JPH05252987A - Anti-igf-ii monoclonal antibody - Google Patents

Abstract

PURPOSE:To obtain a new monoclonal antibody useful for accurately determining and analyzing IGF-II, having high specificity. CONSTITUTION:A monoclonal antibody IgG recognizing human insulin-like growth factor II but not human insulin-like growth factor I, such as 2B11, ID5 or ID9. Monoclonal antibody 2B11 is obtained from hybridoma 2B11 (FERM BP-32,741).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a monoclonal antibody against human insulin-like growth factor II (hereinafter referred to as IGF-II) and a method for producing the same.

[0002]

2. Description of the Related Art IGF-II is a 67-amino acid peptide having insulin-like activity, and is known to be widely present in the living body such as blood and bone tissue. IGF-II has a high homology with human insulin-like growth factor I (hereinafter referred to as IGF-I) at the amino acid level, and accordingly, the anti-IGF-I antibody or anti-IGF-II antibody reported so far. Many of them showed crossability to each other (J. Clin.
Invest., 60 , 646-657 (1977), J. Clin. Endocrinol.
Metab., 50 , 405-406 (1980), J. Clin. Invest., 68 , 1
321-1330 (1981)).

[0003]

[Problems to be Solved by the Invention] Accurately quantifying IGF-II
In order to analyze or isolate IGF-II from in vivo, it is necessary to create superior monoclonal antibodies with higher specificity.

[0004]

[Means for Solving the Problems] To create a plurality of anti-IGF-II monoclonal antibodies having different recognition sites and extremely high specificity. This will enable the development of enzyme-antibody methods that combine multiple antibodies, and thus enable accurate quantification of IGF-II. In addition, such useful antibodies can be used for various analyzes such as Western blotting analysis of IGF-II and purification of IGF-II.

The present invention provides a monoclonal antibody that recognizes human insulin-like growth factor II but not human insulin-like growth factor I. Furthermore, this monoclonal antibody has the amino acid sequence: (NH ₂ —) P
he-Arg-Pro-Ala-Ser-Arg-Va
l-Ser-Arg-Arg-Ser-Arg-Gly
A monoclonal antibody that recognizes (-COOH) and a monoclonal antibody that has different recognition characteristics. The present invention comprises the amino acid _{sequence: (NH 2 -) Ala-} Tyr-A
rg-Pro-Ser-Glu-Thr-Leu-Cy
s-Gly-Gly-Glu-Leu-Val-Asp
-Thr-Leu-Gln-Phe-Val-Cys-
Gly-Asp-Arg-Gly-Phe-Tyr-P
he-Ser-Arg-Pro-Ala-Ser-Ar
g-Val-Ser-Arg-Arg-Ser-Arg
-Gly-Ile-Leu-Glu-Glu-Cys-
Cys-Phe-Arg-Ser-Cys-Asp-L
eu-Ala-Leu-Leu-Glu-Thr-Ty
r-Cys-Ala-Thr-Pro-Ala-Lys
-Ser-Glu (-COOH) (amino acid sequence of the polypeptide (NH ₂ -) represents the amino terminus of a polypeptide, (-. COOH) is showing the carboxy terminus) polypeptide (hereinafter represented by poly (Referred to as peptide-2) is used as an antigen to immunize mammals, hybridomas are produced with antibody-producing cells and myeloma cells of the immunized mammals, and then the cloned hybridomas are selected and cultured, and the antibodies are cultured. The present invention relates to a method for producing a monoclonal antibody belonging to immunoglobulin IgG which recognizes human insulin-like growth factor II but does not recognize human insulin-like growth factor I. It also relates to this monoclonal antibody. The amino acid sequence shown above is 43 of IGF-II.
The second amino acid is converted to leucine, and a plurality of monoclonal antibodies can be obtained using this polypeptide as an antigen.

The resulting monoclonal antibody, namely IG
A monoclonal antibody belonging to the IgG class that recognizes F-II but not IGF-I, for example, that is numbered 2H11 has the amino acid sequence: (NH ₂ —) Phe-Ser-A
rg-Pro-Ala-Ser-Arg-Val-Se
r-Arg-Arg-Ser-Arg-Gly (-CO
It is a monoclonal antibody characterized by recognizing OH). A monoclonal antibody belonging to the IgG class which recognizes IGF-II but does not recognize IGF-I, although having different recognition characteristics, can be obtained. For example, 2B11, ID
Some are numbered 5 or ID 9. By combining these two monoclonal antibodies, IGF-II can be measured by the two-antibody sandwich method. To obtain monoclonal antibody 2H11, 2B11, ID5 or ID9, monoclonal antibody producing hybridoma 2H1
1, hybridoma 2B11, hybridoma ID5 or hybridoma ID9 may be used. In animal immunization methods such as those obtained by the above-described production methods, acquisition of antibody-producing cells, acquisition methods of hybridomas, acquisition methods of hybridomas that have been cloned, and methods of culturing the same to manufacture monoclonal antibodies. The method generally used can be used (NATURE 256 , 495-497 (1975)).

A specific method for producing a monoclonal antibody in the present invention will be described in detail. A. Preparation of Antigen Polypeptide-2 used as an antigen is IGF- reported by Marumoto et al.
Based on the method for producing II (J. gen. Virol. 68, 2599-2606 (1987)), a conventional site-specific mutagenesis technique (Sambrook, J. et a
l. "Molecular Cloning" 2nd ed., vol. 2, 15.51 ~ 15.
80 (1989) and Kunkel, TA et al. Methods in Enz.
ymology 154 , 367-382 (1987)).

B. Immunization of Mouse with Polypeptide-2 Female BALB / mouse can be used, but mouse or rat of other strains may also be used. However, the immunization regimen and the sensitizing concentration with the polypeptide-2 should be set so that lymphocytes stimulated with a sufficient amount of antigen are formed. For example, 0.1 mg of polypeptide-2 /
Calf serum albumin (hereinafter referred to as BSA) and conjugate were subcutaneously administered 4 times at intervals of 2 to 3 weeks, and further 0.1.
1 mg is intraperitoneally administered for the final immunization. Then, several days after the final immunization, spleen cells are taken out for cell fusion.

C. Cell fusion The spleen of the mouse immunized as described above is aseptically removed to prepare a single cell suspension. These spleen cells are fused with the appropriate lined mouse myeloma cells by using an appropriate fusion promoter. The preferred ratio of splenocytes to myeloma cells is in the range of about 20: 1 to 2: 1 (cell number), with about 1 ml of fusion medium suitable for about 10 ⁸ spleen cells. The fusion medium refers to a solution or the like for retaining cells, which contains a fusion promoter used for cell fusion. Bone marrow cells used for cell fusion to obtain hybridomas are mouse-derived P3 × 63 Ag8 cells, P3 × 63 Ag8 U1 cells, P3 × 63.
653 cells, NSI cells and rat-derived 210 RCY Ag1.2.3
Although cells etc. can be used, in the examples P3 × 63 Ag8 U1 cells (Current Topics in Microbiology, 81 , 1-7 (1978))
Was used. Examples of the cell fusion promoter include polyethylene glycol, dimethyl sulfoxide, L-arginine and the like. Specifically, polyethylene glycol having an average molecular weight of about 1,000 to 6,000 is used, but other fusion promoters can be used and two or more can be used in combination. As a medium for dissolving the fusion promoter, distilled water, phosphate buffered saline, various cell culture media, and the like can be used. In the examples, the average molecular weight is about 1,500.
Polyethylene glycol was dissolved in distilled water and used as a fusing medium. Furthermore, an electrofusion method using a cell fusion device (FEBS Lett., 147 , 64-67 (1982)) and HVJ
(Sendai virus) cell fusion method (Nature 25
6, 495-497 (1975)) and the like can also be used.

D. Selection of fused hybridomas A mixture of unfused spleen cells, unfused mouse myeloma cells and fused hybridoma cells is selected in a selective medium that does not support unfused mouse myeloma cells, such as HAT medium. And culture for a sufficient time (about 1 week) to kill unfused cells. Unfused mouse bone marrow cells die in this selective medium. Since unfused spleen cells are non-tumor cells, they also die after a certain period of time (about 1 week). In contrast, fused cells can survive in selective medium under these conditions.

E. Measurement of antibody titer The antibody titer during the mouse immunization period and the detection and confirmation of the antibody produced by the hybridoma cell are detected by the enzyme immunoassay (Enzyme Linked Immunosorbe
nt Assay; ELISA).

F. Selection of Hybridoma Cell Producing Antibody of Interest Hybridoma cells producing antibody of interest are cloned by an appropriate method, for example, limiting dilution.

G. Production of Each Monoclonal Antibody The desired monoclonal antibody can be produced by two methods. The first method is to culture each hybridoma cell in an appropriate medium for a certain period of time to obtain an antibody produced by the hybridoma cell from the culture supernatant. The second method is to inject the hybridoma cells into the abdominal cavity of the mouse, and after a certain period of time, obtain the antibody produced by the hybridoma cells from the blood and ascites of the host mouse. Any hybridoma cell of the present invention is suitable for these two types of antibody production methods.

H. Purification of monoclonal antibodies G. The monoclonal antibody-containing culture solution or ascites fluid obtained by the method described in 1. can be used as a purified sample. For example, ascites can be purified by affinity chromatography using a protein A column and concentrated by an ultrafiltration membrane. Thus, a monoclonal antibody belonging to immunoglobulin IgG that recognizes human insulin-like growth factor II, such as monoclonal antibody 2H11, 2B11, ID5 or ID9, but does not recognize human insulin-like growth factor I, is produced by the above-described production method of the present invention. obtain.

[0015]

EXAMPLES The present invention will be described below with reference to examples. Example 1: Acquisition of antigen (1) Preparation of DNA oligomer for mutagenesis The nucleotide sequence of the DNA oligomer for mutagenesis is as follows. Mut-IGF-II-02: (5 ′) TTCTTCGAGGATACCTC (3 ′) (17mer) Mutation-ligating DNA oligomer Mut-IGF-II-02 was synthesized using a DNA synthesizer and purified by HPLC or the like. Purified DNA oligomer (10 pmol) 5'with T4 polynucleotide kinase
The ends were phosphorylated and used for the synthesis of complementary strands.

(2) Single-chain DN containing deoxyuridine
Preparation of A Plasmid pIGF002 (J. gen. Viol. 68 , 2599-2606 (19
87), Escherichia coli carrying this plasmid
Escherichia coli K12 MC1061 IGF-002 deposited at Institute of Microbial Technology, Ministry of International Trade and Industry, deposit number: FERM BP-93
3) was partially digested with EcoR I and Sal I to obtain approximately 230 bp of IGF-II.
Gene DNA fragment was isolated and digested with EcoR I and Sal I
It was inserted into pUC19. The obtained plasmid pUCIGF-II was transformed with Ec
After digestion with oR I and Hind III, it was treated with mung bean nuclease to isolate an IGF-II gene DNA of about 230 bp. This fragment was inserted into Sma I digested plasmid pUC119
A plasmid p119-IGF-II (14) in which the region encoding the GF-II N-terminal is on the HindI II side and a plasmid p119-IGF-II (24) in which the IGF-II gene was integrated in the reverse direction were obtained.
19-IGF-II (14) was retained in Escherichia coli MV1184 strain. This E. coli was cultured in 2xYT medium (containing ampicillin, tetracycline and streptomycin), and the culture medium was infected with helper phage M13K07 (moi 2-10). 37
Kanamycin was added after standing still at 30 ° C. for 30 minutes, followed by overnight culture and centrifugation to obtain a supernatant. This phage solution was added to E. coli BW 313 strain culture solution (LB) to infect (moi 2-10). 30 at 37 ° C
After standing for a minute, kanamycin was added, and the mixture was further cultured at 37 ° C overnight. The culture solution was centrifuged, 25 ml of 2.5 M NaCl / 20% polyethylene glycol 6000 solution was added to the supernatant, and the mixture was left standing at room temperature for 10 minutes and then centrifuged to obtain a precipitate. Precipitate with 1 mM EDTA / 10 mM Tri
Dissolve in 5 ml of s ・ HCl (PH 8) buffer (hereinafter referred to as TE buffer), first phenol and then phenol / chloroform /
Isoamyl alcohol (25: 24: 1) and finally chloroform / isoamyl alcohol (24: 1) were applied in this order, and the aqueous layer was collected. To the aqueous layer, add 500 μl of 3 M sodium acetate (pH 8.0) and 5 ml of isopropyl alcohol, stir and centrifuge. The precipitate obtained is washed with 70% ethanol, dried under reduced pressure, dissolved in TE buffer and deoxygenated. A single-stranded DNA containing uridine (hereinafter abbreviated as p119-IGF-II.dUssDNA) solution was prepared.

(3) Mutagenesis plasmid p119-M02-IGF-
Preparation of II p119-IGF-II ・ dUssDNA0.02 pmol in annealing buffer
And mutagenized DNA oligomer Mu with phosphorylated 5'end
Mix 1 pmol of t-IGF-II-02 for 15 minutes at 65 ° C, then 37
It was left still at 15 ° C for 15 minutes. 255 μl elongation buffer (Takara Shuzo, Mutan K kit) and 60 U of E. coli DNA ligase
1 U of T4 DNA polymerase was added and the mixture was allowed to stand at 25 ° C for 2 hours. 3 μl of 0.2 M EDTA (pH 8.0) was added and the reaction was stopped by standing at 65 ° C. for 5 minutes.

Escherichia coli BMH71-18 mutS competent cell 3
Mix 0 μl with 3 μl of this DNA solution, let stand at 0 ° C for 30 minutes, then at 42 ° C for 45 seconds, and finally at 0 ° C for 2 minutes, and add 2 ml of 2 × YT medium (containing ampicillin and kanamycin) to 37 ° C. I shook it overnight. Collect the supernatant by centrifugation and
Mix μl with 80 μl of E. coli MV 1184 culture medium and add an appropriate amount.
It was spread on LB agar medium (containing ampicillin) and cultured at 37 ° C. Pick an appropriate number of single colonies and plasmid as usual
After preparing the DNA, the nucleotide sequence of the mutation site was confirmed, and the plasmid containing the IGF-II gene in which the mutation was introduced was incorporated.
p119-M02-IGF-II was obtained.

(4) Plasmid for producing recombinant virus
Preparation of pBM-M02-IGF-II pFIGF II 120 (J. gen. Viol. 68 , 2599 (1987)) was added to Eco
After digestion with RI, make blunt ends with DNA polymerase (Klenow fragment) in the presence of dNTPs and perform about 10 Kbp by agarose gel electrophoresis.
The DNA fragment of was isolated and extracted. DNA with DEAE filter
Was collected, washed and dried as usual, and 1 M Tris.HCl 200 μl
It was dissolved in and treated with bacterial alkaline phosphatase (Takara Shuzo, hereinafter referred to as BAP) to obtain a vector. This was dissolved in an appropriate amount of TE buffer to give a ligation sample. Mutated plasmid p119-M02-IGF-
II was digested with Nco I and Eco RI, respectively, and then blunt-ended with DNA polymerase (Klenow fragment) in the presence of dNTPs.
A DNA fragment of about 220 bp was separated and extracted by agarose gel electrophoresis, the fragment was recovered with a DEAE filter, washed, and then dried. This was dissolved in an appropriate amount of TE buffer to give a ligation sample.

The above vector and DNA fragment were ligated using T4 DNA ligase. Escherichia coli MC1061 strain was transformed with this reaction solution. A part of the culture was spread on LB agar medium containing ampicillin and cultured overnight at 37 ° C. Take an appropriate number of the generated single colony and analyze and confirm the base sequence of the plasmid DNA according to a conventional method,
Escherichia coli carrying the target plasmid pBM-M02-IGF-II was obtained. The construction process from pIGF 002 to plasmid pBM-M02-IGF-II for obtaining recombinant virus for expression is shown in FIGS. 1 and 2.

(5) Recombinant virus for expression vIGF-II-M
Preparation of 02 Silkworm nuclear polyhedrosis virus (viral DNA of BmNPV T3 strain
(ATCC No. 40188)) and pBM-M02-IGF-II in a molar ratio of 1: 100.
The following composition liquids I and II were mixed.

1 ml of the resulting suspension was added to cultured silkworm cells (Bm
Cell culture medium (TC-10 medium containing 10% fetal bovine serum:
J. Invertebr. Pathol., 25 , 363 (1975)) 4 ml, B
The above DNA was introduced into m cells. After 20 hours, the medium was replaced with a fresh medium, and the medium was recovered after further culturing for 5 days. This is centrifuged to collect the supernatant, and the plaque assay method (J.Seric.Sci.Jp
n., 53 , 547 (1984)), and collected the recombinant virus, which was designated as vIGF-II-M02.

(6) Expression of Polyhedron-Polypeptide-2 Fusion Protein in Silkworm Individuals In the silkworm on the 5th day on the 5th day, the expression virus vIGF-II-MO2 suspension 0.5 ml / mouse (10 ⁷ pfu) Percutaneously injected at 25 ° C for 3
After rearing for a day, silkworms expressing the polyhedron-polypeptide-2 fusion protein were obtained.

(7) Recovery of Polyhedral-Polypeptide-2 Fusion Protein from Silkworm Insects 120 silkworms 120 infected with vIGF-II-M02 and bred for 3 days were used.
It was immersed in 0 ml of water, and the worm body was completely crushed with a high-speed homogenizer. The homogenate was then sonicated and filtered using double layered gauze. About 1,200 ml of the filtrate and the gauze washing solution was centrifuged at 10,000 rpm for 20 minutes, the obtained precipitate was suspended in 250 ml of 2.5 M urea solution, and the precipitate was recovered by centrifugation again under the above conditions. This of 150 ml
It was suspended in a 1.0 M guanidine hydrochloride solution and centrifuged under the same conditions to obtain an insoluble fraction containing a polyhedron-polypeptide-2 fusion protein. Next, this fraction was suspended in 360 ml of a 6.0 M guanidine hydrochloride solution and sonicated under ice cooling to dissolve the target protein. To this solution was added 150 ml of ethyl ether, and the mixture was vigorously stirred and then sonicated. When this was centrifuged using a Teflon centrifuge tube, it separated into two phases with the gelled dark green intermediate layer interposed therebetween, so only the lower layer was recovered. The same operation was repeated twice to remove impurities. Then
The same operation was performed using 150 ml of chloroform, and only the upper layer was collected. When this guanidine solution is sufficiently dialyzed against 0.2% formic acid at 4 ° C, a white insoluble matter is produced. Therefore, this is removed by centrifugation (12,000 rpm, 15 minutes), and the centrifugation supernatant is freeze-dried to obtain a crude polygon. A body-polypeptide-2 fusion protein was obtained.

(8) Preparation of polypeptide-2 Crude polyhedron-polypeptide-obtained in the previous example (7)
2 The fusion protein was dissolved in 50 ml of 70% formic acid, thoroughly degassed under reduced pressure, and then replaced with nitrogen gas. Solid cyanogen bromide
200 mg was added and sealed, and the mixture was reacted at room temperature for 24 hours with stirring. The reaction solution was diluted with about 15 times the amount of pure water and then freeze-dried,
The powder obtained was first dissolved in 40 ml of 10 M urea. To this, 20 mM acetate buffer (pH 4.0) was added to adjust the urea concentration to 6.0.
After adjusting to M, the pH was adjusted to 4.0 with acetic acid. The above solution was sent to an SP-Toyopearl 650 M column (manufactured by Tosoh Corporation) (φ 2.5 × 6.0 cm) that had been sufficiently equilibrated with a 20 mM acetate buffer (pH 4.0) containing 6.0 M urea. After adsorbing the target substance and thoroughly washing it with 150 ml of the same buffer, 150 ml of the same buffer and 150 ml of the same buffer containing 0.4 M NaCl
Was eluted with a linear NaCl gradient. The eluate was fractionated in 16 ml fractions with a fraction collector, and each fraction was analyzed by SDS-polyacrylamide gel electrophoresis to confirm the elution position of the target substance and then collected. Then adjust the pH of this solution to about 8.0, add 2-mercaptoethanol to this to a final concentration of 100 mM, and add at room temperature 4
It was left for a time and reduced. Using Spectra / Pore 3 (manufactured by Spectrum Co.) as a dialysis membrane, the above solution was 6.0 M
It was thoroughly dialyzed against 20 mM acetate buffer (pH 4.0) containing urea and 50 mM 2-mercaptoethanol. SP-Toyopearl column (φ1.5) equilibrated with the same buffer solution in advance
This dialysate was pumped to (6.0 cm x 6.0 cm), and after that, it was eluted with a linear concentration gradient of 0 to 0.4 M NaCl in the presence of 50 mM 2-mercaptoethanol in the same procedure as in the above chromatography, and the partially purified reduction was carried out. A type polypeptide fraction was obtained. This was thoroughly dialyzed against 0.1% formic acid using the same dialysis membrane as above, and the dialysate was freeze-dried to obtain 220 mg of powder. To the freeze-dried powder, 30 ml of a 6.0 M guanidine hydrochloride solution was added, and the mixture was stirred under reduced pressure for 30 minutes to be dissolved. Under stirring, 150 ml of 50 mM Tris.HCl buffer (pH 8.4) was slowly added thereto, and the mixture was stirred under reduced pressure for about 1 hour, and immediately thereafter, the mouth of the container was covered with parafilm and sealed. Several holes were opened in this lid with an injection needle, and the air was gradually left to stand still at room temperature for 3 days for air oxidation. This solution was concentrated to about 25 ml by ultrafiltration (Diaflo Membrane YM-5: manufactured by Amicon). Oxidized polypeptide-
The final purification of 2 was performed using preparative HPLC (LC-8A: manufactured by Shimadzu Corp.). The conditions are shown below.

[HPLC 1] Column: Capsule pack C18 (SG type 5 μm, φ 1.5
× 25 cm: manufactured by Shiseido Co., Ltd. Flow rate: 8.0 ml / min Elution: 0.1% Linear concentration gradient of acetonitrile from 20% to 55% in trifluoroacetic acid (50 minutes) Detection: UV absorption chromatography at 210 nm Is repeated 3 times and the holding time is approx.
The target fraction for 24.0 minutes was collected and lyophilized in a batch. This was again finally purified by HPLC using the same column. The conditions are shown below. [HPLC 2] Column, flow rate and detection conditions: Same as above Elution: 0.1% linear concentration gradient of acetonitrile from 26% to 34% in trifluoroacetic acid (40 minutes) Chromatography was repeated twice and retention time was about
The main peak fractions at 21.5 minutes were collected and freeze-dried at one time to obtain about 10 mg of polypeptide-2 powder.

Example 2 Obtaining Monoclonal Antibody (1) Preparation of Antigen Solution 0.4% SDS / 0.1 M phosphate buffer (pH 7.4) 0.1 M phosphorus of pH 7.4 was added to 3 mg of Polypeptide-2 dissolved in 0.5 ml. Bovine serum albumin (BSA) 2.5 dissolved in 0.5 ml of acid buffer
mg was added, 0.5 ml of 20 mM glutaraldehyde was further added, and the mixture was stirred at room temperature for 30 minutes. After sufficient dialysis against phosphate buffered saline, centrifuge and collect the supernatant in IGF-II / BS.
A conjugate.

(2) Immunization method 0.3 mg of IGF-II / BSA conjugate was sufficiently emulsified with an equal volume of Freund's complete adjuvant (FCA, DIFCO LABOLATORIES), and BALB / c female mouse (Charles River Japan, 7 -8-week-old 3 mice were subcutaneously administered to immunize (antigen
100 μg / animal). After another 3 weeks, 0.3 mg of Peptide-2 was added to an equal volume of Freund's incomplete adjuvant (FICA, DIFCO LAB
OLARORIES) and emulsified well, subcutaneous administration (antigen 0.1mg /
) And boostered. Boosters are given every two weeks,
Blood was collected 7 days after each immunization and the antibody titer in the serum was measured.
For the final immunization, select 2 animals with high antibody titers and use peptide-2 / B
A physiological saline solution (0.5 ml) containing 0.1 mg of SA conjugate was intraperitoneally administered. Three days after the final immunization, the spleen was extracted and used for cell fusion.

(3) Cell fusion Aseptic extirpation of spleen from mice and serum-free PRMI 16
After washing with 40 (Nissui Pharmaceutical Co., Ltd.), cracks were put in several places. Splenocytes extruded using the frosted portion of the slide glass were washed with a Tris.NH ₄ Cl solution to remove red blood cells and prepare splenocytes for cell fusion. The obtained splenocytes and mouse myeloma cells P 3 × 63 Ag 8U1 were mixed at a ratio of 5: 1 (spleen cells: myeloma, number of cells), and after sufficiently removing the medium, 1 ml of 50% polyethylene glycol 1500 Cell fusion was carried out by incubating at 37 ° C for 2 minutes. The cells were washed with serum-free PRMI medium and HAT (1 × 10 ^-4
M hypoxantine, 4 × 10 ^-7 M aminopterin, 1.6 × 10
^-5 M thymidine) was added and suspended in 10% fetal bovine serum / PRMI 1640 medium. Add 105 cells / well each and add at 7 ℃ at 7 ℃.
Culture was performed under the condition of% CO ₂ . Obtained hybridoma 2
The hybridoma population containing H11, 2B11, ID5 and ID9 was further monoclonalized by measuring the antibody titer described below and narrowing down the candidates.

(4) Measurement of antibody titer Antibody titer measurement and screening during the mouse immunization period were performed according to the following enzyme-linked immunosorbent assay (ELISA) procedure. 10 in each well of a 96-well flat plate (manufactured by Biotech)
25 mM carbonate buffer (pH 9.0) containing IGF-II at μg / ml 0.05
After adding ml, the mixture was allowed to stand overnight at 4 ° C. to adsorb the antigen. After washing each well 3 times with 0.05% Tween 20 / PBS
0.5 ml of 0.5% BSA / 0.05% Tween 20 / PBS was injected into each well, and the mixture was allowed to stand at 37 ° C for 3 hours. 0.05% T for each well
Wash 3 times with ween 20 / PBS, 0.5% BSA / 0.05% Tween 20
Antiserum or culture supernatant serially diluted with PBS / PBS.
After adding 05 ml / well, the mixture was allowed to stand at 29 ° C. for 90 minutes for antigen-antibody reaction. Each well was washed 3 times with 0.05% Tween 20 / PBS, and 0.05 ml of horseradish peroxidase (HRP) -labeled anti-mouse IgG goat antibody (Funakoshi) diluted 2000-fold with 0.5% BSA / 0.05% Tween 20 / PBS. / well was added and the mixture was allowed to stand at 29 ° C for 1 hour. Add 0.05% T to each well again
After washing 3 times with ween 20 / PBS, add the substrate solution (ABTS, ZYMED) to 0.
05 ml / well was added, and colorimetric measurement was performed at an absorption wavelength of 405 nm.

(5) Selection of antibody-producing cells The population of hybridomas obtained in (3) above is subjected to cell fusion.
After 7 days, the antibody titer of the culture supernatant was measured by ELISA, and the cells in the wells in which antibody production was observed were subjected to primary cloning by the limiting dilution method. After that, a single clone hybridoma was obtained by secondary cloning. Each was designated as hybridoma 2H11, 2B11, ID5 or ID9. Hybridoma 2H11 producing monoclonal antibody 2H11, hybridoma 2B11 producing monoclonal antibody 2B11, and monoclonal antibody I
Hybridoma ID5 that produces D5 and hybridoma 9 that produces monoclonal antibody ID9 have been deposited at the Institute of Microbial Technology, Ministry of International Trade and Industry, Ministry of International Trade and Industry (accession number: FERM BP-32752, accession number: FERM, respectively).
BP-3274I, accession number: FERM BP-3276 and accession number: F
ERM BP-3277).

Example 3: Monoclonal antibody class,
Determination of subclass The subclass of the monoclonal antibody produced by the hybridoma obtained in Example 2 was determined by ELISA.
IGF-II was immobilized on an ELISA plate, hybridoma culture supernatant was allowed to react, and each class and subclass of mouse immunoglobulin (IgG (Fc), IgG1,
IgG2a, IgG2b, IgG3, IgA and Ig
HRP-labeled antibody (The Binding Si)
te Limited) was reacted. As a negative control sample, the culture supernatant of myeloma P3U1 was measured at the same time, and the negative control sample
The case where the absorbance was 2 times or more was regarded as positive. The results are shown in Table-1. Each obtained monoclonal antibody has Ig
It was an antibody belonging to the G1 class.

[0033]

[Table 1]

Example 4 Preparation and Purification of Ascites Monoclonal Antibody To obtain ascites monoclonal antibody, 15 to 20 weeks old
Hybridoma 2H11 was established about 3 weeks after intraperitoneal administration of 0.5 ml of pristane (2,6,10,14-tetra-methyl-pentadecane, Wako Pure Chemical Industries, Ltd.) to BALB / c mice (Charles River Japan). , 2B11, ID5 or ID
9 5 × 10 ⁶ cells were intraperitoneally transplanted. After 2 to 3 weeks, the collected ascites fluid was collected. This ascites is protein A
Column (PROSEP-A 'HIGH CAPACITY', BIOPROCESSING
The product was purified by affinity chromatography. 20 ml of each ascites is added to double the volume of 1.0 M Glycine ・ N
Dilute with aOH / 0.15 M NaCl (pH 8.0) and add this to PROSEP-A.
Pass the antibody through a column (φ 1.0 × 7.5 cm),
The column was washed with about 70 ml 0.1 M Glycine.NaOH / 0.15 M NaCl, and then the antibody was eluted with 0.1 M citrate buffer (pH 3.5). Each eluate was concentrated with an ultrafiltration membrane YM 30 (manufactured by Amicon) and then sufficiently dialyzed against PBS at 4 ° C.
Ascites-type monoclonal antibody 2H11, 2 obtained here
The following examples were carried out using B11, ID5 or ID9.

Example 5: Reaction specificity of monoclonal antibody to IGF-II According to the ELISA shown in Example 2, IGF-II was immobilized on the immobilized antigen.
(J.gen.Virol. 68 , 2599 (1987)) or recombinant IGF-I
(Toyobo Co., Ltd.), each purified monoclonal antibody shown in Example 3 was serially diluted 3-fold from the concentration of 10 μg / ml and reacted with the antigen. Further, after reacting with HRP-labeled anti-mouse IgG goat antibody, the HRP activity of the plate was measured. As a positive control, a commercially available anti-IGF-II monoclonal antibody (Amano Pharmaceutical Co., Ltd.), which is known to have cross-reactivity with IGF-I, was used. However, the 96-well flat plate was manufactured by Coaster. The results are shown in FIG. Monoclonal antibody 2H11, 2B11, ID5
Or ID9 reacts with IGF-II but not with IGF-I at all. A commercially available monoclonal antibody (denoted as commercially available in the table) reacted with both IGF-II and IGF-I.

Example 6 Determination of Epitope (1) Preparation of IGF-II-Derived Peptide Fragment by Pepsin Digestion IGF-II digestion with pepsin was carried out by the method of Smith et al. (J. Bio
l. Chem., 264 , 9314 (1989)). 1
Dissolve 00 μg of IGF-II powder in 250 μl of 0.01 N HCl,
To this, 5 μg of pepsin (Sigma) was added and reacted at room temperature for 48 hours. The peptide fragments in the reaction solution were separated and purified by HPLC, and the eluate containing each peptide fragment was freeze-dried.

The HPLC conditions are as follows. Column: Capsule pack C18 SG 120 (φ4.6 x 250 mm,
Solvent A: Water containing 0.1% trifluoroacetic acid Solvent B: 0.1% acetonitrile containing trifluoroacetic acid Flow rate: 1.00 ml / min, Detection: UV 210 nm Elution condition: 0.1% 5% to 35% in trifluoroacetic acid Elution pattern under these conditions is shown in Fig. 4. Peaks 1 to 4 shown in the figure were used for epitope determination of each monoclonal antibody. The peptide fragment corresponding to each peak is I
The amino acid sequence of GF-II corresponds to the amino acid sequence analysis (Applied Biosystems, Protein Se).
quencer 470A) and amino acid composition analysis.

The amino acid sequences of each peptide are shown below.

Embedded image Peak 1: (NH _2- ) Phe-Ser-Arg-Pro-Ala-Ser-Arg-Val-Se
r-Arg-Arg-Ser-Arg-Gly (-COOH)

[0039]

Embedded image Peak 2: (NH _2- ) Ala-Tyr-Arg-Pro-Ser-Glu-Thr-Leu (-C
(OOH)

[0040]

[Chemical 3]

[0041]

[Chemical 4]

(2) ELISA using each peptide fragment as an antigen
Measurement according to Example 3 The measurement was performed according to Example 3. Each of the peptides shown above was immobilized on a 96-well plate, and 1.1 μg / ml of each antibody was added thereto to react with the antigen. Furthermore, after reacting this with an HRP-labeled anti-mouse IgG goat antibody, HR on the plate
P activity was measured. It is shown in Table 2 as the value of the absorbance (OD). As a reference, commercially available anti-IGF-
II monoclonal antibody (Amano Pharmaceutical), IGF-II as antigen
And IGF-I are shown in Table 2 in the same manner as in the example.

This result shows that the monoclonal antibody 2H11
The IGF-II epitope of is the 28th IGF-II
It is shown to be in the sequence part from Phe to the 41st Gly. Each monoclonal antibody obtained in the present invention is IGF-
Reacts with II and not with IGF-I. A commercially available anti-IGF-II monoclonal antibody (denoted as AMANO in the table) reacts with both IGF-II and IGF-I.

[0044]

[Table 2]

Example 7: Specific assay of IGF-II (1) Sandwich ELISA 6.25 mg of anti-IGF-II monoclonal antibody (ID9) was added to 10 mM.
It was thoroughly dialyzed against a carbonate buffer (pH 9.5). Dissolve 4.0 mg of HRP (Boehringer Mannheim) in 1.0 ml of water,
To this, 0.2 ml of 0.1 M sodium periodate solution was added and reacted at room temperature for 20 minutes, and then 1 mM acetate buffer (pH 4.
It was dialyzed against 4) overnight at 4 ° C. A 0.2 M sodium carbonate solution was added to the dialysate (1.40 ml) to adjust the pH to about 9, and 1.05 ml of this solution was added to the antibody dialysate and reacted at room temperature for 2 hours with stirring. Sodium cyanoborohydride 5 in the reaction solution
mg was added and the mixture was allowed to stand at room temperature for 2 hours to reduce the Schiff base formed by the reaction. Then 0.2 M Tris ・ HCl / 0.1 M
Monoethanolamine (1.0 ml) was added, and the mixture was allowed to stand at room temperature for 90 minutes. Thoroughly dialyze the reaction solution at 4 ℃ against PBS,
The dialysate was used as the HRP-labeled antibody solution.

The quantification method is almost the same as that shown in the third embodiment. First, the anti-IGF-II antibody (2B11) was immobilized on a 96-well flat plate (Coaster) and reacted with various concentrations of IGF-II, IGF-I (Toyobo Co., Ltd.) or insulin (Sigma). The HRP shown above.
After reacting with the labeled anti-IGF-II antibody, HRP activity on the plate was measured. The results are shown in Table-3. The obtained monoclonal antibody specifically reacted with IGF-II and could be quantitatively measured.

[0047]

[Table 3]

(2) Competitive ELISA HRP-labeled IGF-II was prepared by the previously reported maleimide method.
(J. Biochem., Vol. 92, 1413-1424 (1982)). Horseradish peroxidase (3.3 mg) was dissolved in 0.3 ml of sodium phosphate buffer solution (pH 7.0), and the solution was added to this solution.
Add 75 mg of N- (4-acetylmercaptosuccinic anhydride to
Add a solution of l dimethylformamide dissolved at 30 ° C.
Incubation was carried out for 1 hour under stirring. The formed precipitate was removed by centrifugation, and the supernatant was added to a Sephadex G-25 column (1.0 cm) using 0.1 M sodium phosphate buffer (pH 6.0).
The gel filtration was performed by (x38 cm). Fractions containing the target protein were collected and concentrated by ultrafiltration using Diaflo-Membron YM-30 (Amicon) to obtain maleimide-labeled HRP. To bond the maleimide-labeled HRP and IGF-II, a thiol group was introduced into IGF-II by the following method. Dissolve IGF-I (0.8 mg) in 0.6 ml of 0.1 M sodium phosphate buffer (pH 6.5) and add 2.0 mg to it.
A solution of mg S-acetylmercaptosuccinic anhydride in 35 μl of dimethylformamide was added, and the mixture was incubated at 30 ° C. for 30 minutes. Add 40 μl of this solution to the solution.
200 μl with 1 M EDTA, 200 μl 0.1 M Tris · HCl (pH 7.0)
l 1.0 M hydroxyamine / hydrochloric acid was added and incubated at room temperature for 4 minutes. The reaction mixture was subjected to gel filtration with a Sephadex G-25 column (1.0 cm × 41 cm) using 0.1 M sodium phosphate buffer (pH 6.0) containing 5 mM EDTA. Fractions containing the target protein were collected and concentrated to 0.5 ml by ultrafiltration using a Diaflow membrane YM-5 (Amicon). The maleimide-labeled HRP was added to this solution, incubated at 4 ° C. for 20 minutes, 70 μl of 0.1M ethylmaleimide was added, and gel filtration was performed using the same column as above using 0.1M sodium phosphate buffer (pH 6.5). .. Fractions containing the protein of interest were collected and concentrated by ultrafiltration using a Diaflow membrane YM-30. After concentrating to approximately 1 ml, the inner solution is PBS
Labeled IGF-II (1.1 ml) was obtained.

The IGF-II concentration was quantified as follows.
2.5 μg / ml of each of four antibodies (2H11, 2B11, ID) in each well of a 96-well microplate (Coaster)
0.05 ml of the PBS mixed solution of 5, ID 9) was added, and the mixture was allowed to stand at 4 ° C. overnight. Each well was washed with 0.05% Tween 20 / PBS, 0.1 ml of 0.05% BSA / 0.02% Tween 20 / PBS was added, and the mixture was allowed to stand at 37 ° C for 3 hours. After washing 3 times with 0.05% Tween 20 / PBS, 10% with 0.1% PBS / 0.02% Tween 20 / PBS
The above prepared HRP-labeled IGF-II diluted 0.0000 times was added to each well in an amount of 0.025 ml, and various concentrations of IGF-II were added in an amount of 0.025 ml. Incubate this at 4 ℃ overnight and wash each well 3 times with 0.05% Tween 20 / PBS.
P activity was measured. The results are shown in Figure-5. The obtained monoclonal antibody specifically reacted with IGF-II and could be quantitatively measured.

Example 8: Western blotting analysis (1) Preparation of anti-IGF-II monoclonal antibody (2H11) binding resin Anti-IGF-II monoclonal antibody 2H11 (4.5 ml) was added to 5 ml.
0.25 M sodium phosphate buffer (PH 8.0). To this solution, 4 ml of Formyl-cellulofine (manufactured by Seikagaku Corporation) that had been thoroughly washed with 100 ml of water was added, and the mixture was stirred at room temperature for 2 hours. Then, 10 mg of sodium dianoborohydride powder was added, and the mixture was further stirred at room temperature for 3 hours. After filtration, the antibody-bound resin was washed with 100 ml of water. The obtained resin was added with 10 mg of sodium cyanoborohydride powder, and left standing at room temperature for 3 hours. After filtration,
The resin was washed with 100 ml of water to obtain the desired antibody 2H11-binding resin.

(2) Preparation of serum-derived sample for Western blotting Serum of healthy adult or gastric cancer patient with hypoglycemia
300 μl and 1,200 μl of 87.5% ethanol / 2N hydrochloric acid were mixed in a polypropylene container. The mixture was incubated at room temperature for 45 minutes and centrifuged at 15,000 rpm for 10 minutes at 4 ° C. Take 1.2 ml of the supernatant in a separate container and 2 M
Neutralized with ammonium hydrogen carbonate. After ethanol was distilled off under reduced pressure, it was freeze-dried. 1 ml of lyophilized powder
Was dissolved in PBS containing 0.02% Tween 20, and the above antibody (2H11) -binding resin (50 μl) was added thereto, and the mixture was incubated in a vortex mixer at room temperature for 3 hours. IGF-II adsorbed resin was mixed with 1 M NaCl and 0.04% Twee
It was washed once with PBS containing n 20 and then with water. To solubilize and recover from the antigen / antibody complex on the resin,
Laemmli sample buffer (Nature, Vol. 227, 680-
685 (1970)). The desired serum-derived IGF-II is dissolved in this supernatant.

(3) IGF-II for Western blotting
Preparation of Producing Tumor Tissue Derived Sample A mass of IGF-II producing tumor tissue (wet weight, 156 mg) was placed in a polypropylene container and homogenized in 1.5 ml of 70% ethanol / 2N hydrochloric acid with Polytron (KINEMATICA). .. The homogenate was incubated at room temperature for 45 minutes and then centrifuged at 15,000 rpm for 10 minutes at 4 ° C. Finally, the tumor tissue-derived IG of interest was subjected to the same procedure as above.
I got F-II.

(4) Sodium dodecyl sulfate / polyacrylamide gel electrophoresis, membrane transfer, detection Standard IGF-II, IGF-I Serum-derived IGF-II and tumor tissue-derived IG
F-II is a Laemmli sodium dodecyl sulfate / polyacrylamide gel electrophoresis (hereinafter abbreviated as SDS-PAGE) system (Nat
ure, Vol. 227, 680-685 (1970)). SDS-PAGE was carried out without using 2-mercaptoethanol and using a slab gel of 16% polyacrylamide at a constant current of 25 mA. After electrophoresis, the protein on the gel is transferred to Mini Trans-Bl
A PVDF membrane ("Pro Blott", A) was obtained by applying a 50 V constant current for 1 hour in a 10 mM CAPS buffer solution (pH 11) containing 10% methanol using an ot device (manufactured by Bio-Rad).
pplied Biosystems). Membrane-like non-specific binding was blocked by soaking the membrane in PBS containing 5% skim milk and 0.05% Tween 20 for 16 hours at 4 ° C.
The membrane was first treated with 1 μg / ml antibody 2H11 and 0.05% Tween 20.
HRP-conjugated anti-mouse IgG goat IgG (Amarsham) diluted 5,000-fold with PBS containing 0.05% Tw
Each sample was treated with the same buffer containing een 20 at room temperature for 1 hour. IGF-II on the membrane can be detected by ECL Western Blotting De
tectionKit (manufactured by Amersham) was used and the operation was performed according to the manual. The results are shown in Figure-6. Standard IGF-II,
Serum-derived IGF-II was sufficiently detectable, but excess amount of IG
No FI was detected. Furthermore, these results indicate that this system is not only the normal type IGF-II (7.5 KD), but also the large molecular weight type IGF-II (Proc. Natl. . Sci., USA 82, 3169-31
72 (1985)).

[0054]

EFFECTS OF THE INVENTION We have created multiple anti-human IGF-II monoclonal antibodies with different recognition sites and extremely high specificity, and by combining these multiple antibodies, the ELISA method can be performed. Can be quantified.
Such useful antibodies can also be used for diagnostics for measuring human IGF-II blood concentration, various analyzes such as Western blotting analysis of human IGF-II, and purification of human IGF-II. ..

[0055]

[Brief description of drawings]

FIG. 1 is a construction diagram of a plasmid for obtaining a recombinant virus for expression.

FIG. 2 is a construction diagram of a plasmid for obtaining recombinant virus for expression, following FIG.

FIG. 3 is a diagram showing the reaction specificity of each antibody.

FIG. 4 is an HPLC elution diagram of a human IGF-II-derived peptide fragment by pepsin digestion.

FIG. 5 is a standard curve for IGF-II measurement by competitive ELISA.

FIG. 6 shows the results of IGF-II detection by Western blotting analysis in human-derived samples.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI Technical display location C12N 15/18 C12P 21/02 H 8214-4B (C12P 21/08 C12R 1:91) (C12P 21 / 02 C12R 1:91)

Claims (13)

[Claims] 1. A monoclonal antibody which recognizes human insulin-like growth factor II but does not recognize human insulin-like growth factor I. 2. Amino acid sequence: (NH ₂ —) Phe-Se
r-Arg-Pro-Ala-Ser-Arg-Val
-Ser-Arg-Arg-Ser-Arg-Gly
The monoclonal antibody according to claim 1, which recognizes (-COOH). 3. The monoclonal antibody according to claim 1, which has a different recognition property from the monoclonal antibody according to claim 2. 4. Amino acid sequence: (NH ₂ —) Ala-Ty
r-Arg-Pro-Ser-Glu-Thr-Leu
-Cys-Gly-Gly-Glu-Leu-Val-
Asp-Thr-Leu-Gln-Phe-Val-C
ys-Gly-Asp-Arg-Gly-Phe-Ty
r-Phe-Ser-Arg-Pro-Ala-Ser
-Arg-Val-Ser-Arg-Arg-Ser-
Arg-Gly-Ile-Leu-Glu-Glu-C
ys-Cys-Phe-Arg-Ser-Cys-As
p-Leu-Ala-Leu-Leu-Glu-Thr
-Tyr-Cys-Ala-Thr-Pro-Ala-
Immunize mammals with the polypeptide represented by Lys-Ser-Glu (-COOH) as an antigen to prepare hybridomas of the antibody-producing cells of the immunized mammals and myeloma cells, and then selectively obtain the cloned hybridomas. A method for producing a monoclonal antibody which belongs to immunoglobulin IgG characterized in that it is cultured to obtain an antibody, which recognizes human insulin-like growth factor II but does not recognize human insulin-like growth factor I. 5. Amino acid sequence: (NH ₂ —) Ala-Ty
r-Arg-Pro-Ser-Glu-Thr-Leu
-Cys-Gly-Gly-Glu-Leu-Val-
Asp-Thr-Leu-Gln-Phe-Val-C
ys-Gly-Asp-Arg-Gly-Phe-Ty
r-Phe-Ser-Arg-Pro-Ala-Ser
-Arg-Val-Ser-Arg-Arg-Ser-
Arg-Gly-Ile-Leu-Glu-Glu-C
ys-Cys-Phe-Arg-Ser-Cys-As
p-Leu-Ala-Leu-Leu-Glu-Thr
-Tyr-Cys-Ala-Thr-Pro-Ala-
A monoclonal antibody obtained by using a polypeptide represented by Lys-Ser-Glu (-COOH) as an antigen and recognizing human insulin-like growth factor II, but not human insulin-like growth factor I. 6. Amino acid sequence: (NH ₂ —) Ala-Ty
r-Arg-Pro-Ser-Glu-Thr-Leu
-Cys-Gly-Gly-Glu-Leu-Val-
Asp-Thr-Leu-Gln-Phe-Val-C
ys-Gly-Asp-Arg-Gly-Phe-Ty
r-Phe-Ser-Arg-Pro-Ala-Ser
-Arg-Val-Ser-Arg-Arg-Ser-
Arg-Gly-Ile-Leu-Glu-Glu-C
ys-Cys-Phe-Arg-Ser-Cys-As
p-Leu-Ala-Leu-Leu-Glu-Thr
-Tyr-Cys-Ala-Thr-Pro-Ala-
A hybridoma obtained by immunizing a mammal with a polypeptide represented by Lys-Ser-Glu (-COOH) as an antigen, producing a hybridoma of an immunized mammalian antibody-producing cell and a myeloma cell, and further cloning the hybridoma, A hybridoma 2H11 which produces the monoclonal antibody according to claim 1 or 2. 7. Amino acid sequence: (NH ₂ —) Ala-Ty
r-Arg-Pro-Ser-Glu-Thr-Leu
-Cys-Gly-Gly-Glu-Leu-Val-
Asp-Thr-Leu-Gln-Phe-Val-C
ys-Gly-Asp-Arg-Gly-Phe-Ty
r-Phe-Ser-Arg-Pro-Ala-Ser
-Arg-Val-Ser-Arg-Arg-Ser-
Arg-Gly-Ile-Leu-Glu-Glu-C
ys-Cys-Phe-Arg-Ser-Cys-As
p-Leu-Ala-Leu-Leu-Glu-Thr
-Tyr-Cys-Ala-Thr-Pro-Ala-
A hybridoma obtained by immunizing a mammal with a polypeptide represented by Lys-Ser-Glu (-COOH) as an antigen, producing a hybridoma of an immunized mammalian antibody-producing cell and a myeloma cell, and further cloning the hybridoma, Hybridoma 2B11 and hybridoma I producing the monoclonal antibody according to claim 1 or claim 3.
D5, or hybridoma ID9. 8. Hybridoma 2H11 9. Hybridoma 2B11 10. Hybridoma ID5 11. Hybridoma ID9 12. An immunochemical method for specifically detecting human insulin-like growth factor II using the monoclonal antibody according to claim 1. 13. An immunochemical method for specifically detecting human growth factor II using a monoclonal antibody produced by a hybridoma selected from hybridoma 2H11, hybridoma 2B11, hybridoma ID5 and hybridoma ID9.