JP3152429B2 - Anti-hCG-β core monoclonal antibody producing hybridoma, said monoclonal antibody and use thereof - Google Patents

Description

The present invention relates to a human chorionic gonadotropin β subunit core fragment (human chorionic gonadotropin β).
The present invention relates to a novel hybridoma producing a monoclonal antibody recognizing a subunit core fragment (hereinafter, abbreviated as hCG-β core) and hCG-β subunit, and the monoclonal antibody. Furthermore, the present invention relates to an immunochemical measurement method and a measurement reagent characterized by utilizing the monoclonal antibody.

2. Description of the Related Art hCG is a kind of protein hormone produced from villus cells formed at the time of pregnancy, and promotes the secretion of progesterone. Detection of hCG is widely used for early diagnosis of pregnancy. Furthermore, in trophoblastic diseases such as hydatidiform mole, destructive hydatidiform mole, and choriocarcinoma, hCG measurement in body fluids such as urine and blood is extremely important in terms of early detection of these diseases, judgment of therapeutic effects, and management of prognosis. It turns out to be important. However, these diagnoses require the measurement of a trace amount of hCG of about 100 IU / or less, and in this case, the problem is a protein hormone having a structure similar to hCG, that is, luteinizing hormone (hereinafter abbreviated as hLH). In some cases), follicle stimulating hormone (hereinafter sometimes abbreviated as hFSH) and thyroid stimulating hormone (hereinafter sometimes abbreviated as hTSH). In particular, hLH has a high similarity to hCG, and the physiological amount of hLH in urine may reach 150 IU / .However, in order to measure hCG in a small amount of body fluid, hCG and hLH are necessary.
It is necessary to distinguish between and immunologically.

On the other hand, chemical analysis of these protein hormones has revealed that the cross-reactivity is caused by each α-subunit portion having many common structures. Therefore, the β-subunit of hCG having relatively low similarity (hereinafter, hCG-β
(Sometimes abbreviated as abbreviated) to produce an anti-hCG-β antibody to specifically detect hCG.

However, the use of anti-hCG-β antibodies does not completely eliminate cross-reactivity with hLH due to the common amino acid sequence still present between the β-subunits of hCG and hLH. However, a peptide consisting of about 30 amino acids at the C-terminus of hCG-β shows an amino acid sequence that does not exist in hLH. In this portion, hCG and hLH can be completely distinguished. Antibodies against the C-terminal region of hCG-β react only with hCG-β and do not undergo cross-reaction with hLH. Furthermore, research on hCG-β has progressed, and hCG-β
It has been presumed that a β core region (hereinafter sometimes simply referred to as β core) is present. That is, the β core is composed of human chorionic gonadotropin β subunit (hCG-
β) is a glycoprotein having a molecular weight of 12 to 17 K daltons in which two peptides of amino acid residues 6 to 40 and 55 to 92 are disulfide-bonded [Endocrinolog
y), 123 , 572, 1988], clearly not only in ovarian cancer, uterine cancer and other gynecological malignancies, but also in the urine of patients with various malignancies such as stomach cancer and kidney cancer. [Journal of Clinical Endocrinology and Metabolism (J. Clin. Endocrinol. Meta)
b.), 53 , 1014, 1981; Cancer, 45 , 258.
3,1890; Acta Endocri
nol. (Copenh)), 112 , 415, 1986]. However,
There is no established theory regarding its production, secretion, physiological significance, etc., and there is no biological measurement method for β-core. As an immunoassay, a radioimmunoassay using a polyclonal anti-hCG-β antibody called SB6 has been established [American Journal of Obstetrics and Gynecology (Am. JO.
Gynecol., 113 , 751, 1972], the affinity of the antibody is not different between β-core and human chorionic gonadotropin and hCG-β and cannot be distinguished.

In recent years, monoclonal antibodies B202 and B204 that recognize β-core using β-core as an immunogen have been produced [Endocrinology, 123 , 584, 1988], and a β-core radioimmunoassay has been reported [Cancer Research]. (. Cancer Res), 48, 1361, 1988 years;. Cancer Res 48, 13
56, 1988].

[Problems to be Solved by the Invention] SB6 is a polyclonal antibody, and when used in radioimmunoassay, exhibits the same affinity as β-core, hCG and hCG-β, and further has about 10% cross-reactivity with hLH.

B202 and B204 are monoclonal antibodies obtained by immunizing β-core. A. Krichesky
vsky) et al. in the Endocrinolog
y), 123 , 584-593, 1988, produced several monoclonal antibodies against the β-core, mentioning their properties. However, this article states that when hCG-β was used as the immunogen, no antibody against β-core could be obtained.

On the other hand, in the field of clinical examination, the emergence of a monoclonal antibody against a novel β-core having a high affinity that enables a highly sensitive assay is expected.

Also, in the conventional β-core RIA, an assay system is set up for measuring only urine, and biosynthesis, secretion,
It was insufficient to measure body fluid tissue fluids, tissue extracts and tissue culture supernatants, which are essential for studying distribution, metabolism and physiological and biological effects, disease relevance, and the like.

Means for Solving the Problems The present inventors differed from the conventional method for obtaining an anti-β core monoclonal antibody, in which after immunizing an animal with hCG-β as an immunogen, a monoclonal antibody obtained by a cell fusion method was used for hCG-β.
And did not react with protein hormones having a similar structure, and recognized the β core and hCG-β subunit, and found that the use of this monoclonal antibody was particularly important as a diagnostic agent, and completed this study. I came to.

That is, the present invention provides (1) a monoclonal antibody recognizing the hCG-β core region and the hCG-β subunit, which is obtained by fusing a mammalian antibody-producing cell immunized with the hCG-β subunit with a lymphoid cell body. And (2) a hCG- produced by the hybridoma.
a monoclonal antibody recognizing the β core region and the hCG-β subunit; and (3) a substance which immunochemically reacts with the monoclonal antibody using the monoclonal antibody, for example, the monoclonal antibody such as β core itself or hCG-β. Β core region and / or hCG-β that can be recognized by the antibody
It is intended to provide an immunoassay characterized by detecting and measuring a substance containing a subunit.

HCG-β used for preparing the hybridoma of the present invention can be prepared by a method known per se. For example, the method of FJ Morgan et al. [Endocrology (Endocr)
inology), 88 , 1045-1053, 1971], and hCG-β can be purified. More specifically, natural hCG is dissolved in, for example, 10 M urea, dissociated into hCG-α and hCG-β, and the eluted fraction of hCG-β is applied to a column packed with DEAE-Sephadex A-25. Can be obtained.

In immunization, hCG-β, a product obtained by binding hCG-β to a carrier polymer substance, or both of them can be used as an immunogen.

The binding of the peptide used here to the polymer substance for carrier is carried out by a conventional method [eg, Hormon and Metabolic Research.
ch), 8 , 241, 1976]. Examples of the reagent used for the binding include glutaraldehyde and water-soluble carbodiimide. Examples of the carrier polymer substance include bovine thyroglobulin, bovine serum albumin, bovine gamma globulin, and hemocyanin. The use ratio of the peptide to the carrier polymer substance is appropriately 1: 1 to 1: 4, and the reaction pH is around neutral,
Especially around 7.3 often gives good results. Also,
The time required for the reaction is often good for 2 to 6 hours,
In particular, 3 hours is appropriate. The product thus prepared may be dialyzed against water at about 4 ° C. by conventional means, frozen and stored, or may be stored by freeze fitting.

As mammals to be immunized, for example, mice, rats,
Experimental animals such as hamsters can be used to advantage, and mice are particularly preferred. As the antibody-producing cells, spleen cells of an immunized animal are advantageously used. An established human spleen cell line may be used.

Various combinations are possible as a cell of an animal to be immunized and a lymphoid cell line to be a fusion partner, but the most proven in cell fusion experiments, fusion between mouse cells with good conditions in cell fusion efficiency and others, That hyperimmune mouse antibody-producing cells and cell fusion efficiency with antigen, excellent mouse myeloma cells especially hypoxanthine such proliferative - guanine - phosphoribosyl transferase deficient strain (HGPRT ^- Corporation) and thymidine kinase deficiency (TK ^-) Suitable homologous or heterologous (preferably homologous) myeloma having a marker such as [P3-X63-Ag8-U1 (Ichimori et al., Journal of Immunological Methods, 8
0, 55, 1985), X63-Ag8-6.5.3 (Salman, M. (Sh
ulman, M) et al., Nature, 276 , 269, 1978), mouse myeloma cell line SP2 / 0-Ag14 (SP2) Nature, 276 ,
269, 1978].

Alternatively, human lymphoid cells may be used. In this case,
Those obtained from humans and established are advantageously used.

In order to obtain a monoclonal antibody, rats and mice are preferred. For example, when immunizing a mouse, subcutaneous, intraperitoneal, intravenous, intramuscular, intracutaneous, or any other route is possible.
It is preferably injected intravenously (especially subcutaneously). The inoculation interval, inoculation amount, etc. are also highly variable, and various methods are possible. For example, inoculation is performed 2 to 8 times at 2 week intervals, and 1 to 5 days after the final immunization, preferably 2 to 4 times. A method using spleen cells after day is suitably used. Inoculation amount is hCG-
As the β amount, 0.1 μg or more per mouse, preferably 10 μg
g to 100 μg is desirably used.

In the case of using spleen cells as a lymphocyte source, before removing the spleen, it is preferable to perform partial fusion blood sampling before confirming an increase in the antibody titer in the blood before conducting a fusion experiment.

Cell fusion between lymphocytes and lymphoid cell lines, for example, lymphocytes of mice immunized with (especially those derived from spleen cells) hypoxanthine - guanine - phosphoribosyl transferase deficiency (HGPRT ^-) or thymidine kinase deficiency (TK ^-) suitable homologous or heterologous animal (preferably having a marker such as the myeloma, etc. of the same type), is fused with the lymphoid cell line. For the fusion, a fusion agent such as Sendai virus and polyethylene glycol (PEG) is used. Of course, dimethyl sulfoxide (DMS
O) It is also possible to add other fusion promoters. PEG
The average molecular weight is usually 1000 to 10,000, preferably 1000
~ 6000, time is 0.5 ~ 30 minutes, concentration is 10% ~ 80% etc. PEG6000 is 35 ~ 5
By treating at 5% for 4 to 10 minutes, fusion can be performed efficiently. The fused cells (hybridomas) were prepared using a hypoxanthine-aminopterin-thymidine medium [HAT medium; Nature, 256 , 495-497, 1975] and the like.
It can be selectively grown.

Mouse serum and culture supernatants of proliferating cells can be screened for the production of the desired antibody. The antibody titer can be screened as follows. That is, it can be examined by a method such as a radioimmunoassay (RIA) or an enzyme immunoassay (EIA), and various modifications of these methods are also possible. As an example of a preferable measurement method, a method using EIA will be described. An anti-mouse immunoglobulin is immobilized on a solid phase according to a conventional method (for example, using a 96-well makorotiter plate as a solid phase allows rapid measurement using a multiscan or the like, which is advantageous). The culture supernatant to be measured or the serum of a mouse are added, and the mixture is allowed to react at a constant temperature (hereinafter, 4 to 40 ° C.) for a certain time. Then, after the reaction product is thoroughly washed, hCG-β or β core labeled with an enzyme is added, and the reaction is carried out at a constant temperature for a certain time. After thoroughly washing the reaction product, an enzyme substrate is added, and the reaction is carried out at a constant temperature for a certain period of time. Thereafter, the formed color product can be measured by absorbance or fluorescence intensity.
It is desirable to clone the cells of the wells showing growth in the selective medium and exhibiting the antibody activity against the β core by a limiting dilution method or the like. Screening of the supernatant of the cloned cells is carried out in the same manner, and the number of well cells with a high antibody titer is increased to obtain the desired monoclonal antibody-producing hybridoma clone of the present invention.

The anti-hCG-β core monoclonal antibody of the present invention can be obtained by growing the thus cloned hybridoma in a liquid medium or intraperitoneally of a mammal. For example, the monoclonal antibody can be obtained from the culture solution by culturing for 2 to 10 days, preferably 3 to 5 days in a liquid medium or the like containing 0.1 to 40% bovine serum added to RPMI-1640. In addition, by inoculating intraperitoneally into a suitable mammal such as a mouse, growing cells, and collecting ascites, an antibody with a much higher titer than the cell culture supernatant can be efficiently obtained in a large amount. Can be. For this purpose, for example, in the case of a mouse, 1 × 10 ⁴ to 1 × 10 ⁷ , preferably 5 × 10 ⁵ to 2 × 10 ⁶ hybridomas are added to a mouse such as BALB / c inoculated in advance with mineral oil or the like. Is inoculated into the abdominal cavity or the like, and after 7 to 20 days, preferably 10 to 14 days, ascites fluid or the like is collected. Antibodies produced and accumulated in ascites fluid, for example,
Monoclonal antibodies can be easily isolated as pure immunoglobulin by ammonium sulfate fractionation, DEAE-cellulose column chromatography, protein A sepharose column chromatography, or the like.

The anti-hCG-β core monoclonal antibody of the present invention has the following properties.

(1) Reactivity with β core.

(2) It has reactivity to hCG-β.

(3) It has high reactivity to urine of human cancer patients.

(4) It has high reactivity with human serum patient serum.

(5) The class of the antibody is IgG1.

When the antibody is used for detection and measurement described below, the antibody molecule may be a fraction, and examples of the fraction include Fab, Fab ′, and F (ab ′) ₂ .

Thus, the monoclonal antibody of the present invention can be obtained by the EIA method,
Β in systems using immunochemical techniques such as LISA and RIA
It is very advantageous for diagnosis of cancer by recognition of the core region.

These immunochemical techniques can be generally classified into the following methods.

(1) Competition method: A test solution containing an unknown amount of an antigen and a certain amount of an antigen labeled with a labeling agent are subjected to a competitive reaction with a certain amount of a corresponding antibody, and the antibody is bound to a labeling agent or an antibody The activity of the labeling agent not performed is measured.

(2) Sandwich method: A test solution containing an unknown amount of antigen is reacted with an excess amount of antibody held on a carrier (first method).
Reaction), and then add a certain amount of an excess amount of the antibody labeled with a labeling agent to cause a reaction (second reaction). The activity of the labeling agent held on the carrier or the labeling agent not held on the carrier is measured. The first reaction and the second reaction may be performed at the same time or at different times.

In the immunological assay of the present invention, any of (1) and (2) may be used, but the sandwich method of (2) is effective. In the sandwich method, the antibody retained on the carrier is an antibody whose antigenic determinant sites do not overlap each other with the antibody in the antibody to which the labeling agent is bound, and one of the antibodies is a monoclonal antibody that reacts with the β-core of the present invention. . The other antibody may be a monoclonal antibody or a polyclonal antibody as long as it reacts with hCG-β. As a method for producing an antibody that reacts with the hCG-β, a method known per se can be used.For example, in the case of a polyclonal antibody, hCG-β
Using a complex of -β and a carrier protein as an immunogen, a mammal such as a rabbit was immunized several times, and the obtained antiserum was purified by hCG-β solid-phase affinity-column chromatography to purify the anti-hCG-β Antibodies can be obtained.

Known means can be used for purification of the β core used for labeling or standardization. As described in Endocrinology, 123 , 572-583, 1988, β core can be obtained from commercially available partially purified hCG by gel chromatography and immunoaffinity chromatography.

Patient-derived samples subjected to such detection and measurement include, for example, body fluids such as serum, urine, and cerebrospinal fluid, and sometimes tissues and extracts thereof.

As an example of the EIA of the measurement method of the present invention, the case where the standard agent is peroxidase will be specifically described below,
It is not limited to peroxidase.

First, a test sample is added to the antibody held by the carrier to cause a binding reaction, and an antibody conjugated with peroxidase is added thereto and reacted.

A peroxidase substrate is added to the reaction product obtained in the above, and the enzyme activity of the reaction product is determined by measuring the absorbance or the fluorescence intensity of the resulting substance.

Steps (1) to (3) are performed in advance on a known amount of a standard solution of the antibody reactant, and the relationship between the reactant and the absorbance or the fluorescence intensity is prepared as a standard curve.

The absorbance or the fluorescence intensity obtained for the unknown amount of the analyte (test sample) is fitted to a standard curve, and the amount of the substance reacting with the monoclonal antibody in the analyte is measured.

As an example of the RIA of the measurement method of the present invention, the case where the labeling agent is ¹²⁵ I will be specifically described below, but it is not limited to ¹²⁵ I.

First, a test reagent to be measured is added to the antibody held on the carrier to cause a binding reaction, and a ¹²⁵ I-labeled antibody is added thereto and reacted.

Γ-radioactivity of the reaction product obtained in the above is measured.

The above operations (1) to (4) are performed in advance on a known amount of a standard solution of the antibody reactant, and the relationship between the reactant and γ-radioactivity is prepared as a standard curve.

The γ-radioactivity obtained for the unknown amount of the analyte (test sample) is applied to the standard activity, and the amount of the antibody reactant in the analyte is measured.

As a labeling agent, in addition to the aforementioned enzymes and radioisotopes,
Examples include a fluorescent substance and a luminescent substance.

As radioisotopes, for example, ¹²⁵ I, ¹³¹ I, ³ H, ¹⁴
C is preferably a stable enzyme having a large specific activity. For example, (1) carbohydrase [eg, gericosidase (eg, β-galactosidase, β-glucosidase, β-glucuronidase, β-fructosidase, α -Galactosidase, α-glucosidase, α-
Mannosidase), amylase (eg, α-amylase,
β-amylase, isoamylase, glucoamylase,
Taka amylase A), cellulase, lysozyme],
(2) amidase (eg, urease, asparaginase), (3) esterase [eg, cholinesterase (eg, acetylcholinesterase), phosphatase (eg, alkaline phosphatase), sulfatase, lipase], (4) nuclease (eg, deoxygenase) Ribonuclease, ribonuclease), (5) iron / porphyrin enzyme (eg, catalase, peroxidase, cytochrome oxidase), (6) copper enzyme (eg, tyrosinase, ascorbate oxidase), (7) dehydrogenase (eg, alcohol dehydrogenase) Enzymes, malate dehydrogenase, lactate dehydrogenase, and isocitrate dehydrogenase), as fluorescent substances, fluorescamine, fluorescein isothiocyanate, etc., and as luminescent substances, luminol, luminol derivatives, Luciferin, lucigenin and the like can be mentioned.

In order to bind the antibody to the labeling agent, the chloramine T method [Nature, 194 , 495, 1962] is used.
Year], periodate method [Journal of Histochemistry and cytochemistry (Journal of Histochemistry)
tochqmistry and Cytochemistry), 22 , 1084, 1974
Year], maleimide method [Journal of Biochemistry, 79 , 233, 1976]
Are used.

In order to carry out the specific immunochemical measurement method by the sandwich method, a solid phase to which an antibody is physically or chemically bound by a conventional method is added to a test sample containing an unknown amount of a β-core and reacted ( First reaction). After washing the solid phase, a certain amount of the antibody labeled with the labeling agent is added and reacted (second
reaction). Then, usually, the solid phase is thoroughly washed, and the activity of the labeling agent bound on the solid phase is measured. When the labeling agent is a radioisotope, the measurement is performed using a well counter or a liquid scintillation counter. When the labeling agent is an enzyme, the substrate is added and allowed to stand, and the enzyme activity is measured by a colorimetric method or a fluorescent method. Even if the labeling agent is a fluorescent substance or a luminescent substance, the measurement is performed according to a known method. In such an assay method, washing between the first reaction and the second reaction may be omitted, or for further simplification, a test solution, an antibody-bound solid phase, and an antibody labeled with a labeling agent are simultaneously added. You may make it react. That is, since the antibodies used in the present invention have different antigen-determining sites,
It has an excellent feature that it is not affected by the order of addition of the reagents, the time of addition, the presence or absence of a washing operation, and the like.

 Thus, according to the present invention, the following effects can be obtained.

(1) A trace amount of β-core and hCG-β can be measured without being completely affected by peptide hormones such as hCG and hLH.

(2) Urine is particularly effective as a patient-derived sample to be subjected to detection and measurement, but serum and the like can also be used as other samples.

(3) Various malignant tumors, particularly obstetrics and gynecology, and germ-related cancers are selected as the diseases to be measured, and are extremely applicable to diagnosis and prognosis management of these diseases.

In the present invention, for example, a kit for quantification can be used for performing the immunochemical measurement method of the β core region by the sandwich method, and the kit mainly includes the following (1) to (6) Consisting of the following reagents:

(1) Antibody held on a carrier (2) Standardized antibody (3) Standard β core or hCG-β (4) Buffers used for diluting the reagents (1) to (3) and test samples (Any buffer may be used as long as it can be used for diluting the reagent and the test sample. Examples thereof include a phosphate buffer or a glycine buffer having a pH of 6 to 9.) (5) Incubation Thereafter, a buffer used for washing the carrier (any buffer may be used as long as it can be used for washing the carrier, and examples thereof include a phosphate buffer and a glycine buffer). (6) Labeling When an enzyme is used as a reagent, a reagent necessary for the enzyme measurement (for example, in the case of a fluorescence method using peroxidase, p-hydroxyphenylacetic acid and hydrogen peroxide are used as enzyme substrates, and a colorimetric method is used). , O-phenylenediamine and hydrogen peroxide, including a buffer solution (preferably a phosphate buffer solution) used for dissolving the enzyme substrate and an enzyme reaction stop solution.) This kit can be used, for example, by the following method. it can.

Standard β core, hCG-β or test solution about 10 to 200μ
The reaction mixture is diluted with the reagent (4), and a certain amount of the reagent (1) is added. The mixture is reacted at about 0 to 40 ° C. for about 1 to 48 hours.
After the carrier is washed with water, about 10 to 300 µ of the reagent (2) is added, and the reaction is carried out at about 0 to 40 ° C. After the reaction for about 1 to 48 hours, the carrier is washed and the activity of peroxidase bound on the carrier is measured. That is, about 10 to 1000 µ of a substrate solution of peroxidase is added, and about 0.1 to 2 at about 20 to 40 ° C.
After the reaction for a period of time, the enzyme reaction is stopped, and the absorbance or the fluorescence intensity in the reaction solution is measured.

Using the immunochemical analysis method of the present invention, the monoclonal antibody of the present invention reacts with both β-core and hCG-β and has no cross-reactivity with hLH, hFSH and hTSH. β or β core and both hCG-β,
It can be quantified by a single measurement with high sensitivity using a simple technique in a normal clinical test.

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

Example 1 (Production of hybridoma) Five BALB / c mice were treated with 100 μg of hCG-β dissolved in 0.5 ml of physiological saline and Freund's complete
A solution of 0.5 ml of adjuvant suspended in the peritoneal cavity,
Twelve weeks after the week, mice were boosted with 50 μg of hCG-β dissolved in 0.25 ml of physiological saline per mouse in the tail vein. Three days after the final immunization, mouse splenocytes were removed for cell fusion.

The collected spleen cells (1.0 × 10 ⁸ cells) with myeloma cells P3-
X63-Ag8-U1 (1.0 × ¹⁷ ) was mixed in RPMI-1640 medium and centrifuged at 1,000 rpm for 5 minutes. In the obtained settlement
Cell fusion was performed by gradually adding 1 ml of 50% polyethylene glycol 1500 at 37 ° C. for 1 minute, adding 7 ml of 37 ° C. RPMI-1640 over 5 minutes, and then centrifuging. The obtained fused cells
After diluting with 30 ml of HAT medium and dispensing 0.1 ml into a 96-well microplate, culturing was continued while changing the HAT medium by half every 2-3 days. 7-14 days later, about 6 out of 288 wells
0% (170/288) of hybridomas proliferated, of which 40
% (68/170) produced anti-hCG-β antibody. Furthermore, about 30% (17/68) of them showed β-core binding activity.

The cells of the wells showing the highest antibody titer were cloned by the limiting dilution method. That is, as feeder cells
BALB / c mice thymocytes with 10 ⁵ cells / well, the hybridoma was added to one / well, and cultured in HT medium. This manipulation was repeated twice. A clone producing the most stable and large amount of antibody by cloning was selected and named 229. Hybridoma 233 was established by the same operation.

Hybridoma 229 was transferred to FERM BP from September 26, 1989 by the Institute of Microbial Industry and Technology (FRI) of the Ministry of International Trade and Industry.
-2614.

 The measurement of the antibody titer was performed as follows.

The hybridoma culture supernatant was placed on a microplate on which anti-mouse immunoglobulin (IgG + IgM + IgA) antibody was immobilized.
50 μl was added and reacted at 4 ° C. for 1 day. After washing, peroxidase-labeled hCG-β or β-core was added and further reacted at 4 ° C. for 1 day. After washing again, an enzyme substrate o-phenylenediamine (hereinafter abbreviated as OPD) solution was added, and the antibody titer of the hybridoma culture supernatant was determined by measuring the absorbance. The peroxidase-labeled antigen was prepared as follows. 3 mg / ml 4- (N-maleimidomethyl) cyclohexanoic acid / N-succinimide ester solution in 1 ml of 500 μg / ml hCG-β or β core solution
After adding 100 μl, the mixture was reacted at room temperature for 90 minutes, and the reaction solution was subjected to gel filtration through a column of Sephadex G-25 to obtain a maleimidated antigen. To 7 mg of horseradish peroxidase dissolved in 0.02 M phosphate buffer with 0.1 M sodium chloride, 20
A double amount of N-succinimidyl 3- (2-pyridyldithio) propionate was allowed to react at room temperature for 1 hour.
Dithiothreitol 0.2 dissolved in 0.1 M acetate buffer pH 4.5
5 ml was added and reacted at room temperature for 30 minutes. The solution obtained by gel filtration of the reaction solution with Sephadex G-25 was mixed with a maleimidated antigen, and reacted at 4 ° C. for 20 hours.
l) Purified by gel filtration over AcA44.

Example 2 (Production of monoclonal antibody) BAL pretreated by intraperitoneally administering 0.5 ml of pristane in advance
B / c mice were intraperitoneally suspended in 0.5 ml of RPMI-1640.
It was injected × 10 ⁶ cells of the hybridoma 229. After 10 to 14 days, the pooled ascites was collected and purified by protein A-Sepharose to obtain monoclonal antibody 229. The amount of the obtained monoclonal antibody 229 was 3.5 mg per 1 ml of ascites.

Isotype of this monoclonal antibody was determined to be IgG ₁ in the Ouchterlony method. The reactivity is β core, hCG
Cross-reactivity to -β, chorionic and pituitary glycoprotein hormones was determined by examining by EIA. The result is shown in FIG. The monoclonal antibody is β-core, hCG
Responds to almost the same intensity to -β and hardly to human chorionic gonadotropin. Meanwhile, hybridoma 233
Monoclonal antibody 233 obtained in the same manner using
Did not generate a competitive reaction even when added simultaneously with the monoclonal antibody 229 in the above-mentioned EIA. Thus, it was considered that it recognized another β-core epitope different from the recognition site of the monoclonal antibody 229.

Example 3 (Preparation of Measurement Kit) (1) Preparation of Immobilized Antibody 50 polystyrene beads (1/4 inch in diameter, manufactured by Precision Plastic Ball Company (Chicago, USA)) were used at 50 μg / ml in monoclonal amount. The cells were placed in 7.5 ml of a 0.1 M phosphate buffer (pH 7.0) containing an antibody β-core antibody (monoclonal antibody 229) and left overnight at room temperature. The polystyrene beads were washed with a phosphate buffer, added with 0.1% sodium azide, and stored in a refrigerator.

(2) Preparation of enzyme-labeled antibody 20 mg of hCG-β and 20 mg of bovine serum thyroglobulin
Dissolve in 0.05M phosphate buffer pH 7.5, add
Glutaraldehyde was added to a concentration of 0.2% and reacted at 4 ° C. for 2 hours. This reaction solution was added to distilled water 2
After dialysis three times at ° C., lyophilization yielded 35 mg of hCG-β-bovine serum thyroglobulin complex.

The rabbit was immunized subcutaneously with 1 mg of the above-mentioned complex per rabbit together with Freund's complete adjuvant six times at two-week intervals, and 14 days after the final administration, whole blood was collected from the carotid artery to give anti-hCG
-Β rabbit serum was obtained.

Ammonium sulfate was added to 10 ml of anti-hCG-β rabbit serum to give a final concentration of 1.64 M, and salting out was performed.
Centrifuged for 0 minutes. The precipitate is made up of 5 ml of 0.02 M borate buffer p.
It was dissolved in H8.0 and dialyzed against the same buffer. The supernatant was equilibrated with the same buffer, and a column of hCG-β-binding Sepharose 4B (2.0
× 4.8 cm) and eluted with 0.2 M glycine hydrochloride buffer, pH 2.3, to obtain 20 mg of purified anti-hCG-β antibody.

To 20 mg of the obtained anti-hCG-β antibody, 0.4 mg of pepsin derived from porcine gastric mucosa was added, and reacted at 37 ° C. for 16 hours with 0.1 M acetate buffer pH 4.5. After neutralizing the reaction product, it was applied to a column (1.9 × 90 cm) of Ultrogel AcA-44, and eluted with 0.1 M borate buffer, pH 8.0, to obtain 10 mg of F (ab ′) ₂ . F (ab ') ₂ 10mg at 2mM
It was dissolved in a 0.1 M phosphate buffer (pH 6.0) containing EDTA, 2-mercaptoethylamine was added, and the mixture was reacted at 37 ° C for 90 minutes. The reaction was then applied to a Sephadex G-25 column (1 x 30 cm) and eluted with 0.1 M phosphate buffer pH 6.0 containing 2 mM EDTA to yield 7 mg of Fab '.

A solution prepared by dissolving 10 mg of horseradish peroxidase in 1.4 ml of 0.1 M phosphate buffer at pH 6.8 dissolved 9.7 mg of 4- (N-maleimidomethyl) cyclohexanoic acid / N succinimide ester in 80 μN, N-dimethylformamide was added and reacted at 30 ° C. for 1 hour. After centrifuging the reaction product, the supernatant was separated with a Sephadex G-25 column (1 ×
Elution with 0.1 M phosphate buffer pH 6.8 to prepare 7 mg of maleimidated peroxidase.

Fab ′ 7 mg and maleimidated peroxidase 7 mg prepared as described above were mixed with 0.1 M containing 1 ml of 2 mM EDTA.
The reaction was carried out at 4 ° C. for 20 hours in a phosphate buffer pH 6.5. The reaction solution was passed through an AcA44 column (1.5 × 90 cm) and eluted with 0.1 M phosphate buffer pH 6.5. Thus, the desired anti-hCG-
About 9 mg of βFab′-peroxidase-labeled product was obtained.

Example 4 (Diagnosis of cancer) (1) Measurement In a test tube (12 × 75 mm), a test sample or a β-core labeled solution (100 μm) and a 0.02 M phosphate buffer (pH 7.5) containing 0.1% bovine serum albumin (200 μm) were placed. Then, one immobilized antibody (polystyrene beads) was added, and the mixture was allowed to stand at room temperature (20 to 30 ° C.) for 1 hour. After washing twice with 4 ml of purified water, anti-hCG-βFab ′
Mixing about 500 ng of peroxidase with 300 μl of 0.02 M phosphate buffer (pH 7.5) containing 0.1% bovine serum albumin,
It was left at room temperature (20-30 ° C) for 1 hour. After removing the solution portion and washing the polystyrene beads twice as above,
Transferred to another test tube. The activity of the bound peroxidase was measured by the absorbance at a wavelength of 492 nm when the substrate was allowed to stand in the dark at room temperature for 30 minutes using OPD as a substrate in the presence of hydrogen peroxide.

In the enzyme immunoassay reagent of the present invention, on a molar basis,
The cross-reactivity with hCG-β is about 100%, with β core and hCG
It showed almost the same reactivity to -β. On the other hand, the cross-reactivity rates with the glycoprotein hormones human chorionic gonadotropin, human luteinizing hormone, human follicle-stimulating hormone, and human thyroid-stimulating hormone were 2.2%, 0.29%, and 0.01%, respectively.
% And 0.01% or less. The results are shown in FIG.

The measurement limit was extremely high, ie, 2 pg (0.13 fmol) of β-core / tube (20 pg / ml) (FIG. 2).

The reactivity of the monoclonal antibody 281 produced by the hybridoma 281 recognizing the hCG-β core region obtained using the purified β core as an immunogen and the monoclonal antibody 229 of the present invention was compared. When the reactivity with various protein hormones was compared by the sandwich method using each monoclonal antibody as the immobilized antibody, the monoclonal antibody 229 showed almost the same reactivity with β-core and hCG-β. On the other hand, the monoclonal antibody 281 shows reactivity of 14.0% to hCG-β and 1.2% to hCG on a molar basis, when the β core is 100%, and shows human luteinizing hormone, human follicle stimulating hormone and human thyroid stimulating hormone. Was 0.01% or less. From these test results, it is considered that the monoclonal antibody 229 using hCG-β as an immunogen and the monoclonal antibody 281 using β core as an immunogen recognize different epitopes in the β core region.

(2) Urinary β-core level in healthy subjects The urinary β-core level in healthy subjects (n = 231) was measured with the reagent of the present invention, and the value obtained by adding two standard deviations at an average of 0.05 ng / ml was 0.18 ng / ml. ml. Using monoclonal antibody 281, urinary β-core levels averaged 0.0
It was 0.03 ng / ml when 2 standard deviations were added at 1 ng / ml.

(3) Measurement of urine and serum of various disease patients β core and / or hCG-β levels in urine and serum of various disease patients were measured using this measurement kit. As a control, the monoclonal antibody 281
Was used to measure the β core value (Table 1).

The urine sample showed a high value in pregnancy, but 12 of 17 (approximately 71%) of various cancer patients gave a value of 0.2 ng / ml or more, which was a cut-off of a normal level, and were judged to be positive. On the other hand, when the β-core value was measured using the control monoclonal antibody 281, 9 out of 17 cases (about 53%) showed a normal level cut-off value of 0.03 ng / ml or more.
Met.

On the other hand, in serum samples, 8 out of 17 (approximately 47%) of various cancer patients gave a value of 0.2 ng / ml or more, and were judged to be positive. The seropositivity was significantly higher than the previously published value of about 10-20%.

FIG. 3 shows the measurement results in urine of a patient with an obstetrical / gynecological disorder. Ovarian, uterine and choriocarcinomas gave extremely high positive results.

Effects of the Invention Since the monoclonal antibody produced by the present invention reacts with urine and serum of various cancer patients at a high rate, it can be advantageously used as a diagnostic agent for cancer.

In addition, an extremely high-sensitivity measurement can be performed in a short time using the immunochemical measurement reagent using the monoclonal antibody of the present invention, and the level of a healthy person can be known.

Thus, by establishing the measurement test according to the present invention, β
Not only can basic biosynthesis, secretion, and metabolism be promoted, but the measurement method of the present invention can provide a high positive rate and accurate results. Simple and accurate observation of cancer diagnosis and treatment progress has become possible.

[Brief description of the drawings]

FIG. 1 shows the reactivity of β-core (○), hCG-β (●), hCG (■) and various glycoprotein hormones with the monoclonal antibody of the present invention. FIG. 2 shows the standard curve of the β core by the reagent of the present invention (（), hC
G-β reactivity (●) and cross-reactivity with various glycoprotein hormones are shown. FIG. 3 shows the measurement results in the urine of an obstetric / gynecological disease patient.

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI G01N 33/577 C12N 5/00 B // (C12P 21/08 C12R 1:91) (58) Field surveyed (Int.Cl. ⁷ , DB name) C12N 15/06 C12N 5/16-5/28 C12P 21/08 G01N 33/53 G01N 33/574 G01N 33/577 BIOSIS (DIALOG) MEDLINE (STN) WPI (DIALOG)

Claims (4)

(57) [Claims] 1. An hC-fused mammalian antibody-producing cell immunized with hCG-β-subunit and a lymphoid cell line.
A monoclonal antibody-producing hybridoma that recognizes the G-β-core region and the hCG-β-subunit and has the same reactivity with the hCG-β-core region and the hCG-β-subunit. 2. The hCG-β-core region and hCG-β-subunit produced by the hybridoma according to claim (1) are recognized, and the hCG-β-core region and hCG-β-subunit are recognized by the hybridoma. Monoclonal antibodies having the same reactivity. 3. An immunoassay comprising detecting one or both of the hCG-.beta.-core region and the hCG-.beta.-subunit using the monoclonal antibody according to claim (2). 4. A method for determining the presence or absence of a malignant tumor, which comprises detecting the hCG-β core region in a sample from a patient using the immunological assay method according to (3).