JPS59216058A - Determining method of transforming gross factor - Google Patents

Abstract

PURPOSE:To determine an enzyme labeled transforming gross factor (TGF) efficiently by reacting the TGF and a TGF antibody with sample liquid, and separating an enzyme labeled TGF-TGF antibody coupler and unreacted TGF. CONSTITUTION:The sample liquid whose content of TGF is to be measured, such as the urine or serum of a man, and enzyme TGF and TGF antibody are brought into competing reaction by incubation in an immunity reactive medium, e.g. phosphoric acid buffer or veronal buffer solution at about 4-5 deg.C for about 15-72 hours. Then, B-F separation is performed to separate an immunity coupling part, specially, enzyme labeled TGF-TGF antibody coupling part B and an unreacted free part which is not coupled, specially, the free part F of unreacted enzyme labeled TGF. Then, the amount of the enzyme label of either of the separated parts is determined. Thus, the enzyme labeled TGF and TGF antibody obtained by using human TGF are used to determine the TGF in sample liquid easily and extremely accurately, deciding on cancer effectively.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to transforming growth factor (Tra
The present invention relates to a method for quantifying nsforming Growth FacLor (hereinafter referred to as TGF).

TGF is a polypeptide that is produced in human and rodent can cells and causes normal cells to undergo specific cytomorphological alterations (transformation and proliferation). Normal #1 cells of humans and animals do not form colonies in soft-tempered soil (in this case, 11iWL), but when TGF is added to this, they become capable of forming colonies.

1978 The 1-Evening [Proc, Natj
', Acad.

Sci., 75.4001 (1978)) reported the existence of TGF starting from the culture supernatant of hepatocytes. has also been found [Roberts-Cabi et al.; Proc.

NaLl! , Acad, Sci, USA, E-Z,
3 4 9 4 (t9so)].

Recently, D. Al. Tarsik et al. [JNCL, 69°7
93 (1982)] and J. Z. Toguro et al. [Can
cerRes, 43, 403 (1983)] extracted a polymer type TGF with a molecular weight of about 30,000 to 35,000 from the urine of cancer patients, but its detailed physical properties are still unclear.

Therefore, there is no appropriate means to clearly distinguish between TGF and various similar gross factors, and the determination of TGF has only been made based on its ability to form colonies in normal cells. However, it required complicated operations and lacked quantitative properties. In general, there have not been many reports regarding the presence and content of TGF in body fluids such as blood and urine, as well as its fate and development.

The present inventors previously discovered that high-molecular-weight TGF does not exist in normal human urine, but only exists in cancer patient urine, and that this substance has a negative effect on normal cells due to the above-mentioned biological effects. As a result of research on the idea that it is a very important substance with biological activity, we have found that urine from various cancer patients is dialyzed to remove inorganic ions and other low-molecular substances.If necessary, after gel filtration, Human TGF purified to extremely high purity was obtained by performing chromatography and/or adsorption chromatography using a Te cation exchanger (
(Refer to the specification of Japanese Patent Application No. 58-61102).

Furthermore, as a result of continued research, the present inventors found that it was possible to successfully obtain human TGF antibodies against this TGF by injecting this TGF into supplementary animals other than humans, immunizing them, and collecting their blood. As a result of this research, the spleens of immunized salivary mammals were removed and single cells capable of producing antibodies were combined with myeloma cells to produce a human TGF antibody against TGF. We obtained monoclonal antibody-producing cells and succeeded in obtaining human TGF antibodies from the culture. Furthermore, the present inventors
Obtained an enzyme-labeled form of TGF that can successfully perform an immunoreaction with the TGF antibody. Enzyme immunoassay (EIA) using the above-mentioned human TGF antibody and enzyme-labeled TGF
established.

The present invention was completed based on the above findings, and involves reacting enzyme-labeled TGF with a TGF antibody in a test solution, and then separating the enzyme-labeled TGF-TGF antibody conjugate and unreacted enzyme-labeled TGF. TG in the test solution whose purpose is to quantify the amount of either one of the enzyme labels separated after that.
This is a method for quantifying F. Furthermore, in the present invention, the TGF
A solid phase method in which the antibody is used as a solid phase body immobilized on an insoluble carrier; a competitive reaction may be quantified using the solid phase method, or the reaction may be performed using a TGF antibody that remains soluble.

First, the human TGF for obtaining the enzyme-labeled body and human TGF antibody of the present invention may be selected from, for example, lung cancer, hair loss, gastric cancer, etc.
It is easy to purify and collect urine from cancer patients such as pharyngeal cancer, colon cancer, breast cancer, melanosarcoma, and ovarian cancer. To give an example of the method for purifying TGF from the urine of cancer patients, the collected urine of cancer patients is first purified using a dialysis membrane with a molecular weight of about 10,000 or less using water as the external dialysis fluid in order to remove low-molecular impurities. Dialyze. Next, the obtained dialysate solution is repeatedly frozen and thawed as necessary to precipitate impurities, and fine precipitated particles can be removed by centrifugation at mF or through a filter with pores of about 5 microns. good. Furthermore, if necessary, the dialysis fluid should be
Beyogel (Bi.

gel) P 60 + P 1 00 (manufactured by Hi-T F'), Sephadex (Sephadex)
x) Both components exhibiting TGF activity may be collected by gel filtration using a gel filtration agent such as G-50 (manufactured by Pharmacia). At this time, the 1151 minute mark indicating TGF activity exists at a position with an estimated molecular weight of 280 () 0 to 350 UO, and can be identified using the test method described below. The active fraction collected through liquid or gel filtration is subjected to at least one of ion exchange chromatography, molecular plating treatment using an ultrafiltration membrane with a molecular weight of about 10,000 to 20,000, and adsorption chromatography. Alternatively, a purified TGF active fraction is collected by combining these operations.Ion exchange chromatography is performed using, for example, CM-Sephadex (5
ephadex) (manufactured by Pharmacia), SP-Sephadex (manufactured by Farania), CM-52 (manufactured by Whatman), Bio-Rex 70 (manufactured by Bio-Rad), etc. The above TGF-containing solution is added and adsorbed using a column using an ion exchanger as a base material. Then, the TGF active fraction may be recovered by gradient elution using a neutral salt, preferably an aqueous solution of sodium chloride, whose concentration is gradually increased. Next, preferably a molecular weight of 10,000 to 2
Concentration and desalting can be carried out using a 0000 ultrafiltration membrane. Furthermore, as adsorption chromatography, for example, Synchropak PR series (
Silica gel modified with a hydrophobic group such as an alkyl group having 3 to 20 carbon atoms (manufactured by Zinchrome) or Mc I G
The above-mentioned TGF is
Add the containing liquid and let it absorb. Then, gradient elution is performed using a hydrophilic neutral organic solvent, such as an aqueous solution of a lower aliphatic alcohol such as ethanol or propyl alcohol, or a lower aliphatic ketone such as acetone or acetonitrile, in which the concentration is gradually increased. The TGF active fraction may be collected. The TGF active fraction obtained in this way may be obtained as a dry powder by a drying method such as drying if necessary, and this dry powder is white, ninhydrin-reactive, and has a molecular weight of 28,000 to 350 fJL). (Gel filtration method), 5DS - It has physical and chemical properties that are simple in electrophoresis, stable against acids and heat, and inactivated by trypsin and dithiothreitol treatment. It is purified human TGF.

Enzyme-labeled TGF and anti-TGF antibodies are then obtained using such TGF, but the TGF used is not limited to the purification methods described above.

First, enzyme-labeled TGF is obtained (the enzymes to be used are appropriately selected from oxidoreductase, hydrolase, transposase, lyase, imerase, and ligase; examples include lactate dehydrogenase, maleidase, etc.). dehydrogenase, maltose dehydrogenase,
Cucrose-6-posphneetotehydrogenase, alcohol dehydrogenase, glutamate dehydrogenase, α-glycerolphos7ate dehydrogenase,
lagtate oxidase, maleide oxidase, glucose oxidase, alcohol oxidase,
Choline oxidase, xanthine oxidase, amino acid oxidase, amine oxidase, sarcosine oxidase, peroxidase, catalase, NAD
Examples include oxidase, α-amylase, β-galactosidase, lysozyme, lipase, alkaline phosphatase, aminopeptidase, hegisokinase, chryserokinase, pyruvate kinase, and the like. Furthermore, any spacer may be introduced into these enzymes in advance, such as dialdehyde compounds such as sufcine aldehyde, glutaraldehyde, and adipaldehyde, W
- Reactive m conductors such as aminobutyric acid, acid chloride of W-aminoglutamic acid, succinimide ester, p-21-lophenyl ester, reactive derivatives of dicarboxylic acid compounds such as malonic acid, succinic acid, glutaric acid, adipic acid, etc. , diamine compounds such as hexamethylene diamine and decamethylene diamine, reactive derivatives such as 3-C2'-pyridyl-dithio)propionic acid, 3-(2'-benzothiazolyl-dithio)propionic acid, S-acetylmercaptosuccinimide, etc. New functional groups such as aldehyde groups, carboxyl groups, amino groups, and thiol groups are introduced using one or more spacer introduction reagents such as nick anhydride and thiol compounds such as 2-aminoethanethiol. Similarly, instead of introducing into an enzyme, various functional groups may be introduced by using a spacer introduction reagent using TGF. Next, enzyme-labeled TGF using such an enzyme is bonded to the TGF based on the amine group, hydroxyl group, carboxyl group, 1,000-ol group, etc. of the enzyme, or the introduced functional group. obtained using a cross-linking reagent obtained from The crosslinking reagent may be a polyfunctional reagent having two or more groups capable of reacting with functional groups such as amino groups, hydroxyl groups, carboxyl groups, and thiol groups. Dialdehyde compounds such as aldehydes, dicarboxylic acids such as malonic acid, succinic acid, glutaric acid, adipic acid or their reactive derivatives, diisocyanate compounds such as hexamethylene diisocyanate, 2゜4-toluene diisocyanate, hexa diisothiocyanate compounds such as methylene dithiocyanate, maleimidocarboxylic acids or reactive derivatives thereof such as maleimidobenzoic acid, maleimidophenylacetic acid, N.

N'-ethylene bismaleimide, N,N'-0-phenylene dimaleimide and other cimaleimide compounds, bisdiazobenzidine, diethylmalonimidate, dimethyladipineimidate, N,N'-polymethylenebiiodoacetamide and 3-( Examples include thiocarboxylic acid compounds such as 2'-benzothiazolyl-dithio)propion H13-(2'-pyridyl-dithio)propionic acid or reactive derivatives thereof, and these polyfunctional raw reagents are effective in binding the TGF and enzyme used. They may be selected and used in consideration of the functional groups involved, such as amino groups, carboxyl groups, aldehyde groups, hydroxyl groups, and thiol groups. Furthermore, in producing enzyme-labeled TGF, for example, a buffer solution having a pH of 6 to 8.5, methanol, ethanol, dioxanomethyl sulfoxide, etc.

In an organic solvent such as dimethylacetamide or tetrahydrofuran, or a mixed solvent thereof, from 0°C to 4t)'C
TGF and the polyfunctional raw reagent are reacted in a . The ratio to be used in this case is determined based on the molecular weight of the enzyme to be bound per TGFI molecule, usually 1 to 5 molecules. After this reaction is completed, the enzyme is purified if necessary, the enzyme is added thereto, and the reaction is preferably carried out in a buffer having a stable pH for the enzyme. It is sufficient to use an amount to achieve binding in an equimolar ratio or more, usually 1 to 5 molar ratio.Then, the resulting product, the conjugate of enzyme and TGF, is subjected to adsorption chromatography or gel p-filtration. It may be purified and collected using a purification method such as the following.

Furthermore, in obtaining a human TGF antibody, the above-mentioned human TGF antibody
Using GF as an antigen, non-human mammalian animals such as guinea pigs, rabbits, rats, mice, and goats capable of producing antibodies are immunized according to the usual method, and then blood is collected to obtain antiserum. Separate the antibodies. In this case, the TGF used as the antigen is preferably a single protein preparation that has been highly purified as described above. In addition, in obtaining antibodies, for example, the above-mentioned human TGF powder 0.1 to 1 rn
Dissolve t in physiological saline 0.1-5 me, add the same amount of Complete 70 India Adjuvant (Comp
l! After sufficient emulsification, the mixture is injected subcutaneously or subcutaneously into a mammal, such as a rabbit or a mouse, for immunization by several injections every 1 to 3 weeks. Thereafter, blood is collected after a certain period of time from the day of final immunization, allowed to stand, coagulated, and centrifuged to obtain antiserum containing TGF antibodies. In this case, any animal can be used as long as it has the ability to produce antibodies, and in order to obtain a large amount of antibodies, it is preferable to use large animals, and rabbits, mice, and goats are usually used. It is not limited in any way. Furthermore, human I-TGF antibodies can be obtained from antisera containing human TGF antibodies obtained from these animals using commonly used antibody purification methods, such as ammonium sulfate fractionation of the antisera. First, it may be purified and collected by ion exchange chromatography or gel filtration. For further purification, it may be obtained by adsorption by affinity chromatography using an insoluble carrier on which human TGF is immobilized as a base material, followed by elution. Furthermore, another method for obtaining human TGF antibodies is to use myeloma cells and tile cells of non-human mammals that have been immunized with human TGF as an antigen and fuse them, and from this fused cell, monoclonal antibodies against human TGF1 can be produced. A method of isolating cells and producing human TGF monoclonal antibodies using the fused cells, and a method using a mouse as a mammal is often used [Naturve, 25J
495-497 (1975), Nature,
2ヱ6, 397-399 (1978), Ce1l! ,1
4. ．． 9-20 (1978).

Natuve, 266.550-552 (1977).

Eur, J, IrrmunoI! ,,6. 511-5
19 (1976), Chemicaf and E.
ngineering News y Jan.

1, '1979. '15-173゜For example, Ba1b/
Using C mice, an emulsion of TGF-containing saline and complete Freund's adjuvant was subcutaneously injected, and booster immunizations were performed 1 to 3 weeks later, and 3 weeks after the final immunization.
After ~5 days, one tile from the mouse is removed and a suspension of unicellular tile cells is prepared in an appropriate medium. Next, 3 to 10 amounts of these tile cells were treated with 1 amount of mouse-derived myeloma cells, such as PS-NS i/1-Ag4-1, at 37°C and 40 to 50% polyethylene glycol 1.
000-1500 in the presence of a suitable medium, such as RPMI
Medium [J, A, M, A, 19th, '519 (1967
), J, Nat, Cancer Inst.
36. 405, (1966), InVi
tro, i, 89 (1970)], then separated after washing, added to RPMI medium containing fetal bovine serum, and further added hypoxanthine, aminopteridine, thymidine, and fetal bovine serum in minute amounts to this cell suspension. Selective culture was carried out in RPMI medium (HA'T medium) containing RPMI medium (HA'T medium), the supernatant of each culture was collected, 1 the cultured cells with high antibody titers were selected, and 1. Human TGF monoclonal antibody-producing cells are fractionated by cloning by limiting dilution method using cells as feeder cells. Furthermore, these cells were treated with RP containing fetal bovine serum.
MI medium or Tulbecco's modified MEM medium 1 trowel culture
The supernatant was obtained and purified by ammonium sulfate fractionation, ion exchange chromatography, and filtration.
Obtain monoclonal antibodies. Alternatively, human TGF monoclonal antibody-producing cells may be grown as tumors in the bodies of histocompatible animals or athymic nude mice, and then collected and purified. Furthermore, the human TGF monoclonal antibody-producing cells may be cryopreserved in liquid nitrogen (approximately -196°C) in a serum-containing growth medium using a cryoprotectant such as dimethyl sulfoxide or glycerol.

Furthermore, this human TGF antibody or human TGF monoclonal antibody may be used in its soluble state,
Alternatively, it may be used as a homologue immobilized on an insoluble carrier. As such a solid support, TG, which is an insoluble support and the above-mentioned antibody bound together using the above-mentioned polyfunctional reagent, is used.
Any substance that retains immune binding activity to F may be used. The insoluble carrier used may have a group capable of reacting with the functional group of the antibody or multifunctional reagent used, or if necessary, a group capable of reacting with the above-mentioned spacer-introducing reagent may be used. Examples include insolubilized proteins such as albumin and gelatin, insolubilized polysaccharides such as agarose, cellulose, and dextrin treated with ehyglolhydrin, and those treated with cyanogen bromide and amino group introduction. Insoluble semi-synthetic polymer carriers such as insolubilized ones treated with spacer introduction reagents, acrylonitrile, acrylic acid, acrylic esters, methacrylic acid, methacrylic esters, vinyl alcohol, vinyl acetate, styrene, aminostyrene, chloro Examples include insoluble synthetic polymeric carriers such as polymers or copolymers of styrene, sulfostyrene, maleic acid, hexamaric acid, etc., and insoluble inorganic carriers into which amino groups of inorganic compounds such as silicon and aluminum have been introduced. These insoluble carriers usually have a particle size that can be easily isolated at least by means such as filtration, for example, a diameter of 1 mm or more, preferably 5 mm or more, and bead-shaped carriers are often used.

Moreover, instead of a bead shape, a spindle shape similar to the shape of the bottom of an immunoreaction tube may be used. Examples of insoluble carriers having such shapes include those shown in Figures 5, 6, and 7.
8 (see Japanese Unexamined Patent Publication No. 58-5657). In detail, the insoluble carrier is generally indicated by reference numeral l in FIG.

The insoluble carrier 1 is usually made of a polymeric resin material, and is composed of a main body 2 of the insoluble carrier and a mounting leg 3 protruding from the center of the upper surface 2a of the main body 2. Mounting leg 3
Another operating rod (not shown) is attached to the . The main body 2 is formed into a spindle shape (bullet shape), and its outer diameter is formed to maintain a slight clearance from the inner diameter of the reaction tube 4. Further, a projection 5, preferably a wedge-shaped projection, is provided at the top of the lower peripheral surface of the main body 2, and this projection 5 allows the arcuate inner peripheral surface between the arcuate outer peripheral surface 2b of the main body 2 and the immunoreaction tube 4 to 4
A certain gap 6 is maintained between the The height of this protrusion may be such that the width of the main body 2 and the inner wall surface of the immunoreaction tube 4 is the same. Further, the upper surface portion 2a of the main body 2 is provided with a cut groove 8 centered on the mounting leg 3, as shown in FIG. The end of the cut groove 8 is opened at the peripheral edge of the main body 2, and is designed to weaken the surface tension of the reaction liquid on an extension of the upper surface 2a. Further, FIG. 7 is shown as a modification example in which the shape of the reaction tube is different, but the parts having the same structure as in FIG. 5 are given the same numbers, and the explanation will be omitted. For example,
When the test tube used for the immunoreaction is 15×105 mm (inner diameter 12.8 mm±0.1 mm), the insoluble carrier used may have the dimensions shown in FIG. 8 with symbols qA to ■. That is, A: hemispherical part with a radius of 6 mm, B: the height of the cylindrical part is 10 mm, C: the height of the conical protrusion part is 0.4 mm, and 5D: the radius of the cone of the conical protrusion part is 0°5 mm.
5E: Semicircular groove with radius Q and 75 mm, F: Center distance between opposing grooves 7.5 mIn, G: Diameter of convex part for inserting the operating rod 4 mm, H: Operating rod insertion hole diameter 2
mm, i: Height of the convex part for inserting the operating rod: 3 mm Dimensions: 1 trowel The main body of the insoluble carrier consists of a cylindrical part with a height of 10 mm, a diameter of 12 mm, and a hemispherical part with a radius of 6 mm. be. Furthermore, the material of the immunoreaction tube, such as glass or synthetic polymer itself, may be used as an insoluble carrier.

Human TGF is then bound directly or using a polyfunctional reagent based on the reactive group of such an insoluble carrier, and the binding is usually carried out using a buffer solution with a pH of 6 to 8.5, an organic solvent, or the like. in a mixed medium of 0°C to 4
Each may be reacted at 0°C. As another method for producing a solid phase material, adsorption and immobilization may be performed by utilizing the porous adsorption ability of the insoluble carrier used. Furthermore, a second antibody obtained by immunizing another species of mammal, particularly a large animal, with a specific antibody against this TGF antibody, that is, an immunoglobulin fraction of the mammal producing this TGF antibody, is used. Even if a solid phase body is obtained by immobilizing the TGF antibody to an insoluble carrier and then bonding the TGF antibody thereto with an immunological means, the immobilization method hardly deactivates the activity of the TGF antibody. good.

Furthermore, the homologue thus obtained can be easily washed and stored.

Furthermore, if the TGF antibody is used in a soluble state instead of using the above-mentioned solid phase, TGF or a conjugate of enzyme-labeled TGF and TGF antibody and unreacted substances in the quantitative reaction of TGF in the test solution. A specific antibody against TGF antibody is used to separate the TGF antibody. This specific antibody
Also called an antibody, that is, an immunoglobulin fraction of a mammal that produces TGF antibodies is used as an antigen, and this is injected subcutaneously into other species of mammals, especially large animals, using known immunization methods. It is sufficient to immunize the patient, obtain antiserum from the blood, and then obtain antibodies by known purification means. In this case, it is usually convenient to use the antiserum. However, this second antibody may also be purified by a conventional method, immobilized on an insoluble carrier, and used as a solid phase for the second antibody.

Next, in carrying out the present invention, a test liquid whose TGF content is to be measured, such as human urine or serum, enzyme-labeled TGF and TGF antibody are mixed into an immunoreaction medium,
For example, strain 4-5 in phosphate buffer or veronal buffer.
Incubate for about 15 to 72 hours at about 10°C to allow a competitive reaction, and then remove the unreacted portion that has not bound to the immunoconjugated portion, especially the enzyme-labeled TGF-TGF antibody binding portion (Bond: B). B-F separation is performed to separate the free portion, particularly the free portion (Free:F) of unreacted enzyme-labeled TGF. In this B-F separation, in the case of a solid phase method in which the TGF antibody used is a solid phase, after competitive reaction, the solid phase and the reaction liquid layer are separated by means such as filtration, and after washing, The enzyme activity value of either the solid phase body or the filtration may be measured. In addition, in the case of a two-antibody method in which a second antibody is used for B-F separation while the antibody used remains soluble, after a competitive reaction, the second antibody, more preferably an antiserum containing the second antibody, is required. Depending on the situation, add normal serum from the same species of mammal used to create the TGF antibody to
Incubate for an hour, then 3000 rpm.

Centrifuge for about 10 to 30 minutes, separate the immunoreactive fleas and precipitate part (B) from the supernatant (F), and after washing, calculate the enzyme activity value of either the precipitate or the supernatant. Just measure it.

Furthermore, the solid phase body and precipitate which are the binding parts of the above-mentioned immune reaction,
Alternatively, measurement of the enzyme activity of the P solution or supernatant containing unreacted substances (in this case, various known activity measurement methods may be used depending on the properties of the enzyme used).

For example, when an oxidoreductase, especially an oxidase, is used as an enzyme, a reaction is performed using the enzyme's substrate and dissolved oxygen to produce a substrate oxide, hydrogen peroxide, and in some cases ammonia or carbon dioxide gas. It is measured by quantifying the amount of components consumed by the enzymatic reaction, such as oxygen, and the amount of components produced, such as hydrogen peroxide, ammonia, and carbon dioxide. In addition, oxygen, hydrogen peroxide, ammonia, and carbon dioxide gas can be quantified as a result of electrochemical changes caused by electrodes such as oxygen electrodes, hydrogen peroxide electrodes, ion electrodes, and gas electrodes. Sara]
The amount of hydrogen peroxide in the cube is peroxygase and guaiacol, 4-aminoantipyrine and 7-enol, 4-aminoantipyrine and 7-enol,
Aminoantipyrine and dimethylaniline, 3-methyl-
Coloring or fluorescence is produced using 2-benzothiazolinone and a hydrogen donor such as a color reagent such as dimethylaniline or a fluorescent reagent such as homovanillic acid or P-hydroxyphenylacetic acid, and the amount thereof is determined according to a conventional method. You may. Furthermore, dehydrogenase, which uses nicotine adenine dinucleotide (phosphate) [NAD(P)] or its reduced form as a substrate, is used as an oxidoreductase to act on the substrate used to convert its NAD(P) or reduced form. N.A.D.
The amount of reduced N A D (P) can be determined by a reaction that consumes or produces the amount of (P), or the cycling reaction of formazan coloring with a tetrazolium salt and diaphorase (this method can be used). Furthermore, in the case of a hydrolase, it is preferable to use a synthetic substrate that decomposes and releases a chromogenic group that can be detected from the substrate used by the hydrolase.
For example, when the hydrolase is β-galactosidase, the amount of 0-nitrophenyl released by the enzymatic reaction using 0-nitrophenyl-β-D-galactopyranoside as a substrate is measured at a wavelength of 420 nm. It can be measured as absorbance.

In this way, by using enzyme-labeled TGF obtained using human TGF and TGF antibody, it was possible to quantify TGF in a sample solution in an extremely accurate and simple manner, which is useful for cancer diagnosis. This is a novel quantitative method.

Next, the present invention will be specifically described with reference to Examples and Reference Examples, but the present invention is not limited thereto in any way.

Example 1 (1) Creation of anti-human TGF serum TGF (human TGF produced according to Reference Example 1 described below)
) 0.5 rnt to 0.5 ml of saline! IC
After dissolving the mixture, the same amount of Complete 70 India Adjuvant was added thereto, thoroughly emulsified and mixed, and then injected subcutaneously into the extremities and back of domestic rabbits at several points.

The same amount of emulsion was subcutaneously injected 6 times every two weeks, and whole blood was collected 10 days after the final immunization, left at room temperature for 60 minutes to solidify, and then centrifuged at 30 υ Orpm for 10 minutes. ) A TGF antibody-containing antiserum was obtained.

(2) Production method T of β-galactosidase-labeled TGF
GF1rnt in 100mM phosphate buffer (pH 8,0)
100mM EDTA aqueous solution and 241.6 μl of a dimethylformamide solution containing 0.1% 3-(2'-benzothiazolyl-dithio)propionic acid succinimide ester were added and reacted at 5°C for 1 hour. I forced it. The reaction solution was added to a Sephadex G-15 (1.5X 40cm) column, and elution was performed with 50mM acetate buffer (pH 5.0).
The flow-through fraction was collected, and 3-02 was added to the amine group of TGF.
A derivative having a '-benzothiazolyldithio)pyropionyl group introduced thereinto was obtained. Then 1.00 mM phosphate buffer (pH 7.0) containing 0.25 rnl of this derivative.
5+y+l β-galactosidase (Boehringer) 100mM J acid buffer (p
H7,0') 0.42 me was added and reacted at 5°C for 16 hours. The reaction solution was transferred to Sephadex G-150 (
A conjugate of β-galactosidase-labeled TGF (14
．． 7 μy/me and 1 per β-galactosidase molecule
A conjugate of the TGF molecule was obtained, and 96% of the TGF in the conjugate had antigenic activity for one TGF antibody.

+31 h) Antibody titer measurement of TGF antiserum The antiserum obtained in i11 was added to 10mM phosphate buffer (pH 7,4).
(Contains 0.25% bovine serum albumin (BSA), 0.1% sodium azide, 5mMEDTA, 0.9% Ml') - Immune reaction buffer - 100.1000 l
OOOO, diluted 100,000 times, 100μ each
1 was dispensed into small reaction tubes, to which was added β-galactosidase standard; Next, 11'' reaction was carried out, and the resulting precipitate was collected by adding 3 me of physiological saline and centrifuging at 300 Orpm for 10 minutes. The obtained precipitate was added with a substrate for measuring β-galactone cose activity (0.5% 0-nitrophenyl-β-ri-galactopyranoside, 0.1
%BSA, 3mM Mgclz, 0.1% Sodium Azide.

10mM phosphate buffer containing 0.9% NaCf (p
After adding 200 μl of H6,7)) and reacting at 37°C for 30 minutes, a reaction stop solution (100mM glycino-sodium hydroxide buffer (pH 11,0) 2.3me was added to stop the reaction, and the color change was It was measured at a wavelength of 42f) nm. The binding rate by immunoreaction with β-galactosidase-labeled TGF at each dilution concentration of the antiserum was measured, and the results are shown in FIG.

14) Measurement of TGF by liquid phase EIA. TGF was measured at 1.25% per l me using an immunoreaction buffer.
5.0. 20. 80. 320. The solution dissolved at each concentration of 1280 nP was used as a standard TGF solution.

Add 100 μg of each TGF standard solution to a small test tube for immunoreaction.

100μl of β-galactosiguse labeled TGF solution! , and human) 100 μr of a 1:25,000 dilution of TGF antiserum.
was added and reacted at 5'C for 16 hours. Then rabbit IgG 12μ2. Anti-rabbit IgG goat serum was added and reacted at 37°C for 2 hours, and the resulting precipitate was collected by adding 3 me of physiological saline and centrifuging at 3000 rpm for 10 minutes. Add 20'0β of the substrate solution for measuring β-galactosidase activity to the obtained precipitate and add 3
After reacting at 7°C for 60 minutes, add 2.3ηl of reaction stop solution.
The reaction was stopped by adding e, and the color was measured at a wavelength of 420 nm.
Measured at m.

The results are shown in FIG. 2, and this measurement system showed an extremely good quantitative curve. In addition, test liquid (serum,
When measuring the amount of TGF in urine, etc., add 100 μl of this test solution instead of the standard TGF solution, perform the same procedure as above, and compare the concentration in the test solution with the quantitative curve. All you have to do is ask.

Example 2 (1) Preparation of monoclonal antibody against human TGF TGF lr++t was dissolved in 1 ml of physiological saline, and the same amount of Conoply 1..70 was added thereto and thoroughly emulsified and mixed. Emulsifier 0.2 me Ba
It was injected subcutaneously at several points on the back of 1b/C mice. Booster immunizations were performed twice in the same manner at 10-day intervals, and 0.00 ml of physiological saline in which TGF l 00 μl was dissolved (after 2 weeks) was given twice.
2 m, e were administered intravenously. On the 3rd day after the final immunization, one mouse tile was aseptically removed and made into a single cell.The contaminated red blood cells were thawed with 0.83% ammonium chloride solution and the tile cells were suspended. After preparing the solution and washing it several times with the cooled Nox solution, cultured mouse myeloma cells (PS-NSI/1-A) were prepared.
g4-1) at a cell volume ratio of 3:1. Perform centrifugation, and slowly add 1 me of RPMI medium containing 50% polyethylene glycol 1000, pre-warmed to 37°C, over a period of 1 minute while rotating the centrifuge tube to the resulting pellet of tile cells and myeloma cells. Ta. Then 37
After heating for 90 seconds at
After gently adding each of these, centrifugation was performed, and the resulting bell knot was mixed with RPMI medium 34me containing 0% fetal bovine blood 1 (Fe2) to form a cell suspension. Transfer the cell suspension to 3 96-well plates (manufactured by Munk) at 100%
Dispense in micrograms and use a carbon dioxide gas evaporator (37°C, 595%C).
O2, 5%02) Pour 1 cell and start culturing, 1 day later 2
Double concentration HAT medium (100 μM hypoxanthine, 0.
4 μM aminopterin, 16 μM thymidine, 10% FC
Add 100 μl of RPM I medium containing 3.
HAT selective culture was performed, and then half of the medium was replaced with HAT medium at intervals of 1 to 3 days. After 7 to 14 days, collect the culture supernatant of the well where the hybridoma has grown,
Five samples with high antibody titers were selected. Next, using Ba1b/C thymocytes as slider cells, cloning was performed by the limiting dilution method, and 25 monocnal antibody-producing 1a cells were fractionated. Among them, N showed the highest antibody titer.
Using O, l'5 hybridoma, culture from 96 wells to 24 wells, then 50me culture and scale amplification, approximately 10'00mf! The culture supernatant was collected and used as a human 1-TGF monoclonal antibody solution.

(3) H) Measurement of antibody titer of TGF monoclonal antibody. Dilute the culture supernatant with immunoreaction buffer at 10,100°100°C.
100 μl of the 0.10,000-fold diluted solution was added to a small reaction tube, and then a β-galactosidase-labeled TGF solution was added and reacted at 5° C. for 16 hours. To this, 100 μl of a 100-fold dilution of normal mouse serum and 100 μl of anti-mouse ■/gG rabbit serum were added and allowed to react at 37°C for 1 hour. After adding 3 me of physiological saline to the resulting precipitate, The mixture was centrifuged at 3000 rpm for 10 minutes and collected. Add 200 μl of substrate solution for β-galactosidase activity measurement to the obtained precipitate and incubate at 37°C for 3 days.
After reacting for 0 minutes, 2.3 me of a reaction stop solution was added, and the color development was measured at a wavelength of 420 nm. The culture supernatant of the NO,15 hybridoma was bound to 50% of the labeled TGF at a 3000-fold dilution.

(4) Measurement of TGF by solid-phase EIAI 1) HiI-TG
Preparation of F antibody-immobilized carrier Human) TG obtained in (3) above
Polystyrene beads were added to 20me of a solution in which 10μ2 of a monoclonal antibody purified by ammonium sulfate fractionation and DEAE-cellulose chromatography was dissolved in 100mM phosphate buffer (CpH 8,0) from the culture supernatant containing F monoclonal antibody. (5 grains i6.35mm manufactured by Tsukisui Chemical Co., Ltd.
) Add 100 grains at 5°C for 16 hours, then at 37°C for 1 hour.
The antibody was immobilized on the beads by a time reaction.

After washing the beads with physiological saline, they were immersed in an immunoreaction buffer and stored at 5°C to form a homophase.

2) Add 200 μl of immunoreaction buffer and standard TGF solution (1,25 to 1280 n
g/me) 100 fil + and 100 μl of β-galactosidase-labeled TGF solution were added thereto, one homologue bead described in t was added thereto, and the mixture was reacted at 5° C. for 400 hours. After the reaction was completed, 2 me of physiological saline was added and washed with stirring, and the washing liquid was removed by suction using an aspirator. After repeating this operation twice, 500 μl of a substrate solution for measuring β-galactosidase activity was added and reacted at 37° C. for 90 minutes. A reaction stop solution 2me was added, and the color development was measured at a wavelength of 4201 m.

The results are as shown in Figure 3, and this measurement system (1) showed an extremely good quantitative curve. 1TGF
Add 100 μl of the test solution instead of the test solution, perform the same procedure as described above, and compare with the quantitative curve (this allows the T in the test solution to be compared with the quantitative curve.
GF concentration is determined.

Example 3 Preparation of a monoclonal antibody against Ell human TGFI A monoclonal antibody was prepared in the same manner as in Example 2.

Measurement of antibody titer of 12+ human TGF monoclonal antibody Measurement was performed in the same manner as in Example 2.

(Preparation of 31 anti-mouse IgG serum. Serum collected from blood collected from 00 alb/C mice.
m, (', a mouse IgG fraction was prepared by performing normal ammonium sulfate fractionation and DEAE-cellulose chromatography. The same amount of Comple 1... Freund's adjuvant was added and the mixture was thoroughly emulsified and mixed, and then injected subcutaneously at several points on the four fingers and the back of domestic rabbits.

The same amount of emulsion was subcutaneously injected 6 times every two weeks, and whole blood was collected 10 days after the final immunization, left at room temperature for 60 minutes to solidify, and then centrifuged at 300 rpm for 10 minutes. A mouse IgG antibody-containing antiserum was obtained.

(4) Measurement of TGF by solid-phase EIA 1) Preparation of human TGF antibody immobilized carrier Polystyrene-based insoluble carrier (showing the shape shown in Figure 8)
After washing with Skit
The above-mentioned insoluble carrier was inserted into a tube of 0.05 mm (inner diameter 12.8 mm ± O, 1 mm) and reacted at 5°C for 16 hours to form a solid phase body with 8.6 μ2 of IgG immobilized on each insoluble carrier. Obtained. Next, after washing the solid phase body thoroughly with physiological saline, human I-TGF monoclonal antibody 0.08μ2
Insert the immunoreaction buffer Q containing the immunoreaction buffer Q, and a small test tube containing 4me (described above) and react at 5°C for 400 hours to produce an insoluble carrier-mouse gG antibody-human TGF monoclonal antibody conjugate. After thoroughly washing this homologue with physiological saline, it was soaked in an immunoreaction buffer and stored at 5°C to form a solid phase of a human-TGF antibody-immobilized carrier.

2) Measurement method Small test tube (described above) Immune reaction buffer 1001 tl!
.

Mark 4!1 RCP liquid (1,25-1280 ng/me)
l 00 μe, and β-galactosidase labeled T G
100 μl of solution F was added and reacted at 37°C for 4 hours.

After the reaction was completed, 5 ml of deionized water was added, the solid phase was removed while being washed, and 500 μl of the substrate solution for β-galactone activity measurement (described above) was added in advance to a small test tube, and the mixture was incubated at 37°C for 1 hour. Made me react. After adding 2.0 me of the reaction stop solution (described above) to stop the reaction and removing the homophase, the absorbance of the reaction solution at 420 nm was measured.

The results are shown in Figure 4, and this measurement system showed an extremely good quantitative curve.However, when measuring the amount of TGF in the test fluid (serum/ureter), standard TGF solution should be used. Add 100 μl of the test solution instead of the test solution, perform the same operation as above, and compare with the quantitative curve to determine the TGF in the test solution.
In addition, an example of the production of TGF is given below, but it is not limited to this in any way.
TGF may be produced by the method described in the specification and used.

Reference Example 1 Molecular weight Kant 10,000 for lung cancer patients 1,100 + f' to 0.01 M phosphate buffer (pH 8,0)
Dialysis was performed at 4°C for 2 days and nights using a dialysis membrane (manufactured by Sanko Junyaku Co., Ltd.). The dialysis fluid was 6 liters at 2,000 x P.
Centrifuge for 0 minutes to remove the precipitate. Column (capacity 4+,
5 cm x 33 cm), 0.01 M
After equilibration with a phosphate buffer (pH 8,0), the above dialysis solution was allowed to flow down, and the active ingredient was adsorbed. After that, it was thoroughly washed with the same M solution, and then sulfur 1-IJ was added in the same buffer. Um o,
A gradient elution of 5 M was performed.

C flow rate 22.5 me/hr, temperature 4°C, sodium chloride IJium gradient concentration buffer4. , 81 used). TGF was eluted at one position in the same buffer at a concentration of 0.6 to 1.0 M sulfur chloride to obtain an active fraction of 62.0 ++re. In the TGF activity test method described below, normal cells were transformed and colonies were formed in 5 μl of this active fraction. The entire amount of this active fraction was desalted and concentrated using Dia 70-Molecular Sieve Membrane PM-10・C Amicon, 7A East, Limited C (Japan)) and concentrated (repeatedly to 1.2 me. Trifluoroacetic acid was added to make a 0.05% trifluoroacetic acid solution.

A column (capacity 4.1 cm x 250σ) was packed with Synchropack RP-PC (manufactured by Zinchrome Co., Ltd.), which uses silica gel as a carrier to which alkyl groups (C18) are bonded, at a concentration of 0.05%.
Equilibrate with trifluoroacetic acid L1 Mark T on this column
GF, to! , 1/3 of the 170% acetic acid solution is poured and adsorption is performed.
．． After washing with 05% truffle 0 acetic acid water, 0 to 9 in the same solution
Perform gradient elution with 0% acetonitrile and collect the active fraction eluted at the acetonitrile concentration position of 55-75%. The above operation was carried out three times, and the entire amount of the concentrated solution was treated with the molecular peak membrane to obtain an LTGF active fraction of 7.9 me.

This fraction was freeze-dried, then dissolved again in 3.0 me physiological saline, and this solution was subjected to the TGF test method. Normal cells were transformed and exhibited colony-forming ability in 0.4 μl.

Table 1 Materials + 1 NRK-cell 0 lines 49F) 1 unit (2) Dulbecco's modified MEM medium supplemented with 5% bovine baby serum and 1% non-essential amino acids (3) Purified agar (Agar Noble, Dif
(manufactured by Co., Ltd.) (4) Petri dish operation with a diameter of 60 mm Transfer the medium from (1) containing 0.5% agar to 5 Petri plates.
Pressure is applied to obtain a lower layer medium.

Above this R: NRK cells 1 × 10 cells/me 7;
Add 0.3% agar-containing medium containing 2 me to each plate.
Add 0.5 me of a sample dissolved in saline to the upper layer medium and Ll, and culture at 37°C in a carbon dioxide incubator. After 14 days, remove from the incubator.
Petri with a 40x Kenbi mirror, 2 mm x 2
Randomly select 10 m field of view and calculate the number of cells io
Count the number of colonies consisting of 1 or more, and use the total number as the score.

4. Brief description of the drawings Figure 1 shows the antibody titer measurement curve of the TGF antiserum obtained in Example 1.L1 Figure 2 shows the quantitative curve of TGF by liquid phase EIA. Figures 3 and 4 show the quantitative curve of TGF by solid-phase EIA, Figure 5 is an enlarged partial cross-sectional view of the insoluble carrier used in solid-phase EIA, Figure 6 is its plan view, and Figure 7 is the insoluble carrier. FIG. 8 is an enlarged partial sectional view of a modified example of , and FIG. 8 is an actual measurement diagram of the insoluble carrier used in Example 3.

Patent applicant: Toyo Jozo Co., Ltd. -1,-,□J Figure 1 Anti-TGF (Dilution) Figure 2 TGF (ng/mL) Figure 3r2'1 TGF (ng/mL) Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Mr. Commissioner of the Japan Patent Office 1. List and presentation of the case 1982 Patent Application No. 904448
2. Name of the Invention Transforming 1” Quantitative Method of SF Actor 3, Relationship with the Amendment Person Case Special J [Applicant Address: 632-1 Mifuku, Ohito-cho, Tagata-gun, Shizuoka Prefecture Representative: Taka 1) Tetsu Man 4, Agent 6, Detailed explanation of the invention of the specification to be amended 7, Contents of the amendment Contents of the amendment as shown in the attached sheet (1) Ming, 1lIj ancient 12th column, 6th line from the bottom, n
On page 13 of ``,'' lines 2 to 4, II, line 6 from the top, correct ``polyfunctional life'' to ``polyfunctionality.'' 110 (
2) Same, page 18, in the first line from the bottom, ``ri-cum'' is corrected to ``1 silicon''.

(3'i''iL page 21, 9th line from the top, TI
Page 22, line 6 from the bottom, `` M m Page 24, line 9 from the top, correct each word ``present'' to ``rezuku.''

(4) l1il, page 28, 2nd line from the bottom, 1 reaction for ``proactive response'' f: HJ corrects.

(S) Same, page 29, second line from the bottom, Correct "1 small test tube" to "small test tube."

(6) Same, page 32, line 6 from the bottom, “Monochunal”
1 monoclonal," Shimazu corrected.

(7) Same, p. 34, line 8 from the top, ``Clean thoroughly with...'' was corrected to ``Clean thoroughly with...''.

(8) Same, page 34, top J: In line 111, ``test tube'' is corrected to ``1 small test tube''.

(9) On the same page, in the fourth line from the bottom, "removal" has been corrected to "removal."

(+o) Same, page 40, Table 1, replace [S yucl]ropak J in the third row from the top with “3 ynchro
pak J and corrected Shimazu.

- Procedural Nj Orthography (spontaneous) 1. Indication of matter A'4 1982 Patent Application No. 90444 2. Title of the invention 1 - Quantification method for lance-homing gross factor 3. Relationships Patent applicant address: 4, Mifuku, Ohito-cho, Tagata-gun, Shizuoka Prefecture, Agent: 5, Date of amendment order (voluntary): 6. Subject of amendment: Column 7 for detailed explanation of the invention in the specification, Blank spaces in the amendment Attachment rP on page 16, lines 8-9 and page 31, line 12 in the statement II of the correction.
S-NS1J and AruwosotL respectively “P3-NS I,
I first correct g1.

that's all

Claims (1)

[Scope of Claims] (1) Enzyme-labeled transforming gross factor and trans7 Ohming gross factor antibody are reacted in a test solution, and then enzyme-labeled Quantification of transforming gross factor in a test solution, characterized by separating the gross factor antibody conjugate and unreacted enzyme-labeled transforming gross factor, and then quantifying the amount of any of the separated enzyme labels. Law. (21) The assay method according to claim 1, wherein the transforming growth factor antibody is a transforming growth factor antibody immobilized on an insoluble carrier. 131) The transforming growth factor antibody is soluble, (4) A patent for the quantitative determination method described in item 1 of the scope of claim ii'j, which uses a specific antibody against a transforming gross factor antibody in a separate process; (4) a patent for transforming gross factor or human transforming gross factor; The coulometric method according to claim 1. (The assay method according to claim 1, wherein the 51 transfominogloss 7 actor antibody is a monoclonal antibody. (6) The assay method according to claim 1, wherein the enzyme is an oxidoreductase. ( 7) The assay method according to claim 1, wherein the enzyme is a hydrolase. (&) The assay method according to claim 7, wherein the hydrolase is β-calactondase.