JPH10127282A - Monoclonal antibody against obesity gene product, and production and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new antibody consisting of a monoclonal antibody having an affinity specifically to an obesity gene product, and useful for a highly sensitive detection of the obesity gene product, as a body weight adjusting agent, and an obesity-treating and a diagnostic agent for an obesity patient, a hyperphagia patient, etc. SOLUTION: This new monoclonal antibody having a specific affinity against an obesity gene product can determine the obesity gene product in a high sensitivity, perform a diagnosis of diseases such as obesity based on the result of the determination, and also be used for the treatment of an obesity patient, a hyperphagia patient, etc., as a body weight adjusting agent or an obesity- treating agent and for the purification of the obesity gene product. The monoclonal antibody is obtained by administering the obesity gene product, etc., obtained from a human plasma to a mouse, etc., for an immunization, collecting spleen cells after the final immunization, fusing the spleen cells with myeloma cells, selectively culturing in HAT medium for selecting a clone having an antibody producing capability, cloning the cells and culturing the obtained hybridoma cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a monoclonal antibody showing binding properties to an obesity gene product, its production method and use.

[0002]

[Prior Art] 1994: Y. Zhang et al. [Natur
e), 372, 425-432 (1994)] cloned the first mouse and human obesity gene by the positional cloning method. The obesity gene is a protein that regulates obesity (1
(67 amino acid residues), and the mouse and human obesity gene products showed 84% homology at the amino acid level.
The obesity gene mRNA was mainly expressed in adipose tissue. Later, the rat-derived obesity gene was also cloned, and the homology between the human and rat obesity gene products was 83% [T. Murakami and K. Shima, Biochemical and Biophysical Research Communication.
(Biochem. Biophys. Res.Commun.), 209, 944-952 (19
95); Y. Ogawa et al., Journal of Clinical Investigation (J. Clin. Invest.), 96, 1647-1.
652 (1995)]. When an obesity gene product derived from a mouse produced in Escherichia coli is administered intraperitoneally to a C57BL / 6J ob / ob mouse in which the obesity gene is mutated (nonsense mutation at codon 105), weight loss, body fat percentage, and food intake are suppressed. , Decreased blood glucose and insulin levels, increased body temperature, and increased activity [MA Pelleymounter et al., Science, 269, 540-543 (1995); JL Halaas
Et al., Science, 269, 543-546 (1995)]. Obesity gene mRNA is overexpressed in adipose tissue of obese people [F. Lon
nqvist et al., Nature Medicine (Nature Med.), 1,
950-953 (1995)]. In rodents and humans, the obesity gene product in plasma is determined by the Body Mass Index (BMI)
[M. Maffei et al., Nature Med., 1, 11]
55-1161 (1995)].

The aforementioned JL Halaas et al. [Science, 269, 543]
-546 (1995)] uses a rabbit polyclonal antibody to measure the concentration of an obesity gene product in plasma. According to this, 0.5 ml of plasma was applied to antibody-immobilized Sepharose to purify an obesity gene product, which was then subjected to SDS.
-PAGE, and finally chemiluminescence immunoblotting on the membrane using a biotin-labeled antibody. This method is very complicated and has low sensitivity. JL Halaas et al. In WO 96/05309
A polyclonal antibody against a recombinant obesity gene product is obtained from a rabbit, the antibody is purified using an obesity gene product / column, and an enzyme immunoassay (EIA) is prepared using this antibody and a recombinant obesity gene product derived from Escherichia coli. However, this system is used to measure the amount of obesity gene product in human plasma. Regarding radioimmunoassay (RIA), RV Considine et al. [The New England Journal of Medicine (New Engl. J. Med.), 33]
4, 292-295, 1996) and K. Hosoda et al. [Biochem. Biophy
s. Res. Commun., 221, 234-239 (1996)], ¹²⁵
An RIA was made using an I-labeled obesity gene product and a polyclonal anti-obesity gene product serum immunized in rabbits,
The detection sensitivities were 400 pg / ml and 200 pg / ml, respectively.
When measuring trace components in living organisms, highly sensitive and highly specific monoclonal antibodies are often used,
As for the method of quantifying an obesity gene product, no such monoclonal antibody and no quantification method using the same have been reported.

[0004]

The obesity gene product can be regarded as one marker of obesity, and measuring the concentration of the obesity gene product in a body fluid is considered to be clinically useful. In order to prepare a highly sensitive method for quantifying an obesity gene product using a reagent that can be supplied stably, a monoclonal antibody having a certain quality and high sensitivity to the obesity gene product is obtained. This is very important.

[0005]

The present invention provides (1) a monoclonal antibody having a specific affinity for an obesity gene product, and (2) a measurement of an obesity gene product characterized by using the monoclonal antibody. A method, (3) a medicament characterized by containing the monoclonal antibody, (4) a diagnostic agent characterized by containing the monoclonal antibody, and (5) an obesity characterized by containing the monoclonal antibody. A kit for measuring a gene product, (6) a reagent containing the monoclonal antibody, (7) a method for purifying an obesity gene product using the monoclonal antibody, and (8) production of the monoclonal antibody (9) a spleen cell of a mammal immunized with an obesity gene product and a lymphocyte of a homologous or heterologous mammal; Like a cell fusion, fused cells characterized by cloning, culturing method for producing the hybridoma, and (10) the hybridoma,
The present invention relates to a method for producing the monoclonal antibody, comprising recovering the produced monoclonal antibody.

As a method for obtaining a monoclonal antibody having a specific affinity for an obesity gene product and a hybridoma producing the same, a method for obtaining a normal monoclonal antibody or the like using an obesity gene product as an immunogen is used. Can be applied. As the obesity gene product used as an immunogen, an obesity gene product obtained from mammalian plasma or a recombinant obesity gene product is used. For example, in order to produce a monoclonal antibody against the human obesity gene product, an obesity gene product or a recombinant human obesity gene product obtained from human plasma is used,
Preferably, a recombinant human obesity gene product is used. Recombinant human obesity gene products are described, for example, in K. Hosoda et al. [Bioc.
hem. Biophys. Res. Commun., 221, 234-239 (1996)]
, Or according to the method described in WO 96/05309. Laboratory animals such as rats and mice are usually used as mammals to be immunized. When immunizing a mouse, for example, it is preferable to administer the immunogen subcutaneously, intraperitoneally, or intravenously. Also, the immunity interval,
The amount of the obesity gene product as an immunogen is not particularly limited, and various methods may be used. For example, the obesity gene product is inoculated 2 to 8 times at 2 week intervals, and 1 to 5 times after the final immunization. A method using spleen cells after a day, preferably two to four days later, is generally used. When a mouse is used as the mammal to be immunized, the amount of the obesity gene product as an immunogen is preferably 1 μg or more, preferably 10 to 100 μg, per mouse as the amount of protein at one time. It is desirable to perform a fusion experiment after performing partial blood sampling before removing spleen cells from the mammal immunized in this manner, and confirming an increase in blood antibody titer (antibody to an obesity gene product).

Cell fusion between antibody-producing spleen cells and lymphoid cells (eg, myeloma cells) can be performed as follows. The spleen cells (lymphocytes) of the immunized mammal and DNA synthesis pathways such as hypoxanthine guanine phosphoribosyltransferase (HGPRT) and thymidine kinase (TK) from a suitable allogeneic or xenogeneic animal (preferably allogeneic) It is fused with myeloma cells lacking the enzyme. For fusion, it is common to add a fusion agent such as Sendai virus or polyethylene glycol (PEG). PEG having an average molecular weight of about 1,000 to 20,000 is used at a treatment time of 0.5 to 30 minutes and at a concentration of about 10% to 80%. By performing the treatment at ~ 55% for 4 to 10 minutes, the fusion can be performed efficiently. The obtained fused cells (hybridomas) were isolated using hypoxanthine / aminopterin / thymidine medium [HAT medium; G. Kohler and C. Milstein, Nature, 256, 495-497 (1975)].
It can be selectively grown.

[0008] The presence or absence of the target antibody in the serum of the immunized mammal or in the culture supernatant of the grown fused cells is determined by radioimmunoassay (RIA) or enzyme immunoassay (EIA). However, various modifications of these methods are possible. As an example of the measuring method, a method using EIA will be described. An obesity gene product used as an immunogen is immobilized on a solid phase (for example, a 96-well microplate) according to a conventional method, and then a culture supernatant to be measured or serum of an immunized mammal is added thereto for a certain period of time (usually 30 minutes). Minutes to 24 hours), constant temperature (usually,
(0 to 40 ° C). Thereafter, the plate is thoroughly washed, and an antibody to an immunoglobulin of the immunized mammal labeled with an enzyme is added thereto for a certain period of time (usually,
The reaction is carried out at a constant temperature (usually 0 to 40 ° C.) for 30 minutes to 24 hours. After washing the plate well, 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. The reaction time and temperature with the enzyme substrate differ depending on the enzyme used and the enzyme substrate. When horseradish peroxidase (HRP) is used as the enzyme and o-phenylenediamine is used as the enzyme substrate, the reaction time is usually 15 minutes to 2 hours. The reaction temperature is usually 15-30 ° C. When a monoclonal antibody capable of binding to the obesity gene product is present, the absorbance is increased or the fluorescence intensity is increased.

[0009] It is desirable to clone cells that exhibit growth in a HAT medium and produce an antibody having an affinity for an obesity gene product by a soft agar method, a limiting dilution method, or the like. The supernatant of the cloned cells is examined for the presence of an antibody having an affinity for the obesity gene product in the same manner as described above, and by increasing the number of well cells producing antibodies having a high affinity, a monoclonal antibody-producing hybridoma is produced. Can be established. The hybridoma thus cloned is grown in a liquid medium or intraperitoneally in a mammal. For example, in a liquid medium, for example, G
The monoclonal antibody can be obtained from the culture by culturing in an IT medium [Wako Pure Chemical Industries, Ltd.] or the like for 2 days or more (preferably for 7 to 20 days). Also, by inoculating the peritoneal cavity of a suitable mammal such as a mouse to proliferate cells and collecting ascites fluid, a large amount of antibody with a much higher titer than the cell culture supernatant can be efficiently obtained. . For this purpose, for example, in the case of mice, 1 × 10 ⁴ to 1 × 10 ⁷ , preferably 5 × 10 ⁵ to 2 ×, mice such as BALB / c inoculated with mineral oil or the like in advance are added.
10 ⁶ hybridomas were inoculated into, such as the abdominal cavity, 7
Twenty days later, ascites fluid and the like are collected. Antibodies produced and accumulated in a liquid medium or ascites fluid include, for example, ammonium sulfate fraction, DEA
Monoclonal antibodies can be easily isolated as pure immunoglobulins by separation and purification operations such as E-cellulose column chromatography and protein A column chromatography. The monoclonal antibody in the present invention may be an immunoglobulin (preferably IgG), or a fragment (eg, F (ab ′) ₂ , Fab ′ or Fa) that retains affinity for the obesity gene product.
b).

As a method for measuring the obesity gene product of the present invention, not only the sandwich method but also a competitive method or another method can be used as long as it can be measured. The solid phase is not limited to its material shape, and the label may be an enzyme, a radioisotope, a spin labeling agent, a fluorescent substance, a luminescent substance, or the like.
Radioisotopes such as ¹²⁵ I are preferably used. The measuring device may be selected according to the marker. In order to quantify the obesity gene product with high sensitivity, a sandwich method using monoclonal antibodies against two types of obesity gene products having different antigen recognition sites is preferably used. Monoclonal antibodies against two obesity gene products having different antigen recognition sites include, for example, (1) immobilizing one monoclonal antibody on a microplate;
Reacting the obesity gene product, (3) washing the microplate, reacting with another enzyme-labeled monoclonal antibody, and (4) washing the microplate, reacting with the enzyme substrate, and measuring the enzyme reaction product Can be selected. These steps are described in the following EIA
Under the same conditions as those of the sandwich method.

As an example of the measuring method of the present invention, a sandwich method by EIA and a sandwich method by RIA using two types of monoclonal antibodies having different antigen recognition sites are briefly described below, but are not limited to this quantitative method. is not. (1) Sandwich method by EIA: (Step 1) Immobilize a monoclonal antibody (A) against an obesity gene product on a microplate. For immobilization, a monoclonal antibody diluted with a buffer such as Tris buffer is dispensed into each well of a microplate, and 0 to 40 ° C.
For 30 minutes to 24 hours. (Step 2) Block the microplate with a blocking agent such as bovine serum albumin (BSA) or Block Ace. Blocking can be performed by dispensing a buffer solution (for example, a phosphate buffer solution) containing a blocking agent into each well of the microplate, and allowing to stand at 0 to 40 ° C. for 30 minutes to 24 hours. (Step 3) The sample is reacted with the microplate.
The reaction can be performed by dispensing a sample (or a diluent thereof) into each well of a microplate and allowing the mixture to stand at 0 to 40 ° C. for 30 minutes to 24 hours. At this time, BS
A, MOPS [3- (N-morpholino) propanesulfonic acid] buffer containing NaN _{3 and the} like is preferably added. (Step 4) After washing the microplate, the monoclonal antibody (B) against the enzyme-labeled obesity gene product is reacted. Wash the microplate with Tween 20
Physiological saline containing a surfactant such as phosphate buffered saline (PBS) is preferably used. HRP is preferably used as an enzyme as a label. Upon reaction with the antibody, MOPS buffer can be used as a diluent. The reaction can be performed by allowing to stand at 0 to 40 ° C. for 30 minutes to 24 hours. (Step 5) After washing the microplate, the microplate is reacted with an enzyme substrate, and an enzyme reaction product is measured. For washing the microplate, physiological saline or PBS containing a surfactant such as Tween 20 is preferably used. The enzyme substrate varies depending on the type of the enzyme as the label. When HRP is used as the enzyme, o-phenylenediamine is preferably used as the substrate, and the reaction time is usually 15 minutes.
The reaction temperature is usually from 15 to 30 ° C. for from minutes to 2 hours. After terminating the reaction with the substrate with a reaction terminator such as sulfuric acid, the reaction product can be measured. When HRP is used as the enzyme and o-phenylenediamine is used as the substrate, the amount of the reaction product can be measured by measuring the absorbance near 492 nm.

(2) RIA sandwich method: (Step 1) Immobilize a monoclonal antibody (A) against an obesity gene product on a microplate. The above EI
A can be performed in the same manner as in step 1 of the sandwich method using A. (Step 2) Block the microplate with a blocking agent such as bovine serum albumin (BSA) or Block Ace. It can be carried out in the same manner as in step 2 of the sandwich method by EIA. (Step 3) The sample is reacted with the microplate.
It can be carried out in the same manner as in step 3 of the sandwich method by EIA. (Step 4) After washing the microplate, a monoclonal antibody (B) against an obesity gene product labeled with a radioisotope is reacted. For washing the microplate, physiological saline containing a surfactant such as Tween 20 or phosphate buffered saline (PBS) is preferably used. ¹²⁵ I as radioisotope as label
Is preferably used. Upon reaction with the antibody, MOPS buffer can be used as a diluent. Reaction is 0-4
It can be carried out by allowing to stand at 0 ° C. for 30 minutes to 24 hours. (Step 5) After washing the microplate, the radioactivity is measured. Wash the microplate with Tween 20
A physiological saline or PBS containing a surfactant such as a surfactant is preferably used. The radioactivity can be measured by a method known per se.

A reaction curve is prepared by performing the reactions of steps 1 to 5 of the sandwich method by EIA or steps 1 to 5 of the sandwich method by RIA using a known amount of a standard solution of an obesity gene product, and a sample is prepared from the calibration curve. The obesity gene product content in the can be determined. As a sample,
Biological samples include, for example, blood, serum, plasma, urine, cerebrospinal fluid, tissue extracts and the like. The obesity gene product can be quantified by the method described above.

Since the blood concentration of an obesity gene product in an obese patient (usually 10 ng / ml or more) is higher than that in a normal person (several ng / ml), the blood concentration of the obesity gene product must be measured. Can be used to diagnose obesity. The obesity gene product measurement kit and the diagnostic agent of the present invention may include at least one monoclonal antibody of the present invention. Preferably, the monoclonal antibody (A) and the monoclonal antibody immobilized on a solid phase are used. The antibody (A) includes a monoclonal antibody (B) having a different antigen recognition site and used as a secondary antibody. The monoclonal antibody (B) may be labeled with, for example, an enzyme or the like, and the monoclonal antibody (A)
In addition to (B) and (B), the composition may contain various reagents (for example, enzyme substrates, buffers, diluents, etc.). Examples of the form of the reagent containing the monoclonal antibody of the present invention include a reagent containing a monoclonal antibody, a reagent containing a monoclonal antibody labeled with an enzyme, and an enzyme substrate solution, and a reagent containing a buffer solution and the like. .
In addition, the monoclonal antibody of the present invention is used per se or in the field of pharmaceutical preparations, and may be administered together with a pharmacologically acceptable carrier to a weight controlling agent, for example, an obesity therapeutic agent, to obese patients, bulimia patients, etc. Can also. The administration can be generally performed parenterally as an injection, a suppository, or a nasal agent to mammals including humans to be administered. The dose varies depending on the administration subject, symptoms, administration route and the like. For example, when the monoclonal antibody of the present invention is parenterally administered to an obese adult patient, the daily dose is preferably 1 to 50 μg.
/ kg body weight, and can be administered in 1 to 3 times a day. The monoclonal antibody of the present invention contained in the preparation is usually 1 to 100% (w / w). further,
The obesity gene product can be purified using the monoclonal antibody of the present invention. That is, a sample containing an obesity gene product or a crude product of an obesity gene product is loaded on a column to which the monoclonal antibody of the present invention is bound, and after the obesity gene product is adsorbed on the column, the obesity gene product is dissolved in an appropriate elution solvent. By eluting, the obesity gene product can be purified. The eluted obesity gene product can be further purified by subjecting it to dialysis, gel filtration, electrophoresis, various types of chromatography, etc. which are usually used for protein purification.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described more specifically with reference to Reference Examples and Examples, but the present invention is not limited thereto. The hybridomas (Lep 2-B) having the ability to produce monoclonal antibody 2-B and the hybridomas (Lep 2-B) having the ability to produce monoclonal antibody 15-A obtained in the Examples described later.
p15-A) was submitted to the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology (NIBH) from September 4, 1996 under accession number FERM P-15831 and accession number F, respectively.
Deposited as ERM P-15832.

[0016]

【Example】

Reference Example 1 Cloning of human obesity (ob) gene Human obesity ob reported previously (GenBank Accession No. U18915)
The following two primers were synthesized based on the sequence of the gene, and a human adipose tissue cDNA library λgt11 was synthesized.
(Clontech Laboratories, Inc.) from human obesity (o
b) The mature region of the gene was obtained by the PCR method. Sense primer No. 1: 5'-TCCATATGGTGCCCATCCAA
-3 'Antisense primer No. 2: 5'-AGGATCCGTGACCT
TCAAGGC-3 'The amplified DNA fragment was transformed into an E. coli expression plasmid vector pET-3c [Methods in Enzymology (Me
thods in Enzymology, ed. by DV Goeddel), 1st
85, p. 68 (1990), Academic Press] and the constructed plasmid was named pET-hob.

Reference Example 2 Expression of human obesity (ob) cDNA in Escherichia coli MM294 (DE3) Escherichia coli MM29 was expressed using plasmid pET-hob of Reference Example 1.
4 (DE3) and expressed ob under the control of the T7 promoter [Methods in Enzymology, No. 1].
85, 60 (1990)]. The transformed Escherichia coli was cultured, the obtained cells were sonicated, centrifuged, and the precipitate was subjected to SDS-PAGE.
A specific band corresponding to the mature form of ob near CB
Detected by B staining. Since the expressed product formed an inclusion body (inclusion body), an obesity gene product was recovered from the precipitate fraction of the ultrasonically crushed transformant. The precipitate of the mature protein was suspended in 4 M urea and shaken at 4 ° C. overnight to solubilize, and then 20 mM Tris-HC was added.
The urea was removed by dialysis against 1 buffer (pH 7.5). This product (purity 95% or more) was used as a standard product of a modified obesity gene product.

REFERENCE EXAMPLE 3 Refolding of the human obesity (ob) gene product The modified human ob gene product obtained in Reference Example 2 was refolded using oxidized glutathione, which has been reported, in order to return to the natural conformation. [Biotechnology (BIO / TECHNOLOGY), Vol. 9, pp. 157, (1991)].
That is, the dialyzed human ob gene product obtained in Reference Example 2 was subjected to high performance liquid chromatography [column: microbonder sphere (μBONDASPHERE 15μC430).
0A, 3.9 × 300 mm), Nippon Millipore Limited], and the human obesity (ob) gene product
M urea, 0.1 M Tris-HCl, pH 8.0, 0.3
It was dissolved in M dithiothreitol (DTT) and allowed to stand at 4 ° C. overnight. This was added to 50 mM Tris-HCl (pH
Dialysis was performed on 8.5) to remove urea and DTT. Oxidized glutathione (GSSG) was added to this solution to adjust the final concentration to 0.2M. This was allowed to stand at room temperature for 2 hours, and then dialyzed against 50 mM Tris-HCl (pH 8.5) to remove GSSG. Further, L-arginine and GSSG were added to 0.4 M and 4 mM, respectively, and the pH was adjusted to 8.5 with hydrochloric acid, followed by standing at 15 ° C. for 6 days. Thereafter, this solution was equilibrated with 20 mM Tris-HCl buffer (pH 7.5) and HiLoad ^™ 16/60 Superdex was equilibrated.
75 columns [Pharmacia Biotech AB, Uppsala, Sweden]
And collected the product. The obtained product (purity 99% or more) was used as a standard product of a natural obesity gene product.

Example 1 Preparation of hybridoma producing monoclonal antibody capable of binding to obesity gene product 20 μg (0.2 ml) of recombinant obesity gene product (Reference Example 2) dissolved in physiological saline was completely Freund's adjuvant. It was suspended in 0.2 ml and administered intraperitoneally to BALB / c mice. Further, 50 μg (0.2 ml) of the recombinant obesity gene product (Reference Example 2) was suspended in 0.2 ml of Freund's incomplete adjuvant, and boosted twice (2 and 4 weeks later). Three days after the final immunization, spleen cells of the mouse were excised. Collected spleen cells (1.0 × 10 ⁸
) And mouse myeloma cells P3U1 (1.0 × 10
⁷ ) in RPMI-1640 medium, and mixed at 1,000
Centrifuged at rpm for 5 minutes. 50% in the obtained sediment
Polyethylene glycol 6000 (1 ml) was added slowly at 37 ° C. over 1 minute to fuse the cells. continue,
After adding 7 ml of RPMI-1640 medium heated to 37 ° C. over 5 minutes, the supernatant was removed by centrifugation.
The obtained fused cells were diluted with a HAT medium, dispensed into a 96-well microplate in an amount of 0.1 ml, and cultured in a CO ₂ incubator while changing the HAT medium by half every two to three days. Ten days later, the culture supernatant of the hybridoma was subjected to an affinity test for the obesity gene product. Screening of a monoclonal antibody having affinity for the obesity gene product was performed as follows. Recombinant obesity gene product (Reference Example 2) was diluted with carbonate buffer pH 9.6 to 10 μm.
After dilution to a concentration of g / ml, 100 μl was dispensed into each well of the microplate. After leaving at 37 ° C. for 1 hour, the plate was washed and then 1% BSA (phosphate buffer pH
7.4) was dispensed into each well in an amount of 200 μl, and 1 μl was added at 37 ° C.
Each well was blocked for a period of time. After washing the plate, 100 μl of the hybridoma culture supernatant or 100 μl of the HAT medium as a negative control was added to the 96-well microplate, and the plate was left still for 1 hour. After washing, 100 μl of the diluted HRP-labeled anti-mouse immunoglobulin antibody (rabbit) was dispensed, and allowed to stand at 37 ° C. for 1 hour. After washing the plate, an o-phenylenediamine solution was further added to each well and reacted at room temperature for 30 minutes, and then 1N sulfuric acid was added to stop the reaction. 4 with microplate reader
The absorbance at 90 nm (OD _{490 nm} ) was measured, and those giving an absorbance more than twice that of the negative control were _defined as positive wells.
That is, it was determined that a monoclonal antibody against the obesity gene product was produced. After completion of the screening, the clone was obtained by the limiting dilution method as follows. Hybridomas in the positive wells were counted and diluted, and dispensed into a 96-well microplate such that one hybridoma was placed in each well. After culturing in a CO ₂ incubator for 7-14 days, the wells in which hybridomas grew were examined for affinity to the obesity gene product in the same manner as at the time of screening. Thus, finally, a total of 9 clones of hybridomas were established.

Example 2 Preparation of Monoclonal Antibody 0.5 ml of pristane was intraperitoneally administered to a BALB / c mouse in advance. 0.5 in the pristane-treated intraperitoneal cavity
5 × 10 ⁶ hybridomas obtained in Example 1 suspended in ml of RPMI-1640 medium were injected. Ten days later, the ascites pooled was collected and purified by protein A column chromatography to obtain the desired monoclonal antibody.

Example 3 Combination of Monoclonal Antibodies Using 9 clones of the monoclonal antibody against the obesity gene product established in Example 1 and an obesity gene product prepared in Escherichia coli (Reference Example 2), which combination of monoclonal antibodies was highly sensitive Was given. The procedure is as follows. (1) Microplate (Nunc Immunoplate II)
Maxisorp) with Tris buffer (pH 8.5) for 20μ
50 μl of a monoclonal antibody against the obesity gene product diluted to g / ml was dispensed into each well, and left still at 4 ° C. overnight to immobilize the antibody on the microplate. (2) Each well was diluted with phosphate buffered saline (PBS) for 3 hours.
After washing twice, a blocking buffer [10 mM phosphate buffer (pH 7.2), 25% block ace, 0.01%
NaN ₃ ] was dispensed to each well in an amount of 300 μl, and allowed to stand at 4 ° C. overnight to block each well. (3) After washing each well three times with PBS, the primary reaction buffer [50 mM MOPS (pH 7.0), 150 mM
50 μl of NaCl, 1% BSA, 0.1% NaN ₃ ] or 50 μl of an obesity gene product solution diluted to 10 ng / ml with the same buffer and 100 μl of the same buffer were added to each well and reacted at 4 ° C. overnight. . (4) The microplate was washed three times with PBS containing 0.05% Tween 20, and then appropriately diluted [diluent: 5
0 mM MOPS (pH 7.2), 150 mM NaC
l] of the monoclonal antibody against the HRP-labeled obesity gene product was added to each well, and 4 μl at 25 ° C.
Allowed to react for hours. (5) The microplate was washed three times with PBS containing 0.05% Tween 20, and 50 μl of o-phenylenediamine was added to each well as an enzyme substrate. After reacting at room temperature for 30 minutes, 50 μl of 2N sulfuric acid was added to each well.
The reaction was quenched by the addition in small portions. The enzyme product was measured by measuring OD _{490 nm} with a microplate reader. Table 1 shows the measurement results. The horizontal axis indicates the type of the monoclonal antibody used as the labeled antibody, and the vertical axis indicates the type of the monoclonal antibody used as the solid-phase antibody. 10ng / m
From the S / N ratio at 1, the suitability of the combination of the solid phase antibody and the labeled antibody was determined.

[0022]

[Table 1]

Example 4 Preparation of calibration curve-1 Based on the results in Table 1 (S / N ratio at 10 ng / ml), a combination of solid-phase antibody 2-B and labeled antibody 15-A considered to be a preferable combination was selected. The minimum detection sensitivity was determined by the following procedure. (1) Microplate (Nunc Immunoplate II)
Maxisorp) with Tris buffer (pH 8.5) for 20μ
50 μl of the monoclonal antibody (2-B) against the obesity gene product diluted to g / ml was dispensed into each well,
The antibody was immobilized on a microplate by standing overnight. (2) After washing each well three times with PBS, 1% BSA
Was added to each well in an amount of 300 μl / well, and allowed to stand at 4 ° C. overnight to block each well. (3) After washing each well three times with PBS, PBS50
50 μl of a standard obesity gene product (Reference Example 2) solution diluted with μl or PBS and a primary reaction buffer [50 mM
MOPS (pH 7.0), 150 mM NaCl, 1%
100 μl of BSA, 0.1% NaN ₃ ] was added to each well and reacted at 4 ° C. overnight. (4) The microplate was washed three times with PBS containing 0.05% Tween 20, and then appropriately diluted [diluent: 5
0 mM MOPS (pH 7.2), 150 mM NaC
l], 50 µl of a monoclonal antibody (15-A) against the HRP-labeled obesity gene product was added to each well, and reacted at 25 ° C for 4 hours. (5) After washing the microplate three times with PBS containing 0.05% Tween 20, 50 μl of o-phenylenediamine as an enzyme substrate was added to each well, and the plate was added at room temperature for 3 hours.
After reacting for 0 minutes, 50 μl of 2N sulfuric acid was added to each well to stop the reaction. The enzyme product was measured by measuring OD _{490 nm} with a microplate reader. The results are shown in Table 2 and FIG. From the obtained results, assuming that the minimum detection sensitivity is 2 or more in S / N ratio, the minimum detection limit is estimated to be about 0.1 ng / ml.

[0024]

[Table 2]

Example 5 Preparation of Calibration Curve-2 Based on the results shown in Table 1, a calibration curve was prepared using a combination of monoclonal antibodies considered to be a preferable combination and the natural obesity gene product prepared in Reference Example 3 as a standard. Was prepared by the following procedure, and was compared with a calibration curve using the modified obesity gene product prepared in Reference Example 2 as a standard. The protein concentration of the obesity gene product used here was determined by the Lowry method. (1) Microplate (Nunc Immunoplate II)
Maxisorp) with Tris buffer (pH 8.5) for 20μ
50 μl of the monoclonal antibody (2-B) against the obesity gene product diluted to g / ml was dispensed into each well,
The antibody was immobilized on a microplate by standing overnight. (2) After washing each well three times with PBS, 1% BSA
Was added to each well in an amount of 300 μl / well, and allowed to stand at 4 ° C. overnight to block each well. (3) After washing each well three times with PBS, PBS50
μl, 50 μl of a modified obesity gene product (Reference Example 2) solution diluted with PBS or 50 μl of a natural obesity gene product (Reference Example 3) solution diluted with PBS and a primary reaction buffer [50 mM MOPS (pH 7.0), 150 mM
100 μl of NaCl, 1% BSA, 0.1% NaN ₃ ] was added to each well and reacted at 4 ° C. overnight. (4) The microplate was washed three times with PBS containing 0.05% Tween 20, and then appropriately diluted [diluent: 5
0 mM MOPS (pH 7.2), 150 mM NaC
l], 50 µl of a monoclonal antibody (15-A) against the HRP-labeled obesity gene product was added to each well, and reacted at 25 ° C for 4 hours. (5) After washing the microplate three times with PBS containing 0.05% Tween 20, 50 μl of o-phenylenediamine as an enzyme substrate was added to each well, and the plate was added at room temperature for 3 hours.
After reacting for 0 minutes, 50 μl of 2N sulfuric acid was added to each well to stop the reaction. The enzyme product was measured by measuring OD _{490 nm} with a microplate reader. The results are shown in Table 3 and FIG. From the obtained results, it is considered that the calibration curves of the denatured obesity gene product and the natural obesity gene product are almost the same, and the reactivity of the combination of the monoclonal antibodies used to both antigens is equal.

[0026]

[Table 3]

[0027]

The obesity gene product can be quantified with high sensitivity by using the monoclonal antibody against the obesity gene product of the present invention, and a disease such as obesity can be diagnosed based on the result. Further, the monoclonal antibody of the present invention can be administered to a body weight regulator, for example, a therapeutic agent for obesity to obese patients, bulimia patients, etc. by itself or together with a pharmacologically acceptable carrier. further,
Obesity gene products can also be purified using the monoclonal antibodies of the present invention.

[0028]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence TCCATATGGT GCCCATCCAA

SEQ ID NO: 2 Sequence length: 21 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence AGGATCCGTG ACCTTCAAGG C

[Brief description of the drawings]

FIG. 1 shows a calibration curve using a modified obesity gene product as a standard.

FIG. 2 shows a calibration curve (□) using a denatured obesity gene product as a standard and a calibration curve (+) using a natural obesity gene product as a standard.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI G01N 33/53 G01N 33/53 D 33/577 33/577 B (72) Inventor Kazukazu Nakao Oedakita, Nishikyo-ku, Kyoto-shi, Kyoto 412 Kutsukake-cho

Claims (12)

[Claims] 1. A monoclonal antibody having specific affinity for an obesity gene product. 2. A hybridoma capable of producing a monoclonal antibody having a specific affinity for an obesity gene product. 3. A method for measuring an obesity gene product, which comprises using a monoclonal antibody having a specific affinity for an obesity gene product. 4. A monoclonal antibody (A) having a specific affinity for an obesity gene product, and a monoclonal antibody (A) having a specific affinity for an obesity gene product. 4. The method according to claim 3, wherein the monoclonal antibody (B) is different from the above. 5. The method according to claim 3, which is an enzyme immunoassay or a radioimmunoassay. 6. A method for producing a monoclonal antibody, which comprises culturing a hybridoma capable of producing a monoclonal antibody having a specific affinity for an obesity gene product, and collecting the produced monoclonal antibody. Method. 7. An obesity gene product, comprising fusing mammalian spleen cells immunized with an obesity gene product with lymphoid cells of a homologous or heterologous mammal, and cloning the fused cell. For producing a hybridoma capable of producing a monoclonal antibody having specific affinity. 8. Use of a monoclonal antibody having a specific affinity for an obesity gene product,
A method for purifying an obesity gene product. 9. A medicament comprising a monoclonal antibody having a specific affinity for an obesity gene product. 10. A diagnostic agent comprising a monoclonal antibody having a specific affinity for an obesity gene product. 11. A kit for measuring an obesity gene product, comprising a monoclonal antibody having a specific affinity for an obesity gene product. 12. A reagent comprising a monoclonal antibody having a specific affinity for an obesity gene product.