JPH0441308B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for quantifying transforming growth factor (hereinafter referred to as TGF). TGF is a polypeptide produced by human and rodent cancer cells that causes specific cytomorphological changes (transformation and proliferation) in normal cells. Normal human and animal cells do not form colonies on soft agar;
When TGF is added, the cells become capable of forming colonies. 1978 Z. Todaro et al. [Proc. Natl.
Acad.Sci., 75 , 4001 (1978)] reported the existence of TGF, starting from the culture supernatant of sarcoma cells. It has also been discovered from [Roberts et al.; Proc. Natl.
Acad.Sci.USA, 77 , 3494 (1980)]. Recently, D. I. Tajitsuk et al. [JNCL, 69 , 793]
(1982)] and Z. Todaro et al. [Cancer
Res., 43 , 403 (1983)] extracted a high-molecular-weight 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 for clearly distinguishing between various gross factors that are similar to TGF, and the determination of TGF has been based on the normal cell colony forming ability of TGF. However, it required complicated operations and lacked quantitative properties. Furthermore, there have been few 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 first discovered that high-molecular-weight TGF does not exist in normal human urine, but only exists specifically in cancer patient urine. As a result of our research, we found that the urine of various cancer patients was dialyzed to remove inorganic ions and other low-molecular substances, and if necessary, gel filtration was performed, and then the urine was analysed. Human TGF purified to extremely high purity was obtained by performing chromatography and/or adsorption chromatography using an ion exchanger.
59-186994). Furthermore, as a result of continued research, the inventors found that this
I learned that human TGF antibodies against this TGF can be easily obtained by injecting TGF into non-human mammals to immunize them and collecting their blood.
Furthermore, as a result of the research, the spleen of the immunized repaired animal was removed, and monoclonal antibody-producing cells as human TGF antibodies against TGF were obtained by fusing single cells capable of producing the antibody with myeloma cells, which were then cultured. We succeeded in obtaining human TGF antibodies using this method. Furthermore, the present inventors obtained a radioisotope (hereinafter referred to as RI)-labeled TGF that can perform a good immune reaction with the obtained human TGF antibody, and
The above-mentioned human TGF antibodies and
We established an RI immunoassay (hereinafter referred to as RIA) using RI-labeled TGF. The present invention was completed based on the above findings, and involves reacting RI-labeled TGF with a TGF antibody in a test solution, and then separating the RI-labeled TGF-TGF antibody conjugate from the unreacted RI-labeled TGF. In this method, the transforming growth factor is positive in ninhydrin reaction, is single in SDS-electrophoresis, is stable against acid and heat, and is resistant to trypsin and This is a method for quantifying TGF in a test liquid, which is characterized in that it is inactivated by dithiothreitol treatment and has physical and chemical properties showing an estimated molecular weight of 28,000 to 35,000 by gel filtration. Furthermore, in the present invention, the competitive reaction may be quantified by a solid-phase method using the TGF antibody as a solid phase substance immobilized on an insoluble carrier, or the TGF antibody that remains soluble may be used to quantify the competitive reaction. In the separation, a competitive reaction may be quantified by a two-antibody method using a specific antibody against the TGF antibody. First, human TGF for obtaining the RI-labeled TGF and human TGF antibody of the present invention is obtained from the urine of patients with cancer such as lung cancer, choriocarcinoma, gastric cancer, pharyngeal cancer, pharyngeal cancer, breast cancer, melanosarcoma, and ovarian cancer. It is easy to refine and recover as a raw material. To give an example of the means for purifying TGF from the urine of cancer patients, the collected urine of cancer patients is first purified using a 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. and dialysis.
Next, the obtained dialysate solution may be repeatedly frozen and thawed as necessary to precipitate impurities, and fine precipitated particles may be removed by centrifugation or through a filter having pores of about 5 microns. . Furthermore, if necessary, the dialysis fluid may be prepared from Biogel p-60, p-100 (manufactured by Bio-Rad),
Gel filter with a gel filter such as Sephadex G-50 (manufactured by Pharmacia).
What is necessary is to collect fractions showing TGF activity. On this occasion,
The fraction showing TGF activity has an estimated molecular weight of 28,000.
It exists at a position of ~35,000 and can be identified by the test method described below. Next, the active fraction collected through the urine dialysis fluid or gel filtration is subjected to ion exchange chromatography, molecular sieve treatment using a molecular sieve membrane, such as an ultrafiltration membrane with a molecular weight of about 10,000 to 20,000, or adsorption chromatography. A TGF active fraction purified by at least one or more operations or a combination of these operations is collected. Examples of ion exchange chromatography include CM-Sephadex (manufactured by Pharmacia),
SP-Sephadex (manufactured by Pharmacia), CM-52 (manufactured by Watmann), Bio-
Bio-Rex 70 (manufactured by Bio-Rad)
The above TGF-containing solution is added and adsorbed using a column based on a cation exchanger such as .
Then, the TGF active fraction may be recovered by performing gradient elution using an aqueous solution of a neutral salt, preferably sodium chloride, whose concentration is successively increased. Next, concentration and desalting may be performed preferably using an ultrafiltration membrane having a molecular weight of about 10,000 to 20,000. Furthermore, for adsorption chromatography, for example, silica gel modified with a hydrophobic group such as an alkyl group having 3 to 20 carbon atoms, Synchropak PR series (manufactured by Synchrome), and MCI Gel.
Using a column with a base material such as polystyrene-based hyperpolar adsorption resin such as CH20P series (manufactured by Mitsubishi Chemical Industries, Ltd.) and Amberlite (manufactured by Amberlite) XAD series (manufactured by Amberlite), the above
Add a TGF-containing solution and allow it to be adsorbed. 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 thus obtained may be obtained as a dry powder by drying means such as freeze-drying, if necessary, and this dry powder is white, positive for ninhydrin reaction, and has a molecular weight of 28,000 to 35,000.
(gel filtration method), is single in SDS-electrophoresis, is stable against acids and heat, has physical and chemical properties that are inactivated by trypsin and dithiothreitol treatment, and is extremely highly purified. This is human TGF. Then RI labeling using such TGF
TGF, used to obtain TGF antibodies
TGF is not limited to the purification methods described above. First, to obtain a human TGF antibody, using the aforementioned human TGF as an antigen source, a non-human mammal,
For example, animals capable of producing antibodies, such as guinea pigs, rabbits, rats, mice, and goats, are immunized in accordance with conventional methods, blood is collected to obtain antiserum, and the antibodies are further isolated. At this time, it is used as an antigen.
Although TGF is preferably a single protein preparation highly purified as described above, it is not necessarily limited to this. To obtain antibodies, for example, dissolve 0.1 to 1 mg of the human TGF powder described above in 0.1 to 5 ml of physiological saline, add the same amount of Complete Freund's adjuvant, and emulsify thoroughly. After that, the mammal to be used, such as a rabbit or a mouse, is injected subcutaneously or intradermally, and immunized 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 human TGF antibodies. In addition, any animal can be used in this case as long as it has the ability to produce antibodies.
To obtain large amounts of antibodies, it is preferable to use large animals, usually rabbits, mice, or goats.
It is not limited in any way. Furthermore, human TGF antibodies can be obtained from antiserum containing human TGF antibodies obtained from these animals by a commonly used antibody purification method, such as ammonium sulfate fractionation of the antiserum. Then, it may be purified and collected by ion exchange chromatography or gel filtration. For further purification, it is possible to obtain the protein by adsorbing it by affinity chromatography using an insoluble carrier on which human TGF is immobilized as a base material, and then performing elution. Furthermore, as an alternative method to obtain human TGF antibodies, human
The spleen cells of a non-human mammal immunized with TGF as an antigen are fused with myeloma cells, the monoclonal antibody-producing cells against human TGF are isolated from the fused cells, and the fused cells are used to produce human TGF monoclonal antibodies. Among the methods for producing antibodies, methods using mice as mammals are often used [Nature, 256 , 495-497
(1975), Nature, 276 , 397-399 (1978), Cell,
14, 9-20 (1978), Nature, 266 , 550-552
(1977), Eur.J.Immunol., 6 , 511-519 (1976),
Chemical and Engineering News, Jan.1.1979,
15-17]. For example, TGF is administered subcutaneously to Balb/C mice.
An emulsion of physiological saline and complete Freund's adjuvant was injected, and multiple booster immunizations were performed 1 to 3 weeks later. The spleen of the mouse was removed 3 to 5 days after the final immunization, and the spleen was inoculated in an appropriate medium. Prepare a single cell suspension of cells. Next, 1 amount of mouse-derived myeloma cells, e.g., P3-NS1/1-Ag4-I, was added to 3 to 10 amounts of these splenocytes in 40 to 50% polyethylene glycol at 37°C.
1000 to 1500 in a suitable medium, such as RPMI medium [JAMA, 199 , 519 (1967), J. Nat. Cancer
Inst., 36 , 405 (1966), In Vitro, 6 , 89 (1970)
]
The cell suspension was fused in RPMI medium containing fetal bovine serum, and a small amount of this cell suspension was added to RPMI medium containing hypoxanthine, aminopterin, thymidine, and fetal bovine serum (HAT medium). , collect the supernatant of each culture, select cultured cells with a high antibody titer, and clone using the limiting dilution method using mouse Balb/c thymocytes as feeder cells. Sort human TGF monoclonal antibody-producing cells. Furthermore, these cells were cultured in RPMI medium containing fetal bovine serum or Dulbecco's modified Eagle's medium, and the supernatant was obtained, which was subjected to ammonium sulfate fractionation, ion exchange chromatography, gel filtration, and affinity chromatography. A purified human TGF monoclonal antibody is obtained. Alternatively, human TGF monoclonal antibody-producing cells can be grown as tumors in histocompatible animals or athymic nude mice, and then collected.
May be purified. Furthermore, these human TGF monoclonal antibody-producing cells may be cryopreserved at about -196° C. in liquid nitrogen 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 as a solid phase solidified on an inert carrier. Insoluble fragments such as F(ab') ₂ obtained by further treating these antibodies with papain, Fab' obtained by further reducing this, and F(ab) ₂ obtained by treating the antibody with pepsin, etc. It may also be used as a solid phase bound to a carrier. Any solid phase may be used as long as it retains the immunobinding activity of TGF obtained by binding the insoluble carrier and the above-mentioned antibody using the above-mentioned multifunctional reagent. Also, in the combination of the two,
An arbitrary spacer may be introduced in advance into either or both of the insoluble carrier and the antibody. For example, a spacer may be introduced into either or both of the insoluble carrier and the antibody. Acid chloride, succinimide ester, p-
Reactive derivatives such as nitrophenyl esters, dicarboxylic acid compounds or their reactive derivatives such as malonic acid, succinic acid, glutaric acid, adipic acid,
Diamine compounds such as hexamethylene diamine and decamethylene diamine, thiocarboxylic acid compounds such as 3-(2'-pyridyl-dithio)propionic acid and 3-(2'-benzothiazolyl-dithio)propionic acid, or their reactive derivatives, S- 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 acetylmercaptosuccinic anhalide and thiol compounds such as 2-aminoethanethiol. You may.
Next, the amino groups of this insoluble carrier and antibody,
A conjugate of an insoluble carrier and an antibody can be obtained using a crosslinking reagent that can bind the two based on a hydroxyl group, a carboxyl group, a thiol group, or a further introduced functional group. The crosslinking reagent to be used 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, such as succinic aldehyde, glutaraldehyde, adipoline dialdehyde compounds such as aldehydes; dicarboxylic acids such as malonic acid, succinic acid, glutaric acid, and adipic acid or their reactive derivatives; diisocyanate compounds such as hexamethylene diisocyanate and 2,4-toluene diisocyanate; diisothiocyanate compounds such as methylene diisothiocyanate, maleimidocarboxylic acids or reactive derivatives thereof such as maleimidobenzoic acid, maleimidophenylacetic acid, N,N'-ethylene bismaleimide, N,
dimaleimide compounds such as N'-0-phenylene dimaleimide, bisdiazobenzidine, diethylmalonimidate, dimethyladipineimidate, N,N'-polymethylene bis iodoacetamide and 3-(2'-benzothiazoline-dithio) Thiocarboxylic acid compounds or reactive derivatives thereof such as propionic acid, 3-(2'-pyridyl-dithio)propionic acid, N-[2-(2'-pyridyl-dithio)ethyl]-3-(2'-benzothiazolyl- Dithio compounds such as dithio)propionamide, 1-(2'-benzothiazolyl-dithio)-2-(2'-pyridyl-dithio)ethane, etc., and these polyfunctional reagents are compatible with the insoluble carrier used and the antibody. The functional groups involved in bonding, such as amino groups, carboxyl groups, aldehyde groups, hydroxyl groups, and thiol groups, may be considered and used in selection. Furthermore, the insoluble carrier to be 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 a spacer-introducing reagent as described above. Examples include insolubilized proteins such as albumin and gelatin, insolubilized polysaccharides such as agarose, cellulose and dextrin treated with epichlorohydrin, and spacers for cyanogen bromide treatment and amino group introduction. Insoluble semi-synthetic polymer carriers, such as insoluble ones treated with introduction reagents or spacer introduction reagents for thiol group introduction, acrylonitrile, acrylic acid, acrylic esters, methacrylic acid, methacrylic esters, vinyl alcohol, vinyl acetate , styrene, aminostyrene,
Examples include insoluble synthetic polymeric carriers such as polymers or copolymers of chlorostyrene, sulfonestyrene, maleic acid, fumaric acid, etc., and insoluble inorganic carriers into which amino groups of inorganic compounds such as silicon and aluminum have been introduced. These insoluble carriers are
Generally, particles with a particle size that can be easily isolated by means such as filtration are preferred, for example, particles with a diameter of 1 mm or more, preferably 5 mm or more, and bead-shaped particles 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 are shown in FIGS. 5, 6, 7, and 8 (see Japanese Patent Laid-Open No. 58-5657). Specifically, the insoluble carrier is generally indicated by reference numeral 1 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. Another operating rod (not shown) is attached to the mounting leg 3. 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 4a, a gap 6 is maintained between the two. The height of this protrusion is
It is sufficient that the widths of the main body 2 and the inner wall surface of the immunoreaction tube 4 are 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 end edge of the main body 2, and is designed to weaken the surface tension of the reaction liquid on the extension of the upper surface 2a. In addition, Fig. 7 is shown as a modification example when the shape of the reaction tube is different, but the same numbers are given to the parts whose structure is the same as in Fig. 5.
Delighting is omitted. To give an example, the test tube used for immune reaction is 15 x 105 mm (inner diameter 12.8 mm ± 0.1 mm).
mm), the insoluble carrier used is
The dimensions of each part indicated by symbols A to I in the figure can be used. That is, A: hemispherical part with a radius of 6 mm, B:
Height of cylinder part 10mm, C: Height of conical projection part 0.4
mm, D: radius of cone of conical projection 0.5 mm, E:
Semicircular groove with a radius of 0.75 mm, F: Center distance between opposing grooves 7.5 mm, G: Diameter of the protrusion for inserting the operating rod 4 mm, H: Diameter of the operating rod insertion hole 2 mm, I : The main body of an insoluble carrier consisting of a cylindrical part with a height of 10 mm, a diameter of 12 mm, and a radius part of 6 mm, as shown by the height of the convex part for inserting the operating rod of 3 mm. Furthermore, the material of the immunoreaction tube, such as glass or synthetic polymer itself, may be used as an insoluble carrier. The human TGF antibody is then bound directly or using a multifunctional reagent to 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. What is necessary is just to make each react at 0 degreeC - 40 degreeC in a mixed medium. 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 specific antibody for this TGF antibody, i.e., this TGF
A second antibody obtained by immunizing another species of mammal, particularly a large animal, with an immunoglobulin fraction of an antibody-producing mammal is used, and this second antibody is immobilized on an insoluble carrier, and then the second antibody is immobilized on an insoluble carrier. By binding this TGF antibody by immunological means
The solid phase may be obtained by immobilization means that hardly deactivate the activity of the TGF antibody. Furthermore, the solid phase body thus obtained may be washed and stored. Furthermore, instead of using the solid phase described above,
When using the TGF antibody in a soluble state, TGF or RI in the quantitative reaction of TGF in the test solution
A specific antibody against the TGF antibody is used to separate the conjugate of labeled TGF and TGF antibody from the unreacted material. This specific antibody, also called a second antibody, uses the immunoglobulin fraction of a mammal producing TGF antibodies as an antigen, and subcutaneously injects it into other species of mammals, especially large animals, using known immunization methods. The antibody can be immunized by intradermal injection, antibody serum can be obtained from the blood, and the antibody can then be obtained by known purification means. In this case, it is usually convenient to use the antibody in an anti-blood state. 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. Furthermore, in the RI-labeled TGF used in the present invention,
¹²⁵ I and ¹³¹ I are usually used as RI, and ¹²⁵ I is generally used. Also, these iodine
For example, to bind iodine to TGF,
Chloramine-T is directly bound within the molecule of
Bolton & Hunter (Bolton & Hunter), which is an indirect iodine attachment method that attaches an iodine phenyl group to a TGF molecule.
Methods such as law are often used. In particular, in the chloramine-T method, iodine is used in the form of sodium iodide, and in the Bolton-Hunter method, iodine is used in the form of sodium iodide.
Hunter's reagent, e.g. 3-(4-hydroxy-5
-[ ^125I ]iodophenyl)propionate is often used in the form of succinimide ester. When labeling TGF using such RI, the above-mentioned chloramine-T method or Orton-T method can be used.
It can be carried out by a conventional method based on the Hunter method, and after the reaction is completed, purification methods such as gel filtration can be used.
A purified product of RI-labeled TGF may be collected. moreover
Labeling means for RI is not limited to the above-mentioned method, and various known methods may be used for labeling. Next, in carrying out the present invention, first, TGF
A test solution whose content is to be measured, for example, 50 μ to 1 ml of human urine or serum, and RI-labeled TGF and TGF antibody are placed in an immunoreaction medium, such as phosphate buffer or veronal buffer, 100 μ to 5 ml. 4
Incubate at ~5°C for about 15 to 72 hours to allow competitive reaction. Next, by this reaction, the unreacted free portion that is not bound to the immunologically bound portion, especially the RI-labeled TGF-TGF antibody binding portion (Bond: B), especially the unreacted RI-labeled TGF free portion (Free: B-F separation is performed to separate F). 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,
After washing, remove either the solid phase or the liquid phase.
The radioactivity of RI-labeled TGF may be measured. Also B
- In the case of F separation, in the case of a two-antibody method using a second antibody while the antibody used remains soluble, the second antibody after a competitive reaction, more preferably an antiserum containing the second antibody, if necessary. Add normal serum from the same species of mammal used for TGF antibody production and incubate for 1 to 12 hours, then at 3000 rpm.
After centrifugation for about 10 to 30 minutes, the precipitated portion B and supernatant F, which are bound by immunoreaction and are separated, may be separated, and after washing, the radioactivity of either the precipitate or the supernatant may be measured. Furthermore, when measuring the radioactivity of solid phase bodies and precipitates, which are the binding parts of the above immune reaction, or liquids and supernatants containing unreacted substances, radioactivity measurement devices such as Geiger-Muller counters and scintillation counters are used. You can do it at the counter. In this way, obtained using human TGF
By using RI-labeled TGF and TGF antibody, it was possible to quantify TGF in a test fluid extremely accurately and easily, and it is useful for determining cancer. 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) Preparation of anti-human TGF serum
Dissolve 0.5mg of TGF) in 0.5ml of physiological saline, add the same amount of Complete Front Adjuvant to this, thoroughly emulsify and mix, and then inject into the skin of the fingers and toes of domestic rabbits and several subcutaneous locations on the back. did. The same amount of emulsion was subcutaneously injected six times at two-week intervals, and whole blood was collected 10 days after the final immunization and left at room temperature for 60 minutes to coagulate.
Human TGF was centrifuged at 3000 rpm for 10 minutes.
Antibody-containing antiserum was obtained. (2) Preparation of ¹²⁵ I-labeled TGF by the chloramine-T method Add 50 μ of 0.5 M phosphate buffer (PH7.5) containing ¹²⁵ I-NaI with 2 mCi of radioactivity,
10μ of an aqueous solution containing 2μg of TGF, and 0.35
After adding 20μ of % chloramine T solution and stirring for 30 seconds, add 0.45% sodium bisulfite solution.
The reaction was stopped by adding 50μ. Next, the reaction solution contained 5% human serum albumin (HSA).
After adding 500μ of 0.1N acetic acid solution, it was added to a Sephadex G-25 (1.0 x 50cm) column.
It was eluted with a 0.1N acetic acid solution containing 0.5% HSA, and ¹²⁵ I-labeled TGF was obtained in the flow-through fraction. (3) Antibody titer measurement of human TGF antiserum The antiserum obtained in (1) was dissolved in 10mM phosphate buffer (PH7.4) (0.25% bovine serum aluminium (BSA), 0.1
% Sodium Azide, 5mMEDTA, 0.9%
NaCl-containing) - immunoreaction buffer -, 100,
Diluted 1000, 10000, and 100000 times, respectively.
Dispense 100μ into Shionogi tube for RIA,
Add 100 μl of ¹²⁵ I-labeled TGF solution to this and incubate at 5°C.
The reaction was allowed to proceed for 16 hours. Next, Rabbit Ig
Add 12 μg of G and anti-rabbit IgG goat serum.
The reaction was allowed to proceed at 37°C for 2 hours, and the resulting precipitate was collected by adding 3 ml of physiological saline and centrifuged at 3000 rpm for 10 minutes, and the radioactivity of the precipitate was measured. ¹²⁵ I labeling at each dilution concentration of antiserum
The binding rate by immunoreaction with TGF was measured and the results are shown in FIG. (4) Measurement of TGF by liquid phase RIA Standard TGF solutions were prepared by dissolving TGF at concentrations of 1.25, 5.0, 20, 80, 320, and 1280 ng per ml using an immunoreaction buffer.
100μ of each TGF standard solution in Shionogi tube for RIA
, 100 μl of ¹²⁵ I-labeled TGF, and 100 μl of a 30,000-fold dilution of human TGF antiserum were added and incubated at 5°C for 16
I let it react over time. Next, Rabbit Ig
12 μg of G was added with anti-rabbit IgG goat serum.
The reaction was allowed to proceed at 37°C for 2 hours, and after adding 3 ml of physiological saline to the resulting precipitate, the mixture was centrifuged at 3000 rpm for 10 minutes and collected, and the radioactivity of the precipitate was measured. The results are shown in FIG. 2, and this measurement system showed an extremely good quantitative curve.
When measuring the amount of TGF in a test solution (serum, urine, etc.), use 100μ of the test solution instead of the standard TGF solution.
The concentration of TGF in the test liquid may be determined by adding the above, performing the same operation as above, and comparing with the quantitative curve. Example 2 (1) Preparation of monoclonal antibody against human TGF 1 mg of TGF was dissolved in 1 ml of physiological saline, and the same amount of Complete Freund's Adjuvant was added and thoroughly mixed to emulsify. emulsifier 0.2ml
It was injected subcutaneously at several points on the back of Balb/c mice.
Booster immunizations were performed twice in the same manner at 10-day intervals, and two weeks later, 0.2 ml of physiological saline in which 100 μg of TGF had been dissolved was administered intravenously. On the third day after the final immunization, the spleen of the mouse was removed aseptically and made into a single cell, and the contaminated red blood cells were removed.
Dissolved in 0.83% ammonium chloride solution. This suspension of splenocytes was washed several times with chilled Hank's solution, and then mixed with mouse myeloma cells (P3-NS1/1-Ag4-1) cultured separately at a cell volume ratio of 3:1. Perform centrifugation and add the resulting pellet of splenocytes and myeloma cells in advance.
50% polyethylene glycol heated to 37°C
1 ml of RPMI medium containing 1000 was slowly added over 1 minute while rotating the centrifuge tube. Then 37℃
After heating for 90 seconds with
After gently adding 34 ml of RPMI medium containing 10% fetal calf serum (FCS) to the pellet obtained by centrifugation, a cell suspension was prepared. Dispense the cell suspension into 96-well plates (manufactured by Munk) in 100μ increments and sifter with carbon dioxide gas (95% air, 5% _CO2 ; temperature 37℃, humidity 100%).
to start culturing, and one day later, the concentration was increased to 2 times the concentration.
Start HAT selection culture by adding 100 μM of HAT medium (RPMI medium containing 100 μM hypoxanthine, 104 μM aminopterin, 16 μM thymidine, 10% FCS), and then add half of the medium to HAT at intervals of 1 to 3 days. Replaced with medium. After 7 to 14 days, culture supernatants from wells in which hybridomas had grown were collected, and five hybridomas with high antibody titers were selected. Next, using Balb/c thymocytes as feeder cells, cloning was performed by the limiting dilution method to disperse 25 monocnal antibody-producing cells. Using the No. 15 hybridoma that showed the highest antibody titer among them, we scaled up the culture from 96 wells to 24 wells and then 50ml to obtain approximately 1000ml of culture supernatant, which was then used in human It was used as a TGF monoclonal antibody solution. (2) Antibody titer measurement of human TGF monoclonal antibody Culture supernatant was mixed with immunoreaction buffer at 10, 100,
Add 100μ of the 1000- and 10000-fold diluted solution to a small reaction tube, then add ¹²⁵ I-labeled TGF solution,
The reaction was carried out at 5°C for 16 hours. This was supplemented with 100 μl of a 100-fold dilution of normal mouse serum, and anti-mouse Ig.
100μ of Rabbit G serum was added and reacted for 1 hour at 37°C, and the resulting precipitate was collected by adding 3ml of physiological saline, centrifuged at 3000 rpm for 10 minutes, and the radioactivity of the precipitate was measured. . No.
Culture supernatants of 15 hybridomas bound 50% of ¹²⁵ I-labeled TGF at approximately 4000-fold dilution. (3) Measurement of TGF by solid phase RIA (1) Preparation of human TGF antibody immobilized carrier From the culture supernatant containing the human TGF monoclonal antibody obtained in (2) above, ammonium sulfate fraction,
100 mg of monoclonal antibody purified by DEAE-cellulose chromatography
Solution dissolved in mM phosphate buffer (PH8.0)
100 polystyrene beads (manufactured by Sekisui Chemical Co., Ltd., particle size: 6.35 mm) were added to 20 ml and reacted at 5°C for 16 hours and at 37°C for 1 hour to immobilize the antibody on the beads. After thoroughly washing the beads with physiological saline, they were immersed in an immunoreaction buffer and stored at 5°C to form a solid phase. (2) Measurement method In a small reaction test tube (Shionogi Tube), add 200μ of immunoreaction buffer and standard TGF solution (1.25~
1280ng/ml) 100μ and ^125I labeled TGF solution
100μ of the solid phase was added thereto, and one particle of the above solid phase material was added thereto, followed by reaction at 5°C for 40 hours. After the reaction was completed, 2 ml 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, the beads were transferred to a new tube and their radioactivity was measured. The results are shown in FIG. 3, and this measurement system showed an extremely good quantitative curve. In addition, TGF in the test liquid (serum, urine, etc.)
When measuring the amount, the TGF concentration in the test solution can be determined by adding 100μ of the test solution instead of the standard TGF solution, performing the same procedure as above, and comparing with the quantitative curve. Example 3 (1) Preparation of monoclonal antibody against human TGF A monoclonal antibody was prepared in the same manner as in Example 2. (2) Measurement of antibody titer of human TGF monoclonal antibody Measurement was performed in the same manner as in Example 2. (3) Preparation of anti-mouse IgG serum Serum collected from 100 Balb/c mice
From 50 ml, perform normal ammonium sulfate fractionation and DEAE-cellulose chromatography to extract mouse IgG.
Fractions were prepared. The same amount of Complete Freund's Adjuvant was added to 1 ml of physiological saline containing 5 mg of mouse IgG, thoroughly emulsified and mixed, and then injected into the skin of the fingers and toes of domestic rabbits and at several subcutaneous locations on the back. The same amount of emulsion was subcutaneously injected 6 times at two-week intervals, and whole blood was collected 10 days after the final immunization, left at room temperature for 60 minutes to solidify, and then centrifuged at 3000 rpm for 10 minutes to collect mouse IgG. Antibody-containing antiserum was obtained. (4) Measurement of TGF by solid-phase RIA (1) Preparation of human TGF antibody immobilized carrier After washing the polystyrene-based insoluble carrier (the shape shown in Figure 8) with Schitt XN, the IgG content of anti-mouse IgG rabbit serum was washed. 100mM phosphate buffer (PH8.0) containing 32μg 0.4
Small test tube 15×105mm (inner diameter 12.8mm±
Insert the above insoluble carrier into a tube (0.1 mm) and heat at 5°C.
The reaction was carried out for 16 hours to obtain a solid phase material in which 8.6 μg of IgG was immobilized on each insoluble carrier. Next, after thoroughly washing the solid phase with physiological saline, 0.08 μg of human TGF monoclonal antibody was added.
was inserted into a small test tube (described above) containing 0.4 ml of immunoreaction buffer containing
Create a TGF monoclonal antibody conjugate,
After thoroughly washing this solid phase with physiological saline, it was immersed in an immunoreaction buffer and stored at 5°C.
It was used as a solid phase of a TGF antibody immobilized carrier. (2) Measurement method: Add immunoreaction buffer to a small test tube (described above).
100μ, standard TGF solution (1.25-1280ng/ml)
100μ of ¹²⁵ I-labeled TGF solution were added, and the mixture was reacted at 37°C for 4 hours. After the reaction was completed, 5 ml of physiological saline was added, the solid phase was removed while washing, and the solid phase was added to a new small test tube and its radioactivity was measured. The results are shown in FIG. 4, and this measurement system showed an extremely good quantitative curve. In addition, TGF in the test liquid (serum, urine, etc.)
To measure the amount, add 100μ of the test solution instead of the standard TGF solution, perform the same procedure as above, and compare with the quantitative curve to determine the TGF concentration in the test solution. In addition, an example of producing TGF is given below, but it is not limited to this in any way. good. Reference example 1 1100ml of urine from a lung cancer patient was mixed with 0.01M phosphate buffer (PH
8.0) was subjected to dialysis for 2 days and nights at 4°C using a dialysis membrane with a molecular weight of 10,000 (manufactured by Sanko Pure Chemical Industries, Ltd.).
The dialysed fluid was centrifuged at 12,000 xg for 60 minutes to remove the precipitate. SP-Sephadex (Pharmacia,
Japan Co., Ltd.) was packed into a column (capacity 4.5cm x 33cm), equilibrated with 0.01M phosphate buffer (PH8.0), the above dialysis solution was allowed to flow down, the active ingredient was adsorbed, and then the same After thorough washing with a buffer solution, elution with a tilted needle of 0.01M to 1.5M sodium chloride was performed in the same buffer solution. (Flow rate 22.5 ml/hr, temperature 4°C, sodium chloride gradient buffer 4.8 used). TGF was eluted at a sodium chloride concentration of 0.6 to 1.0 M in the same buffer, and 62.0 ml of active fraction was obtained. In the TGF activity test method described below, this active fraction caused normal cells to undergo degeneration and form colonies at 5μ. The entire amount of this active fraction was added to Diaflow Molecular Sieve Membrane PM-10 (Amicon,
Desalting and concentration were repeated using Far East, Limited (Japan) to make 1.2 ml, and trifluoroacetic acid was added to this to obtain a 0.05% trifluoroacetic acid solution. A column (capacity 4.1 mm x 250 mm) was packed with Synchropack RP-P (manufactured by Synchrome), which uses silica gel as a carrier to which alkyl groups (C18) are bonded.
After equilibration with % trifluoroacetic acid, 1/3 of the above TGF and trifluoroacetic acid solution was poured into one column and adsorbed. After washing with 0.05% trifluoroacetic acid water, a gradient elution of 0 to 90% acetonitrile in the same solution was performed to obtain acetonitrile. Take the active fraction eluted at a concentration of 55-75%. The above operation was performed three times, and the entire volume of the concentrated preparation was treated with the molecular sieve, and the TGF activity fraction was 7.9
ml was collected. After freeze-drying this fraction, 3.0 ml was added again.
This solution was subjected to the TGF test method, and at 0.4μ, normal cells were transformed and showed colony-forming ability. [Table] TGF activity test method materials (1) NRK-cells (49F strain) (2) Dulbecco's modified Eagle medium supplemented with 5% fetal bovine serum and 1% non-essential amino acids (3) Purified agar (Agar Noble, Difco) (4) Petri dish operation with a diameter of 60 mm Place 5 pieces of the medium in (1) containing 0.5% agar in one Petri dish.
Add ml each to make the lower layer medium. On top of this, 0.3% containing 1 x ¹⁰³ NRK cells/ml
Layer 2 ml of agar-containing medium on each Petri dish to form an upper layer medium, add a sample dissolved in 0.5 ml of physiological saline on top, and place in a carbon dioxide incubator.
Incubate at 37℃, remove from incubator after 14 days,
Obtain a 2 mm x 2 mm field of view on a Petri dish using a 40x Kenbi mirror.
Randomly select 10 locations, count the number of colonies consisting of 10 or more cells, and use the total number as the result.

[Brief explanation of the drawing]

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

Claims (1)

[Scope of Claims] 1. A test solution is made to react with a radioisotope-labeled transforming growth factor and a transforming growth factor antibody, and then a radioisotope-labeled transforming growth factor-transforming growth factor is reacted with a sample solution. A method for separating an actor-antibody conjugate and an unreacted radioisotope-labeled transforming growth factor, and then quantifying one of the separated radioisotope-labeled substances, wherein the transforming growth factor is positive for ninhydrin reaction, SDS−
Single in electrophoresis, stable to acids and heat, inactivated by trypsin and dithiothreitol treatment, estimated molecular weight by gel filtration
1. A method for quantifying a transforming growth factor in a test liquid, characterized by having physical and chemical properties of 28,000 to 35,000. 2. The quantitative method according to claim 1, wherein the transforming growth factor antibody is a transforming growth factor antibody immobilized on an insoluble carrier. 3. The quantitative method according to claim 1, wherein the transforming growth factor antibody is soluble and a specific antibody to the transforming growth factor antibody is used in the separation. 4. The quantitative method according to claim 1, wherein the transforming growth factor is a human transforming growth factor. 5. The quantitative method according to claim 1, wherein the transforming growth factor antibody is a monoclonal antibody. 6. The quantitative method according to claim 1, wherein the radioactive isotope is ¹²⁵ I (and ¹³¹ I).