] as a second antibody.
/ well and left at room temperature for 2 hours. After washing well with PBS, ABTS substrate solution [2,2'-azinobis(3-
550 mg of diammonium ethylbenzothiazoline-6-sulfonic acid was added to 0.1 M citrate buffer (pH 4.2).
) 1 liter of solution, to which 1 μl/ml of hydrogen peroxide was added immediately before use] was used to develop color, and the OD41
Absorbance at 5 nm was measured.

(2) Preparation of mouse myeloma cells 8- Culture the azaguanine-resistant mouse myeloma cell line P3-U1 in a normal medium, secure 2 x 10 7 or more cells at the time of cell fusion, and use them as a parent strain for cell fusion. did.

(3) Selection of monoclonal antibodies that can be used in the sandwich quantitative method Rat splenocytes obtained in Example 1 (1) and Example 1 (
2) Mix with the myeloma cells obtained in step 10:1, centrifuge (1,200 rpm, 5 minutes), discard the supernatant, loosen the precipitated cells, and stir. At 37℃, polyethylene glycol-1,
000 (PEG-1,000) 2g, MEM medium 2m
A mixture of 0.2 ml and 0.7 ml of dimethyl sulfoxide
Add ~1 ml/103 rat splenocytes and add 1-2 ml of MEM medium several times every 1-2 minutes, then add MEM medium to bring the total volume to 50 ml. centrifugation
(900 rpm, 5 minutes) After discarding the supernatant and gently loosening the cells, the cells were gently suspended in 100 ml of HAT medium by suction and blowing with a measuring pipette. Transfer this suspension to a 96-well culture plate at 100 μl/
Dispense into wells and place in a 5% CO2 incubator for 37 hours.
The cells were cultured at 5% CO2 for 10-14 days. This culture supernatant is examined by the assay method shown below, and the wells with positive activity are selected and further changed to HT medium and normal medium, and cloning is repeated twice to produce rat monoclonal antibody KM714 that can be used in the sandwich quantitative method. Hybridoma strain KM714 was established. Using a rat monoclonal antibody typing kit (manufactured by Zymet),
Rat monoclonal antibody KM7 by enzyme immunoassay
Class 14 was determined to be IgM.

[0023] 10 μg of antibody KM565 obtained in assay method reference example (4)
/ml was dispensed into a 96-well plate (manufactured by Greiner) at 50 μl/well and left at 4° C. overnight to coat the plate as the first antibody. 4 on PBS
The cells were washed twice and then blocked with 1% BSA-PBS. 50 μl of an endothelin solution diluted with BSA-PBS to a concentration of 10 ng/ml was added thereto, and the mixture was reacted overnight at 4°C. After washing 6 times with PBS-Tween, the culture supernatant obtained above was used as a second antibody at 50 μl/
The mixture was dispensed into each hole and allowed to react at room temperature for 2 hours. Then PBS
- After washing six times with Tween, the peroxidase-labeled anti-rat immunoglobulin antibody was reacted at room temperature for 1 to 2 hours. After washing 6 times with PBS-Tween, A
Color was developed using a BTS substrate solution, and absorbance at OD415 nm was measured.

(4) Purification of monoclonal antibody KM714 in 8-week-old nude female mice treated with pristane (3)
The hybridoma strain KM714 obtained in
106 cells/mouse were each injected intraperitoneally. 10-21
Days later, the hybridoma turned into ascites cancer. Ascitic fluid was collected (1 to 8 ml/mouse) from mice with accumulated ascitic fluid, and the solid content was removed by centrifugation (3,000 rpm, 5 minutes). After salting out with 50% ammonium sulfate and dialysis with PBS added with 0.5M sodium chloride, Cellulofine G
SL2000 (manufactured by Seikagaku Corporation) (bet volume 7
50 ml) column at a flow rate of 15 ml/hour.
Fractions were collected and used as purified monoclonal antibodies.

(5) High-sensitivity quantification of endothelin by sandwich quantification method Quantification of endothelin using mouse anti-endothelin monoclonal antibody KM565 obtained in Reference Example and rat anti-endothelin monoclonal antibody KM714 obtained in Example 1 (4) was carried out as follows.

Mouse anti-endothelin monoclonal antibody KM565 10 μg/ml was added to 50 μl/well 96
It was dispensed onto a well plate, left overnight at 4°C, and coated on the plate as the first antibody. Washed 4 times with PBS, then blocked with 1% BSA-PBS. BS there
50 μl of a 0-50 ng/ml endothelin solution diluted with A-PBS was added and reacted overnight at 4°C. PBS
- After washing 6 times with Tween, unlabeled rat anti-endothelin monoclonal antibody KM714 was added as a second antibody.
Or biotinylated labeled rat anti-endothelin monoclonal antibody KM714 10 prepared by the method shown below.
50 μl/well of μg/ml was dispensed and allowed to react at room temperature for 2 hours. Then, unlabeled antibody KM71 was used as a second antibody.
4, the plate was washed six times with PBS-Tween and then reacted with a biotinylated anti-rat immunoglobulin antibody (manufactured by CALTAG) at room temperature for 1 hour. After washing six times with PBS-Tween, avidin-peroxidase (manufactured by Vector) was reacted at room temperature for 1 hour. After washing 6 times with PBS-Tween, color was developed using ABTS substrate solution and OD41
Absorbance at 5 nm was measured. In addition, when biotinylated labeled antibody KM714 is used as the second antibody, PBS-T
After washing six times with ween, avidin-peroxidase (manufactured by Vector) was reacted at room temperature for 1 hour. After washing 6 times with PBS-Tween,
Color was developed using an ABTS substrate solution, and absorbance at OD415 nm was measured. As a result, endothelin is highly sensitive (
>13 pg/ml). The results are shown in FIGS. 1 and 2.

Comparative Example 1 Comparison with existing monoclonal antibodies As existing monoclonal antibodies, monoclonal antibody MCA ET-01 (manufactured by Yamasa Soy Sauce Co., Ltd.) that recognizes the N-terminus of endothelin and monoclonal antibody MCA ET-02 that recognizes the C-terminus of endothelin (Manufactured by Yamasa Soy Sauce Company)
Endothelin was quantified using The method was carried out in accordance with the method of Example 1 (5) described above. That is, a plate was coated with KM565 or MCA ET-01 as a first antibody, and after blocking, a diluted endothelin solution was reacted. Next, biotin-labeled KM714 or biotin-labeled MCA ET-02 was used as a second antibody.
reacted. Finally, avidin-peroxidase was reacted and color was developed using a substrate solution. The results are shown in Table 1.

[0028] The combination of KM565 and KM714 had the best quantitative performance, followed by MCA ET-01 and KM71.
4 combination, MCA ET-01 and MCA E
The order was the combination of T-02. KM565 and MC
With the combination of AET-02, endothelin could not be quantified. The above suggests that KM565 recognizes the same endothelin N-terminus as MCA E.
MCA ET- has superior reactivity than T-01, and KM714 seems to recognize the same endothelin C-terminus.
This shows that the reactivity is superior to that of 02.

[0029]

[Table 1]

Reference Example Purification of Mouse Anti-Endothelin Monoclonal Antibody KM565 (1) Animal Immunization and Preparation of Antibody-Producing Cells Mouse Immunity Endothelin [Peptide Institute Inc.
)] and the carrier protein thyroglobulin [manufactured by SIGMA, hereinafter referred to as Thy] were mixed at a ratio of 1:5, and 0.02M glutaraldehyde was used in the presence of 0.1M ammonium acetate. Methods known in the art (Proceedings of the Society
of Experimental Biological Medicine (Proc. Soc. Exp. Biol
．． Med. ), 148, 784-789 (1
975)] to prepare a complex, which was dialyzed against PBS and used as an antigen. In addition, KLH [Calbichem
ochem) manufactured by Co., Ltd.] as the carrier protein,
KLH- which was purified with a Sephadex G25 column after reacting endothelin with MBS (manufactured by Nacalai Tesque)
Mixed with MBS at a ratio of 1:5 and mixed with MBS in a known manner [Cell
), 28, 477-487 (1982)] and dialyzed against PBS to obtain an antigen. Balb/
c Mice were injected with 50 μg of endothelin-thyroglobulin complex (hereinafter abbreviated as ET-Thy) or endothelin-KLH complex (hereinafter abbreviated as ET-KLH) with 2 mg of aluminum gel and pertussis vaccine (manufactured by Chiba Serum Institute) 1 ×109 cells/mouse and 2 weeks later, 50 μg of ET-Thy or ET-KL
H was administered once a week for a total of 6 times. Blood was collected from the fundus venous plexus, and its serum antibody titer was examined by the enzyme immunoassay method shown in Example 1. The spleen was removed from mice that showed a sufficient antibody titer 3 days after the final immunization. The spleen was cut into MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.), loosened with tweezers, and centrifuged (120
0 rpm, 5 minutes), discard the supernatant and add Tris-
Red blood cells were removed by treatment with ammonium chloride buffer (pH 7.65) for 1 to 2 minutes, washed three times with MEM medium, and used for cell fusion.

(2) Preparation of mouse myeloma cells 8-Azaguanine-resistant mouse myeloma cell line P3-U1 was cultured in a normal medium, and at the time of cell fusion, 2 x 107 or more cells were secured and used as a parent strain for cell fusion. did.

(3) Reference example of hybridoma production (1
) The mouse splenocytes obtained in Reference Example (2) and myeloma cells obtained in Reference Example (2) were thoroughly washed with MEM medium or PBS,
After mixing the cells so that the number of cells was mouse splenocytes: myeloma cells = 10:1 and centrifuging (1,200 rpm, 5 minutes), the supernatant was discarded, and the precipitated cells were thoroughly loosened.
Polyethylene glycol-
1,000 (PEG-1,000) 2g, MEM medium 2ml and dimethyl sulfoxide 0.7ml mixture 0
．． Add 2-1 ml/103 mouse splenocytes, add 1-2 ml of MEM medium several times every 1-2 minutes, and then add MEM medium to make the total volume 50 ml. After centrifugation (900 rpm, 5 minutes), the supernatant was discarded, the cells were gently loosened, and then gently suspended in 100 ml of HAT medium by suction and blowing with a measuring pipette. Transfer 100 μl of this suspension to a 96-well culture plate.
Dispense into each hole and incubate in a 5% CO2 incubator for 3
The cells were cultured at 7°C for 10-14 days under 5% CO2. Measure the antibody titer against endothelin in the culture supernatant obtained, select wells with positive activity, change to HT medium and normal medium, repeat cloning twice, and use the enzyme immunoassay method described above to detect endothelin. Hybridomas that specifically reacted with were selected. Finally, endothelin, ET
-Mouse anti-endothelin monoclonal antibody K that reacts with KLH, ET-Thy but does not react with KLH, Thy
Hybridoma strain KM565 producing M565 was established. The subclass of mouse monoclonal antibody KM565 was determined to be IgG1 by enzyme immunoassay using a mouse monoclonal antibody typing kit (manufactured by ZYMED).

(4) Purification of mouse anti-endothelin monoclonal antibody KM565 Reference example (3) was applied to 8-week-old nude female mice treated with pristane.
) Hybridoma strain KM565 obtained in 5 to 10
×106 cells/mouse were injected intraperitoneally. 10-2
One day later, the hybridoma turned into ascites cancer. Ascitic fluid was collected from mice with ascites (1 to 8 ml/mouse), centrifuged (3,000 rpm, 5 minutes) to remove solids, and salted out twice with 50% ammonium sulfate and 40% ammonium sulfate. 0.04M phosphate buffer, 0.03M
Dialysis was performed with NaCl (pH 8.0). After that, D.E.
The column was passed through AE Sepharose (Whatman, DE52), eluted, and the IgG fraction was collected.
It was used as a purified monoclonal antibody.

[0034]

According to the present invention, the amount of endothelin in samples such as serum and urine can be measured with high sensitivity, and can be applied to clinical diagnosis of various human diseases.

[Brief explanation of the drawing]

[Figure 1] Mouse anti-endothelin monoclonal antibody KM565 obtained in Reference Example and rat anti-endothelin monoclonal antibody KM714 obtained in Example 1 (unlabeled)
The standard straight line measured by the sandwich quantitative method in combination with

[Figure 2] Mouse anti-endothelin monoclonal antibody KM565 obtained in Reference Example and biotinylated labeled rat anti-endothelin monoclonal antibody KM7 obtained in Example 1.
The standard straight line measured by the sandwich quantitative method in combination with No. 14 is shown.

Claims (7)

[Claims] Claim 1: An endothelin antigen in a specimen is bound to an immobilized anti-endothelin monoclonal antibody, and then an unlabeled or labeled anti-endothelin monoclonal antibody is bound to the bound endothelin antigen. When an anti-endothelin monoclonal antibody is used, an anti-immunoglobulin antibody labeled with a labeling substance is further bound to the bound unlabeled anti-endothelin monoclonal antibody, and the amount of the bound labeling substance is measured. quantitative method. 2. The method for quantifying endothelin according to claim 1, wherein the immobilized anti-endothelin monoclonal antibody is mouse anti-endothelin monoclonal antibody KM565. 3. The method for quantifying endothelin according to claim 1, wherein the unlabeled or labeled anti-endothelin monoclonal antibody is rat anti-endothelin monoclonal antibody KM714. 4. An endothelin antigen in a specimen is bound to a immobilized mouse anti-endothelin monoclonal antibody, and then an unlabeled rat anti-endothelin monoclonal antibody KM714 is bound to the bound endothelin antigen, and further labeled with a labeling substance. A method for quantifying endothelin, which comprises binding a rat anti-immunoglobulin antibody to the bound rat anti-endothelin monoclonal antibody KM714, and measuring the amount of bound labeling substance. 5. The mouse anti-endothelin monoclonal antibody is mouse anti-endothelin monoclonal antibody KM.
565. The method for quantifying endothelin according to claim 1. 6. Rat anti-endothelin monoclonal antibody KM714. Claim 7: Hybridoma cell line KM714 (F
ERM BP-3265).