JPH0321560B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to substances related to Recognins, and in particular to substances related to Recognins, which are novel acidic peptides useful in the diagnosis and treatment of malignant tumors or cancer. Recognins are obtained by treating normal or abnormal cells or tissues (e.g. tumor cells, artificial cancer cells) and are supported on a support or without being supported with body fluids. Chemoreciprocals, ie, antibodies, can be produced by contacting them. These chemoreciprocals can be tested in vivo or in vitro (e.g., quantitative precipitation tests, Ouchterlony antigen-antibody gel diffusion tests in agar,
It is a substance that reacts with recognins with immunochemical specificity (immunofluorescence test). Recognins are substances with the following properties: (a) pK
It is an acidic peptide collected from a fraction with a value of about 1 to 4; (b) Forms a single linear precipitate with specific antibodies in quantitative precipitation tests and antigen-antibody gel diffusion tests in Ukhtalony agar. (c) Soluble in water and aqueous solutions at acidic or neutral pH; (d) Slightly soluble in water and aqueous solutions at alkaline pH; (e) Has a spectral absorption peak wavelength of 280 mμ; (f) It has a molecular weight of about 3000-25000; (g) It has a high content of glutamic acid and aspartic acid, and a high ratio of glutamic acid and aspartic acid to histidine. Recognins specifically include various acidic peptides having the above-mentioned properties, the representative ones being astrocytin and malignin. Astrocytin: - Astrocytin is obtained from brain tumor tissue, preferably brain glioma tissue. First, protein components including astrocytin precursors are extracted from the tissue,
A preferred extraction method in this case involves treating the tissue with a neutral buffer under homogenization or other suitable destructive conditions to solubilize the protein fraction containing the astrocytin precursors. At this point, the astrocytin precursor is still bound to large molecular weight substances such as proteins, glycoproteins, lipoproteins, nucleic acids, and nuclear proteins. The solubilized extract is clarified to remove insoluble particles and low molecular weight impurities are removed by evaporative concentration techniques. The remaining solution is processed to separate the astrocytin precursor from other impurities to obtain a protein fraction with a pk value of about 1-4. In this case, the treatment is carried out, for example, by pouring the solution into a chromatography column and eluting it with a solvent whose acidity is increased stepwise. Neutral or acidic fractions with pk values up to about 4 are discarded, fractions with pk values of about 1 to 4 are collected, and the collected fractions are processed to yield a material with a molecular weight of about 8000. In this case, the treatment includes, for example, filtering the fraction to remove low molecular weight substances, that is, substances with a molecular weight of about 1000 or less,
This is then carried out by filtering again to remove substances with a molecular weight of about 25,000 or more. Molecular weight approx.
The 1000-25000 fractions are processed, for example, by thin layer gel chromatography to obtain astrocytin. In this way, astrocytin is extracted from brain glioma tissue using a neutral buffer, followed by repeated homogenization and high-speed centrifugation, and a fraction with a pk value of approximately 1 to 4 is separated from the resulting extract. It can be obtained by collecting high molecular weight substances of up to about 230,000 from this separated fraction and isolating substances with a molecular weight of about 8,000 from them. Astrocytin obtained by the above method forms a single line precipitate with its specific antibody in a quantitative precipitation test or an Uftaloni gel nucleic acid test, and is soluble in water or an aqueous solution with an acidic or neutral pH,
It is characterized by being sparingly soluble at alkaline pH, with an absorption peak wavelength of approximately 280 mμ as measured by a spectrophotometer, and a molecular weight of approximately 8,000. Astrocytin is also characterized by a very high proportion of glutamic acid and aspartic acid residues, and a very high proportion of these acids to histidine. Malignin:- Malignin is an artificial cancer cell, i.e.
It can be obtained from cancer cells cultured in vitro and approximately
It has a molecular weight of 10,000 and an amino acid residue composition similar to, but different from, astrocytin. That is, the ratio of glutamic acid and aspartic acid is high, and the ratio of these acids to histidine is high. Malignin is extracted from artificial cancer cells grown in a fermentation medium by repeating homogenization and high-speed centrifugation with a neutral buffer, and a fraction with a pk value of approximately 1 to 4 is separated from this extract. It can be obtained by collecting a high molecular weight substance of about 230,000 or less from the fraction and isolating a substance with a molecular weight of about 10,000 from it. The amount of malignin produced in the artificial cell culture and the percentage of total protein produced in the artificial cell culture is increased by carrying out the artificial cancer cell culture in a large volume incubator. The malignin obtained by this method forms a single line precipitate with its specific antibody in quantitative precipitation tests and Uftalony gel diffusion tests, is soluble in water or aqueous solutions at acidic or neutral PH, and is sparingly soluble at alkaline PH. It is characterized by having an absorption peak wavelength of about 280 mμ as measured by a spectrophotometer and a molecular weight of about 10,000. In addition, recognins can be complexed with an appropriate carrier, such as bromoacetylcellulose, to form bromoacetylcellulose-recognin, and when injected into mammals, anti-recognin, which is a specific antibody, can be produced. , the anti-recognin specifically attaches to the recognin precursor.
For example, one antirecognin, antimalignin, is toxic to brain tumor cells in vitro. Recognins such as astrocytin and malignin are useful as substances that can be introduced into biological systems to suppress external reactions; for example, a certain substance can be coated with a certain type of recognin. Another example is the introduction of recognin to generate chemoreciprocals or antibodies in biological systems. Recognins may also be used to nutritionally promote the growth of specific biological systems. Other uses of recogni include the production of target reagents consisting of recognin and carrier complexes to facilitate application in biological systems. This recognin complex has, for example, the physical and chemical properties of recognin itself. The carrier should be selected from one that is capable of forming a complex with recognin and is substantially inert to biological systems. Any known substance that forms a stable complex with a polypeptide or protein can be used as a carrier to be complexed with recognin. One example is cellulosic materials such as bromoacetylcellulose. In addition to being inert in biological systems, the carrier should not alter the unique physical and chemical properties of recognin useful for the purposes described herein. Complexes consisting of recognin and its carrier are useful for producing, isolating, and identifying chemoreciprocals or antibodies in biological systems with which they are contacted. The recognin-carrier complexes are also useful in stimulating the production of antibody precursors in biological systems into which they are introduced. Antirecognins, such as antiastrocytin and antimalignin, belong to the chemoreciprocal class. These substances are produced by injecting recognin into biological systems. An immunologically effective amount of Recognin is contacted with body tissues or fluids to elicit an antibody response according to conventional antibody production techniques. Antirecognis can be used to deliver substances such as diagnostics, nutrients, and therapeutics to specific cells or sites in biological systems, where the substances are present in complex form with antirecognins in the biological system. introduced into the system. Anti-recognins are also useful in diagnosing the presence of tumor cells in living tissues, in which case anti-recognins conjugated with labeled substances such as dyes or radioactive substances are applied to living tissues. do. Yet another use of anti-recognins is to inject immunologically effective amounts of recognins into mammals or other biological systems.
The goal is to increase the production of other useful chemoreciprocals. Traditionally, glycoprotein complexes are produced from brain tissue;
Antibodies against it had been formed. That is,
When rabbits are injected with an isolated substance known as 10B glycoprotein made from the diseased brain of Tay-Sachs, antibodies are produced. This Tysahas antibody is used for immunofluorescence studies of tumor-bearing brains. However, only reactive normal non-tumor glia, not tumor glia, are colored by this antibody. In contrast, when astrocytin is produced from tumor tissue and antibodies against astrocytin (anti-astrocytin) are produced and used in immunofluorescent studies of the brain, normal (non-tumor)
Only tumor glia, not neuroglia, are colored by anti-astrocytin. Therefore, it is clear that the anti-Tysahus antibody and anti-astrocytin are different substances not only in the tissue of origin and antibody characteristics, but also in the specific cell type of coloration. Other chemoreciprocals include targeting reagents complexed with chemoreciprocals.
For example, a targeting reagent as a product of astrocytin complexed with a carrier such as bromoacetylcellulose is used to contact the anti-astrocytin. This type of material can be complexed and used to deliver diagnostic, nutritional, and therapeutic agents to specific cells or locations in biological systems.
These compounds can also be used for purification treatments. For example, anti-astrocytin can be produced by hydrolyzing bromoacetylcellulose-astrocytin-anti-astrocytin with acid or proteinase enzymes. Targeting reagents also serve to increase the amount of a target substance in a biological system by bringing an immunologically effective amount of the target into contact with body tissues or fluids. Further, other chemoreciprocals include target-adding globulins. This is achieved by contacting the targeting reagent with body fluids to form a complex from which the targeting globulin is released. Two useful examples include Slow-Target-additive globulin.
Attaching-Globulin (S-TAG) and Fast-Target Additive Globulin (S-TAG)
Traget-Attaching-Globulin (F-TAG)). S-TAG is produced by reacting serum or other body fluid with a targeting reagent (e.g., bromoacetylcellulose-malignin) for about 2 hours or more at a low temperature, e.g., about 4°C, and cleaving the S-TAG from the product, e.g. It is obtained by cleavage with dilute acid for about 2 hours at a temperature of .degree. The product S-TAG produced according to this method is soluble in aqueous buffers, forms a single precipitate with the corresponding recognin in an antigen-antibody gel diffusion test in agar, and is not dialyzable on cellophane membranes. ,about
Retained by a Millipore filter that retains molecules with a molecular weight of 25,000 or more, the macroglobulin range is approximately
It is characterized by having a differentially solidifying molecular weight as determined by thin layer gel chromatography of 50,000 and multiples thereof, and having a spectrophotometric absorption peak wavelength of about 280 mμ. A method for producing F-TAG is to react serum or other body fluid with a targeting reagent (e.g., bromoacetylcellulose-malignin) for about 10 minutes at a low temperature, e.g., 4°C, and cleave F-TAG from the product, e.g. It consists of cleavage with dilute acid for about 2 hours at a temperature of °C. The product F-TAG produced according to this method is soluble in aqueous buffers;
It produces a single precipitate with its equivalent recognin in the antigen-antibody gel diffusion test in agar, is not dialyzable on cellophane membranes, and is retained on Millipore filters that retain molecules with a molecular weight of about 25,000 or more, and from the macroglobulin range to about 50,000. and multiples thereof, and are characterized by having different solidifying molecular weights as determined by thin layer gel chromatography, and having a spectrophotometric absorption peak wavelength of about 280 mμ. TAG products determine the concentration of S-TAG and F-TAG produced by known amounts of mammalian serum and other body fluids and correlate these concentrations with values established as indicative of cancer. Therefore, it is useful for detecting cancerous tumors in living mammals.
TAG products are also useful in diagnosing the presence of tumor cells at a tissue site, which consists of applying TAG conjugated to a labeled substance, such as a dye or radioactive substance, to the site, thereby identifying the tumor. Coloring or radioactive labeling occurs only in cells. Additionally, TAG products were found to be cytotoxic to tumor cells. TAG products also provide diagnostic,
Introducing nutritional and therapeutic agents complexed with TAG products is useful in delivering these agents to specific cells or sites. When a cell completely occupies a particular location, normal cell division in plants or animals is restricted or inhibited. (a) The mechanism by which a normal cell "recognizes" that it has filled a space it could occupy, and (b) the mechanism by which this recognition mechanism in turn inhibits cell division, are both unknown. When normal recognition and sensing occur, the concentration of the precursor increases;
A group of compounds that are involved in the recognition and sensing of particles and cells that connect cells to each other have begun to be produced. These compounds are called recognins. Attempts to produce these compounds from normal cancer cells revealed that they did not exist as such, and
Cancer cells lose (a) the ability to recognize that they have occupied their normal volume, and/or (b) the ability to stop dividing when they have occupied their normal volume, while at the same time changing their molecular structure. I found out what happened. These new compounds are named recognins. Recognins are novel compounds with physicochemical properties that mimic the morphological characteristics of cancer cells in that they are unable to recognize and stop cell division. The use of recognins goes beyond determining the mechanism of action of cancer. This is because it provides direct products and methods useful in the diagnosis and treatment of cancer, as well as its prevention. A method has been discovered in which artificially cultured cells can be used to produce malignins.
One of the advantages of the method disclosed herein is that malignins and new products made therefrom can be efficiently produced in virtually unlimited amounts. The present invention has advantages in the field of cancer research and is readily applicable to any biological system in which one wishes to influence growth and metabolism. Thus, by producing a specific compound or a compound of an appropriate cell type artificially cultured,
Furthermore, by further manufacturing products from these substances, it is possible to initially exert a specific influence on tissues, cells, organelles, sub-organelle molecules or molecular assemblies in biological systems. thus,
Specific nutritional efficacy during critical periods of development, specific diagnostic, prophylactic, and therapeutic methods, and configuration of engineered bioelectrical systems (such as in tissue or organ transplants) are all initially affected. be able to. These artificial bioelectrical systems can now be made to have the properties of specific recognins, malignins or their chemoresiprocals of the normal tissues or compositions adjacent to them, so that they are no longer 'recognized' as 'foreign bodies'. , thus avoiding reactions to foreign objects that include rejection. Another aspect of the invention is to produce a specific antibody-like product (anti-astrocytin) of high value against a specific brain product (astrocytin), which can be used to treat the nervous system of any species. It can be used as a specific complex at a specific point and as a specific delivery carrier for this. Malignins and astrocytins are recognins. Yet another aspect of the invention is the production of two new products TAG from biological fluids. This involves two reactions: the first reacts the biological fluid with a synthetic complex containing a malignin-like physicochemical form, called the target, and the second cleaves a specific TAG from the complex. By measuring the TAG thus formed, a quantitative indication of whether or not a tumor is present in the living body can be obtained from the biological fluid of the living body, allowing a diagnostic test for the tumor. TAG products and antimalignins are physicochemically complementary to malignin, so they are named chemoreciprocals. Furthermore, the two can be clearly distinguished quantitatively and qualitatively by the time allowed for the reaction of the serum with the specific target reagent used and by the time allowed for cleavage of the complexed product. It was discovered that TAG products could be manufactured. After testing the quantities of these products that could be produced from a large number of different people with brain tumors and various other medical disorders, as well as those without any obvious signs of disease, for certain individuals. It became clear that the amount of these two new products that could be produced was an indication of whether or not an individual had a brain tumor. Therefore, a new serologic diagnostic test for brain tumors has been discovered. In addition to the use of these new products in diagnosing brain and other tumors from serum and other biological fluids, TAG and Recognin compounds can be used to diagnose brain tumors and surrounding tissues removed during brain tumor surgery. This is explained by the evidence that it preferentially attaches to glial tumor cells at the biological site. of this tumor cell
Preferential labeling with TAG and anti-recognins is demonstrated by standard immunofluorescence techniques. Thus, the new method can also be used to determine with certainty by histological examination whether tumor cells have penetrated to the extremes of the removed tissue and the tumor is This may indicate that the tumor cells may still be present in the brain or other organs, or may have disappeared from the area around the removed tissue, indicating that all of the tumor may have been removed from the brain or other organs. can. In addition, prepared as described
TAG and antimalignins were found to be cytotoxic to glioma brain tumor cells generated in tissue culture in vitro. This high affinity for tumor cells in other media, here generated in tissue culture, further demonstrates the specific binding potential of the new product TAG, with respect to the synthetic product target and histological location. Regarding tumor cells in
As the property of TAG indicates, TARGET−
This explains the adoption of the name ATTACHING-GLOBULINS (TAG). Furthermore, the cytotoxicity of TAG and anti-recognins to tumor cells provides a new diagnostic test for serum from patients suspected of having a tumor. Thus, for example, serum or other body fluids from these patients can be reacted with a target to produce TAG, and the product TAG can be tested for cytotoxicity on tumor cell growth in tissue culture. The concentration and degree of cytotoxicity of TAG expressed by TAG, which can be produced from the serum of a given individual, is not only of diagnostic benefit, but also can be used to determine the course of the disease before and after surgery for a particular patient. It is also worth exploring. Combining radioactive and dye detectors with TAG can create novel TAG products that are useful in vivo in diagnosing tumors and determining their precise location. Thus, injection of suitably labeled TAG into the cerebrospinal fluid, either intraarterially or intravenously, or directly into the brain tissue or its fossa, can be performed by radioactive means or by visualization of bound dyes. , which can be explained by the presence of a brain tumor. This is because TAG specifically attaches only to tumor cells.
Furthermore, this method can accurately visualize the location of brain tumors. This can be seen as an improvement on this in-vivo diagnostic method that uses anti-astrocytin made from rabbit blood to label brain tumors. This is because using TAG made from human serum avoids the possibility of foreign protein reactions.
TAGs and anti-recognins have chemical properties that allow them to preferentially attach to astrocytin precursors, including tumor cells both in vitro and in vivo, such as with radioactive, proton scavenging agents or other In combination with toxic physical or chemical agents, these toxic substances preferentially localize due to the specificity that they add to tumor cells compared to the normal cells adjacent to them. These products can be used therapeutically as well as diagnostically. This selectivity is universally recognized as at least one decisive factor in achieving effective chemical or physical treatment of tumors, and as a hitherto unattainable factor. It will be done. Thus, TAG has been shown to be effective in preferentially attaching to tumor cells, and for these reasons should hold promise as a novel therapeutic product. In the serum of patients with malignant tumors, FAST-TAG (FAST-TAG)
- TAG), i.e.
SLOW-TAG (S-TAG) can be produced in relatively larger amounts from a given amount of serum than in patients without such tumors. This means that
Suggestions are that the concentration of one of the naturally occurring precursors of TAG (P-TAG) is increased or that there are other factors that may cause S-TAG to perform relatively better in vitro than F-TAG. It is something to do. The action of postulated but unproven cellular ``antigens'' and circulating ``antibodies'' on actual synthetic product targets and their precursors of TAG and, in turn, on those that may be present in vivo. Possible functional relationships between the two have not yet been elucidated. Thus, for example, in an antibody-like manner, F-TAG and S-TAG produce a single, separate family of reactions with astrocytin in antigen-antibody gel diffusion in agar. So, if you inject a target into a rabbit, after it reacts with the target,
This will increase the yield of TAG products made from rabbit serum. The discovery that there may be normal levels of circulating antibody-like precursors for cellular antigens hidden in nondividing cells raises questions about the possible effects of the pair. Here, we propose that TAG precursors (P-TAG) and target substances that act to control cell proliferation and cell death exist in living organisms. For example,
Exposure of cellular components not normally exposed directly to serum proteins may occur during cell division. As a result of this exposure of cellular constituents, the constituents become converted into target-like substances, to which the addition of P-TAG-like molecules from the serum occurs, which is thought to stimulate or inhibit cell division. It will be done. Alternatively, injured or malfunctioning non-dividing cells may expose target substances to which the addition of P-TAG-like molecules is reversible. However, under certain cellular conditions, the addition of P-TAG-like molecules can lead to cell destruction (e.g., ANTIGLIOMA-
TAG is markedly cytotoxic to glial tumor cells growing in tissue culture). As such, it may be said to model the normal mechanisms for the control of cell division and for the repair or removal of individual cells within the body throughout the life of the organism. When the exposure of cellular components is abnormally increased and abnormally large amounts of cellular targeting material are formed, as occurs in rapidly dividing cancer cells such as in brain gliomas,
It may occur that the concentration of one type of serum P-TAG is increased relative to the other. Whatever the actual action of the precursor, one major type of TAG, SLOW, can be produced in vitro from the serum of patients with malignant tumors by the method described here. -TAG(S-
TAG) is the basis for the serological diagnostic tests described in the Examples below. Example 1 Generation of crude astrocytin precursor-containing fractions: - Human glioma swollen tissue is excised and surface blood vessels and normal brain tissue are separated as much as possible. Take a representative amount of 11 g of isolated swollen tissue and
Divide into 6 1.5g portions and 2 1.0g portions. Each part is then processed as follows. Each portion is homogenized in a neutral buffer solution by ultrasound or other mechanical means. For example, mix each portion with 0.005M phosphate buffer (PH7) in a warning mixer.
Homogenize to 100 c.c./g tissue. To prevent protein denaturation, the homogenization process should be performed under cold conditions. For example, the mixer should be pre-chilled in a cold room at 0-5°C and operated for about 3 minutes. Then, in order to clarify the homogeneous material, it is centrifuged using, for example, frozen ultracentrifugation at 80,000 times gravity for 30 minutes. The soluble supernatant is decanted and kept cold. The insoluble residue is homogenized again using an additional 100 ml of neutral buffer, centrifuged as above, and the resulting second soluble extract is combined with the first. In the supernatant, protein is 50%
The yield is very good when homogenization and centrifugation are repeated until below micrograms/ml solution. This is accomplished in 5 extractions for most tissues. The solutions thus obtained are combined and concentrated by thorough evaporation in subsequent dialysis. Cool down,
Obtain 15 ml of solution by dialysis against 0.006 M phosphate buffer. The volume of this solution is recorded, a portion is used for total protein analysis, and the remainder is separated to obtain a protein fraction with pK1-4. Chromatography, described below, is the preferred fractionation method. The solution is fractionated on a 2.5 x 11.0 cm DEAE cellulose (Cellex-D) column equilibrated with 0.005M sodium phosphate buffer in a cold room (4°C).
The elution solvent is changed stepwise by using the following solvents (solutions): Solution (1) Dissolve 4.04 g of NaH ₂ PO ₄ and 6.50 g of Na ₂ HPO ₄ in 15000 ml of distilled water (0.005 mol, PH 7); (2) Dissolve 8.57g of NaH ₂ PO ₄ in 2480ml of distilled water; Solution (3) 17.1g of NaH ₂ PO ₄
Dissolve in 2480 ml of distilled water (0.05 mol, PH4.7);
Solution (4) Dissolve 59.65 g of NaH ₂ PO ₄ in 2470 ml of distilled water (0.175 mol); Solution (5) Dissolve 101.6 g of NaH ₂ PO ₄ in 2455 ml of distilled water (0.3 mol, PH4.3); Solution ( 6)
Dissolve 340.1 g of NaH ₂ PO ₄ in 2465 ml of distilled water (1.0 mol, PH 4.1); Solution (7) 80% phosphoric acid (H ₃ PO ₄ )
Add 283.64g to 2460ml of distilled water (1.0mol, PH
1.0) Add 6-10ml of nerve tissue extract. Pass this through the column. Then add solution (1) on top and add solution (1) 300
Attach a reservoir to receive the ml and let it fall into the column by gravity. Eluate 3 using an automatic fraction collection device
Collect ml. Then, use a different elution solution with the next elution tube number. Solution (2): With tube 88, carry the solution on the column to the top of the resin, then add 50 ml of solution (2) from above and install the reservoir: With tube 98, carry the solution on the column to the top of the resin, and then add 50 ml of solution (2) from above and attach the reservoir. , solution (3) 75ml
Add from above and attach the reservoir; Solution (4): Tube
At 114, the solution is carried over the column to the top of the resin;
Then add 150 ml of solution (4) from above and attach the reservoir; Solution (5): Using tube 155, bring the solution onto the column and onto the top of the resin, then add 125 ml of solution (5) from above, Install the reservoir; Solution (6): Tube 187
Then, bring the solution onto the column and onto the top of the resin, then add 175 ml of solution (7) from above and attach the reservoir; continue elution until reaching tube 260 to complete the elution. Use freshly prepared resin for each volume of tissue extract. Each tube is subjected to quantitative protein analysis. Combine the eluate in tubes 212-230. This contains the crude product from which astrocytin is obtained. In the past, data on a crude product called fraction 10B has been made public [Protein Metabolism of the Nervous].
System pp555-69 (Pleum Press, 1970); Journal of Neurosurgery, Vol.
33, pp. 281-286 (September 1970)). Here we fractionate a specific product called astrocytin 10B
achieved separation from Crude fraction 10B is prepared as 0.1-10 mg of product per gram of initial fresh nervous system tissue. It contains, in addition to the astrocytin precursor, a large number of hexoses, namely glucose, galactose, mannose; hexose amines, including glucose amine, galactose amine and mannose amine; and occasionally other sugars, fucose, ribose and perhaps lanose. They contain varying amounts of covalently bonded hydrocarbon residues, including. It also contains high molecular weight protein products, some lipids and nucleic acids. Example 2 Production of purified astrocytin from crude astrocytin precursor-containing fractions: - The astrocytin precursor-containing fraction is further isolated from contaminants. As a preferred specific example, Example 1
The material obtained was transferred to a representative column (40 cm (length)
(diameter), 196ml capacity) using Sephadex G-
Chromatography on 50 resin. The working pressure is
40mm・Hg; flow rate is 35ml/Hr, buffer is 0.05M phosphate buffer solution (PH7.2). First flow
The flow through peak contains impurities along with the astrocytin precursor, whereas the following peaks contain only impurities. In a preferred embodiment, the product in the first flow-through peak above is then concentrated on a Sephadex G-15, and then the same solutions (1)-(7) as in Example 1 are used and Pass through a column of Selex-D using an elution step. The astrocytin product is present as a sharp peak in the same tubes (numbers 212-230) as above and is present in the SELEX-D
Chromatographically behaves without the presence of large amounts of contaminants. Low molecular weight contaminants are then removed by conventional means such as Millipore disc filtration. In a preferred method, the astrocytin product is purified by Millipore
Pellicon Disc No.1000, 13mm (this one is
Capturing substances with a molecular weight greater than 1000,
Permeates substances with a molecular weight less than 1000. ) to separate salts and other small molecular weight contaminants. Astrocytin products remain on the pericon disc;
It is recovered by washing with solution (1) of Example 1. Astrocytin is then obtained by isolating a compound with a molecular weight of about 8000 from the solution. As the method, it is preferable to use a thin layer gel (TLG) chromatograph as described below. As a device,
Botsflingen Mannheim GmbH; Pharmacia Huain Chemicals &
A commercially available version designed by CAMAG (Switzerland) is used. 2.5g of Sephadex G-200 resin (of the highest quality)
Prepare phosphate buffer PH6.8 (6.6-7.0 ₎ of _0.5M NaCl in _{0.02MNa2HPO4KH2PO4} in _85ml . Swell at room temperature for 2 to 3 days with gentle mixing from time to time. (Magnetic and other stirring means should not be used.) The swollen gel is stabilized at freezing temperatures for 3 weeks. However, bacterial and fungal growth inhibits the swelling gel. If you want the gel to hold for a longer time, a small amount of bacteriostatic agent (sodium azide 0.02%) should be added. Using 2.5 g of dry gel, make two glass plates 20 x 20 cm and 0.5 mm thick. The plates can be dried for 10 minutes at room temperature, stored in a humid chamber for about two weeks, or used immediately after appropriate pre-equilibration. (usually,
It takes at least 12 hours at night. ). The main function of equilibrium is to normalize the ratio between the stationary and mobile layers. With the plate pre-equilibrated in horizontal position, the substance to be measured is applied with a micropipette as a spot or streak at the starting line. 10-20 ml of the 0.2-2% protein solution is placed on the edge of a microscope cover slide (18 x 18 mm) against the gel surface. After a few seconds, the solution soaks into the gel. All samples were first placed on a cover slide;
Then adapt quickly. If not enough material is used, it is difficult to locate individual spots after separation. If too many substances are applied, clear separation cannot be achieved. Dilute the sample with buffer for ease of manipulation;
Sample separation is performed using a technique in which the plate is tilted at an angle of 22°. A flow rate of 1-2 cm/hour is very suitable. Labeling substances (cytochrome C, hemoglobin, myoglobin or albumin classified and identified by bromophenol blue) are applied at different positions across the plate and reference proteins are used to calculate the relative distance (mobility) of unknowns. function as. After applying the sample, the plate is replaced in the apparatus with the paper core slightly downward to provide good contact with the gel layer. The paper core must not be immersed. Wipe off excess moisture. The liquid solvent in the reservoir is kept constant at 1 cm from the top of the container. The operation is usually completed in 4 to 7 hours depending on the progress of the separation. Separates colored substances directly. Separated protein spots were collected after completing the chromatographic separation by transferring to a paper sheet replica of the TAG plate and pre-washed with methanol + H ₂ O + acetic acid.
90:5:5 top color becomes easily visible by staining for 48 hours. Paper sheet is 3mm,
It's paper. A sheet of 20 x 18 cm paper is placed on top of the gel layer and rolled to ensure proper and sufficient contact with the gel. so that air is not trapped under the paper (replica), and
Be careful not to disturb the gel layer. The liquid layer is penetrated through the gel layer with paper, removed after about 1 minute, immediately dried in an oven at 60°C for 15 minutes, and dyed using a conventional dyeing method. Staining in 10% sodium carbonate
0.03% diazotized sulfanilic acid (Powley reagent)
This is achieved by spraying the replica paper with Staining can also be achieved using a saturated solution of amide black in methanol-acetic acid (using 90:10 v/v): staining time is 5-10 minutes. To decolorize, mix 2 volumes of methanol and acetic acid (90%) mixed with 1 volume of water.
10) Wash. It is difficult to dye the background low without extensive washing. If only astrocytin is to be stained, the plate itself may be dried at about 60° C. (in an oven with air circulation). For isolation, plates are air-dried at room temperature. Overheating will cause cracks, which can be avoided by using 50-60°C, a temperature that normally dries Sephadex G-200 plates in 15-30 minutes. Dry plates in a mixture of methanol + H ₂ O + acetic acid (75:20:5).
Allow to swell for 10 minutes, dye in saturated amino black in the same solvent system for 5 hours, then wash by soaking in the same solvent for 2 hours before drying.
To determine the molecular weight, measure the distance from the starting line to the center of each band with an accuracy of 0.05 mm on the direct print (replica) or on the densitogram. The measurement results are expressed as the migration distance (dm) of cytochrome C or myoglobin (used as a control protein) relative to the migration distance (dp) of the test protein.
The relationship between the distance traveled by the standard material relative to the test material is expressed by the _formula :
(−R _n =dp/dm). A linear calibration line is obtained by plotting the logarithm of the molecular weight of the standard used against R _n . The molecular weight of the unknown protein can be obtained from this straight line. For very accurate results, before applying to the plate,
Equal parts of the protein sample solution can be mixed with a standard, in this case cytochrome C. the above
By the TLG method, astrocytin products are observed as discrete spots at a distance of about 0.83 +/- 0.02 compared to standard cytochrome C, giving a molecular weight of about 8000 for astrocytin. This method separates several individual products from astrocytin based on slight differences in molecular weight.
Thus, three products with molecular weights of about 64,000, 148,000 and 230,000 and often a 32,000 molecular weight product are found which are carried as contaminants to this point and are removed in the TLG process described above. The astrocytin product is absorbed into the gel in dry form,
Dissolve in solution (1) and separate the resin by centrifugation or other similar means. The astrocytin product produced at this stage is soluble in distilled water, soluble in neutral, acidic pH, and alkaline.
Insoluble at PH and spectrophotometer absorption peak wavelength
It has 280mμ. This is a polypeptide with a molecular weight of approximately 8000 as described above. The covalently bound amino acids are shown to have the following average composition of amino acids by hydrolysis with 6NHCl followed by automated quantitative measurements. Number of residues (approx.) Aspartic acid 9 Threonine 5 Serine 6 Glutamic acid 13 Proline 4 Glycine 6 Alanine 9 Valine 4 1/2 Cysteine 2 Methionine 1 Isoleucine 2 Leucine 8 Tyrosine 2 Phenylalanine 3 Lysine 8 Histidine 2 Arginine 4 total 88 Cysteic acid , hydroxyproline, norleucine, ammonia, isodesmosine, desmosine, hydroxylysine, lysinonorleucine, and gamma-aminobutyric acid are not present in detectable amounts, and glucosamine is present in trace amounts. From 11 g of the starting brain tumor tissue in Example 1, about 3 mg of purified astrocytin is obtained by the above method. Example 2A REELER Recognin Production: - Reeler's disease is a genetic disease in which animals are unable to perform stable and coordinated motor muscle activity, and at certain deployment times certain neurons At a certain location, it is unable to move to the cerebellum and exhibits a reeling state. Electron microscopy studies have shown that certain glial cells provide vertical rod-like axes;
It has been shown that new cells migrate to new positions under normal conditions. In Riehler's disease, the inability of new neurons to climb up these glial fibers is thought to be due to some disorder in the neurons or glia, or both. Recognin from mouse reeler brains is obtained according to the methods of Examples 1 and 2 and compared with recognin made from normal mouse brains. The molecular weight of Recognin made from the entire brain of a normal mouse is 8000.
It is. The molecular weight of “Reeler” Recognin is 3600.
~5000. "Reeler" ligognin is unusual as indicated by its very small molecular weight. Furthermore, the amount of recognin that can be produced from the cerebellum of reeler mouse brains is much lower than that of normal mice. This is shown in the table.

[Table] The table shows that there is a significant decrease in recognin concentration in the cerebellum of reeler mice, with the same amount or more recognin in other parts of the brain. Either recognin did not move from other parts of the brain to the cerebellum, or it increased slightly elsewhere in the brains of reeler mice, reflecting some kind of compensatory behavior. Pathological recognin in the brains of reeler mice confirms the role of recognin in cognition and shows that the migrating brain cells have to achieve the proper location for recognition. It is related to the inability to have physical contact. In a similar manner, antirecognins directed against recognins produced from the brains of mouse reelers may be produced and used in mice in a similar manner to the use of antiastrocytin and antimalignin, as described herein. Example 3 Production of Malignin Precursor in Artificial Cancer Cell Media Fermentation: - In general, sterilization procedures are carefully carried out. All solutions (e.g., Hank's balanced salts (BSS), F-10 nutrient medium, fetal calf serum, trypsin solution) were stored at approximately 35°C in a water bath before use.
Incubate for 20 minutes or longer. Cells are separated from tumor tissue and grown in vitro for multiple generations by using an appropriate medium as described below. A beaker is used that has been previously cleaned with a sterile solution, such as 12-propanol and Amfil or Creolin solution. In a preferred embodiment, engineered cancer cells (i.e., cells grown in vitro for multiple generations)
Grow in 250ml flasks. Place the liquid medium in which the cells will grow into a pre-cleaned beaker. The cells are then gently washed for approximately 30 seconds using Hank's BSS or other similar solution. Avoid stirring.
Clean all walls and surfaces. The solution is cleared from cells by centrifugation at 3000 rpm for approximately 10 minutes in the cold. Pour the medium into a beaker as above.
Add a small amount of buffered proteinase enzyme solution and wash quickly to avoid digestion of cells. A preferred method is to add 1-2 ml of trypsin solution (EDTA) and wash for only 10 seconds. Drain the trypsin solution. Add the same volume of fresh trypsin solution and incubate until the cells are seen to detach from the chamber wall by microscopic observation. This usually takes 5-10 minutes. 50% solution of fetal calf serum in 100 ml of a suitable growth medium, e.g. F-10 nutrient medium.
Add ml. Transfer 25 ml of fresh medium with cells to a new growth chamber and propagate. Place both chambers in an incubator at 35°C for approximately 7 days. According to the previous procedures in this example, the engineered cancer culture cells are divided into two fresh media approximately every 7 days. This entire procedure is repeated for each growth chamber as often as desired at approximately 7 day intervals. In this way, the number of viable cells in vitro doubles approximately every 7 days. After about 7 days of growth, cells are extracted for making malignin. For example, the proliferating cells in each of the 250 ml proliferation chambers described above are regenerated as follows. Transfer the culture medium to a centrifuge tube and centrifuge at 3000 rpm under cooling.
Centrifuge for 10 minutes. Discard the medium. Cells remaining in the growth chamber are detached from the chamber walls and flushed into the centrifuge with a neutral buffer solution. Wash the cells twice with neutral buffer solution, centrifuge again at 3000 rpm in the cold, and discard the medium. Suspend the washed cells in 10 ml of neutral phosphate buffer until ready for extraction of the crude malignin precursor-containing fraction. Example 4 Generation of a crude malignin precursor-containing fraction: - Washed cells, prepared in Example 3, suspended in a neutral buffer, are mechanically processed under conditions that avoid denaturation of most proteins. Smash. As a preferred method,
Treat the washed cells with ultrasound for 20 seconds in the cold.
After sonication, the cell debris is centrifuged for 30 min at 30,000 rpm and the supernatant is decanted. buffer solution 10
Wash remaining cell debris using a ml portion. Sonication and centrifugation are performed as described above, and the supernatants are combined. Repeat this operation again. Combine the supernatant liquid and evaporate thoroughly to reduce the volume to approximately 30ml.
Reduce the volume to about 6-7 ml. The specimen is used for total protein analysis and the remainder is fractionated according to the method described in Example 1 above for astrocytin precursors. Example 5 Production of a purified malignin product from a crude malignin-containing fraction: - The malignin product is further isolated from contaminants by the method of Example 2 for astrocytin. In the TLG step of the preferred embodiment, the malignin product is approximately 0.91 +/- compared to standard cytochrome C.
It is observed as individual spots at a distance of −0.02, giving malignin a molecular weight of approximately 10,000. The malignin product produced at this stage is soluble in distilled water, soluble at neutral or acidic PH, insoluble at alkaline PH, and exhibits absorption peaks on a spectrophotometer.
It has 280mμ. This is a polypipetide with a molecular weight of approximately 10,000. The table shows the molecular weight of malignin produced in culture media that has been stabilized in successive generations of media, as shown by thin layer gel chromatography measurements. The reproducibility of molecular weight measurements is remarkable in view of the inherent limitations of TLG chromatography.

[Table] The covalently bound amino acids of malignin are hydrolyzed with 6NHCl and then shown by quantitative measurements to have the following average composition of amino acids: Number of residues (approximately) Aspartic acid 9 Threonine 5 Serine 5 Glutamic acid 13 Proline 4 Glycine 6 Alanine 9 Valine 6 1/2 Cysteine 1 Methionine 2 Isoleucine 4 Leucine 8 Tyrosine 3 Phenylalanine 3 Lysine 6 Histidine 2 Arginine 5 Total 89 amino acids Cysteine, hydroxyproline, norleucine, ammonia, isodesmosine, desmosine, hydroxylysine, lysinonorleucine and gamma-aminobutyric acid are not present in detectable amounts. The yield of pure malignin from the 12 250 ml reaction chambers of Example 3 was approximately 1 mg mignin.
It is. The unique structure of malignin and astrocytin is
These compositions are ascertained by exhaustive computer searches comparing them to substantially known protein substances. Amino acid compounds of malignin and astrocytin, the absolute and relative amounts of each amino acid component in terms of the total number of amino acid residues per mole, and the absolute and relative amounts of each amino acid component in terms of the molecular weight of the molecule. were subjected to matrix computer analysis against the most extensive literature on protein organization in the world, that of the National Biomedical Research Foundation (Washington DC). In matrix analysis comparing hundreds or thousands of proteins and protein fragments, no organization is found that is identical or even very similar to that of astrocytin or malignin. In any case, the structurally related proteins, their individual amino compounds and molecular weights are shown in the table for comparison. They programmed a computer to determine each degree of structural similarity among countless possibilities. In this way, for example, larger or smaller molecular weight proteins, or 85
Proteins with less than or more than 95 residues, or proteins with less than 12 or more than 15 glutamic acid residues, or proteins with less than 6 or more than 11 asparagine residues, etc. are included. does not match the amino acid of

[Table] The above fingerprint has 22 variables to match. It appears that some materials are compatible with one, four, or five variables, but none are compatible with all 22 variables. In fact, there is no good fit for more than 14 variables (8 of which show differences). Therefore, the closest match is cytochrome
b ₅ (human). As seen in the table,
Its alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, histidine, methionine, tyrosine, tryptophan residues can all be identified as significantly different from astrocytin and malignin. Cytochrome b ₅ contains seven histidine molecules, whereas astrocytin and malignin contain only two of these molecules, so it is unlikely that they have the same chemical structure. The most unusual thing about the composition of astrocytin and malignin is the high concentration of glutamic acid. One would expect to find only 5-6 residues out of 89. The next most closely matched are the ferodoxins in Leucaene glauca and alpha alpha, which differ in having 4 and 6 amino acids, respectively, and both have 96 residues, respectively.
It differs in that it has 1 and 97 pieces. Another suitable example is the E. coli 26 acyl carrier protein, which has a residue count of 11 amino acids.
It differs significantly from malignin and astrocytin in that it has only 77 residues. Other brain proteins (neuroficin and gonadotropin-releasing hormone in cows and pigs) were recorded (see table), but the number of this one was stored in the computer.
Out of 100,000 protein fragments, none of them fit very well. Although respiratory proteins can contain metal and/or heme components in their intact state, protein fragments in their isolated state, such as apocytochrome b ₅ , do not contain either metal or heme components. According to the trace analysis results of astrocytin and malignin, iron, sulfur, phosphorus,
Contains no magnesium (0.01% or less),
It has typical absorption spectral characteristics at 280 mμ. As a result of forming heme substances together with apoprotein and measuring their absorption spectra, we found that 400 and
Typical absorption was observed between 450 mμ. It is noteworthy that although astrocytin and malignin are structurally distinct from all other proteins and protein fragments, their structures are closely related to that of respiratory proteins. Yes, it's probably very important. It is well known in the art that many important relationships can be drawn both from a developmental genetic point of view and from a functional point of view in its chemical structure. One of the major problems in cancer (i.e. This study provided light for solving the problem of how to satisfy the energy relationship of greedy and rapidly proliferating malignant cells. Regardless of the theoretical importance of such a discovery,
The above data showed that the proportion of malignin increased in more malignant cells and that anti-malignin substances not only bind to malignin-like chemicals in cancer cells, but that such bound anti-malignin is also toxic to cancer cells. Both of the data shown below have important significance. Since the anti-malignin product of the present invention is selectively bound to heme compounds in cancer cells, if the malignin-like compound is a respiratory protein and therefore has a respiratory function in its system. It is easy to understand that if it does, it can kill the cancer cells. The potential for therapeutic efficacy of antimalignins and other similar chemoreciprocals is greatly enhanced by the structural knowledge of malignin and astrocytin and the already established relationship between malignin content and malignancy. Example 5A Increase in yield, degree of malignancy and proportion of malignin by increasing its volume and surface area during the fermentation process: 1000 ml flasks were used instead of 250 ml flasks, and the amounts of all reagents were increased by a factor of 3. , Example 3
The same operations as in steps 5 to 5 were performed. Space for growth of malignant cells from 250ml
Increasing to 1000 ml almost doubled the yield of malignin product after 7 days of growth from inoculation. For each flask size, the total protein yield (in mg) and the malignin produced, expressed as a percentage of the total protein of the fermented material of successive generations, are shown in the table. When using a 250ml flask, the average amount of total protein produced was 17.5mg;
When a 1000 ml flask was used, the average amount of total protein produced was 40.4 mg.

【table】

[Table] Surprisingly, by increasing the space and surface area for cell proliferation, the amount of malignant cell proliferation (degree of malignancy) increases per 7-day development period, expressed as a percentage of total protein. The amount of malignin produced also increases significantly. During the 7-day growth period, the percentage of malignin to total protein increased from an average value of 10.7% when using a 250 ml flask to 1000 ml.
The average value increased to 28.3% when using a flask. FIG. 1 shows the relationship between the proportion of malignin produced and the degree of malignancy (that is, a function of the amount of protein produced relative to the amount of malignant cell proliferation in vitro for 7 days). Figure 1 shows that as the flask size increases, the proportion of malignin product increases approximately three times. Moreover, FIG. 1 shows the relationship between malignin and the degree of malignancy. The broken line is a line indicating an ideal linear relationship. This empirical relationship is important because as the cell proliferation becomes more unstoppable (pathologically), the proportion of malignin present increases. The normal function of recognin itself is related to contacting cells and inhibiting their proliferation, as described above. If the growth of malignant cells is more pathological, the inhibitory effect of contact will decrease and malignin will increase as it becomes the more preferential protein. Example 5A illustrates that artificial cancer cell cultures grown in large growth vessels unexpectedly increase the proportion of malignin produced, ie, the percentage of malignin in total protein. . A large capacity growth container such as that used in this invention is one in which the ratio of container volume to total cell culture medium volume according to Example 3 is greater than about 8:1 (eg, the range is 7:1 to 10:1). It means that. In Example 5A, the ratio was about 8:1. Example 6 Production of target reagent from recognins:- Astrocytin produced in Example 2 or malignin produced in Example 5 is mixed with a carrier to produce a target reagent. In a preferred embodiment, astrocytin or malignin is dissolved in 0.15M sodium dihydrogen phosphate-citrate buffer (PH4.0). Bromoacetyl resins, e.g. bromine per gram of cellulose
Bromoacetyl cellulose (BAC) with 1.0-1.5 mmol is prepared in 0.15 M sodium dihydrogen phosphate buffer (PH 7.2) and stored in a cool place. PH7.2
The pH of the resulting buffer is adjusted to 4 by adding the 0.15 M sodium dihydrogen phosphate-citrate buffer (PH 4.0). A solution of astrocytin or malignin solution and BAC (10:1 ratio of BAC to recognin) is stirred at room temperature for 30 hours and then centrifuged. Preferably, all sites on the BAC used to bind to recognin are involved in binding. This is accomplished as follows. The supernatant obtained in the step just described is lyophilized and the protein content is determined to determine the amount of astrocytin or malignin not bound to BAC. Combined BAC-Astrocytin (or BAC
- malignin) was resuspended in 0.1M bicarbonate buffer (PH8.9), stirred at 4℃ for 24 hours, and BAC
and forming chemical bonds between astrocytin or malignin. After 24 hours, the suspension is centrifuged and the supernatant is analyzed for protein. composite BAC
-Astrocytin or BCA-malignin is further resuspended in 0.05M aminoethanol-0.1M bicarbonate buffer (PH8.9) to occlude unreacted bromine. This suspension is centrifuged and the supernatant is retained without analysis due to the presence of aminoethanol. This is centrifuged and the resuspension is washed three times with 0.15M sodium chloride until no absorption is observed at 266 mμ in a spectrophotometer. This BAC-astrocytin or BAC-malignin complex is stirred in 8M urea at 38°C for 2 hours, and after centrifugation, it is added to the washing solution until no absorption of 266 mμ appears (usually 3
times) and washed with 8M urea. The complex is washed twice with 0.15M sodium chloride to remove urea. This complex was stirred in 0.25M acetic acid at 37°C for 2 hours,
Prove its stability. After centrifugation, the resulting supernatant is necessarily free of absorption at 266 mμ. Therefore, this chemical complex: BAC-astrocytin or BAC-malignin is stable and can be used as a reagent in methods such as those described below. In this stable reagent type, it can be called a complex obtained by synthetic methods, whose physical and chemical properties are stable when understood as a serum component in a potentially reactive state - astrocytin or malignin. can be considered as a bonding precursor of For storage, the targeting reagent is centrifuged and washed with 0.15M sodium dihydrogen phosphate buffer (PH 7.2) until neutral. Targeting reagents can even be used from bromoacetyl carriers with ligands other than cellulose, such as bromoacetylated resins or paper. Example 7 Preparation of antisera against astrocytin, malignin and targets: - Antisera against astrocytin, malignin or target reagents are produced by inducing antibody responses against these reagents in mammals. A method satisfactory for this purpose is described below. 1 mg of recognin (astrocytin and malignin) is injected into the pad of the foot of a white male rabbit with standard Freund's adjuvant, followed by a similar injection intraperitoneally after 1 week, then 10 days, and if necessary for a further 3 weeks. .
Specific antibodies can be detected in the serum of these rabbits as early as 1 week to 10 days after the first injection. To obtain the target antigen, a procedure similar to that described above is performed in which a target amount containing 1 mg of astrocytin or malignin (measured by the phorin-Lowry protein detection method) is injected. A special antibody against astrocytin is called anti-astrocytin. Anti-Malignin is a special antibody against malignin.
It is called. Similarly, a special antibody against a target reagent is called an anti-target. The standard antigen-antibody-in-agar (Ouchterlony) gel diffusion test method for antigen-antibody reactions reveals that the specific antibodies obtained with the specific antigen exhibit a single sensitive linear relationship in reaction with that antigen. It is. The presence of specific antibodies in serum can also be tested by the precipitin standard quantitative test method for antigen-antibody reactions. This test allows a good precipitin quantification curve to be obtained, from which the number of μg of the specific antibody can be calculated. Furthermore, the presence of specific antibodies in serum can be verified by absorbing the specific antibody: anti-astrocytin onto bromoacetyl-cellulose-astrocytin (BAC-astrocytin) as described above. That is, an antiserum containing a special anti-astrocytin can be reacted with BAC-astrocytin. Serum to BAC−
When passed over astrocytin, only specific antibodies against astrocytin bind to its specific antigen astrocytin. Since astrocytin covalently binds to bromoacetyl-cellulose, the specialized antibody: anti-astrocytin binds to BAC-astrocytin to produce BAC-astrocytin-anti-astrocytin (BACA-anti-astrocytin). This is demonstrated by testing serum residues separated from BAC-astrocytin by washing. A standard agar antigen-antibody diffusion test revealed that there were no residual antibodies in the serum that could react with astrocytin. Therefore,
It is clear that all the special antibodies (anti-astrocytin) previously shown to exist in the serum were absorbed by BAC-astrocytin. Furthermore, when the anti-astrocytin is released from binding to BAC-astrocytin, it is separated from any contaminating antibodies. This release of anti-astrocytin can be carried out by washing the BACA-anti-astrocytin with 0.25M acetic acid (at 4° C. for 2 hours), which does not destroy the BAC-astrocytin bond shown above. In addition, the presence of a special antibody in the serum is determined by the special antibody: anti-malignin, as described above, bromoacetyl-cellulose-malignin (BAC-malignin).
The proof can be obtained by absorbing the above. That is, an antiserum containing a special anti-malignin can be reacted with BAC-malignin. When serum is passed over BAC-malignin, only specific antibodies against malignin bind to the specific antigen malignin. Malignin is bromoacetyl
Because it binds covalently to cellulose, the special antibody: anti-malignin binds to BAC-malignin and forms BAC-malignin-antimalignin (BACM
- anti-malignin). This is due to cleaning
This is demonstrated by testing serum residues separated from BAC-malignin. A standard antigen-antibody diffusion test in agar revealed that there were no residual antibodies in the serum that could react with malignin. Therefore, all BAC-
It is clear that it was absorbed by malignin. Furthermore, when the antimalignin is released from binding to BAC-malignin, it is separated from any contaminating antibodies. This release of antimalignin does not disrupt the BAC-malignin bond shown above. Wash the BACA-antimalignin complex with 0.25M acetic acid (4°C).
2 hours). By standard antigen-antibody-in-agar gel diffusion test methods for antigen-antibody reactions, specific antibodies against the target antigen obtained by the antiserum, such as anti-astrocytin or anti-malignin (i.e., astrocytin or It is clear that malignin itself exhibits a monolinear relationship in its reaction with its antigen (as well as the antiserum obtained by injection). It should be noted that some rabbits retain a certain amount of anti-target prior to injecting the target. These anti-target substances are particularly effective when reacted with targeting reagents as described in human serum tests.
Approximately equimolar amounts (two types) of TAG, S-TAG and F-TAG are produced (see Examples below). Example 8 Target Attachment from Biological Fluid
Detection of malignant tumors by quantitative in vitro production of globulin (TAG): - The targeting reagent prepared according to Example 6 was washed to remove unbound recognin, which may be present due to deterioration. do. The satisfactory method is as follows. The target reagent is stirred with acetic acid at 37°C for 2 hours, centrifuged, the supernatant is decanted, and the optical density of the supernatant at 266 mμ is measured. If absorption at that wavelength is present, repeat the washing as described above until the material no longer dissolves. The target is then resuspended in phosphate buffered saline (PH 7.2) (see standard methods for testing target reagents).
If it is not possible to obtain whole rabbit serum tested to contain S-TAG and F-TAG with other target preparations, standard S-
TAG and F-TAG can be used). Slow binding (S-
TAG): Frozen serum that has been stored for several days should not be used. Serum is carefully prepared from freshly obtained whole blood or other body fluids by conventional methods. The method that should be satisfied is as follows. The collected blood is left in a glass test tube at room temperature for 2 hours to allow clotting. Separate the clot from the tube wall using a glass stir bar and leave the blood at 4°C for a minimum of 2 hours (or overnight). Clots are separated from serum by centrifugation (20000 rpm) for 45 minutes at 4°C, serum is decanted into centrifuge tubes, and further centrifuged for 45 minutes (2000 rpm) at 4°C. The serum was decanted and a 1% solution of methiolate (1 g in 95 ml of water and 5 ml of 0.2M bicarbonate buffer (PH10)) was added at 1% to the serum.
(capacity) limit. Add 0.2 ml of each serum obtained by the above method or another method to 0.25 ml of each target suspension reagent containing 100 to 200 mg of recognin per 0.25 ml of target reagent to prepare two test samples. The suspension is mixed at 4° C. in such a way that pellet formation is avoided. After shaking vigorously for 1-2 seconds, for example using a small rubber cap, the tube is gently decanted and shaken in a Thomas shaker for about 2 hours or more. The targeting reagent and its bound proteins are separated from the serum. According to the satisfactory method discovered in this invention, the tube was centrifuged (2000 rpm) at 4°C for 20 minutes, the supernatant was decanted, and then 0.2-0.3 ml of 0.15M saline solution was added.
The resulting pellet is washed by repeating the procedure three times: remixing with the pellets, shaking at room temperature, and centrifuging to separate the supernatant. The remaining protein attached to the target is then separated and this protein is quantified. For example, add 0.2 ml of 0.25M acetic acid, and shake the suspension in an incubator at 37°C for 1 to 2 hours. Centrifuge the tube (2000 rpm) for 30 minutes at 4°C. Decant the supernatant, being careful to avoid migrating particulates, and measure the optical density of the supernatant at 280 mμ. Serum protein (S
-TAG) Divide the above optical density measurement by a factor of 1.46 to obtain a result in μg per ml. There shall not be a difference of more than 5% between two test materials. Other methods for testing protein content, such as phorin-
The Lowry test method can be used, but the range of values for the control sample concentration and (S-TAG)-(F-TAG) concentration must be specified in the standard method. Fast-binding (F-TAG) values are tested as follows: serum stored frozen for more than a few days should not be used. By the usual method,
Carefully prepare serum from freshly obtained whole blood or other body fluids. The method described at the beginning of this example is sufficient for preparing serum. Serum samples prepared by the above or other methods are
Leave at 4°C for 10 minutes less than the total time that the S-TAG serum assay agent is in contact with the target reagent described above (e.g., ``2 hours'' S-TAG
If you do TAG measurement, it will take 1 hour and 50 minutes). In this method, the temperature histories of the S-TAG and F-TAG measuring agents are balanced. A 0.2 ml sample of temperature-equilibrated serum is added in duplicate to each 0.25 ml divisor of target suspension reagent containing 100 to 200 micrograms of recognin per 0.25 ml of target reagent.
The suspension is mixed at 4°C for approximately 10 minutes, avoiding granulation. For example, use a small rubber cap shaken rapidly for 1-2 seconds, then tilt the tube slightly and shake these on a Thomas shaker for about 10 minutes.
Just shake for a minute. Target reagents and proteins attached thereto are separated from the serum. One method found to be satisfactory is the following. The tubes were then centrifuged at 2000 rpm for 20 min at 4°C, the supernatant was decanted, and the granules formed by centrifugation were removed.
Wash three times by remixing with 0.2-0.3 ml of 0.15 M saline and shaking at room temperature, centrifuge and discard the supernatant. The protein that remains attached to the target is then cleaved and quantitatively measured. S-
The above method in this example for measuring TAG concentration is satisfactory. Other effective techniques for measuring protein content may be used, such as the Folin-Lowry assay, but the range of control and standards for determining the tumor value of S-TAG minus F-TAG concentration may be used. must be determined. The final result is expressed as micrograms of TAG per ml of serum, which is S-TAG minus F.
−Equal to TAG. TAG values in non-brain tumor patients and other controls currently range from zero (or negative number) to 135 micrograms per ml of serum. For results greater than 100 mμ/ml, repeat measurements are displayed. Some other tumors may exhibit high TAG values, especially if there are secondary (metastasized) tumors within the brain. TAG values in brain tumor patients currently range from 136 to more than 500 micrograms per ml of serum. In an initial "blind" study of 50 blood samples performed according to the procedure of this example utilizing targeting reagents prepared from astrocytin and bromoacetylcellulose, 11 of 11 brain tumors;
Of the 32 normal ones, 28 were correctly identified. 4
Of the cases that appeared to be normal (ie, non-brain tumor controls), one case was found to have thyroid cancer, which had apparently been well treated several years earlier. The remaining three normal cases were individuals between the ages of 60 and 70 who were in poor health and probably had undiagnosed cancer.
Of the remaining seven cases, three out of three cases of Hodgkins disease were correctly identified. One case in the tumor range (136-500μgTAG/ml) corresponds to a patient with severe gliosis, and one in the non-tumor range (0-500μgTAG/ml).
The three cases falling within the range of 135 μg TAG/ml corresponded to patients with an unclear but non-tumorous intracranial mass diagnosis, and osteosarcoma (a non-brain tumor) and melanocytic sarcoma (a non-brain tumor), respectively. Subsequent studies were conducted according to the method of this example using malignin and target reagents prepared from malignant brain tumors and all normal subjects. In further studies conducted according to the methodology of this example, the total number of human tumor specimens tested was 50.
The number ranged from 114 to 114. The utility of these products and procedures is shown in Table 5, which records the results of these tests. Example 9 Diagnosis of tumor cells by immunofluorescence The compounds anti-astrocytin, anti-malignin and S-TAG are shown to preferentially adhere to tumor cells. This specificity allows dyes or radioactive substances to be used in the histology department as anti-astrocytin or anti-astrocytin.
When coupled to malignin or S-TAG, these compounds can be used in the diagnosis of tumor cells. Standard labeling techniques are therefore used. The usage of S-TAG is as follows. One method that has been found satisfactory is St.
This is a variation of the St.Marie method. A human brain tumor specimen is frozen and cut into 5-micron-thick pieces. These are stored in a wet container at -70°C for 4-8 weeks before dyeing. The conjugate is a standard antiserum such as goat anti-rabbit conjugate. The conjugate is labeled with fluorescent or other standards by techniques known in the art. A commercially available fluroetscein-labeled goat anti-rabbit conjugate may be used. The fluorescent technique used is standard, in which a 1:200 to 1:400 solution of TAG is incubated on the tumor section for 30 minutes or more and then washed to remove adhesion.
Excluding TAG. Three washes with phosphate buffered saline are recommended. Bind with fluorescein-labeled conjugate, incubate, wash, and microscopically examine. Normal cells and their processes are not stained in both tumor sections and control sections of normal non-tumor brain. Fluorescence is brightly present in tumor glia and their processes. Example 10 Anti-astrocytin, anti-malignin and S-TAG against tumor cells growing in tissue culture
Experiments to demonstrate that a substance is cytotoxic A standard test for measuring cytotoxicity is used. Generally, calculate the number of cells in a fixed calculation box, which is usually prepared to contain about 100 live cells. These cells are then treated with the reagent to be tested and the number of cells still alive is calculated. Of the S-TAG solution obtained by the method of Example 8,
In a standard test for cytotoxicity to cells in tissue culture from patients with well-characterized glioblastogenesis, S-TAG is as low as 0.2 and 0.02 μg S-TAG per ml of solution. Even high dilutions of 1:100 and 1:1000, which correspond to small volumes, kill all cells in the calculation box.
Similar results are obtained with high dilutions of anti-astrocytin and anti-malignin. Both the specificity shown in Example 9 and the cytotoxicity shown in Example 10 are highly relevant to the therapeutic potential of anti-astrocytin, anti-malignin and S-TAG against human brain tumors. Their therapeutic use requires a proven histological diagnosis in addition to the actual diagnostic possibility of both these phenomena on surgically removed tumor tissue.

[Table] Example 11 Hydrolytic cleavage of recognin A solution of recognin, in this case astrocytin or malignin, is chilled at pH 1-2. After 7 to 14 days, the product shows a decrease in molecular weight of about 200 by thin layer chromatography. If this solution is allowed to stand for a longer period of time, approximately 200 molecular weight units will be cleaved in 7 to 10 days. That is, for astrocytin there is a decrease in molecular weight from 8000 and for malignin from 10000, with a subsequent decrease of about 200 units in each case. The physiochemical specificity of astrocytin is maintained for each product up to a molecular weight of 4000. The physiochemical specificity of malignin is maintained in each product up to a molecular weight of 5000. This is demonstrated in an antigen-antibody gel diffusion test in agar for anti-astrocytin and anti-malignin, respectively. This cleavage can also be accomplished enzymatically with trypsin and other proteases with the same results. The molecular weights of these compounds prepared by hydrolytic cleavage of recognin are approximately defined by the following formula: For a product with the physiochemical specificity of astrocytin: 4000 + 200X = Y For a product with the physiochemical specificity of malignin: 5000 + 200X' = Y where Y is the molecular weight of the product and X' is 0
is an integer between ~19. Example 12 Fabrication of Artificial Tissues or Organs with Recognin Hard-walled tubes of plastic, metal or other suitable hard material are injected with high concentrations (i.e. astrocytin or malignin in this case) of recognin.
10 mg/ml) viscose solution until all surfaces are coated. Alternatively, the Recognin solution is passed under pressure into and around the tube until all surfaces are thoroughly coated. The tube is then air dried, dried under reduced pressure, or lyophilized. The coating operation is repeated several times to coat many molecular layers of recognin. The tube is thus ready to be placed into a cavity or tissue containing precursors such as astrocytin or malignin in adjacent tissues or body fluids of a living mammal. This artificial tissue or organ is used to minimize or eliminate the irritating responses of foreign materials without Recognin coating. Other shapes of artificial tissues or organs can be similarly made.

[Brief explanation of drawings]

FIG. 1 is a graph showing the amount of malignin produced (as the amount of total protein produced (mg)) as a function of the degree of malignancy (as % of the total protein amount) in the present invention.

Claims (1)

[Scope of Claims] 1. A complex composition of a carrier, recognin, and an antibody to recognin, which is characterized in that an antibody to recognin is bound to a target reagent obtained by binding recognin having the following characteristics to a carrier. :(a) Normal or diseased cells or tissues are extracted with a neutral buffer, and the resulting solubilized protein extract has a pk value of 1 to 4.
(b) A single linear precipitate can be formed with a specific antibody in a quantitative precipitation test and an antigen-antibody gel diffusion test in Uftalony agar; c) Soluble in water and aqueous solutions at acidic or neutral PH; (d) Slightly soluble in water and aqueous solutions at alkaline PH; (e) Has a spectral absorption peak wavelength of 280 mμ; (f) Molecular weight 3000 ~25000; (g) high content of glutamic acid and aspartic acid, high ratio of glutamic acid and aspartic acid to histidine; 2. Claim 1, wherein recognin is a substance called astrocytin, which has the following properties:
The complex composition described in Section 1: (f') has a molecular weight of about 8000; (g') amino acid composition: 9 aspartic acid, 5 threonine, 6 serine, 13 glutamic acid;
4 proline, 6 glycine, 9 alanine, 4 valine, 2 1/2 cysteine, 1 methionine, 2 isoleucine, 8 leucine, 2 tyrosine, 3 phenylalanine, 8 lysine, 2 histidine, 4 arginine, total 88 (can be detected) Amounts of cysteic acid, hydroxyproline, norleucine, ammonia, isodesmosine, desmosine, hydroxylysine,
Lysinonorleucine and γ-aminobutyric acid are not present. ) has an approximate number of residues. 3. A complex composition according to claim 1, wherein the recognin is a substance called malignan having the following properties: (f'') having a molecular weight of about 10,000; (g'') aspartic acid as the amino acid composition; 9,
5 threonine, 5 serine, 13 glutamic acid, 4 proline, 6 glycine, 7 alanine, 6 valine, 1/
2 cysteine 1, methionine 2, isoleucine 4,
Leucine 8, Tyrosine 3, Phenylalanine 3,
6 lysine, 2 histidine, 5 arginine, total 89
(There are no detectable amounts of cysteic acid, hydroxyproline, norleucine, ammonia, isodesmosine, desmosine, hydroxylysine, lysinonorleucine, gamma-aminobutyric acid.) 4. The carrier is a cellulose-based material,
A composite composition according to claim 1. 5. The composite composition according to claim 4, wherein the carrier is bromoacetylcellulose. 6. An antibody composition of a recognin formed by contacting a body fluid with a recognin having the following properties or a targeting reagent comprising the recognin bound to a carrier: (a) normal or diseased cells or tissues in a neutral buffer; A fraction with a pk value of 1 to 4 is separated from the resulting solubilized protein extract, and the acidic peptide is collected from this fraction; (b) Quantitative sedimentation test and antigen-antibody gel diffusion in Uftalony agar In the test, a single linear precipitate can be formed with a specific antibody;
(c) Soluble in water and aqueous solutions at acidic or neutral pH; (d) Slightly soluble in water and aqueous solutions at alkaline pH; (e) Has a spectral absorption peak wavelength of 280 mμ;
(f) It has a molecular weight of 3000-25000; (g) It has a high content of glutamic acid and aspartic acid, and a high ratio of glutamic acid and aspartic acid to histidine. 7. Claim 6, wherein the recognin is a substance called astrocytin, which has the following properties:
Antibody composition described in Section 1: (f') having a molecular weight of about 8000; (g') amino acid composition of aspartic acid 9;
5 threonine, 6 serine, 13 glutamic acid, 4 proline, 6 glycine, 9 alanine, 4 valine, 1/
2 cysteine 2, methionine 1, isoleucine 2,
Leucine 8, Tyrosine 2, Phenylalanine 3,
88 lysine, 2 histidine, 4 arginine, total 88
(There are no detectable amounts of cysteic acid, hydroxyproline, norleucine, ammonia, isodesmosine, desmosine, hydroxylysine, lysinonorleucine, gamma-aminobutyric acid.) 8. The antibody composition of claim 6, wherein the recognin is a substance called malignan having the following properties: (f'') having a molecular weight of about 10,000;
(g'') Amino acid composition: 9 aspartic acid, 5 threonine, 5 serine, 13 glutamic acid, 4 proline, 6 glycine, 7 alanine, 6 valine, 1/2
cysteine 1, methionine 2, isoleucine 4,
Leucine 8, Tyrosine 3, Phenylalanine 3,
6 lysine, 2 histidine, 5 arginine, total 89
(There are no detectable amounts of cysteic acid, hydroxyproline, norleucine, ammonia, isodesmosine, desmosine, hydroxylysine, lysinonorleucine, gamma-aminobutyric acid.) 9. The antibody composition according to claim 6, wherein the antibody is an antigen-binding globulin and is separated from a complex formed by reacting a targeting reagent with a body fluid. 10. The antibody composition according to claim 6, which is an antigen-binding globulin having the following properties:
(b) Soluble in aqueous buffer; (b) Forms a single linear precipitate with recognin in Uftalony agar antigen-antibody gel diffusion test; (c) Not dialyzed with transparent cellulose membrane; (d) Molecular weight 25,000 or more (e) The molecular weight measured by thin layer gel chromatography in various aggregation states is about 50,000, and the molecular weight is in the macroglobulin region and is a multiple of the above molecular weight. (f) Has a spectral absorption peak wavelength of 280 mμ;
(g) Binds preferentially to glioma tumor cells in histological sections of brain tumors. 11. Claim 6, which is a slow antigen-binding globulin obtained by reacting a body fluid with a target reagent at a low temperature that does not cause a decomposition reaction for about 2 hours or more, and treating the resulting substance with a dilute acid. The antibody composition described in Section 1. 12. The fast antigen-binding globulin according to claim 6, which is a fast antigen-binding globulin obtained by reacting a body fluid with a target reagent at a low temperature that does not cause a decomposition reaction for about 10 minutes, and treating the obtained substance with a dilute acid. Antibody composition.