JPH06327492A - Modified alpha1-antitrypsin-resistant monoclonal antibody, hybridoma producing same and method for measuring modified alpha1-antitrypsin with same - Google Patents

Abstract

PURPOSE:To provide a method for accurately and simply detecting or determining only m-AT. CONSTITUTION:A monoclonal antibody which reacts with modified alpha1-antitrypsin and not with a1-antitrypsin and denatured alpha1-antitrypsin.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an anti-modified α ₁ -antitrypsin monoclonal antibody, a hybridoma producing the same, and a method for measuring modified α ₁ -antitrypsin using the same. The present invention is useful for diagnosis of cancer, myeloid acute leukemia and rheumatism.

[0002]

BACKGROUND ART α ₁ -antitrypsin (hereinafter sometimes referred to as AT) is one of the most abundant protease inhibitors present in serum and exhibits inhibitory activity against various serine proteases. It is considered to have a physiological function of protecting the tissue damage caused by elastase. It also has the property of being an acute phase reactive protein, and it is known that it tends to increase in cancer as well (for example, Shunsuke Ueda et al .: Acute phase proteins from the viewpoint of molecular evolution, infection / inflammation / Immunity, 14: 339, 1984). Furthermore, when looking at the relationship between AT and the progression of malignant tumors,
It has also been confirmed that it will become even higher in metastatic and terminal cancers. However, such a tendency is AT
Not limited to, alpha ₁ - acid glycoprotein, alpha ₁ - antichymotrypsin, which is also seen more or less acute phase reactive protein in general, such as C-reactive protein (CRP), quantifying the AT It does not make cancer diagnosis possible. Regarding protease inhibitor activity, it is known that the total activity of AT in cancer patients is low (Rajender K. Chawla et al .: Cancer Res., 44, 2
718-2723, 1984). In addition, it has been reported that some ATs of cancer patients have the same molecular weight as ATs in normal humans and do not have protease inhibitor activity (Ra.
jender K. Chawla et al .: Cancer Res., 47, 1179-118
4, 1987). However, no material difference between this inactivated AT (hereinafter sometimes referred to as denatured AT) and normal AT has been clarified, and therefore specific detection is difficult.

Further, regarding a substance that decomposes AT, it is inactivated by cleaving the C-terminal side serine protease inhibitor active site, that is, modified α ₁ -antitrypsin (modified-AT; hereinafter referred to as m-AT). However, it is said that there is no difference in antigenicity from AT (Japanese Clinical Volume 43, Autumn Extra Edition (1985)).

Regarding this m-AT, it was reported that GP41 in the urine of drug-treated patients with myeloid acute leukemia (the same as m-AT in terms of amino acid sequence) serves as a monitor for the therapeutic effect (Biol. Chem. Hoppe. -Syeler Vol. 373, pp.581-
588, July 1992), and m in joint exudates of patients with rheumatism.
-A report that AT exists (literature: Zhang Z. et al.,
Biochemical Society Transactions, Vol. 18, pp. 898
-899 (1990)). Therefore, if m-AT can be detected immunologically and specifically, myeloid acute leukemia and rheumatism can be easily diagnosed.

As an attempt to quantify m-AT, the above G
In the report on P41, m-AT was changed to SDS-PAGE.
And immunostaining with an anti-AT antibody (immunoblotting on a membrane) and scanning the concentration have been attempted (Biol. Chem. Hoppe-Syeler Vol. 373, pp.581-588, J.
uly 1992) has a drawback that the operation is complicated.
Further, in this report, preparation of an anti-GP41 monoclonal antibody (anti-m-AT antibody) is also described. However, since the prepared antibody has cross-reactivity with AT, it can be used only for m-AT. It cannot be measured specifically. Other than the above reports, no literature has been known that attempts to quantify m-AT. By cleaving the active site of AT, m-
Since AT is produced, m-AT does not show protease inhibitory ability. Focusing on this point, for example, the total AT amount and the serum trypsin inhibitory capacity (STI) measured by the single radial immunodiffusion method (SRID).
A method of calculating by comparing with the amount of active AT according to C) is also conceivable. However, the quantitative value of inactivated AT by this method includes not only m-AT inactivated by cleavage of the active site but also the above-mentioned modified AT amount in the measured value. Normally, m-AT is metabolized in the body faster than α ₁ -antitrypsin-elastase complex but faster than AT (Alan
E.Mast et al .: Biochemistry, 30 , 1723-1730), it is estimated that the amount of m-AT corresponds to only a small part of inactivated AT. Further, the STIC value is approximately regarded as the activity value of AT because AT exists in an extremely large amount, but strictly speaking, not only AT but α ₁ -antichymotrypsin, α ₂ -macroglobulin, inter α trypsin inhibitor, Since it is the total activity of all serine protease inhibitors in serum such as C1 inhibitor, it does not give an accurate quantitative value. Therefore, an attempt to quantify m-AT by comparing the total AT amount with the active AT amount is not realistic because the influence of noises other than m-AT or noises other than AT is large. Further, in the course of research until the completion of the present invention, the present inventors have found that a monoclonal antibody M801 that reacts with m-AT but does not react with AT (Microtechnology Research Institute, No. 12942, Japanese Patent Application No. 5-132600). (Patent application 4-
143479)) was prepared. However, this antibody had the property of reacting strongly with denatured AT.

As a result of the study conducted by the present inventors, denatured AT may be produced in the preserved sample, and m-AT in the preserved sample may be generated.
Monoclonal antibody M80
In the ELISA method using 1, the denatured AT was observed as noise, which adversely affected the measurement results.

[0007]

The object of the present invention is to provide m-
It is to provide a means for accurately or simply detecting or quantifying AT alone.

[0008]

As a result of earnest research, the inventors of the present invention have surprisingly found that although it reacts with m-AT, A
The present invention has been completed by succeeding in producing a monoclonal antibody that does not react with T and modified AT.

That is, the present invention responds to m-AT,
A monoclonal antibody that does not react with AT and modified AT is provided. The present invention also provides a hybridoma producing the above-described monoclonal antibody of the present invention. Furthermore, the present invention provides a method for immunologically measuring m-AT in a sample by utilizing the specific reaction between the above-mentioned monoclonal antibody of the present invention and m-AT. As will be described later, the present inventors have found for the first time that the use of the antibody of the present invention surprisingly enables accurate diagnosis of cancer. Therefore,
INDUSTRIAL APPLICABILITY The present invention can contribute to the diagnosis of cancer in addition to the diagnosis of myeloid acute leukemia and rheumatism, which were conventionally considered to use m-AT as a marker.

The present invention will be described in detail below.

The anti-m-AT monoclonal antibody of the present invention is characterized by having no reactivity with AT and reacting with m-AT which is a partially degraded form of AT. Further, no reactivity with modified AT is observed. Modified AT is
As described above, it may be generated in the stored sample, which is a problem when trying to measure m-AT in the stored sample.
Since the monoclonal antibody of the present invention does not react with denatured AT, m-AT in a stored sample can be accurately measured.

[0012] Since m-AT has the inhibitory activity domain of AT cleaved and the cleaved C-terminal fragment having a molecular weight of about 4000 and the remaining part are non-covalently bonded, m-AT is compared with AT in the amino acid sequence. There is no AT-specific part. Therefore, even if an attempt was made to produce an antibody that reacts with m-AT, it was considered that it always cross-reacted with AT or modified AT, and in the case of GP41 (Biol. Chem. Hoppe-
Syeler Vol. 373, pp.581-588, July 1992), and the present inventors' attempts so far have been so. However, the present inventors surprisingly responded well to m-AT after earnest research, but AT
We succeeded in producing an m-AT-specific monoclonal antibody that does not react with modified AT at all. Probably A
By cleaving the serine protease inhibitor active site of T, a part of the remaining three-dimensional structure is m-
It is considered that AT is altered as compared with that of AT, and the present inventors presume that the monoclonal antibody of the present invention recognizes a portion having this altered three-dimensional structure as an epitope. There is.

As an immunogen for producing the anti-m-AT monoclonal antibody of the present invention, AT is treated with a specific protease to specifically cleave the serine protease inhibitor active domain present at the C-terminal side of the AT molecule. The m-AT prepared in this way can be used. Specifically, m-AT is, for example, Hoppe-Seyler's Z.
It can be isolated by the method described in Phisiol. Chem. 363, 1377-1388, 1982. That is, purified AT and chymotrypsinogen A were reacted in equimolar amounts at 37 ° C. for 4 days and then subjected to gel filtration chromatography at 48 k.
It is possible to separate m-AT having a molecular weight of D. Also,
The present inventors have found another method for separating m-AT with good quantitativeness. That is, after reacting AT and elastase in a molar ratio of 1: 1, AT-elastase complex is isolated by anion exchange chromatography, and a small amount of elastase is added to the complex. After reacting this for a certain period of time, m-AT can be isolated by performing anion exchange chromatography again. This method is detailed in the examples below.

Then, the mA obtained as described above is used.
Animals are immunized using T as the immunogen. Immunization methods are basically known methods such as Monoclonal Antibod.
ies: Principles and Practice, James W. Goding, pp.
56-87, Academic Press Inc., the anti-m-AT monoclonal antibody of the present invention is preferably immunized by treating m-AT with glutaraldehyde. A method used as a source (glutaraldehyde treatment method) or a method of immunizing an animal with an antiserum obtained by immunizing an animal with m-AT and then immunizing an animal with an m-AT (antiserum method) can be efficiently obtained. it can. These methods are detailed in the examples below.

After collecting immunogenic cells (spleen cells, lymphocytes, etc.) from animals immunized with m-AT by the above-mentioned method, a known method (eg Monoclonal Antibodies: Pri
nciples and Practice, James W. Goding, pp.56-87, A
The monoclonal antibody of the present invention can be obtained by the method described in Academic Press Inc.). That is, splenocytes are removed from the immunized animal and fused with myeloma cells. Upon fusion, polyethylene glycol (PEG) can be used as a fusion accelerator. After the fusion operation, only the fused cells (hybridomas) are selected by culturing in a usual selection medium, for example, HAT medium, and then the hybridomas producing the desired antibody are screened and cloned. The screening can be carried out by a general method, for example, an ELISA method, but preferably, a method in which the antigen is not immobilized on the solid phase, that is, the target antibody is immobilized on the solid phase via an anti-immunoglobulin antibody. It is preferable to use a method in which the antigen is reacted with the antibody (so that the liquid phase condition is achieved). This screen responds well to m-AT, but to AT and denatured A
Select one that does not react with T. In addition, cloning can be performed, for example, by the limiting dilution method, whereby a monocloned hybridoma can be obtained. The monoclonal antibody produced by the obtained hybridoma can be obtained by a method in which the hybridoma is intraperitoneally administered to the mouse and recovered from the ascites, or a method in which the hybridoma is cultured and separated from the culture supernatant. Further, it can be purified using a general method, that is, ammonium sulfate precipitation, gel filtration, ion exchange chromatography and the like.

As described in detail in the Examples below, 13 kinds of hybridomas were obtained by the above method. Seven of these hybridomas were cloned to obtain stable clones with high specificity for m-AT. M2406, M2409, M3603, M
It was named 3606, M3610, M3616, M3618. Among them, hybridomas producing M3616 and M3606 have been deposited at the Institute of Biotechnology, Institute of Industrial Science, respectively, and the deposit numbers are FERM P-13477 and FERM P-1347, respectively.
It is 6.

Using the anti-m-AT monoclonal antibody of the present invention, m-AT in a sample can be specifically immunologically detected or quantified by a conventional method. Any of the immunoassay methods conventionally used in this field can be adopted. For example, immunoassay can be performed by the two antibody method, the competition method, the agglutination method, or the like. The two-antibody method is known as a sandwich method, and the two-antibody method is preferably used for the purpose of high-sensitivity measurement. Two
When the measurement is carried out by the antibody method, the monoclonal antibody of the present invention (first antibody) and a polyclonal antibody against the immunoglobulin of the animal used for immunization or a monoclonal antibody against m-AT (which may be the monoclonal antibody of the present invention) are used. One of the different antibodies (second antibody) is immobilized on the solid phase, and the other is immobilized directly by radioisotope, enzyme, fluorescent substance, etc. or indirectly through avidin, biotin (or their derivatives), etc. It can be used by labeling. That is, the antibody can be used by labeling with ¹²⁵ I, horseradish peroxidase, β-galactosidase, alkaline phosphatase and the like. The measurement procedure can be performed as follows, for example. Add the monoclonal antibody of the present invention to a 96-well microtiter plate and
After physically adsorbing at 0 ° C. overnight, the solution is removed, and blocking is performed by adding a blocking agent such as BSA to prevent nonspecific adsorption. Regarding the reaction between the immobilized antibody and the test liquid, the test liquid is added to the wells of the plate and reacted for a certain period of time. This reaction is usually carried out preferably at 37 ° C. for 1 hour or at 4 ° C. overnight. After completion of the reaction, the liquid is removed and then washed, and horseradish peroxidase-labeled antibody is added and reacted. The labeled antibody need only react with m-AT, and need not be specific to m-AT, that is, only m-AT. Therefore, for example, an anti-AT polyclonal antibody that also reacts with m-AT (commercially available), an anti-GP41 monoclonal antibody that also cross-reacts with the aforementioned AT, or a research process until the completion of the present invention A monoclonal antibody that does not react with AT prepared by the present inventors but reacts with m-AT and modified AT (an anti-BP monoclonal antibody described in Japanese Patent Application No. 4-143479. For example, M801, Microtechnology Research Institute No. 12942).
No.) may be labeled and used. The reaction conditions may be such that the antigen-antibody reaction is carried out and the enzyme activity is maintained, but it can usually be carried out at room temperature for 1 hour. After the reaction of the labeled antibody is completed, the wells are thoroughly washed, o-phenylenediamine is added as an enzyme substrate to react, and after a certain period of time, sulfuric acid is added to stop the reaction and the absorbance of the solution is measured and quantified. .

The above anti-BP monoclonal antibody can be prepared by the following procedure.

[0021] Galactose transferase (GT) and an m-AT-like substance (Binding Prot) that is natively associated with GT
Ein, BP) aggregate (GT-BP), ascites of cancer patients,
For example, ascites from an ovarian cancer patient, Cancer Research Volume 48,
According to the method described in page 5325, 1988, GT is separated as a coexisting substance by α-lactalbumin affinity chromatography, and most of GT and a small amount of BP and GT are contained in the sample thus obtained. Since there is an aggregate of the above, the latter can be separated by adsorbing only the aggregate by elution by affinity chromatography of an anti-AT antibody to obtain only the latter. In this case, the anti-AT antibody may be either a monoclonal antibody or a polyclonal antibody, but in the case of a monoclonal antibody, a polyclonal antibody is preferable because it is necessary to select an antibody that also reacts with an m-AT-like substance. . However, the step of separating only the latter is not necessarily required for the purpose of producing a monoclonal antibody against the m-AT-like substance. Rather, it is preferable not to do so because there is a risk of denaturing the aggregate (particularly the m-AT-like substance portion) due to elution conditions.

The monoclonal antibody against the m-AT-like substance (anti-BP monoclonal antibody) can be used to immunize a mammal by a general method using the immunogen isolated according to the above-mentioned procedure. That is, the above immunogen can be administered intraperitoneally or intravenously together with an adjuvant such as the Libi-adjuvant system.

Next, a monoclonal antibody can be obtained by a method known in the art by cell fusion between spleen cells collected from an immunized animal and myeloma cells. That is, splenocytes are removed from the immunized animal and fused with myeloma cells. Upon fusion, polyethylene glycol (PEG) can be used as a fusion accelerator. After the fusion operation, only the fused cells (hybridomas) are selected by culturing in a usual selection medium, for example, HAT medium, and then the hybridomas producing the desired antibody are screened and cloned. The screening can be performed by a general method such as an ELISA method. In addition, cloning can be performed, for example, by the limiting dilution method, whereby a monocloned hybridoma can be obtained. The obtained hybridoma can be obtained by a method of intraperitoneally administering to a mouse and collecting from the ascites, or a method of culturing the hybridoma and separating it from the culture supernatant. Further, it can be purified using a general method, that is, ammonium sulfate precipitation, gel filtration, ion exchange chromatography and the like.

The above-mentioned BP was separated from the ascites of cancer patients by α-lactalbumin affinity chromatography as a heteromultimer with galactosyltransferase, and was treated with sodium dodecylsulfate or 8M urea to be in a single molecule state under SDS-PAGE reduction conditions. It shows a molecular weight of about 48 kDa on lower electrophoresis and has an isoelectric point of about 4.6 to 5.0.
And reacts with anti-α ₁ -antitrypsin polyclonal antibody, has no protease inhibitor activity,
It is a protein capable of association with galactosyltransferase and self-association.

[0023]

INDUSTRIAL APPLICABILITY The present invention provides a monoclonal antibody which reacts with m-AT but does not react with AT or modified AT at all. By using the monoclonal antibody of the present invention, only m-AT can be measured accurately and easily. Moreover, the present inventors have found that the use of the antibody of the present invention for the first time is also useful for cancer diagnosis. Therefore, the present invention is considered to contribute to the diagnosis of cancer, myeloid acute leukemia and rheumatism.

[0024]

【Example】

1. Preparation of m-AT Preparation of m-AT AT (manufactured by Athens Research Co., hereinafter, all ATs used in this example are the same) 1 mg (1 ml) of elastase (manufactured by Elastin Products) 0.5 m
g (0.5 ml) were mixed and reacted at 37 ° C. for 1 hour. This was subjected to anion exchange chromatography (column; Mono-Q5 / 5HR, manufactured by Pharmacia) to give a pH of 7.
8 Tris-HCl buffer was eluted with a NaCl gradient. AT-elastase complex eluted near 100 mM NaCl was collected, 0.05 mg of elastase was added thereto, and the mixture was reacted at 37 ° C. for 6 hours. The mixture was again subjected to gradient elution with the same buffer solution by anion exchange chromatography (the same column) to obtain 200 mM NaCl.
The m-AT fraction eluted near was separated and dialyzed against phosphate buffered saline (PBS).

SDS-Polyacrylamide Electrophoresis The obtained sample was measured by sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE). SDS-PAGE was performed using Fast System (Pharmacia) using 12.5% polyacrylamide gel, and silver staining was performed. As a result, the molecular weight was about 48 kD.
The single band of a is shown, and it was confirmed that m-AT was separated and purified.

2. Preparation of anti-m-AT antibody Immunization protocol Five immunization methods shown below were carried out. (1) Normal immunization method m-AT (200 μg / ml · PB prepared in Example 1
Approximately 50 μg of the S solution) sample was mixed with equal amount of complete Freud's adjuvant to form an emulsion, which was intraperitoneally administered to BALB / c mice (female, 8 weeks old), and the same immunogen and incomplete Freund's adjuvant were again administered 3 weeks later. Similarly, an immunization operation was performed by intraperitoneal administration. Two weeks after the administration, blood was collected, the antibody titer was measured by enzyme-linked immunosorbent assay (ELISA) to confirm that the antibody was positive, and then 300 μg of m-AT sample dissolved in PBS was intravenously injected. Three days later, the spleen was removed from the mouse and cell fusion was performed as follows.

(2) Glutaraldehyde treatment method 50% glutaraldehyde solution was added to the separated and purified m-AT (200 μg / ml in PBS) to give a final concentration of 0.1% (v / v), and then at room temperature for 2 hours. It was made to react.
After the reaction, dialyze thoroughly against PBS (4 ° C, 3 times overnight),
Unreacted glutaraldehyde was removed. This is 12.
When confirmed by 5% SDS-PAGE, a faint band with a molecular weight corresponding to most and a dimer was recognized around 48 kDa.

This prepared glutaraldehyde-treated m-
50 μg of AT was mixed with Libi adjuvant system (manufactured by Libi Immunochem Research) or complete Freund's adjuvant to form an emulsion, and BALB / c mice (female, 8 weeks old) were immunized (peritoneal administration). After a further 3 weeks, the same dose was administered using the same adjuvant (Freund's incomplete adjuvant). Two weeks after this administration, blood was collected, and the antibody titer was measured by an oxygen immunoassay (ELISA) to confirm that the antibody was positive, and then 300 μg of m-AT (untreated with glutaraldehyde) sample dissolved in PBS
Was injected intravenously. Three days later, the spleen was removed from the mouse,
Cell fusion was performed as follows.

(3) Cyclophosphamide method 20 μg of AT was mixed with LIBI adjuvant system (manufactured by LIBI Immunochem Research) or complete Freund's adjuvant to form an emulsion, which was immunized to BALB / c mice (female, 8 weeks old). (Peritoneal administration). 10 minutes, 24 hours and 48 hours after the immunization, 2 ml / each.
Cyclophosphamide (100 mg / ml in PBS)
1 kg) was intraperitoneally inoculated. After another two and four weeks, the same amount of AT was administered using the same adjuvant (Freund's incomplete adjuvant) and the subsequent cyclophosphamide inoculation was repeated. Blood was collected 2 weeks after 3 times of the immunotolerization procedure by AT and oxygen immunoassay (ELIS
After confirming that the antibody titer was negative by measuring the antibody titer according to A), 50 μg of m-AT was mixed with Libi adjuvant system (Libi Immunochem Research Co.) or complete Freud's adjuvant to form an emulsion, and BALB /
C mice (female, 8 weeks old) were immunized (peritoneal injection). After another two weeks, the same dose was administered using the same adjuvant (Freund's incomplete adjuvant). Two weeks after this administration, blood was collected, the antibody titer was measured by oxygen immunoassay (ELIAS) to confirm that the antibody was positive, and then 300 μg of a m-AT sample dissolved in PBS was intravenously injected. Three days later, the spleen was removed from the mouse and cell fusion was performed as follows.

(4) Antiserum method A solution of 20 μg of AT in PBS was mixed with complete Freund's adjuvant to form an emulsion, and BALB / c mice (female, 8 weeks old) were immunized (peritoneal administration). Three weeks later, booster immunization was similarly performed using incomplete Freund's adjuvant. Two weeks after this, blood was collected and the antibody titer was measured by enzyme-linked immunosorbent assay (ELISA) to confirm that the antibody was positive, and then about 500 μl of antiserum was obtained from the mouse.

The antiserum was diluted 10-fold with PBS, and 300 µl / was newly added to BALB / c mice (female, 8 weeks old).
The animals were injected intravenously. Then, 30 minutes later, m-AT20
μg (PBS solution) was mixed with complete Freund's adjuvant to form an emulsion, which was immunized to the mouse (peritoneal administration)
did. After two more weeks, the antiserum was intravenously injected in the same manner, and then incomplete Freund's adjuvant was used.
-AT was administered at the same dose. Two weeks after this administration, blood was collected, the antibody titer was measured by enzyme-linked immunosorbent assay (ELISA) to confirm that the antibody was positive, and then the sample was dissolved in PBS.
-300 μg of AT sample was injected intravenously. Three days later, the spleen was removed from the mouse and cell fusion was performed as follows.

(5) Spleen injection immunization method BALB / c mice (female, 8 weeks old) were anesthetized with diethyl ether, the upper left abdomen was shaved, and the epidermis was incised by about 15 mm. About 50 μg of a sample of m-AT (200 μg / ml / PBS solution) prepared in Example 1 was inoculated into the spleen through the endothelium in an amount of 200 μl, and then sutured. After 4 to 5 days, the spleen was removed from the mouse and cell fusion was performed as follows.

ELISA : Anti-mouse immunoglobulin (A + G + M) (manufactured by Zymet) diluted in PBS was dispensed into wells of a 96-well microtiter plate (manufactured by Nunc) at a concentration of 5 μg / ml in 50 μl aliquots. Adsorbed overnight at 4 ° C. After removing the liquid and washing the wells with PBS, 100 μl of PBS containing 1% bovine serum albumin (BSA) was added to each well and incubated at 37 ° C. for 1 hour. After removing the BSA solution, 50 μl of a solution containing the antibody (that is, culture supernatant or antiserum) was added, and the mixture was incubated at 37 ° C. for 1 hour. After removing the solution containing the antibody and thoroughly washing with PBS, m-AT diluted in 1% BSA / PBS solution was added and reacted at 37 ° C for 1 hour. In the case of hybridoma screening, AT and heat-denatured AT are equally parallel
(Prepared by heat treatment at 100 ° C. for 5 minutes in PBS.
All modified ATs used in the following examples were prepared by this method), and a 1% BSA / PBS solution was mixed and reacted at 37 ° C. for 1 hour. After the reaction was completed, the solution was removed and thoroughly washed with PBS, and then 1% BSA / P was added.
100 μl of horseradish peroxidase-labeled anti-AT polyclonal antibody (manufactured by Kappel) diluted in BS solution
Each well was added to each well and reacted at room temperature for 1 hour. After removing the solution and thoroughly washing with PBS, a citrate / phosphate buffer solution (pH 5.0) (0.02% H) in which a concentration of 1.5 mg / ml of o-phenylenediamine was dissolved as a substrate.
100 μl of ₂ O ₂ was added to each well to develop color. 50 μl of 9N sulfuric acid was added to each well to stop the color development reaction, and the absorbance at a wavelength of 492 nm was measured using a microplate reader MPRA4 (manufactured by Tosoh Corporation).

Cell fusion and screening The spleen was aseptically removed from the immunized mouse, dissociated into single cells with a stainless mesh, and the mouse myeloma cell line P3 × 63-Ag8-653 cells in 1/5 the amount of the splenocytes and RPMI1640 medium. (Nissui Pharmaceutical Co., Ltd.) was mixed and centrifuged, 50% polyethylene glycol 1500 (manufactured by Boehringer) was added to the cell pellet, and fusion operation was performed. Thereafter, the fused cells, which were gradually diluted with the PRMI1640 solution and the supernatant was removed by centrifugation, were mixed with HAT medium (0.01 mM hypoxanthine, 1.6 μM thymidine, 0.04 μM aminopterin) containing 20% fetal calf serum (FCS). And RPMI1640 medium containing 1), 4 × 10 ⁵ cells per well were seeded in a 96-well microplate, and cultured at 37 ° C. in 5% CO ₂ . Two weeks later, the culture supernatant of the colony-growing wells was used to screen wells that reacted with m-AT and hardly reacted with the mixed solution of AT and denatured AT by the ELISA method. m-AT
The value of [absorbance when antigen is m-AT] / [absorbance when antigen is AT / denatured AT mixture] in ELISA (however, When both denominator and numerator values were 0.5 or less, negative (= 0) was used as the specificity index. An attempt was made to screen as a hybridoma with a specificity index of 2 or more.
The screened hybridomas were transferred to a 24-well microplate and cultured for 3 to 4 days, and then the reactivity was confirmed by the ELISA method again. As a result, 13 hybridomas were obtained that reacted strongly with m-AT in two cell fusions out of a total of 38 cell fusion operations and had low reactivity with AT and modified AT (Table 1). In Table 1, the number of cell fusions is the number of samples for which cell fusion was attempted, and for example,
In the case of (1), it was shown that none of the 17 desired mice could obtain the desired hybridoma. M3603,
When screening M3606, M3610, M3616, and M3618, cells obtained by the immunization method (4) were dispensed into a total of five 96-well microplates. M3
603 and M3606 are the first sheet, M3609 and M3610
The desired hybridoma was evenly obtained from any of the plates, the second plate, M3616 the third plate, and M3618 the fifth plate. FIG. 1 shows the specificity index of the third plate obtained with M3616 for reference. In addition to M3616, there are some wells showing a specificity index of more than 2, but in the second screening after transferring them to a 24-well microplate, the specificity index was 2 or less in all cases. It was just an artifact of some kind. The screened antibody-producing colonies were cloned three times by the limiting dilution method to obtain stable antibody-producing clones. Finally, 7 clones were obtained, and M2406,
M2409, M3603, M3606, M3610, M
They were named 3616 and M3618.

M36 ** is the immunization method (4), M2
4 ** was obtained in the immunization method (2),
The target antibody was not obtained at all by other immunization methods.

Purification of monoclonal antibody M2409, M3606, M3 out of 7 clones
10% FC for each of the three clones of 616
The cells were cultured in RPMI1640 medium containing S, centrifuged (200 g, 5 minutes), washed with RPMI1640 medium, centrifuged again, and then 1 × 10 ^{7 in} RPMI1640.
Suspended at a concentration of 4 cells / ml. 0.5 ml of this was inoculated into the abdominal cavity of BALB / c mice, which had been previously injected with pristane (2, 6, 10, 14-tetramethylpentadecane) 7 days before. After about 2 weeks, ascites was collected from the swollen abdominal mouse, cells were removed by centrifugation (200 g, 5 minutes), and ammonium sulfate was added to make a 50% saturated solution, and the precipitate was separated. It was dissolved in PBS.

This was thoroughly dialyzed against a 1.5 M glycine buffer (pH 8.0) containing 3 M potassium chloride, and a protein A column (20 mm × 5 mm) (Pharmacia).
Was applied to the column to sufficiently adsorb it, and then eluted with 3M citrate buffer. The pH of the citrate buffer is M2409, M3
616 had a pH of 6.0 and M3606 had a pH of 5.0.

3. Characterization of Monoclonal Antibody Monoclonal Antibody Class and Subclass The antibody class and subclass of each monoclonal antibody was determined using a mouse monoclonal sub-isotyping kit (manufactured by AMERICAN QUALEX). As a result, all the obtained monoclonal antibodies were determined to be IgG1.

Specificity The specificity of the three types of monoclonal antibodies obtained was determined by the following test (ELISA).

5 μg of anti-mouse immunoglobulin (A + G + M) (manufactured by Zymet) diluted in PBS was placed in a well of a 96-well microtiter plate (manufactured by Nunc).
50 μl was dispensed at a concentration of / ml and adsorbed at 4 ° C. overnight. After removing the solution and washing the wells with PBS, 1%
100 μl of PBS containing bovine serum albumin (BSA) was added to each well and incubated at 37 ° C. for 1 hour. After removing the BSA solution, 50 μl of a solution containing the antibody (that is, culture supernatant or antiserum) was added, and the mixture was incubated at 37 ° C. for 1 hour. After removing the solution containing the antibody and washing thoroughly to PBS, m-AT, which was prepared as a 3-fold dilution series, was added to a 1% BSA / PBS solution and reacted at 37 ° C for 1 hour. Similar dilution series were prepared for AT and denatured AT, and they were separately added in parallel and reacted. After the reaction was completed, the solution was removed and thoroughly washed with PBS, and then 1%
Horseradish peroxidase-labeled anti-AT polyclonal antibody diluted in BSA / PBS solution (Kappel)
Was added to each well by 100 μl and reacted at room temperature for 1 hour. After removing the solution and thoroughly washing with PBS, a citrate / phosphate buffer solution (pH 5.0) in which a concentration of 1.5 mg / ml of o-phenylenediamine was dissolved as a substrate
100 μl (containing 0.02% H ₂ O ₂ ) to each well
Each was added to develop a color. The color reaction was stopped by adding 50 μl of 9N sulfuric acid to each well, and the absorbance at a wavelength of 492 nm was measured using a microplate reader MPRA4 (manufactured by Tosoh Corporation). The results are shown in FIGS.

4. Analysis of M3616 reactive substance in sample Affinity column of monoclonal antibody M3616
Preparation 2 ml of Affi-Gel 10 (manufactured by Bio-Rad) using an agarose gel carrier was rapidly washed 3 times with cold distilled water (4 ° C.), and a monoclonal antibody M3616 in a PBS solution was used.
(20 mg) was added, and the mixture was shaken at 4 ° C. for 4 hours. After the reaction, 100 mM glycine-H containing 1M sodium chloride
Cl buffer (pH 2.8), 100 mM diethylamine solution (pH 11.5) containing 1M sodium chloride, 3M
50 mM phosphate buffer containing potassium thiocyanate (p
The cells were washed successively with H7.2) and equilibrated with PBS.

[0042] After equilibration by addition of 2 fold concentrated PBS equal volume ascites about 2ml of isolation cancer patients in a test m-AT, and insoluble matters were removed by centrifugation. This supernatant was passed through a monoclonal antibody M3616 affinity column (7 mm × 50 mm) and thoroughly washed with PBS containing 1M sodium chloride. Then, it was eluted with 50 mM phosphate buffer (pH 7.2) containing 3 M potassium thiocyanate. The eluted M3616 reaction substance was monitored by ELISA measurement of the collected fractions (FIG. 5). The eluted protein fraction (fraction 2) was collected and dialyzed thoroughly against PBS to obtain 1 ml. this,
Ultra Filter USY-1 (manufactured by Advantech)
Was concentrated to 65 μl.

SDS-Polyacrylamide Electrophoresis The molecular weight of the obtained sample was confirmed by SDS-PAGE. Fast system (Pharmacia)
12.5% polyacrylamide gel was used, and the staining was performed by the silver staining method. As a result, a sample derived from ascites of a cancer patient and trapped in the affinity column of the monoclonal antibody M3616 was confirmed to have a single band with the same molecular weight of about 48 kDa as m-AT, and no other molecular weight band was observed.

Isoelectric Focusing The isoelectric point of the obtained sample was confirmed by isoelectric focusing. The measurement was performed using a fast system (Pharmacia) using IEF4-6.5 gel, and the staining was performed by the silver staining method. After the electrophoresis, the gel was plotted on a PVDF membrane (manufactured by Millipore) under mild conditions (37 ° C, 4 hours) and then immunostained as follows. That is, 1% B
HRP by soaking in SA / PBS solution and blocking
1% BSA diluted with the labeled monoclonal antibody
-A reaction was carried out by contacting with a PBS solution. After that, remove the solution, wash with PBS, and use the Konaikanomustin Kit IS.
Color was developed with -50B (manufactured by Konica). as a result,
The obtained sample had a p value similar to that of m-AT in silver staining.
Multiple bands of I4.5-5.0 were confirmed, and immunostaining was also associated.

From these results, M3616 was found in the sample.
It can be seen that there is no substance that cross-reacts with.

5. Detection of m-AT Preparation of calibration curve for m-AT As a method for measuring the total amount of m-AT, anti-m-AT monoclonal antibody M3616 and m-AT which does not react with AT
-Japanese Patent Application No. 4-1434 which reacts with AT and modified AT
The two-antibody method using the anti-BP monoclonal antibody M801 described in 79 (Microtechnology Research Institute, No. 12942) was used.

A calibration curve for m-AT was prepared as follows.

M3616 at a concentration of 10 μg / ml in PBS
100 μl each was added to a 96-well microtiter plate (Nunc) and adsorbed at 4 ° C. overnight.
After washing this with PBS, 200 μl of 1% BSA / PBS solution was added to each well and allowed to stand at 37 ° C. for 1 hour to block the solution. Next, a dilution series was prepared by doubling the m-AT prepared in Example 1 to 100 μl each.
Each well was added to each well and allowed to stand at 37 ° C. for 1 hour. After the reaction
Each well was thoroughly washed 3 times with PBS. Then 1
100 μl of horseradish peroxidase-labeled M801 diluted in 100% BSA / PBS solution was added to each well,
The reaction was carried out at room temperature for 1 hour. After completion of the reaction, it was thoroughly washed with PBS, and then a citrate-phosphate buffer solution (pH 5.0) (0.02% H ₂ O ₂ ) in which o-phenylenediamine having a concentration of 1.5 mg / ml was dissolved was used as a substrate. 100 μl each was added to each well, and color reaction was allowed to proceed for 10 minutes at room temperature. 50 μl of 9N sulfuric acid was added to each well to stop the reaction, and the absorbance at a wavelength of 492 nm was measured.
A graph of -AT concentration-absorbance was prepared. The result is shown in FIG.

Measurement of m-AT in Specimen As a method for measuring the total amount of m-AT, a two-antibody method using anti-m-AT monoclonal antibody M3616 and anti-BP monoclonal antibody M801 was used.

M3616 at a concentration of 10 μg / ml in PBS
100 μl each was added to a 96-well microtiter plate (Nunc) and adsorbed at 4 ° C. overnight.
After washing this with PBS, 200 μl of 1% BSA / PBS solution was added to each well and allowed to stand at 37 ° C. for 1 hour to block the solution. Next, the sera of healthy subjects and cancer patients (CEA (carcinoembryonic antigen) or CA19-9 high-digestive system cancer) were diluted 150-fold with 1% BSA / PBS solution, and 100 μl was added to each well. It left still at 1 degreeC. After the reaction, each well was thoroughly washed with PBS three times. Next, 100 μl of horseradish perosidase-labeled M801 diluted in a 1% BSA / PBS solution was added to each well and reacted at room temperature for 1 hour. After the reaction is complete, wash thoroughly with PBS and then use a substrate with a concentration of 1.5.
Citric acid-phosphate buffer solution (pH 5.0) (0.02% H ₂ O ₂ ) in which mg / ml o-phenylenediamine was dissolved.
100 μl) to each well and add at room temperature for 1
Color reaction was allowed for 0 minutes. 50 μl of 9N sulfuric acid was added to each well to stop the reaction, the absorbance at a wavelength of 492 nm was measured, and the concentration was converted from FIG. The result is shown in Fig. 7.
Shown in.

As is clear from the results shown in FIG. 7, the m-AT in the sample shows a low value in a healthy person, but in many cases in a cancer patient, it shows a high value. The anti-m-AT monoclonal antibody of the invention is the mA-A in the test solution.
This enables accurate measurement of T concentration, and as a result contributes to the diagnosis of cancer.

[0052]

[Table 1]

[Brief description of drawings]

FIG. 1 is a diagram showing the specificity index of each clone when the monoclonal antibody of the present invention is screened.

FIG. 2 m-of the monoclonal antibody M3616 of the present invention
It is a figure which shows the reactivity with respect to AT, AT, and modified AT.

FIG. 3: m- of the monoclonal antibody M3606 of the present invention
It is a figure which shows the reactivity with respect to AT, AT, and modified AT.

FIG. 4: m-AT, A of monoclonal antibody M2409
It is a figure which shows the reactivity with respect to T and modified | denatured AT.

FIG. 5 shows m-in a sample by affinity chromatography using the monoclonal antibody M3616 of the present invention.
It is a figure which shows the m-AT density | concentration contained in each fraction at the time of refine | purifying AT.

[FIG. 6] ELISA by a two-antibody method using the monoclonal antibody M801 and the monoclonal antibody M3616 of the present invention
It is a figure which shows the calibration curve of m-AT measured by.

FIG. 7: ELISA by the two antibody method of the monoclonal antibody M801 and the monoclonal antibody M3616 of the present invention
In serum of healthy subjects and cancer patients measured by
It is a figure which shows T concentration.

Front page continuation (51) Int.Cl. ⁵ Identification code Office reference number FI Technical display location G01N 33/573 Z 8310-2J 33/577 B 8310-2J // C12N 15/06 (C12P 21/08 C12R 1 : 91)

Claims (5)

[Claims] 1. Reacting with modified α ₁ -antitrypsin,
alpha ₁ - antitrypsin and modified alpha ₁ - monoclonal antibody does not react to antitrypsin. 2. A monoclonal antibody M3616 or M3
The monoclonal antibody according to claim 1, which is 606. 3. A hybridoma producing the monoclonal antibody according to claim 1 or 2. 4. A hybridoma M3616 (FERM
P-13477) or M3606 (FERM P-13
476). The hybridoma according to claim 3. 5. A method for immunologically measuring modified α ₁ -antitrypsin in a sample by utilizing a specific reaction between the monoclonal antibody according to claim 1 and modified α ₁ -antitrypsin.