JPH0195799A - Monoclonal antibody and method for immunochemical measurement - Google Patents

Abstract

PURPOSE:To measure human arginase, in human blood serum, etc., by synthesizing an antiarginase monoclonal antibody capable of binding to human arginase using an arginase of a mammal other than humans as an antigen. CONSTITUTION:A splenic cell obtained from a mouse immunized with purified or crude arginase is mixed and fused to a myelomatous cell of the same strain mouse in the presence of a cell fusion agent, such as polyethylene glycol, and then cultivated. The resultant hybridoma is selectively propagated using a hypoxanthine-aminopterin-thymidine culture medium, etc., to select cells capable of producing an antiarginase antibody. The obtained cells are subsequently propagated in an ordinary liquid culture medium, etc., to provide an antiarginase monoclonal antibody, which is then used to immunochemically measure a human arginase.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an anti-arginase monoclonal antibody and an immunochemical assay method using the same.

[Prior art and its problems Co-arginase (E, C, 3, 5, 3, 1, L-arginine amidinohydrolase) converts L-arginine into L-
An enzyme that breaks down ornithine and urea, and is present in large amounts, especially in the liver. Measuring arginase is useful for diagnosing liver diseases, hyperammonemia, etc. Conventionally, arginase is measured by measuring urea, which is one of the reaction products of L-arginine and arginase [Clinical Chemistry (
Cl1n, Chem, ) Volume 13, Page 900, 19
67], and a method for measuring another reaction product, mono-ornithine [Journal of Biological Chemistry (J, Biol.

Chem, Volume 199, Page 91, 1952], 1
A method using 4c-labeled glinidino-arginine as a substrate [Analytical Biochemistry (A
nnal, Biochem, ) Volume 102, 20
6, 1980], etc. are known. However, these methods of measuring enzyme activity require a large amount of sample and require centrifugation to remove protein.
Due to the necessity of operations to remove urea, ornithine, etc. present in the blood, processing a large number of test subjects requires a great deal of effort, time, and reagents.

[Means for solving the problem] The present inventors attempted to develop an immunochemical measurement method for arginase that can be used in mass screening for diagnosis of liver diseases and genetic metabolic abnormalities in children, and as a result, a novel method was developed. We have discovered a monoclonal antibody and a method for immunochemically measuring the above-mentioned arginase using it. That is, the present invention relates to an anti-arginase monoclonal antibody obtained using arginase of a mammal other than humans as an antigen, and a human arginase monoclonal antibody, and a human arginase monoclonal antibody that is used to detect human arginase monoclonal antibodies in human serum or other antibodies. This is an immunochemical measurement method characterized by immunochemically measuring the concentration of human arginase in human body fluids.

Although arginase from mammals other than humans has similar enzymatic activity to human arginase, it is thought that their antigenicity is not exactly the same due to differences in their amino acid sequences, tertiary structures, etc. However, it is thought that both types of arginase share some antigen-determining sites that are recognized by antibodies. Therefore, some antibodies against arginase from mammals other than humans recognize the antigen-determining site that is common to human arginase. It is thought that there are antibodies that do this. Therefore, the anti-arginase monoclonal antibody of the present invention is obtained by selecting a hybridoma that binds to human arginase and produces a vine antibody from a group of hybridomas (fusion cells) obtained using arginase from mammals other than humans, and from this selected hybridoma. This is the monoclonal antibody obtained. Note that in selecting this hybridoma, purified human arginase is usually required. Arginase from mammals other than humans is
For example, arginase obtained from organs such as rackets, mice, pigs, sheep, cows, horses, sheep, monkeys, etc., especially livers, is suitable, but the type of mammal is not limited to these. The arginase may be crude arginase, but preferably purified arginase is used. Arginase can be purified using a known method (eg, Schimke's method described below). Monoclonal antibodies may be of the usual IgG class, as well as IgM or other classes.

Monoclonal antibodies were prepared using the method of Milstein et al. [Nature, Vol. 256, p. 495.

1975]. For example, mouse tile cells obtained by immunization with arginase as an antigen are fused with mouse myeloma cells, a hybridoma that secretes a human arginase-binding monoclonal antibody is selected, and a monoclonal antibody is obtained from the hybridoma. More specifically, monoclonal antibodies are obtained as follows: mice immunized with purified or crude arginase (e.g., BALBZC system, etc.)
and myeloma cells from syngeneic mice (
Example: N5-1. P3Ul, etc.) and a cell fusion agent (e.g.
(polyethylene glycol, Sendai virus, etc.), fuse, and culture. Hybridomas were grown in pipoxanthine-aminopterin-thymidine medium [H
AT medium; Nature, Vol. 256,
p. 495, 1975, etc., to selectively proliferate, and then anti-arginase antibody producing cells are selected. Whether or not the culture solution contains the desired antibody that binds to human arginase can be assayed using a known oxygen immunoassay method. Hybridomas that produce highly specific antibodies for human arginase are further monocloned by limiting dilution. The obtained target hybridoma is grown in a normal liquid medium or in the peritoneal cavity of a mammal. Monoclonal antibodies produced by hybridomas are concentrated and purified by known methods (eg, salting out with ammonium sulfate, DEAE cellulose chromatography, protein A column chromatography, etc.).

The present invention also provides a method for immunochemically measuring human arginase using the anti-arginase monoclonal antibody. The above monoclonal antibodies may be used as they are, or may be fragments such as Fab' fragments and F(ab')2 fragments, or other modified products.

In the method of immunochemically measuring human arginase using an anti-arginase monoclonal antibody as described above, test samples to be measured include human serum and other body fluids. Specifically, there are urine, serum, plasma, etc., and serum is especially frequently used.

As an immunochemical measurement method, a competitive method, Sand-Deutsch method, agglutination method, etc. can be used. A summary of these methods is shown below.

■Competitive method: A test solution containing an unknown amount of arginase and a certain amount of arginase labeled with a labeling agent are competitively reacted with a certain amount of the corresponding anti-arginase antibody, and the antibody is bound to the labeling agent or the antibody. The amount of labeling agent that is missing is measured, and the amount of arginase in the test solution is determined from the measured value.

■Sandwich method: A test solution containing an unknown amount of arginase is added to an excess amount of anti-arginase antibody held on a carrier and reacted (first reaction). Next, the antibody used in the first reaction has an antigen recognition region. A certain amount of labeled antibodies of different colors are added and reacted (second reaction), and the amount of labeled agent retained on the carrier or not retained on the carrier is measured, and from that value. The amount of arginase in the test solution is determined. Note that the first reaction and the second reaction may be performed simultaneously,
The steps may be performed sequentially at intervals.

■Agglutination method: Anti-arginase antibodies are bound to the surface of a carrier such as red blood cells or latex particles, and the antibody-sensitized particles are brought into contact with a test solution containing an unknown amount of arginase, and the antibody-sensitized particles are bound via arginase. to cause agglomeration. Since aggregation occurs depending on the amount of arginase in the test solution, the degree of aggregation is measured by a slide method, microtimer method, colorimetric method, etc., and the amount of arginase in the test solution is determined from the results. .

Labeling agents used in the above method include, for example, radioactive isotopes, enzymes, fluorescent substances, luminescent substances, and the like. Specifically, for example, there are radioactive isotopes such as +2sJ, +311°31, and 14c. As the enzyme, one that is stable and has a large specific activity is preferable;
urease, asparaginase], ◎Esterase [e.g. cholinesterase, phosphatase, sulfatase, lipaseco, ■Nuclease [e.g. deoxyribonuclease, ribonuclease J, ■Iron/porphyrin enzyme [e.g. calatase, peroxidase, ■Copper enzyme [e.g. tyrosinase] , ascorbate oxidase],
■Dehydrogenase [e.g. alcohol dehydrogenase, malate dehydrogenase, lactate dehydrogenase, incitric acid dehydrogenase]
, etc. Examples of fluorescent substances include fluorescamine and fluorescein isothiocyanate, and examples of luminescent substances include luminol, luminol derivatives, luciferin, and lucigenin. As a method for binding these labeling agents with antibodies or human arginase, the known chloramine T method [Nature
).

Vol. 194, p. 495, 1962], periodic acid method [Journal of Histochemistry and Cytochemistry, Vol. 22, p. 1084, 1974]
, maleimide method [Journal of Biochemistry,
79, p. 233, 1976 can be used.

The carrier includes gel particles (e.g. agarose glue),
Dextran gel, polyacrylamide gel, cellulose particles, ion-exchanged cellulose (e.g. di-ethylaminoethyl cellulose, carboxymethyl cellulose), physical adsorbents (e.g. di-glass spheres, aminoalkylated glass spheres)
, silicon pieces, polystyrene resins (eg, polystyrene spheres, polystyrene particles), plates for immunoassay, ion exchange resins (eg, weakly acidic cation exchange resins, weakly basic anion exchange resins), and the like. As a method for retaining the antibody on a carrier, known methods can be used, such as the Bromcean method and the glutaraldehyde method (Metabolism, Vol. 8, p. 696, 1971). Can be done. Alternatively, as a simpler method, it may be physically adsorbed onto the surface of the carrier. The present invention will be specifically explained below with reference to Reference Examples and Examples, but the present invention is not limited thereto.

Reference Example 1 (Purification of human arginase) Schjmke
The method [isorad inenzymology (Metho)
d in Enzymology), Volume 17.

Human arginase was collected and purified according to [Page 314]. That is, 400 g of human liver was shredded, and 1200 m
A 0.01M Tris buffer containing 0.0.IMkcl and 0.05M manganese chloride was added, and the mixture was homogenized for 1 hour under water cooling to prepare a suspension. Next,
After centrifugation, ammonium sulfate was added to the supernatant to give a final concentration of 35 wt%, and the mixture was stirred for 1 hour under water cooling. After further centrifugation, ammonium sulfate was further added to the supernatant to give a final concentration of 60 wt%, and the mixture was stirred for 1 hour under water cooling. Next, centrifugation was performed to obtain a precipitate, and 0.01M Tris buffer was added to the precipitate to dissolve it, and after dialysis against the same buffer, heat treatment was performed at 60°C for 20 minutes.
After cooling with water, centrifugation was performed to obtain a supernatant.

Next, chromatographic purification was performed using the same buffer as above and applied to a DEAE cellulose column (2.6 cm x 50 cm). The unadsorbed fraction was further treated with CM-Sepharose CL-6B [Pharmacia (Sweden)]
Co., Ltd.] column (2.6 cm x 50 cm). The adsorbed fraction was eluted with a linear concentration gradient of 0 to 0.5 M NaCl to obtain a fraction containing human arginase.

This fraction was dialyzed against 0.01M Tris buffer and adsorbed onto a CM-Sepharose CL-6B column again.

Elution was performed with 0.1M arginine solution, the fraction containing human arginase was concentrated, and further heptaphryl 52
00 (manufactured by Pharmacia) column (2.6cm
0 cm) to obtain 43 mg of purified human arginase.

Reference Example 2 (Preparation of labeled antibody) (a) ePurified human arginase obtained in Example 1 0.3m
g to 1 m of physiological saline (dissolved in 2), add 1 mQ of complete adjuvant of Ooid, mix well to prepare an emulsion, and inject this into both thigh muscles of the goat and several subcutaneous places on the back. Injected.

Repeat this procedure 5 times every 2 weeks (total antigen amount: 1.5 mg)
), blood was collected one week after the final immunization to obtain antiserum at 2°C.

After 50 cc of antiserum was salted out using the ammonium sulfate method to prepare a globulin fraction, it was purified using a DEAE cellulose column (2.6 cm x 50 cm) to obtain 1 g of anti-human arginase antibody.

(b) Preparation of alkaline phosphatase-labeled anti-human arginase antibody complex 1 mQ of a solution of alkaline phosphatase (0.5 mg) diluted in 0.1 M phosphate buffer (p)I 6.8) obtained in (a) above 2 mg of anti-human arginase antibody was dissolved. Next, add 0.0% of 3% glutaraldehyde solution to this solution.
Add 1 mQ and react at room temperature for 60 minutes, then add 0.5M N
Dialysis was performed overnight against Tris buffer (pH 7, 8) containing aC1. Furthermore, the mixture was fractionated using a Sephacryl 5200 column to obtain an alkaline phosphatase-labeled anti-human arginase antibody complex.

Reference Example 3 (Purification of rat arginase) Rat liver 1
Rat arginase was collected and purified from 35 g using the same method as in Reference Example 1 to obtain 13 mg of purified rat arginase.

Example 1 (1) Preparation of anti-arginase monoclonal antibody 100 μQ of purified rat arginase obtained in Reference Example 3 was dissolved in 500 μQ of physiological saline, and 500 μQ of Freund's complete adjuvant was added thereto and mixed well to prepare an emulsion. was administered intraperitoneally to BALB/C mice. Furthermore, immunization with the same amount of the above antigen-containing emulsion twice every two weeks,
Lungs were removed 4 days after the final immunization. Next, RPMI
After washing well with 1640 medium, 1 x 10 of the lung cells were
8 pieces and 5×10' pieces of mouse myeloma (P2O3) were mixed and centrifuged at 120 pm for 15 minutes to form a pellet. Next, add polyethylene glycol 4000 to RPM
After diluting by adding 1 mQ of a 50% solution of I1640 and gradually adding RPM116408mQ, centrifugation was performed at 11,000 rpm for 5 minutes to disperse the cells in 40 m of HAT medium. Next, add 1 portion of the above cell dispersion to a 90-well plate [manufactured by Nunc (Denmark)].
00 mQ each, and then HA on the 4th and 6th day.
When the antibody titer against rat arginase was measured for the culture supernatant 14 days after injecting 50 μQ of T medium, 16 wells out of a total of 288 wells were found to be positive.

Next, these positive hybridomas were cloned by repeating the limiting dilution method, and finally 11 types of hybridomas producing anti-rat arginase monoclonal antibodies were obtained.

These were injected intraperitoneally into BALB/C mice treated with mineral oil, and ascites was collected 2 to 3 weeks later, from which an antibody-containing solution was obtained. This solution was salted out with ammonium sulfate to obtain globulin fractions.

(2) Selection of human arginase-binding anti-arginase monoclonal antibodies The following reagents were prepared, and antibodies capable of binding to human arginase were selected from the 11 anti-arginase monoclonal antibodies obtained in the previous section.

HRP labeled antibody: Horseradish peroxidase (HRP
) Labeled anti-mouse immunoglobulin antibody [manufactured by Kabbell (USA)] Buffer A: p)I7 containing 0.15M NaCl
．． 4 of 0.02M phosphate buffer.

Buffer B: 0.02M phosphate buffer containing 1% bovine serum albumin (BSA), and 0.15 NaCl.

) IRP activity measurement reagent: 0.02% hydrogen peroxide and 0.1
0.1 M citric acid-phosphate buffer, pH 4.8, containing 5% phenylenediamine.

First, 5 μg of the purified human arginase solution (25 μg/ml) obtained in Reference Example 1 prepared by diluting with buffer A was added to each well of an immunomicroplate I for EIA [manufactured by Nunc].
The solution was injected in μρ portions and left at room temperature for 1 hour. Next, buffer A
After washing with water, 100 μQ of buffer B was added and stored in a cool place until use to prepare an antigen-binding microplate.

The monoclonal antibody obtained in the previous section (1) was diluted with buffer B (1 μs/ml) and 50 μQ of the antibody solution obtained was injected into each well and reacted at 37° C. for 1 hour. After washing each well with saline, add HRP-labeled antibody to buffer B for 400 min.
50 μQ of the diluted solution was injected and reacted at 37° C. for 1 hour. After washing with physiological saline, 100 μQ of HRP activity measurement reagent was added and reacted at room temperature for 10 minutes.
After the reaction was stopped by adding 100 μQ of 1, the absorbance at 490 nm was measured using an automatic colorimeter for microplates, using a blank as a control. The results are shown in Table 1.

As a result of the above measurement, two monoclonal antibodies (RID81
and R2G11) were confirmed to have binding properties to human arginase.

Table 1 RID811gG1 10 RIGII IgG1 - R2DI9 1gG2a - R2H101gG1 - R2G51 1gG1 - RIC21gM - R2B6 1gM - R2G11 1gM + R3A3 1gM - R3F2 1gM - R3H61gM - (3) Add human arginase-binding monoclonal to each well of the immunoplate for measurement EIA. Antibody (RID81, or R
2G11) diluted with the above buffer A (25 μg
/mf2) was added in 50 μQ portions and left at room temperature for 1 hour. Next, after washing with buffer A, add 100μ of the buffer B.
Q was added and stored in a cool place until use.

On the other hand, the purified human arginase obtained in Reference Example 1 was diluted with the buffer B to prepare human arginase standard solutions of various concentrations. 100 μQ of this standard solution was added to each well of the immunoplate to which the monoclonal antibody was bound, and the reaction was allowed to proceed at 370° C. for 2 hours. Next, after washing each well with physiological saline, 100μ of the alkaline forsterase-labeled anti-human arginase antibody complex prepared in Reference Example 2 was added.
Q was added and reacted at 37°C for 2 hours, followed by washing with physiological saline. Next, the enzyme substrate solution ([1
1 g of 0.05M carbonate buffer (p) containing 1 g of mM MgCl
Add 100 μQ of 2 mg/mQ p-nitrophenyl phosphate solution dissolved in 9.8) and react for 30 minutes at room temperature.
100 μQ of a reaction terminator [3NNa Kano] was added and the reaction was carried out. Absorbance at 405 nm was measured using a blank as a control. The standard curve obtained using RID81 monoclonal is shown in FIG.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the relationship between human arginase concentration and absorbance obtained in Example 1. Figure 1

Claims (1)

[Scope of Claims] 1. An anti-arginase monoclonal antibody obtained using arginase of a non-human mammal as an antigen and capable of binding to human arginase 2. An anti-arginase monoclonal antibody obtained using a non-human mammal arginase Using an anti-arginase monoclonal antibody obtained using arginase as an antigen and capable of binding to human arginase, the concentration of human arginase in human serum or other human body fluids can be determined immunochemically. An immunochemical measurement method characterized by: