WO1996028733A1

WO1996028733A1 - Antibody specific for arginase originating in liver and use of the same

Info

Publication number: WO1996028733A1
Application number: PCT/JP1996/000644
Authority: WO
Inventors: Hiroshi Murayama; Hirohisa Kato; Atsuo Nagata; Masaki Ikemoto; Kunihiro Ueda; Toru Mori; Masayuki Totani
Original assignee: Yamasa Corporation
Priority date: 1995-03-16
Filing date: 1996-03-14
Publication date: 1996-09-19

Abstract

An antibody which binds specifically to an arginase originating in liver but substantially not to an arginase originating in erythrocyte; a method for specifically assaying an arginase originating in liver contained in a sample with the use of this antibody; and a kit to be used in this method. The specific assay of the arginase originating in liver is useful in the diagnosis of liver diseases wherein hepatocytes are broken or damaged or in monitoring the postoperative course of liver transplant.

Description

Description Antibody specific to liver-derived arginase and its use

The present invention relates to an antibody specific for liver-derived arginase, a method for specifically detecting or quantifying liver-derived arginase in a sample using the antibody, and a kit for performing the method. . Background technology ''

Arginase is an enzyme that breaks down arginine into ordinine and urea, and has been confirmed to be present in the liver, erythrocytes, and the like. The concentration of liver-derived arginase in the blood is usually about 2 ng Zm £, and when liver cells are destroyed or damaged, arginase quickly flows out into the blood, and liver-derived arginase in the blood. Significantly increased. Therefore, it has been reported that liver-derived arginase in blood is useful as a diagnostic marker for liver diseases (such as fulminant hepatitis and liver cancer) in which liver cells are destroyed or damaged. In addition to the above-mentioned liver diseases, its usefulness as an index for observing the postoperative course after liver transplantation has been reported.

Conventionally, several methods have been known as methods for measuring luginase in blood. For example, a method has been reported for measuring urea and L-orditin, which are the reaction products of arginase (Clin. Cem.-13.900 (1967). J. Biol. Chem. .. 199, 91 (1952)). Also, methods for immunologically measuring the amount of arginase itself in blood have been reported (Japanese Patent Application Laid-Open Nos. 194,262, 195,7999, and 199,791). No. 07676666, Clinical Chemistry, 39 (5). 794-799 (1993)).

Normally, when blood is collected from animals, including humans, and when the Z or the collected blood is pre-treated, the rupture or damage of some or all of the red blood cells (commonly referred to as "hemolysis") is completely eliminated. Therefore, liver-derived arginase and erythrocyte-derived arginase are mixed in most samples. However, the antibodies used in conventional immunoassays are liver-derived antibodies. Since it is not possible to distinguish between arginase and erythrocyte-derived arginase, the conventional immunoassay method can measure the total amount of arginase in blood, but specifically measures only liver-derived arginase. Can not.

Accordingly, an object of the present invention is to provide a method for preparing an antibody specific to liver-derived arginase and specifically detecting or quantifying liver-derived arginase in a sample using the antibody. is there. Disclosure of the invention

The present inventors have conducted intensive studies to achieve the above-mentioned object, and as a result, specifically bind to liver-derived arginase, and do not substantially bind to erythrocyte-derived arginase. The antibody was successfully prepared, and a method for measuring only liver-derived arginase in a sample using the obtained antibody was established, thereby completing the present invention.

Therefore, the present invention relates to an antibody (the antibody of the present invention), which specifically binds to arginase derived from liver and does not substantially bind to arginase derived from erythrocytes.

The present invention also relates to a method for specifically detecting or quantifying liver-derived arginase in a sample (the method of the present invention), which comprises using the antibody of the present invention.

Furthermore, the present invention relates to a kit (kit of the present invention) for carrying out the method of the present invention, which comprises the antibody of the present invention as an antibody reagent. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a standard curve for the sandwich ats (1) of Example 4. In FIG. 1, Arginase indicates human arginase derived from liver, and E-Arginase indicates human arginase derived from erythrocytes.

FIG. 2 shows a standard curve in the sandwich atsese (2) of Example 5. In FIG. 2, L-ARG indicates human arginase derived from the liver.

Figure 3 shows the results of the study of dilution linearity.

Corrected form (Rule 91) Figure 4 shows the results of measurement of liver arginase in the serum of a patient immediately after a liver transplant operation. In FIG. 4, AKBR indicates the blood ketone body ratio.

FIG. 5 shows the results of measurement of liver arginase in the serum of patients 15 to 37 days after liver transplantation. In FIG. 5, AST indicates aspartate aminotransferase, and ALT indicates laninaminotransferase. BEST MODE FOR CARRYING OUT THE INVENTION

The antibody of the present invention is an antibody characterized in that it specifically binds to liver-derived arginase and does not substantially bind to erythrocyte-derived arginase. As long as the antibody has such characteristics, it may be any of a polyclonal antibody and a monoclonal antibody, and among them, a monoclonal antibody is preferable.

Among these monoclonal antibodies, those having the following properties are particularly preferred.

(1) Coupling constant

The binding constant of the antibody to liver-derived arginase is at least 1 × 10 ⁸ (Liter Z mol).

(2) Specificity

Assuming that the reactivity with liver-derived arginase is 100%, the reactivity with erythrocyte-derived arginase is 5% or less.

Although the measurement sensitivity varies depending on the method of assembling the measurement system, it is considered that having such a binding constant is useful for measuring liver-derived arginase in a sample with high sensitivity. The above specificity is necessary for specifically measuring liver-derived arginase. In other words, the concentration of liver-derived arginase in a healthy human blood sample is around 2 ng Zm, whereas the concentration of erythrocyte-derived arginase in a hemolyzed blood sample varies depending on the degree of hemolysis. However, it is said to be 10 to 200 ng / m (average about 50 ng Zm). Therefore, in order to specifically measure liver-derived arginase in a healthy human blood sample, it is desirable to use an antibody having the above-mentioned cross-reactivity.

In this specification, “substantially no binding” is apparent from the above description of the specificity. Thus, assuming that the reactivity with liver-derived arginase is 100%, this means that the reactivity with erythrocyte-derived arginase is 5% or less. Such a monoclonal antibody can be prepared by appropriately applying a known method (for example, a cell fusion method, a transformation method, and the like) as described in detail in Examples below. In particular, in screening for a hybridoma that produces the desired monoclonal antibody, first, a primary antibody screen is searched for a specific antibody-producing hybridoma for arginase derived from liver, and then a secondary screening is performed for liver-derived arginase. It is important to search for differences in binding to both arginases using arginase derived from erythrocytes and erythrocytes, and select those with a large difference. The desired antibody of the present invention can be selected by such a screening method.

The antibody used for the measurement may be the antibody itself, but an antibody active fragment may also be used. The fragment of the antibody may be F (ab ') ₂ , Fab', or Fab, which has characteristics as an antibody. These antibody fragments should be prepared using a known method (for example, see molecular immunology 1 [1 “New Chemistry Laboratory Course 12”, pages 185-195, 1991). Can be.

Further, if necessary, the antibody is modified into a form suitable for the assay system (eg, an immobilized antibody, a labeled antibody, etc.), and used in the measurement method of the present invention.

Such modification of the antibody can be performed according to a conventional method. That is, as the material of the carrier used for preparing the solid-phased antibody, for example, polyvinyl chloride, polystyrene, styrene-divinylbenzene copolymer, styrene-maleic anhydride copolymer, nylon, polyvinyl alcohol , Synthetic organic polymers such as polyacrylamide, polyacrylonitrile, polypropylene, and polymethylene methacrylate; dextran derivatives (such as Sephadex); agarose gels (such as sepharose and biogel); and cellulose (such as paper discs and filter paper). And inorganic polymer compounds such as glass, silica gel, and silicone.These may be compounds having a functional group such as an amino group, an aminoalkyl group, a carboxyl group, an acyl group, or a hydroxyl group. . The material of the carrier is Tall ones are preferred.

The shape of the carrier can be flat (microplate, disk, etc.), particulate (beads, etc.), tubular (test tubes, etc.), fibrous, membrane-like, particulate (latex particles, etc.), capsule-like, An endoplasmic reticulum or the like is exemplified, and a carrier having an appropriate shape according to the assay method can be selected. In addition, it can be used as a carrier for immobilizing antibodies, such as liposomes (monolayer or multilayer lipid membrane).

The binding between the antibody and the carrier can be performed by a known method such as a physical adsorption method, an ionic bonding method, a covalent bonding method, or an inclusive method (for example, “immobilized enzyme” (edited by Ichiro Chibatake, March 20, 1980). , Published by Kodansha)). In particular, the physical adsorption method is preferred because it is simple. Further, the above-mentioned bonding may be performed directly, or may be performed via another substance (crosslinking agent) between both substances.

Labeling agents used for preparing labeled antibodies include radioisotopes (eg, ³² P, ³ H, ¹⁴ C, ¹²⁵ I), enzymes (eg, 3-galactosidase, peroxidase, alkaline phosphatase). Fatase, glucose-16-phosphate dehydrogenase: lyase, ribonuclease, glucose oxidase, lactate oxidase, alcohol oxidase, mono-minoxidase, etc., coenzyme 'prosthetic groups (eg, FAD, FMN. ATP, biotin, heme, etc., fluorescein derivatives (eg, fluorescein itchiosinate, fluorescein thiol bamil, etc.), rhodamine derivatives (eg, tetramethyl rhodamine B itchisocyanate, etc.) Fluorescent colors such as, umbelliferone and 1-anilino-1-naphthalenesulfonic acid Colloidal metal particles such as elemental, luminol derivatives (for example, noreminol, isorminol, N- (6-aminohexyl) -N-ethylisoluminol, etc.), gold, silver, platinum or compounds of these metals Etc. can be used.

The binding between an antibody and a labeling agent is described in a book [for example, “Seismic Chemistry Laboratory Lecture 5 Immunobiochemical Research Method”, Tokyo Kagaku Dojin, Ltd., published in 1996, No. 102-112. Page, Enzyme Immunoassay (Biochemical Experimental Methods 11), 196--252 (1992), Molecular Immunology III (New Chemistry Experimental Course 12), 1 27- 154 (1992), Enzyme Immunoassay, Second Edition, 82-144 (1992)] What is necessary is just to carry out by selecting suitably from such known methods.

The Atsey method employed in the method of the present invention is not particularly limited as long as it is a known method employed in the immunoassay, and any of the Atsey methods such as a competitive method, a sandwich method, an agglutination method, and a block overlay method can be used. Can be adopted.

For details of the measuring means in the adopted assay method, for example, the following literature may be referred to.

(a) Hiroshi Irie "Radio Immuno Atsushi" (Kodansha Co., Ltd., issued May 1, 1980)

(b) Eiji Ishikawa et al. “Enzyme immunoassay” (2nd edition) (Medical Publishing Co., Ltd., published on Feb. 15, 1982)

(c) Clinical Pathology Special Issue No. 53 “Immunoassy Technology and Application for Clinical Testing” (Clinical Pathology Publishing Association, published in 1983)

(d) “Biotechnology Encyclopedia” (CMC Co., Ltd., issued on October 9, 1998)

(e) Japanese Patent Publication No. Hei 6-43998, Japanese Patent Application Laid-Open No. 55-15010, Japanese Patent Application Laid-Open No. 52-148620, and Japanese Patent Application Laid-Open 9 1 2 9 6

(f) rMet ods in ENZYMOLOGY Vol. 70 '' (Immunochemical techniques (Part A)

(g) rMethods in ENZYMOLOGY Vol. 73 '' (Immunochemical techniques (Part B))

(h) rMethods in ENZYMOLOGY Vol. 74 ”(I unochemical techniques (Part 0)

(i) rMethods in ENZYMOLOGY Vol. 84 ”(I picture unochemical techniques (Part D: Selected Immunoassay))

(j) rMethods in ENZYMOLOGY Vol. 92 '' (Immunochemical techniques (Part E: Monoclonal Ant ibodies and General Immunoassay Methods))

[(f)-(: i) are published by Academic Press]

Among such atsie methods, the sandwich method can be particularly exemplified as a suitable atsie method. Among such sandwich methods, a sandwich method using a polyclonal antibody and a monoclonal antibody is preferable in consideration of measurement sensitivity and specificity. In addition, a sandwich method using two types of monoclonal antibodies can also be adopted as a preferable method. As is clear from the examples described later, a highly specific model is used.

6

Corrected form (Rule 91) Combinations using noclonal antibodies and low specificity monoclonal antibodies are particularly preferred.

The test sample to be measured is not particularly limited as long as it contains a liver-derived kininase. The test sample may be diluted with a buffer solution or the like, if necessary, and used in the method of the present invention. When blood and its fractions (serum, plasma, etc.) are used as the test sample, the destruction or damage of red blood cells should be prevented as much as possible when collecting blood and pre-processing the collected blood. It is important.

The kit used for measurement is characterized by containing the antibody of the present invention as a component reagent of the kit, and the other component reagents of the kit include the necessary reagents (depending on the assay method used). Washing solution, substrate solution, reaction stop solution, etc.) may be appropriately combined. When the sandwich method is adopted as the Atsey method, for example, a kit comprising the following constituent reagents can be exemplified.

①Immobilized first antibody

② Labeled second antibody solution

③ Antigen solution of known concentration

In the above kit, the antibody is an antibody of the present invention that specifically binds to liver-derived arginase, and the combination thereof may be any one of a polyclonal antibody-monoclonal antibody or a monoclonal antibody-monoclonal antibody. Absent. In addition, the first antibody and the second antibody may recognize the same antigenic determinant on liver-derived arginase, or may recognize different antigenic determinants.

In the above kit, a labeled anti-immunoglobulin antibody or the like may be used instead of the labeled secondary antibody solution, and labeling may be performed indirectly, in which case, the following kit is exemplified. Is done.

①Immobilized first antibody

②Second antibody solution

③ Labeled anti-immunoglobulin antibody solution

0) Antigen solution of known concentration Using such a kit of the present invention, liver-derived arginase in a test sample can be detected or quantified by an operation method known per se.

For example, when the above-mentioned kit is used, a method suitable for reacting the test sample with the immobilized first antibody, then reacting the labeled second antibody, and then measuring the employed labeling substance Either measure the labeled substance by (two-step method) or react the immobilized antibody I, test sample and labeled second antibody simultaneously, and then use a method suitable for measuring the adopted label. By measuring the labeling substance (one-step method), it is possible to detect or quantify liver-derived arginase in the test sample. Example

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited thereto.

Example 1: Preparation of various arginase

As the liver-derived human arginase (hereinafter referred to as “liver arginase”), lycopinnant human liver arginase prepared by a recombinant DNA technique was used (Japanese Patent Laid-Open Publication No. 2-117383, Biochem. J. 270, 697-703 (1990), Proc. Natl. Acad. Sci. USA. 84.412-415 (1987)).

Erythrocyte-derived human arginase (hereinafter, referred to as "erythrocyte arginase") was extracted and purified from human erythrocytes (Ann. Π in. Biochem., 26, 547-553 (1989)) ο

Example 2: Preparation of monoclonal antibody

1) Preparation of monoclonal antibody-producing hybridoma

50 g of liver arginase with Freund's complete adjuvant at 6-8 weeks of age

BALB / c mice were dosed intraperitoneally four times every 2-3 weeks. Two weeks after the fourth immunization, blood was partially collected and the antibody titer against liver arginase was examined by the following ELISA method.

That is, liver arginase was diluted to 1 gZm with PBS, dispensed 50 times into a flexible assay plate (Falcon), and allowed to stand at 4 ° C. After washing 3 times with PBS, 0.5% skim milk solution is dispensed at 200 / ml and room temperature For 1 hour. The skim milk solution was removed, and the serum of the mouse, which was serially diluted with PBS containing 1% BSA, was dispensed in 50 ί portions, and allowed to stand at room temperature for 1 hour. After washing three times with PBS, a solution prepared by diluting horseradish peroxidase (HRPO) sensible goat anti-mouse IgG antibody (Zymed) to 1Z500 with PBS containing 1% BSA was added to 50%. The mixture was dispensed at a time and left at room temperature for 1 hour. Substrate solution (0.3 mM 3,3 ', 5,5'-tetramethylbenzidine dihydrochloride, 0.05% hydrogen peroxide in 0.2M citrate buffer, pH 3.8) Each was dispensed, and allowed to stand at room temperature for 5 minutes to develop color. The reaction by adding 1 N sulfuric acid one 00 stops, the most antibody titer of 4 5 0 nm liver arginase 1 0 g dissolved in _c saline and absorbance was measured at using a microplate follower Tome Isseki one A final immunization was performed by intravenous administration to the mouse, which had been high.

Three days after the final immunization, the spleen of the mouse was removed, and this spleen cell and mouse myeloma cell P3X63Ag8U.1 (P3U1) (ATCC CRT-15997) were added to Keller and Milun Utain. Cells were fused according to the method. That is, spleen cells and myeloma cells were mixed at a ratio of 10: 1, and the pellet obtained by centrifugation was gradually added with 1 mi of RPMI 1640 solution containing 50% polyethylene glycol to remove cells. Fused. Further, RPMI 1640 medium was added to this to make 1 Om ^, and the pellet obtained by centrifugation was changed to P3U1 in RPMI 164 medium containing 10% fetal calf serum (FCS). Then, the suspension was suspended at a concentration of 3 × 10 ⁴ 1100 μ u, and each plate was dispensed into 10 pieces of 10 pieces of 96-well mic mouth tie plate.

One day later,? One eight-choice medium was added to 100 ^, and half of the medium was replaced with fresh HAT medium every 3 to 4 days.

Seven days after the fusion, the culture supernatant was sampled, and screening was performed using the culture supernatant instead of the mouse serum of the above ELISA method. First, as a primary screening, a specific antibody positive for liver arginase was searched (see Table 1 below), and using liver arginase and erythrocyte arginase as the secondary screening, the difference in binding to both arginases was examined. did. As a result of the screening, a gel deemed to have high specificity for liver arginase was selected and subjected to cloning at least twice by the limiting dilution method. As a result, antibody-producing hybridoma 11 clones against liver arginase (2E11, 4C9, 5C8, 5F1, 6G3, 6G7, 8C9, 8G3, 9 E9, 9G2, 10E11) were established, dispensed into glass vials, and stored frozen in liquid nitrogen. Among them, Hypri-Doma 6G3 and Hypri-Doma 4C9 were developed by the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology, Institute of Biotechnology and Industrial Technology (address: 1-3-1 Higashi, Tsukuba, Ibaraki, Japan (: 〒30 5)) ARG 6G3 and ARGB 4C9 were deposited internationally on January 25, 1995 based on the Bush Treaty on January 25, 1995, and the deposit numbers were issued on January 25, 1995. FERM BP-497 and FERM BP-497, respectively.

Table 1 Number of specific antibody positive gels Z number of proliferation gels Z total number of gels

6 0 9 4 0 9 4 0

2) Production and purification of monoclonal antibodies

Each established hybridoma was cultured, and 3 × 10 ⁶ mice were administered intraperitoneally to mice to which pristane (Aldrich) had been administered in advance, and ascites was collected 8 to 14 days later.

The ascites fluid collected was mixed with 1.5 M glycine hydrochloride buffer containing 3 M sodium chloride, mixed with H8.9 and H: 1, and then filtered through a 0.22 m membrane filter. The solution was applied to a Protein A Sepharose CL-4B (Pharmacia) column equilibrated with the liquid. After washing with a sufficient amount of the same buffer, the antibody was eluted with 0.1 M citrate buffer, pH 6.0. The eluate was dialyzed against PBS, and the purity was confirmed by SDS-polyacrylamide gel electrophoresis to obtain a purified monoclonal antibody. Example 3: Properties of monoclonal antibody

1) Iso type

After coating liver arginase, add the culture supernatant of each hybridoma to a 96-percent PVC plate that has been blocked with 0.5% skim milk, and use the MONOAB-ID kit (Zymed) to detect the antibody. An isotype search was performed. The results are shown in Table 2. Table 2

2) Coupling constant

The binding constant of the monoclonal antibody to liver arginase was determined by adding unlabeled liver arginase to ¹²⁵ I-labeled liver arginase 100 ^ (approximately 20, OOO cpm) and 0, l CdgZm monoclonal antibody 100 at different concentrations. After standing at room temperature for 4 hours, it was determined by measuring ¹²⁵ I-labeled liver arginase bound to the monoclonal antibody (Immunological Experiment Procedures IX, supplemented in 1990, pp. 2691-2705) Etc.). Measurement of bound ^'25 I labeled liver arginase monoclonal antibodies precipitated by the addition of a solution 500 was diluted 200-fold mouse serum 1 0 0〃 and catcher formic anti-mouse I gG with 3% PEG, and centrifuged After removing the supernatant, the radioactivity was measured. Liver arginase labeled with ¹²⁵ I was obtained by mixing 0.3% chloramine T in a test tube containing a mixture of liver arginase solution 20 (20 ^ g) and Na ¹²⁵ I \ u £ (0.1 mC i). solvent solution 5, were successively added 0.5% sodium metabisulfite solution 1 0, 1% potassium iodide dissolved solution 5, 1% BSA solution 5〃F, subjected to PD 1 0 column (Farman §), free ¹²⁵ Prepared by separation from I.

Table 3 shows the binding constants of the monoclonal antibodies thus determined.

Table 3

Hybr idoma 2E11 4C9 5C8 5F1 6C3 6G7 Coupling constant 1.84x10 ^s 1.66X10 ⁹ 3.63X10 ⁹ 3.66X10 ⁹ 2.57X10 ³ 3.52X10 ⁹

C mo)

Hybr idoma 8C9 8G3 9E9 9G2 10E11

Coupling constant 6.60x10 ^s 2.18X10 ⁹ 3.27X10 ⁹ 4.18xl0 ⁹ 3.22X10 ⁹ Example 4: Sandwich Atssey (1)

1) Enzyme labeling of polyclonal antibody

It was prepared according to a known method (Clinical Chemistry. 39 (5). 794-799 (1993)). That is, the anti-arginase antiserum was precipitated with 60% ammonium sulfate and then applied to a DEAE-cellulose column. Further, the obtained fraction was applied to a Sepharose 4B column on which arginase was immobilized, and subjected to affinity purification. The purified antibody was filtered through a 0.1 M sodium acetate buffer (pH 4.5) containing 0.1 NaC, pepsin was added, the plate was allowed to stand at 37 ° C for 30 minutes, and then 2M Tris-HCl buffer ( The solution was neutralized at pH 8.0). This was applied to a Sephadex G-50 column. To the obtained F (ab ') ₂ fraction was added 19 volumes of 0.1 M 2-mercaptoethylamine, 0.1 M sodium phosphate buffer (pH 6.5) containing 5 mM EDTA. The reaction was carried out for 90 minutes. This was applied to a Sephadex G-50 column, and the Fab 'fraction was collected.

The Fab * thus obtained was reacted with HRPO into which a maleimide group had been introduced, and labeled. That is, HRPO (6 ng Zm) dissolved in 0.1 M sodium phosphate buffer (pH 7.0) was reacted with 5 OmM SMCC for 30 minutes, and 0.1 M sodium phosphate containing 5 mM EDTA was used. The mixture was applied to Sephadex G-50 equilibrated with a buffer (pH 6.0), and the fraction containing HRPO was collected. The obtained fraction was reacted with Fab 'for 30 minutes, and stopped by adding 50 mM N-ethyl maleimide. The reaction solution was applied to Sephadex G-50 equilibrated with 0.1 M sodium phosphate buffer (pH 6.5), and fractions containing Fab'-HRPO were collected. The labeled antibody thus obtained was used for the following measurement.

2) Standard curve

The anti-liver arginase monoclonal antibody 4C9 was diluted to 5 g / m with 75 mM carbonate buffer (pH 9.5), dispensed into 96-well plates (Falcon) at 50 ° C.ー晚 Stood still. After washing with PBS containing 0.05% Tween 20 three times, 200% of 2% BSA was dispensed and left at room temperature for 1 hour. After removing the BSA solution and washing three times with PBS containing 0.05% Tween 20, a liver arginase standard solution (liver arginase is 0, 2.5, 5, 10, 25, Diluted with PB containing 2% BSA to 50, 100 ng gZm) or hemolyzed serum (10.5, 17.3, 31.5, 155, 28 with erythrocyte arginase) 3,

470, 850, and 140 ng gZm) were dispensed at 50 times and left at room temperature for 1 hour. After washing three times with PBS containing 0.05% Tween 20, HRPO-labeled anti-arginase Fab 'antibody was diluted with PB containing 2% BSA to 1 /

The solution diluted to 500 was dispensed in 50 portions, and left at room temperature for 1 hour. After washing three times with PBS containing 0.05% Tween 20, the substrate solution (0.3 mM 3, 3 ', 5, 5'-tetramethylbenzidine dihydrochloride, 0.05% hydrogen peroxide solution) A 0.2 M citrate buffer solution (pH 3.8) was dispensed in 100 ^ portions, and allowed to stand at room temperature for 10 minutes to develop color. The reaction was stopped by adding 100 N of 1 N sulfuric acid at a time, and the absorbance at 450 nm was measured using a microplate photometer. Fig. 1 shows the obtained standard curve.

3) Specificity

According to the procedure for obtaining the standard curve, liver arginase and erythrocyte arginase were measured using each monoclonal antibody as an immobilized antibody, and a standard curve was prepared. The reactivity to erythrocyte arginase when the reactivity with liver arginase was set to 100% was calculated from the obtained standard curve.

As a result, as shown in Table 4, the reactivity to erythrocyte arginase was found to be at most about 500 times lower than that of liver arginase. Considering the ratio of hepatic arginase to erythrocyte arginase in serum samples, the reactivity with erythrocyte arginase should be at least 5% or less when the reactivity with hepatic arginase is 100%. Seems to be desirable.

Table 4

Hybridoma 2E11 4C9 5C8 5F1 6G3 6G7

Reactivity 100 0.2 32 30 100 30

Hybridoma 8C9 8G3 9E9 9G2 10E11

Reactivity 35 4 18 22 22 In addition, a polyclonal antibody that had been affinity-purified in the same manner as in 1) above was used as an immobilized antibody, and a monoclonal antibody that was detected in the same manner as in 1) in Example 5 described below was used as a detection antibody, and The specificity of the monoclonal antibody was determined by the same method.

As a result, the monoclonal antibody 5F1 was 4.5% reactive with erythrocyte arginase when the reactivity with liver arginase was set at 100%, which is a preferred antibody for use in the present invention. It can be said that.

As described above, the specificity of the monoclonal antibody may differ depending on the ability to use the antibody as a solid-phased antibody or whether the antibody is used as a labeled antibody. Then, an antibody having a reactivity with erythrocyte arginase of 5% or less when the reactivity with liver arginase determined by either one of the above methods is 100% may be used.

Example 5: Sandwich Atssey (2)

1) Enzyme labeling of monoclonal antibody

The monoclonal antibody was labeled with HRP〇 by the periodate method. That is, the monoclonal antibody was dialyzed once at 4 ° C. against a 0.1 M sodium carbonate buffer (pH 9.5) to prepare a concentration of 8 mg / m 2. HRPO (Toyobo) was prepared with distilled water to a concentration of 4 mgZm ^, added with 0.1 M sodium periodate solution 200, and allowed to stand at room temperature for 20 minutes. After overnight dialysis against 1 mM sodium acetate buffer (pH 4.0), add 0.2Μ sodium carbonate buffer (pH 9.5) to the HRPO solution, and add ρ Η 9.0 and ρ Η 9. Adjusted to between 5 and at the same time 0.5 μl of the monoclonal antibody solution was added and stirred. After gently stirring at room temperature for 2 hours, 50 mg / m of 4 mg / m sodium borohydride was added, and the mixture was allowed to stand at 4 ° C for 2 hours. The solution was dialyzed once against 2 liters (L) of PBS, and the obtained product was used for the following measurement.

2) Standard curve

Dilute 6G3 or 8G3 of anti-liver arginase monoclonal antibody to 5 g / m with 75 mM carbonate buffer (pH 9.5), and aliquot 50 9 into 96 プレー elplate (Falcon). It was poured and left still at 4 ° C. After washing 3 times with PBS containing 0.05% Tween 20, 2% 83-8 was dispensed at 200 "f each and left at room temperature for 1 hour. It was left still. Remove the BSA solution and use liver arginase standard solution (liver arginase or 0, 1, 2.5, 5, 10, 25, 50, 100 ng gZm with PB containing 2% BSA). Dilution) or hemolyzed serum (prepared to give erythrocyte arginine or 2, 35, 63, 104, 189, 311, 933, 1700 ng gZm) The mixture was dispensed by 0 and left at room temperature for 2 hours. After washing three times with PBS containing 0.05% Tween 20, the HRP0-labeled anti-hepatic arginase monoclonal antibody 4C9 diluted to 1 gZm with PB containing 2% BSA was dispensed 50 times. The mixture was allowed to stand at room temperature for 2 hours. After washing three times with PBS containing 0.05% Tween η20, the substrate solution (0.3 mM 3, 3 ', 5, 5'-tetramethylbenzidine dihydrochloride, 0.05% hydrogen peroxide 0.2 M citrate buffer solution (pH 3.8) containing water was dispensed 100 times each, and allowed to stand at room temperature for 10 minutes to develop color. The reaction was stopped by adding 1 N sulfuric acid 100 by 100, and the absorbance at 450 nm was measured using a microplate photometer. The obtained standard curve is shown in FIG.

As a result, when comparing the sandwich method using the combination of 6G3 and 4C9 with the sandwich method using the combination of 8G3 and 4C9, the combination of 6G3 and 4C9 was considered in terms of measurement sensitivity and specificity. Found that the sandwich method was superior.

3) Dilution dilute property

According to the procedure for obtaining the standard curve, patient serum was diluted with OngZm ^ standard solution, and the dilution linearity was examined.As a result, a straight line passing through the origin as shown in Fig. 3 was obtained. It became clear what to show.

4) Recovery

According to the procedure for obtaining the standard curve, a standard solution was added to the patient's serum, and the recovery was examined. As a result, a favorable result of 96.8% was obtained.

5) Determination of serum of healthy human

According to the procedure for obtaining the standard curve, the liver arginase concentration of the serum of a healthy individual was measured, and the result was 2 ngZm on average.

6) Measurement of patient serum

According to the procedure for obtaining the standard curve, two patients who underwent liver transplant surgery over time Liver arginase in the collected serum was measured. In the first case, as shown in Fig. 4, the liver function immediately after the operation was examined. The blood ketone ratio (AKBR), an indicator of liver function, decreased immediately after surgery, indicating a decrease in liver function. In contrast, arginase levels increased, indicating a potential indicator of liver function.

In the second case, rejection after surgery was examined as shown in FIG. Postoperative rejection resulted in elevated levels of liver marker enzymes such as AST (aspartate aminotransferase) and ALT (arananaminotransferase). Correspondingly, liver arginase levels also increased, indicating its potential as an indicator of rejection. Industrial applicability

As is clear from the above examples, the antibody of the present invention is an antibody that does not substantially bind to erythrocyte-derived arginase and specifically binds to liver-derived arginase. By using the above antibody, it is possible to specifically measure liver-derived arginase in the sample. Therefore, the antibodies, methods and kits of the present invention are useful for diagnosing liver diseases such as rupture or damage of liver cells or for monitoring postoperative progress after liver transplantation.

Claims

Claims: An antibody which specifically binds to liver-derived arginase and does not substantially bind to erythrocyte-derived arginase. The antibody according to claim 1, wherein the antibody is a monoclonal antibody. The antibody according to claim 1, wherein the antibody is a monoclonal antibody and has the following properties.

(1) Coupling constant

The binding constant of the antibody to liver-derived arginase is greater than 1 × 10 ⁸ (liter zmol).

(2) Specificity

A method for specifically detecting or quantifying liver-derived arginase in a sample, comprising using the antibody according to any one of claims 3 to 3.

Claims 1 to 3 are used as the solid-phased antibody, and those obtained by binding the antibody according to any one of the claims to a carrier are used as the labeled antibody, or an antibody that binds to arginase. 5. The method according to claim 4, wherein an antibody obtained by labeling the antibody according to any one of the above is used to specifically detect or quantify liver-derived arginase in a sample by Sandwich tissue.

5. The method according to claim 4, wherein the sample is one in which liver-derived arginase and erythrocyte-derived arginase coexist.

A kit for specifically detecting or quantifying liver-derived arginase in a sample, comprising the antibody according to any one of claims 1 to 3 as an antibody reagent.

An immobilized antibody obtained by binding the antibody according to any one of claims 1 to 3 to a carrier and an antibody that binds to arginase or an antibody obtained by labeling the antibody according to any one of claims 1 to 3 9. A liver antibody-derived arginase in a sample is specifically detected or quantified by Sandwich tissue analysis using a labeled antibody. The kit