JPH022935A - Angiotensin measuring method - Google Patents

Abstract

PURPOSE:To make it possible to measure angiotensin simply and quickly at high sensitivity and at a low cost by recognizing the angiotensin, using two kinds of monoclonal antibodies having the different recognizing parts, and performing immunity measuring method utilizing a sandwich method. CONSTITUTION:To obtain two kinds of monoclonal antibodies having the different recognizing parts of angiotension, e.g. the following antibodies are prepared: the monoclonal antibody which recognizes a peptide part on the side of amino end (N end) of the angiotensin; and the monoclonal antibody which recognizes a peptide part on the side of carboxy end (C end). To obtain the monoclonal antibody which recognizes the peptide part on the side of the N end, e.g. a peptide chain having an amino acid arrangement which is the same as that of the peptide part on the side of the N end of the angiotensin or the entire length of the angiotensin is used. Then, the C end is bonded to protein and the like of a carrier. An animal is sensitized with the material and antibody formation cells are used for fusion.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to the use of angiotensin (angloLensin).
) is related to the measurement of angiotensin in a sample using a monoclonal antibody against it.

[Prior art] Various factors are involved in blood pressure regulation, but the two that play a major role as humoral factors are the renin-angiotensin system (hereinafter referred to as the R-A system) and the renal system. It is kallikrein-kinin.

The R-A system has many functions such as circulatory regulation and maintenance of body fluid and electrolyte balance. Hypertension in which the R-A system is directly involved in the pressor mechanism includes hypertension caused by renin-producing tumors and renovascular hypertension.

This includes malignant hypertension. On the other hand, the R-A system is suppressed in primary hyperaldosteronism.

There are three types of angiotensin, I, n, and m, and they are peptide hormones with the structure shown in the diagram below. In particular, angiotensin (hereinafter referred to as AII) is the final active substance of the R-A system. Known as the most powerful pressor substance in the body.

Angiotensin? +W-produced angiotensin I (hereinafter referred to as AI) Nll, -
Asp-^rg-Vat-Tyr-I Ic-111s
-Pro-Phe-111s-I, cu-COOII U Nll, -As p-A rg-Va I -Ty
r-I I e-111s-Pro ro-Phe-COO
II Angiotensin ■ N112- Arg-Vat-Tyr-l Ic-11
1s-Pro-Pltc-COOIIIAII is produced by hydrolysis from AI by angiotensin converting enzyme present mainly in the lungs through the reaction shown below.

Changes in the R-A system are seen in various diseases that cause abnormalities in water and electrolyte metabolism, including hypertension, and knowing these changes is not only important for clinical diagnosis, but also for elucidation of pathological conditions and treatment. It is also considered to be helpful from the perspective of prognosis.

In addition, pathological conditions that show an increase in AII include renovascular hypertension, paraspherocytoma, and hyperthyroidism.
Pathological conditions that show a decrease in AII include primary hyperaldosteronism, DOC-producing tumors, Cushing's disease 1iN J! (Yuji Ueno, Wakayama Medical University)

Plasma renin activity is currently widely used as an indicator of R-A system activity, and is usually measured by the amount of AI produced after incubating plasma for a certain period of time. The renin substrate and angiotensin-degrading enzyme influence the production of A, which has the greatest effect on the RA system. Therefore, in order to understand the final stage activity of the R-A system, plasma A
All IIa degrees! It is considered more direct to define

Regarding the measurement of angiotensin concentration in plasma,
Radioimmunoassay (hereinafter referred to as RIA) is used.

However, this RIA also utilizes a competitive method, and a sandwich method using monoclonal antibodies has not yet been established.

[Problems to be solved by the invention] The present invention solves the problems of the conventional methods as described above,
The purpose is to provide a simple dll + standard method. That is, the present invention provides amino acids 7 to 1, which have been considered difficult or impossible.
The purpose of this invention is to establish a sandwich system for angiotensin, which is a peptide with 0 residues, and to solve the above problems.

[Means and effects for solving the problem] The present inventors
As a result of intensive study on the above-mentioned problems, we have arrived at the present invention. That is, the present invention converts angiotensin in a sample into 71P
This is a method for measuring angiotensin, which recognizes angiotensin and is characterized by performing the immuno-III standard method by a sandwich method using two types of monoclonal antibodies with different recognition sites.

The monoclonal antibody of the present invention is preferably obtained by fusing antibody-producing cells of an animal immunized with angiotensin with myeloma cells to obtain a hybridoma that produces a monoclonal antibody that recognizes the angiotensin, and then or by culturing a cell line derived therefrom and collecting a monoclonal antibody that recognizes angiotensin from the culture medium. Hybridomas producing monoclonal antibodies that recognize angiotensin can be obtained by cell fusion method [G, Köhler (Kol+1
cr) and C. Milstein (formerly 1stein).

Nature, 25 (vol. i, 49
5, 1975, 1.

As a method for obtaining two types of monoclonal antibodies with different recognition sites for angiotensin, for example, a monoclonal antibody that recognizes the peptide portion at the amino terminal (hereinafter referred to as N-terminus) side of angiotensin, and a monoclonal antibody that recognizes the peptide portion at the amino terminal (hereinafter referred to as N-terminus) side of angiotensin, and a monoclonal antibody that recognizes the peptide portion at the amino terminal (hereinafter referred to as N-terminus) of angiotensin, and
A monoclonal antibody that recognizes the peptide portion on the terminal) side may be prepared. Specifically, in order to obtain a monoclonal antibody that recognizes the N-terminal peptide portion, for example, a peptide chain having the same amino acid sequence as the N-terminal peptide portion of angiotensin or the entire length of angiotensin is used, and the C-terminus is used as a carrier. Antibody-producing cells may be used for fusion by binding to a protein, etc., sensitizing an animal, and using the antibody-producing cells for fusion.

Although it is possible to obtain a 1'' antibody by binding the N-terminus to a carrier, binding the C-terminus is more effective. Finally, hybridomas that produce a monoclonal antibody that recognizes the N-terminal peptide portion of angiotensin may be screened.

To obtain a monoclonal antibody that recognizes the C-terminal peptide portion, a similar idea is used, using a peptide chain having the same amino acid sequence as the C-terminal peptide portion of angiotensin or the entire length of angiotensin, and attaching the N-terminus to a carrier. It can be bound to proteins and treated in the same way. Although it is possible to obtain the antibody of interest by binding the C-terminus to a carrier, binding the N-terminus is more effective.

Finally, hybridomas that produce a monoclonal antibody that recognizes the C-terminal peptide portion of angiotensin may be screened.

In the present invention, it is desirable to immobilize one of the antibodies on a carrier, but known methods can be used for immobilization, and the carrier may be polystyrene or polyethylene.

Beads and microplate caps made of polyvinyl chloride, latex, agarose, cellulose, glass, etc. are used.

Furthermore, the method and means for labeling the other antibody, the detection method thereof, etc. are not limited in any way, and d1
can be determined. Enzymes such as peroxidase and alkaline phosphatase are commonly used as labeling agents. As a substrate for determining the enzyme activity,
As a substrate for horseradish peroxidase, 2,2°-
Azinodi-(3-ethylbenzthiazolinesulfonic acid)
Niammonium salt-H202,5-aminosalicylic acid-
H202 and the like are used as substrates for alkaline phosphatase, such as p-nitrophenyl phosphate.

In addition, in methods using radioactive substances as labeling agents,
I, H, etc. may be used, and in a method using a fluorescent substance, fluorescein inthiocyanate or the like may be used.

In addition to these reagents, a lysis agent is also required for measurement.

Known suppositories such as detergent 1 reaction terminators are used.

[Effects of the Invention] In the present invention, two types of monoclonal antibodies with different recognition sites are obtained for angiotensin by the above method, and by using these antibodies, an immunoassay method using a sandwich method becomes possible. Ta.

The sandwich method allows angiotensin to be measured more sensitively, more easily, more quickly, and less expensively than conventional methods.

[Examples] The present invention will be explained in more detail with reference to Examples, but the present invention is not limited only to these Examples.

(a) Preparation of a monoclonal antibody that recognizes the C-terminal peptide portion of AII (b) Bovine serum albumin (hereinafter referred to as BSA) SH
Preparation of BSA (1, 3 mg into a vial), Lilo, LM
NaCl-containing f-T O, 1M phosphate buffer (pH 7.5
), 10 μg of 20IIIM N-succinimidyl-3-(2-pyridyldithio)propionate (hereinafter referred to as 5PDP) ethanol solution was added, and the mixture was stirred at room temperature for 30 minutes.
After dialysis against 0.1M acetate buffer (pH 4, 5) containing NaCl, the mixture was transferred to a vial, 0.5M dithiothreitol aqueous solution was added thereto, left at room temperature for 20 minutes, and then 0.1
Contains 0.1M mM EDTA! J>Acid buffer (p
H[i, O) l:, at 4°C, - night i! i+I? was carried out to introduce an SH group into BSA.

(b) Introduction of maleimide group into AII Take 1 rag of A II in a vial and dissolve it in 1 ml of 0.1M phosphate buffer 1tJt (pH 8,0). (hereinafter referred to as MBS) in dimethylformamide solution (25.3 mg/m+) 62
．． Add 5 μII and stir at room temperature for 30 minutes, then 0.3 ml
After adding dichloromethane and centrifuging at 3000 rn for 5 minutes, the supernatant was taken and a maleimide group was introduced into AII.

(c) Preparation of AID-BSA The two solutions prepared in (a) and (b) were heated at 4°C.

- An-BSA conjugate was prepared by coupling overnight.

(d) Sensitization After emulsifying 1008 g of Aff-BSA and an equal amount of Freund's complete adjuvant, the emulsified emulsification was administered into the abdominal cavity of a BALB/c strain mouse.

Four weeks later, An-BSAIO μg was emulsified in the same manner as the first time and administered intraperitoneally. Four weeks after the second administration, 50 μg of An-BSA emulsified in an equal volume of Freund's incomplete adjuvant was administered twice every two weeks, and three days after the fourth administration, An-BSA was administered twice. A 10
0 μg in phosphate buffered saline (hereinafter referred to as PBS)
What is dissolved in the final step! 7. did.

(e) Preparation of hybridoma II! of the mouse immunized above! l! G, Kohler (G, Kohler)
) and C.

Milstein (C, formerly 1sLcin) method (N az
ure, Vol. 256, p. 495, 1975) to obtain a hybridoma that produced a monoclonal antibody that recognized the C-terminal peptide portion of AII.

For screening to obtain hybridomas, two amino plates commercially available from Sumitomo Bakelite were used.
After activation with % glutaraldehyde/PBS for 2 hours at room temperature, 100 μg of An (5 μg/ml) was added.
Dispense 1 Ω/wet, 0.2% gelatin/PB320
After blocking with 0 μg, screening was performed using the hybridoma culture medium after cell fusion to obtain hybridomas producing antibodies against An.

(b) Preparation of a monoclonal antibody that recognizes the N-terminal peptide portion of All (a) Preparation of An-ovalbumin (hereinafter referred to as OVA) The All-OVA conjugate was prepared by the known mixed acid anhydride method.

That is, AII was dissolved in 2 ml of water-free dioxane after removing peroxides, and equimolar amount of triethylamine was added to the solution, cooled by placing it in ice water at 8°C, and then AII and Kasamole of isobutyl chlorocarbonate were added. The mixture was stirred at a low temperature for 10 to 20 minutes to promote the formation of mixed acid anhydride.

OVA was dissolved in 10 ml of distilled water, NaOH was added to bring the pH to 9-9.5, and then 8 ml of dioxane was added with stirring at 8°C. The previously prepared mixed acid anhydride solution was added dropwise little by little to the BSA solution. During this time, stirring was continued and NaOH was added to maintain the pH at 9 or higher.

After adding the mixed acid anhydride solution, continue stirring for 30 minutes.
The reaction solution was left at 4° C. overnight, and then dialyzed against a 50% dioxane solution and further dialyzed against distilled water to remove unreacted AII to produce AID-OVA.

(b) BALB/c mice were immunized using sensitized An-OVA. As a method, an emulsified mixture of Alow OV A too μg and an equal amount of Freund's complete adjuvant was administered intraperitoneally, and 4 weeks later, an emulsified mixture of 50 μg of All-0VA and an equal amount of Freund's incomplete adjuvant was administered. , 1 week later, this time 50 μg of An-OV A
was dissolved in PBS and used for cell fusion 30 days after administration.

(c) Preparation of hybridomas Cells were combined in the same manner as described above to obtain no hybridomas (11).

(d) 5 residues Nl+, -, in which one cysteine residue was bonded to the four amino acid residues on the N-terminal side of screening AII for coupling.
8sp-Arg-Vat-Tyr-Cys-COOII
3+1 was produced by peptide synthesis, coupled with BSA, and used for screening.

For details, refer to BSAG, G mark g in 0.1M phosphate buffer (pH
6,5) and a dioxane solution of MBS (25,3).
After adding 02.5 μg of mg/ml) and stirring at room temperature for 30 minutes,
T SK Gel G-3000S W (inner diameter 7.5x
BSA purified by Length GOOmm) (manufactured by Tosoh Oki)
Fractions were collected. Add the previously synthesized peptide consisting of 5 amino acid residues (10IIIM EDTA-containing #0. [dissolved in Jl/+1 in 4 phosphate buffer]) to this 1, and let it cumulate overnight at 4°C. T.S.
K Gel G-3000S W (inner diameter 7.5 x length (i
ool) and used for screening.

In addition, AII and AS+)-Arg-Vat-Tyr
does not physically form on the polystyrene plate.

(c) Preparation of insolubilized antibodies 5 μg/ml dissolved in 0.1 M sodium carbonate buffer (pH 9,6) was added to each well of an untreated 96-well microtiter plate (Nunqui Immunopre-1, manufactured by Intermed). 250 ItΩ of a mouse anti-angiotensin monoclonal antibody (one that recognizes the N-terminal peptide portion of AII prepared in (b)) was added and incubated overnight at 4°C. Next, remove the solution from each well and add 3 mL of PBS solution containing 0.04% Tween-20.
After washing twice, blocking treatment was performed with 3300 μg of 0.2% gelatin/PB and stored at 4°C.

(d) Preparation of alkaline phosphatase-labeled antibody 4.3 mg of alkaline phosphatase was added to O, 15M phosphate buffer (
(pH 7,5) was dissolved in 1 ml, and to this was added ethanol (f, 100 tt I of a solution in which SPDP was dissolved to a concentration of 4 mg 11), and the mixture was reacted at 4° C. overnight. After that, it was dialyzed against 0.1M phosphate buffer (pH 7.5), and 5p
The reaction product of DP and alkaline phosphatase (hereinafter referred to as PD
P-alkaline phosphatase) was obtained.

Next, Img/ml of angiotensin monoclonal antibody (one that recognizes the C-terminal peptide portion of AU prepared in (a)) dissolved in 0.15M phosphate buffer (pH 7,5) was added to l+nl with dioxane. 50 μg of S-acetylmercaptosuccinic anhydride dissolved in 8.7 mg/ml was added and reacted at 30° C. for 1 hour. Furthermore,
This was dialyzed overnight against O.1M phosphate buffer (pH 7.0) to obtain an S-acetylmercaptosuccinylated anti-angiotensin antibody.

Then, FDP-alkaline phosphatase and S-acetylmercaptosuccinylated anti-angiotensin antibody were
The mixture was mixed at a ratio of 1:1, 1,150 molar volume of 1M hydroxylamine dissolved in 0.1M phosphate buffer was added, and the mixture was left at 4°C overnight. Add this to TSK Gel G
Purified alkaline phosphatase-labeled mouse anti-angiotensin monoclonal antibody was obtained by gel filtration using -3000SW.

(e) Measurement of angiotensin in serum by enzyme-linked immunosorbent assay The microtiter plate on which the antibody prepared by the method described in (e) of this example was immobilized was returned to room temperature, and then added to PBS.
- After washing 3 times with Tween solution, O1 old ~ 20 μg
/ml of normal human serum containing purified angiotensin ■ was added to each well in an amount of 10 μΩ as a standard substance. Each well was then injected with an alkanophosphatase-labeled antibody in PBS containing 1% bovine serum albumin (5D/
ml) to 100. czN was added at 37°C for 2 hours.
After incubation, remove the lysate and then add PBS.
- Washed three times with tween solution. Additionally, 5mM p-
Nitrophenylphosphate was added as a substrate to each well, and after reacting for 30 minutes at room temperature, 1. NNa
The reaction was stopped by adding 100 μg of OH solution.

After stopping the reaction, wavelength 40511111% for each well.
The absorption intensity at a reference wavelength of 130 Or+m was determined using an automatic microtiter plate reader (MPR-A4 manufactured by Tosoh Corporation), and a calibration curve was prepared by plotting the concentration and absorption intensity of the standard substance. Similar ΔIll determination was performed twice and the results are shown in FIG.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the relationship between An concentration and absorbance in Examples. Ct +i'l Applicant Tosoh Corporation

Claims (1)

[Claims] (1) In the method of measuring angiotensin in a sample, angiotensin is recognized and two different recognition sites are used.
A method for measuring angiotensin, which is characterized in that an immunoassay is performed by a sandwich method using different types of monoclonal antibodies.