JPH0753105B2 - A hybridoma producing a monoclonal antibody that recognizes α-hANP - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a ring structure N of α-human atrial natriuretic polypeptide (α-hANP).
More specifically, it relates to a hybridoma producing a monoclonal antibody that recognizes about half of the end, and more specifically, α-hANP
The present invention relates to a hybridoma producing a monoclonal antibody that recognizes a portion contained in the [7-16] fragment and also containing Met [12].

[0002]

2. Description of the Related Art Atrial natriuretic polypeptide (at
rial natriuretic polypeptide (ANP) is a polypeptide produced by atrial myocytes and contained in granules, which has a strong diuretic action and a natriuretic action. Such polypeptides have been found not only in humans but also in rats and are called hANP and rANP, respectively. hANP and rANP are further classified into three types, α, β and γ. α-hANP consists of 28 amino acid residues, and Cys [7] at the 7th position from the N-terminus and Cys [23] at the 23rd position are disulfide-bonded, and the sequence between them forms a ring structure (Biochem. Biophys. Res.
Commun. (Abbreviated as BBRC below) 118 , 131-139, 198
Four). In α-rANP, the 12th residue from the N-terminal is Met in α-hANP, whereas I-in A-rANP is
Only differ in that it is le (BBRC 117 , 839-865,
1983). β-hANP is an antiparallel dimer of α-hANP (JP-A-60-184098). γ-hANP consists of 126 amino acid residues, and 99 to 126 amino acids at the C-terminal thereof correspond to α-hANP.

As an assay method for ANP, a radioimmunoassay using an antiserum has already been established (Science 22
8 , 323-325, 1985; Nature 314 , 264-266, 1985; BBRC
124 , 815-821, 1984; BBRC 124 , 663-668, 1984; BBRC 1
25 , 315-323, 1984). Of these, antiserum CR-3 is AN
It is known to recognize the C-terminal fragment of P [17-28].

[0004] Further, monochrome-recognizing α-hANP
11A-A11 is known as a null antibody. The antibody was obtained by using atriopeptin II, which is a kind of ANP of rat, as an antigen, and the epitope exists between Cys [7] -Ser [25] containing disulfide bond,
That is, it is considered to be a part of the ring structure of ANP. However, the affinity of the antibody is Met [12, human] and Ile.
Recognizes rANP and hANP without any difference between [12, rat] (Life Science, Vol. 38, 1991-1997, 1986).

[0005]

In the conventional immunoassay for ANP, it is necessary to extract ANP from a sample, and a monoclonal antibody with high affinity which enables highly sensitive assay without extraction is desired. It had been. A
As a monoclonal antibody that recognizes NP, the above 11
Although A-A11 was obtained, the antibody recognizes not only hANP but also rANP. Also, as mentioned above, AN
Since P antiserum CR-3 recognizes the C-terminal side of ANP,
If an antibody that specifically recognizes the N-terminal fragment of ANP is obtained, a sandwich immunoassay using such two kinds of antibodies becomes possible. Therefore, a monoclonal antibody with high affinity that specifically recognizes the N-terminal fragment of hANP has been desired.

[0006] As immunoassays for hANP, conventionally known are radio immunoassays (hereinafter abbreviated as RIA) utilizing radioisotopes and enzyme immunoassays (EIA) utilizing enzymes. ,
In the case of RIA, there is a tendency to avoid using it due to problems with facilities and equipment, and highly sensitive E regardless of where it is used.
Rather, the development of IA is desired. As an enzyme labeling method in EIA, a method of using glutaraldehyde as a cross-linking agent has hitherto been generally used, but recently, there have been problems in terms of polymerization and low yield, and it is not used so much. . Recently, N, N'-O-phenylenedimaleimide and N- have been used as cross-linking agents to replace glutaraldehyde.
N-hydroxysuccinimide ester of (4-carboxycyclohexylmethyl) maleimide and the like are known [J. Immunoassay 4 , 209-327 (198).
3)] has not been reported to be applied to hANP, and it cannot be said that a highly sensitive immunoassay for hANP has been developed yet.

[0007]

[Means for Solving the Problems] The present inventors have a high-affinity monochrome-type which specifically recognizes the N-terminal fragment of hANP.
As a result of earnest research to create a null antibody, α-h
Monochrome that recognizes the N-terminal fragment of the ring structure of ANP-
A hybridoma producing a null antibody was obtained, and a highly sensitive assay method for α-hANP utilizing the monoclonal antibody was completed.

(1) Preparation of Monoclonal Antibody-Producing Hybridoma α-hANP is a polypeptide consisting of 28 amino acid residues and, since it is a relatively small molecule, its ability to induce antibody production (immunogenicity) Is low. Therefore, it is combined with bovine serum albumin, bovine thyroglobulin, etc. for use as an antigen. The obtained complex is emulsified in an appropriate adjuvant such as Freund's complete adjuvant and used for immunization of mice.

Immunization is carried out by repeatedly inoculating the above emulsion to the abdominal cavity, subcutaneously or vein of a mouse several times every several weeks. Three to five days after the final immunization, the spleen is removed and used as antibody-producing cells. As the parent cells for this time simultaneously fused with antibody-producing cells obtained hybridomas, hypoxanthine - with appropriate markers such as guanine - phosphoribosyl trans Hue hydrolase deficiency (HGPRT - ^-) or thymidine kinase deficiency ^(TK) A myeloma cell line is prepared and fused with antibody-producing cells to prepare a hybridoma.

As a medium for producing hybridomas,
Eagle MEM, Dalbetzco modified medium, RPMI-16
40, such as 40, which is commonly used, is approximately 15
% Fetal calf serum (FCS) is used.

First, parental cells, myeloma and splenocytes, are prepared at a ratio of about 1: 6.5. 50 of polyethylene glycol (PEG) which is often used as a fusion agent
It is said that the fusion rate is high when using%. Fusion strain is H
Select by AT selection method. The resulting hybridoma was screened by using the culture supernatant, the membrane fluorescent antibody method, ELI.
A known hybridoma method such as SA method (Enzyme Linked Immunosorbent Assay) and immunohistological staining method is performed to select a hybridoma clone secreting the desired immunoglobulin. 96 to examine the unity of hybridomas
Feeder lay (feeder lay)
er), about 10 ⁶ cells / well of normal splenocytes are sown, and not more than one hybridoma is sown per well, and the growing clones are screened again. By repeating this subcloning, a single hybridoma is obtained.

(2) Production of Monoclonal Antibody Next, in order to produce the monoclonal antibody of the present invention,
The hybridoma obtained above was placed in a culture vessel (in vitro
o) or in vivo. When culturing in an in vitro system, the medium may be the normal medium described above with FCS added, and after culturing in this medium for 3 to 5 days,
Monoclonal antibody is obtained from the culture supernatant. For in vivo culture, the hybridoma is inoculated into the abdominal cavity of a mammal,
Ascites was collected 7 to 14 days later, and the
To obtain a null antibody. When culturing in an in vivo system, in vitro
It is preferable since a much larger amount of antibody can be efficiently obtained as compared with in-system culture.

Purification of the monoclonal antibody from the thus obtained culture supernatant or ascites is carried out by appropriately combining known methods such as ammonium sulfate fractionation and DEAE Sepharose column.
For example, it can be performed as described in Examples below.

Monoclonal antibody KY obtained by the present invention
-ANP-I is α-hA as shown in Reference Example below.
It specifically recognizes NP and shows little affinity for rANP. The epitope is the N-terminal half of the ring structure of α-hANP, specifically Cys [7] to Gly [16].
Is assumed to recognize the part included in
Since it does not react with ANP, it is considered to be a part containing Met [12]. The epitope is a common part not only to α-hANP but also to β-hANP and γ-hANP, and the antibody has reactivity to β-hANP and γ-hANP.

The present antibody also showed a high affinity (Ka = 4.5 × 10 ⁹ M ⁻¹ ) for α-hANP. From the standard curve of α-hANP shown in FIG. 1, 90% and 50% inhibitory concentrations (IC ₉₀ , IC ₅₀ ) were 10 pg / tu.
And 100 pg / tube.

Monoclonal antibody KY-ANP of the present invention
The hybridoma KY-ANP-I producing -I is 19
From 20th August 1987 United Kingdom Porton Down, Salisbury. S
P4 OJG. PHLS Center for Applied Microbiology &
Research, European Collection of Animal Cell Cultu
It has been deposited with res (ECACC) under accession number 87082001 under the Budapest Treaty.

(3) Binding of antibody to carrier As the solid phase for immobilizing the antibody, a commercially available carrier for antigen-antibody reaction used in a usual immunoassay, for example, glass or synthetic resin particles ( Beads) or spherical materials (balls), tubes, plates and the like can be used. An antibody that recognizes the N-terminal side or C-terminal side of α-hANP is adsorbed on these carriers. Adsorption is usually carried out by allowing the mixture to stand overnight at room temperature in a phosphate buffer at pH 6 to 10, preferably around neutrality. The carrier on which the antibody is adsorbed is stored in a cool place in the presence of a bactericide such as sodium azide.

Monoclonal antibodies and polyclonal antibodies can be bound to carriers by the same treatment.

(4) Rabbit anti-α-hANP [17-28] serum α-hANP [17-28] prepared by the carbodiimide method
-Bovine thyroglobulin complex is administered to a rabbit to immunize it several times, and 10 to 14 days after the final immunization, blood is collected to prepare a rabbit anti-α-hANP [17-28] serum.

(5) Preparation of Enzyme-Labeled Antibody Rabbit IgG, F (ab ') ₂ and Fab' The antiserum prepared above was fractionated with sodium sulfate and then passed through a column of DEAE-cellulose. I
Prepare gG. The obtained IgG was digested with pepsin and F
(ab ') ₂ fragment, which is further reduced with 2-mercaptoethylamine to obtain the desired anti-α-hANP [17-28] Fab'
Is obtained. For preparation of Fab ′ from IgG, Journal of Immunoassay [J. Immunoassay], 4 , 2
09-327 (1983), a similar method can be used in the present invention.

Antibody Enzyme Labeling Antibody Labeling enzymes for the antibody include alkaline phosphatase,
Although β-D-galactosidase, peroxidase, glucose-oxidase and the like can be used, horseradish peroxidase is particularly preferably used in the present invention. Further, as the cross-linking agent, N, N'-o-phenylene dimaleimide, 4- (N-maleimidomethyl) cyclohexanoic acid / N-succinimide ester, 6-maleimidohexanoic acid / N-succinimide ester, 3
-(2-Pyridyldithio) propionic acid / N-succinimide ester, 4,4'-dithiodipyridine, and other known crosslinking agents can be used. The reaction of these cross-linking agents with the enzyme and the antibody may be performed according to a known method depending on the properties of each cross-linking agent. Also, the antibody may optionally be a fragment thereof, such as Fa
b ', Fab and F (ab') ₂ are used. In the present invention, it is preferable to use 4- (N-maleimidomethyl) cyclohexanoic acid / N-succinimide ester or 6-maleimidohexanoic acid / N-succinimide ester as the crosslinking agent. In addition, an enzyme-labeled product can be obtained by the same treatment regardless of whether the antibody is polyclonal or monoclonal. Therefore, the enzyme-labeled antibody obtained by using the above-mentioned two cross-linking agents has the general formula (I);

[0022]

[Chemical 1]

[Wherein A is an antibody or fragment thereof that recognizes the N-terminal side or C-terminal side of α-hANP,
B represents a labeling enzyme, and R represents 4-methylenecyclohexyl or pentamethylene. ] Can be represented.

The enzyme-labeled antibody thus obtained is
Preferably, purification by affinity chromatography enables a more sensitive immunoassay system.
The purified enzyme-labeled antibody is stored in a cool and dark place after adding thimerosal or glycerin as a stabilizer or by freeze-drying.

As a combination of antibodies for measuring α-hANP using the immunological assay reagent prepared above, C is used when an antibody recognizing the N-terminal side of α-hANP is immobilized. The antibody that recognizes the end side is an enzyme-labeled antibody, and C
When the antibody recognizing the end side is immobilized, the antibody recognizing the N end side may be used as the enzyme-labeled antibody. Generally, since a relatively large amount of antibody is required for immobilization, a monoclonal antibody (for example, NK-ANP-I of the present invention) that can stably obtain a large amount of antibody is suitable for immobilization. Polyclonal antibodies obtained from antisera can also be used without any inconvenience. The enzyme-labeled antibody may be either a monoclonal antibody or a polyclonal antibody, as long as it recognizes a site different from that recognized by the immobilized antibody. For example, when KY-ANP-I of the present invention is used as the immobilized antibody, the above-mentioned antiserum CR-3 is used as the enzyme-labeled antibody.
Alternatively, the antiserum F36 in the examples can be applied, or the reverse combination may be used. Of course, a monoclonal antibody that recognizes the C-terminal side of α-hANP and an antiserum that recognizes the N-terminal side can also be applied to the present invention.

[0026]

EXAMPLE Preparation of hybridoma Synthetic α-hANP (1.5 mg) and bovine thyroglobulin (5.4 mg) are dissolved in 2 ml of distilled water. 30 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide dissolved in 1 ml of distilled water is added dropwise to this solution at room temperature over 10 minutes, and the mixture is stirred at room temperature for 24 hours. The solution is dialyzed against 3 L of distilled water 6 times for 3 days. This dialysate was dispensed into 5 and stored at -20 ° C (BBRC
124 , 815-821, 1984).

The above aliquot-saved solution (300 μg of α
-Containing hANP), distilled water was added to make 1.2 ml, and this was suspended in 1.2 ml of Freund's complete adjuvant. About 2 ml of them was intraperitoneally and subcutaneously injected into 10 BALB / c female mice (200 μl per mouse),
After a further 3 weeks, the same method was used for booster immunization. Five weeks after the booster, the antibody in the blood was measured, and 50 μg of α-hANP was added to the tail vein of the two mice showing the strongest antibody reaction.
Was further boosted with the above solution. 4 days later, 2
Spleen cells were harvested from a single mouse for cell fusion.

The collected spleen cells (1.3 × 10 ⁸ cells) and myeloma cells X63-Ag8.653 (2 × 10 ⁷ cells) were mixed in Dulbecco's medium (DMEM) and mixed at 1500 rpm and 4 ° C. for 5 minutes.
It spun down for a minute. The obtained pellet was heated to 37 ° C. and thawed, and then 50% PEG4000 (PEG1 g / DMEM1
1 ml) was added dropwise at 37 ° C. over 1 minute. 37 ° C
After allowing to stand for 2 minutes at 10 ° C., 10 ml of DMEM at 37 ° C. is added over 5 minutes for dilution, and centrifugation is performed at 4 ° C. with 15% FCS-containing DMEM.

The obtained cells were seeded on a 96-well plate, and H
The cells were cultured in AT medium for 2 weeks and further in HT medium for 1 week. Hybridomas grew in approximately 30% (212/768) of all 768 wells, of which 4% (8/212) produced anti-α-hANP antibody.

The cells in the well showing the highest antibody titer were cloned by the limiting dilution method. That is, as feeder cells, BALB / c mouse thymocytes were added to the wells at 10 ⁶ cells / well and hybridomas were added at 1 cell / well, and the cells were cultured. This operation was repeated twice.

By this cloning, the clone which produces the most stable and large amount of antibody was selected, and KY-ANP-
It was named I.

The antibody titer in the antiserum of the immunized mouse and the culture supernatant of the hybridoma was measured as follows.

The antiserum of the immunized mouse or the culture supernatant of the hybridoma was used as a sample, and the sample dilution solution was 100 μm.
l, Assay buffer- (RIA buffer-) 300μ
l, ¹²⁵ I-α-hANP (10000 cpm) 100μ
The mixture of 1 is reacted at 4 ° C. for 24 hours. This was mixed with 1 ml of dextran-coated charcoal and mixed at 4 ° C.
React for 5 minutes. Then 3000 at 4 ° C for 30 minutes
The antibody titer in the sample diluent was determined by centrifuging at rpm and measuring the radioactivity of the supernatant with a γ-counter.

The above ¹²⁵ I-α-hANP was prepared by the chloramine T method. That is, α-hANP (1 μg) and Na
Mix ¹²⁵ I (1 mCi) and mix with 10 μl chloramine T
(5.25 mg / ml) and 10 seconds later 20 μl sodium pyrosulfite (4.5 mg / ml). Furthermore, 1% of 2% gelatin was added, and Sep-Pak C was added.
18 (manufactured by Waters).

Preparation of Monoclonal Antibodies BALB / c mice were pretreated by intraperitoneal administration of 0.5 ml of pristane twice at 1-2 week intervals. The mouse was injected intraperitoneally with 5 × 10 ⁶ hybridoma KY-ANP-I suspended in 200 μl DMEM. The obtained ascites fluid was purified by protein A-sepharose CL-4B column, and the monoclonal antibody KY-
ANP-I was obtained.

Various Properties of KY-ANP-I The isotype of this monoclonal antibody was determined according to the Octaloni method, and it was determined to belong to the IgG ₁ subclass. The affinity was determined by preparing a Scatchard plot by a radio binding assay, and the result was Ka = 4.5 × 10 ⁹ M ⁻¹ .

Epitope was determined by examining RIA for cross-reactivity to various ANP-related peptides. The result is shown in FIG. α-ANP [17-28] and α-
Since it hardly reacts with ANP [1-6], it is presumed that the epitope is included in α-ANP [7-16]. Furthermore, α
Since it does not react with -hANP [8-22] or α-rANP, it is presumed that the epitope needs to form a ring structure and contains Met. That is, it was concluded that the epitope recognized by the monoclonal antibody of the present invention is approximately half of the N-terminal side of the ring structure of α-hANP.

Further, according to the above antibody titer measuring method, KY-AN
As a result of examining the reactivity of P-I with β-hANP and γ-hANP, KY-ANP-I recognizes not only α-hANP but also γ-hANP, and β-hANP has a cross-reactivity of 80%. It reacted with.

Anti-α-hANP [17-28] Bovine Tiroglob
Preparation of phosphorus serum α-hANP [17-28] was prepared by the known carbodiimide method (BBRC
117 , 695, 1984) to bind bovine thyroglobulin. Aqueous solution containing 3.1 mg of α-hANP [17-28].
0.7 ml of an aqueous solution containing 19 mg of bovine thyroglobulin in 5 ml
Aqueous solution 1 containing 40 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride.
Add ml and stir for 2 hours under ice cooling. The reaction solution was dialyzed 3 times against 2 L of distilled water at 4 ° C. and then freeze-dried to obtain 21 mg of a complex. The binding molar ratio of α-hANP [17-28] to bovine thyroglobulin was 30 as determined by amino acid analysis.

Immunization and Blood Collection 0.25 mg of the above α-hANP [17-28] -bovine thyroglobulin complex was dissolved in 0.25 ml of physiological saline and suspended in an equal volume of Freund's complete adjuvant. Subcutaneously injected into 20 or more places on the back of the rabbit. This is repeated 6 times every 3 weeks, and 10 days after the final administration, whole blood is collected from the carotid artery to obtain antiserum F36.

Preparation of rabbit IgG 1 ml of anti-α-hANP serum (F36) contained 0.18 sodium sulfate
g is slowly added with stirring and the stirring is continued at 22-25 ° C after the additive has completely dissolved. Then, the mixture was centrifuged at a temperature of 22 to 25 ° C. and a rotation speed of 10,000 rpm for 10 minutes, and the precipitate was dissolved in 1 ml of sodium phosphate buffer (pH 6.3; 17.5 mmol / L) and the same buffer was added.
Dialyze in. The supernatant was previously mixed with the same buffer (pH 6.
3; 17.5 mmol / L) and passed through a DEAE cellulose column equilibrated. The wet capacity of DEAE cellulose required to pass 10 mg of protein in the supernatant is 1 ml. IgG
The yield of 7 mg was calculated from the absorbance at 280 nm with the absorbance at 280 nm being 1.5 g ^-1 L, cm ^-1 and the molecular weight of IgG being 150,000 (Journal of Immunoassay [J. Immunoassay], 4 , 209-327 (1
983)).

[0042] F (ab ') ₂ Preparation rabbit IgG 10 mg sodium acetate buffer - (pH
4.5, O.1 mol / L) 1 ml is dialyzed at 5 ° C. To the dialyzed IgG solution, 0.05 ml (volume 1/20) of aqueous sodium chloride solution (2 mol / L) is added. In this solution,
Add pepsin (0.2 mg / 10 mg IgG) derived from porcine gastric mucosa and dissolve. The mixture is allowed to react at 37 ° C for 15-24 hours. Then, with an aqueous solution of sodium hydroxide (1 mol / L)
The pH was adjusted to 8, and the Sephadex G-150 column (1.
1.5 x 45 cm for 0 to 1.5 ml, for 2.0 to 2.5 ml
2.0 x 45 cm) with sodium borate buffer
Elute at (pH 8.0, 0.1 mol / L). The yield of F (ab ') ₂ is calculated from the absorbance at 280 nm to the absorbance at 280 nm.
The molecular weight of 1.48 g ^-1 L, cm ^-1 and F (ab ') ₂ was calculated to be 92,000, which was 6 mg (Journal of Immunoassay [J. Immunoassay], 4 , 209-327 (1)).
983)).

Preparation of Fab ′ 3 mg of F (ab ′) _{2 was} added to sodium phosphate buffer (pH 6.
Dissolve in 0.45 ml (0, 0.1 mol / L). A 2-mercaptoethylamine / sodium phosphate buffer solution (pH 6.0, 0.
1 mol / L), and react at 37 ° C for 1.5 hours. The reaction mixture was then applied to a Sephadex G-25 column (1 x 30 cm) and sodium phosphate buffer-
Elute with (pH 6.0, 0.1 mol / L; containing 5 mmol / L EDTA). Fab 'yield was calculated from the absorbance at 280 nm.
The absorbance at 280 nm was calculated to be 1.48 g ^-1 .Lcm ^-1 and the molecular weight of F (ab ') ₂ was 46,000, and was 2.5 mg (Journal of Immunoassay [J. Immunoassay],
4 , 209-327 (1983)).

Anti-α-hANP [17-28] Fab'-peroxida
Preparation of ze-labeled product Horseradish peroxidase 2 mg (50 nmol) in sodium phosphate buffer (pH 7.0, 0.1 mol / L) 0.3 ml
And 6-maleimidohexanoic acid / N-succinimide ester (0.65 mg (2100 nmol)) and N, N-dimethylformamide (0.03 ml) were added to the solution.
Stir at ℃ for 0.5 to 1 hour to react. Then
The reaction mixture was centrifuged to remove excess reagent as a precipitate, and the supernatant was passed through a Sephadex G-25 column (1.0 x 45 cm) to obtain sodium phosphate buffer (pH 6.0, 0.1 mol). / L) and the flow rate of 30 to 40 ml / h, and the volume of each fraction is 0.5 to 1.0 ml. Sephadex G-50 (fine, Pharmacia) column with a fine mesh filter at the bottom (1.0 x 6.4 c)
m, 5 ml) is equilibrated with the above buffer and
Centrifuge for 2 minutes at 00g. The column is loaded with the above reaction product (0.5 ml) and centrifuged in the same manner. The obtained fraction is used as a microconcentrator (CENTRICON-30,
Concentrate by centrifugation at 2000 g in Amicon Corp) at 4 ° C.

1.8 mg (45 nmol) of the maleimide peroxidase conjugate thus prepared was dissolved in sodium phosphate buffer (pH 6.0, 0.1 mol / L), and Fab 'of about 2.0 mg ( 43 nmol) 5 mmol / L ED
Sodium phosphate buffer containing TA (pH 6.
0, 0.1 mol / L) Add a solution dissolved in 0.2-0.4 ml, and add 4
The reaction is carried out at 20 ° C for 20 hours or at 30 ° C for 1 hour. Maleimide-peroxidase conjugate in the reaction mixture and
The final concentration of Fab ′ is 50-100 μmol / L. The reaction mixture was loaded onto a Ultrogel AcA 44 column (1.5 x 45 c
m) through a sodium phosphate buffer (pH 6.5,
Elute at 0.1 mol / L). The flow rate should be 0.3 to 0.5 ml / min and each fraction should be about 1.0 ml. Thus the desired anti-α-
hANP [17-28] Fab'-peroxidase-labeled substance about 2.5 mg
(J.Immun
oassay] 4 , 209-327 (1983)).

HANP [17-28] -non-specific rabbit IgG conjugate 0.2 ml sodium phosphate buffer (0.1 mol / L, pH
Dissolve hANP [17-28] (0.5 mg) in 7.0) with N,
It is reacted with 105 mmol / L of 6-maleimidohexanoic acid / N-succinimide ester (0.01 ml) dissolved in N′-dimethylformamide at 30 ° C. for 30 minutes. The reaction product was Sephadex G-10 using 0.1 mol / L sodium phosphate buffer (pH 6.0) containing 5 mmol / L EDTA.
Gel filtration through the column (1.0 x 45 cm). hANP [1
The average value of the maleimide group introduced in [7-28] was 0.6 / molecule.

0.1 mol / L sodium phosphate buffer
Non-specific rabbit IgG dissolved in (0.6 ml, pH 7.5)
(10 mg) is reacted with 210 mmol / L anhydrous S-acetylmercaptosuccinic acid (0.03 ml) dissolved in N, N′-dimethylformamide at 30 ° C. for 30 minutes. The reaction mixture was added with 0.1 mol / L Tris-HCl buffer (0.1 ml,
pH7.0), 0.1 mol / LEDTA (0.02 ml) and 1 m
ol / L Hydroxylamine hydrochloride (0.1 ml, pH 7.0) was added, and the mixture was reacted at 30 ° C. for 5 minutes. The reaction product is 5 mmol / L of E
0.1 mol / L sodium phosphate buffer containing DTA
Gel filtration through a Sephadex G-25 column (1.0 x 45 cm) using (pH 6.0). The average value of the thiol group introduced into the non-specific rabbit IgG was 11 / molecule.

Part of the above maleimide-hANP [17-28] (2.
0 ml) with mercaptosuccinylated non-specific rabbit IgG (2.4 mg) in 0.1 mol / L sodium phosphate buffer (pH 6.0, 0.25 ml) containing 5 mmol / L EDTA at 30 ° C. Let react for minutes. 0.01 ml of 0.1 mol / L
N-ethylmaleimide was added to the reaction to block the remaining maleimide groups. The mixture was gel filtered through a Sephadex G-25 column (1.0 x 45 cm) with 0.1 mol / L sodium phosphate buffer (pH 7.0). The average number of hANP [17-28] molecules bound to non-specific rabbit IgG was calculated to be 8.7 /
It was a molecule.

Anti-hANP [17-28] Fab'-horseradish peroki
Purification of sidase- labeled product hANP [17-28] -non-specific rabbit IgG conjugate (2 mg), bovine thyroglobulin (10 mg), mouse serum protein (20 m
g) and cyanogen bromide-activated cepharose 4B (1), respectively.
g). Anti-hANP [17-28] Fab'-horseradish peroxidase labeling in 0.8 ml 0.1 mol / L sodium phosphate buffer (pH 6.5) containing 1 g / L gelatin and 50 mg / L thimerosal. Column (0.55 x 4.0 cm) of bovine thyroglobulin-sepharose 4B.
And mouse serum protein-Sepharose 4B column (0.
55 × 4.0 cm) with 10 mmol / L sodium phosphate buffer (pH 7.5) containing 1 g / L gelatin and 0.1 mol / L sodium chloride at a flow rate of 0.5 ml / h, and then hANP [ 17-28] -Non-specific rabbit IgG-Sepharose 4
It was purified by eluting with 3.2 mmol / L hydrochloric acid (pH 2.5) from the B column (0.35 × 2.0 cm). The eluate (1 ml) containing 0.35 mg of the purified label was immediately added to 0.1 ml of 1 mol / L sodium phosphate buffer (pH 7.0) and 0.01 ml of 10 ml.
It was mixed with 0 g / L gelatin. The amount of labeled substance was about 0.35 mg calculated from the peroxidase activity.

Monoclonal anti-α-hANP-coated polyester
Preparation of Renboru Polystyrene ball (diameter 3.2 mm, Precision Plastic Ball Company (Precision Plastic
Ball Co.) (Chicago, USA) 50 pieces are put into 1 ml of 0.1 M sodium phosphate buffer (pH 7.0), and 1
Monoclonal anti-α-hANP-IgG ₁ (KY-AN
PI) and leave at room temperature overnight. This polystyrene ball is washed with sodium phosphate buffer (pH 7.0), 0.1% sodium azide is added, and the mixture is stored in a refrigerator.

Collection of Plasma Blood was collected from the anterior cubital vein of a healthy male subject (26-32 years old) who fasted overnight at 9:00 am in the supine position. In addition, 40 mg of furosemide (furosemide) was intravenously administered to the same subject,
Blood is sampled in the same manner after walking for 1 hour. Blood was collected in chilled plastic syringes for aprotinin and EDT.
Transfer A to a pre-cooled disposable silicon-coated glass tube and centrifuge (500 xg) to separate plasma.
The final concentrations of aprotinin and EDTA were respectively
1,000 kallikrein inactivating units (KIU) / ml and 1 m
It is g / ml.

Enzyme immunization of α-hANP by sandwich method
Assay method Monoclonal anti-α-hANP IgG ₁ (KY-ANP-I) -coated polystyrene ball is added to α-hANP standard solution or plasma (total volume 0.15 ml) and left standing at 4 ° C for 24 hours. . α-hAN
P standard solution is 10 mM sodium phosphate buffer (pH
7.0; lmg / ml gelatin, 0.3M sodium chloride, 0.2mM cystine, 1mM EDTA, 1mg / ml sodium azide, and 1,000 KIU / ml aprotinin for final volume
Dilute to 0.15 ml. In addition, plasma (50 μl)
Is 0.1 ml of 10 mM sodium phosphate buffer- (pH
7.0; containing 1 mg / ml gelatin, 0.4 M sodium chloride, 0.3 mM cystine, 1.5 mM EDTA, 1.5 mg / ml sodium azide, and 1,000 KIU / ml aprotinin).

After removing the solution portion from the reaction mixture, the polystyrene ball was washed twice with 2 ml of 10 mM sodium phosphate buffer (pH 7.0; containing 0.1 M sodium chloride), and then purified by affinity chromatography. 50 ng of α-hANP [17-28] Fab'-peroxidase conjugate and 10 mM sodium phosphate buffer (pH 8.
0; l mg / ml gelatin, 0.2 mM cystine and 1 mM EDT
0.15 ml (including A), and leave at 4 ° C. for 24 hours. The solution portion is removed, the polystyrene ball is washed twice as above, and then transferred to another test tube. The activity of bound peroxidase was measured by allowing the 3- (4-hydroxyphenyl) propionic acid as a substrate to stand at 30 ° C. for 60 minutes and then measuring the fluorescence intensity. Fluorescence intensity is 200ng / ml
The measurement is based on kine / 50 mM sulfuric acid.

Specificity In EIA by this method, the standard dilution curve of α-hANP is: α-hANP [4-28], α-hANP [5-28] and α-hANP
Since it agrees with the curve of [7-28], it can be seen that the structural change on the N-terminal side has no effect. Conversely, the lack of the C-terminal amino acid significantly reduces the reactivity. However, it does not react with the terminal fragments α-hANP [17-28] and α-hANP [1-6]. The cross-reactivity with β-hANP and α-rANP was 4.7% and 0.01% on a molecular basis, respectively. These results are consistent with the specificity of the antibody used. Mouse monoclonal IgG immobilized on polystyrene ball
₁ shows specificity to the N-terminal half of the cyclic structure of α-hANP, and rabbit Fab ′ bound to peroxidase is α-h
Shows specificity for ANP [17-28].

Effect of Plasma The bound peroxidase activity (non-specific binding) measured in the absence of plasma and α-hANP was 1 pmol of monoclonal anti-α-hANP IgG ₁ (KY-ANP-I. ) And peroxidase activity measured in the presence of pretreated plasma. Four.
Α-hANP added to plasma containing 3 to 332 pg / ml ANP
The recovery rate of (10-200 pg / ml) is 81-94%. The plasma dilution curve is parallel to the standard curve for α-hANP obtained in the absence of plasma, as long as the plasma volume per tube is in the range 1-50 μl. Therefore, the amount of plasma used is 50
In the case of less than μl, almost no plasma damage was observed. The result is shown in FIG.

EIA sensitivity The measurement limit of α-hANP is 30 fg (10 amol) / tube. The sensitivity of plasma α-hANP when using 50 μl of plasma is 0.6 pg / ml (0.2 fmol / ml). This sensitivity in EIA is one to two orders of magnitude higher than in existing RIA.

Reproducibility of EIA The reproducibility of the product of the present invention is 5-15 for α-hANP in plasma.
It was determined at 5 different levels within the range of 8 pg / ml.
The reproducibility within and between experiments was coefficient of variation 3.2-9.4% (n = 20) and 5.4-12.0%, respectively.

E in steady state and hypovolemic state
Plasma α-hANP level and its R in normal subjects by IA
Comparison with IA Steady-state α-hANP level in normal subjects was 2 in EIA.
It was 4.5 ± 5.9 pg / ml. Furosemide
e) (40 mg) was intravenously injected, and the plasma α-hANP level in the hypovolemia state after walking for 1 hour was 15.3 ± 3.7 pg / ml.
Fell to. Plasma α-hAN measured simultaneously by RIA
P levels were 28.2 ± 5.7 pg / ml and 18.2 respectively.
It was 3 ± 3.2 pg / ml. The following correlation exists between α-hANP levels measured by EIA (y) and RIA (x).

Y = 0.78x + 1.3, r = 0.92, n = 24

As is clear from the above data, EIA in the method of the present invention has a very high sensitivity, and the plasma α-hANP concentration can be measured without extracting operation even in a circulatory hypovolemia state.

Plasma α-hANP Levels in Heart Disease Patients Plasma α-hANP levels in heart disease patients measured by the reagent for measuring α-hANP of the present invention are shown in Table 1 below.

[0062]

[Table 1]

Application by EIA One-Step Method The EIA in the present invention can also be applied by a one-step method.
FIG. 3 shows a standard curve of α-hANP and a plasma dilution curve by one-step EIA. In this test, hANP, enzyme-labeled antibody and antibody-coated polystyrene ball were mixed at the same time and reacted at 4 ° C. for 24 hours. Peroxida
The ze (POD) activity was measured at 30 ° C. for 30 minutes. Other conditions are exactly the same as the two-step method.

[0064]

In the conventional measurement of α-hANP by RIA, it was necessary to extract α-hANP from a sample, but α-hANP using the monoclonal antibody of the present invention was used.
If an immunological assay reagent for ANP is used, α-hANP in a sample can be directly measured without extracting α-hANP. For this measurement, not only the two-step method in which the immobilized antibody is reacted with α-hANP and then the labeled antibody is added, and the reaction is performed by simultaneously adding α-hANP and the labeled antibody to the immobilized antibody. The one-step method can also be applied, and simple measurement is possible. By establishing this method for measuring α-hANP, heart disease, renal disease, hypertension (essential
Secondary), edematous disease (cirrhosis, nephrosis, idiopathic edema, etc.), dehydration, and other diseases accompanied by abnormal fluid balance, and simple and accurate observation of the course of treatment has become possible.

[Brief description of drawings]

FIG. 1 α-hANP, α-rANP, and α-hA
3 shows the cross-reactivity of the NP fragment with the monoclonal antibody of the present invention.

FIG. 2 shows a standard curve of α-hANP by EIA (◯) and a plasma dilution curve (●, ▲, ■).

FIG. 3 shows a standard curve of α-hANP (◯) and a dilution curve of plasma (●, ▲, ■) by one-step EIA.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location G01N 33/53 D 33/577 B (C12P 21/08 C12R 1:91)

Claims (4)

[Claims] 1. A Mouse Myeloperoxidase - Ma cells and alpha - hybrid by fusing spleen fine <br/> cells of mice immunized with human atrial natriuretic polypeptide - to prepare a Ma, the hybridoma - from Ma The monoclonal antibody, which is obtained by selecting a hybridoma that produces a monoclonal antibody that recognizes approximately half of the N-terminal side of the ring structure of α-human atrial natriuretic polypeptide. A hybridoma that produces. 2. The monoclonal antibody is characterized by being included in an α-human atrial natriuretic polypeptide [7-16] fragment and recognizing a portion containing Met [12]. The hybridoma according to 1. 3. The hybridoma according to claim 1, wherein the myeloma cell has a hypoxanthine-guanine-phosphoribosyltransferase deficiency or a thymidine kinase deficiency. 4. Hybrid-ma KY-ANP-I (ECA
The hybridoma according to claim 1, which is CC87080801).