DE2546670A1 - Astrocytin and malignin from tumour tissue - for prepn of products for cancer diagnosis and therapy - Google Patents

Abstract

The novel recognins astrocytine (I) and malignin (II) are prepd. as follows: (a) (I) is obtained by extracting disintegrated cerebral glioma tissue with a neutral buffer, separating dissolved proteins with a pK range of 1-4 from the extract, and isolating a product with a mol. wt of ,000 from the proteins; (b) (II) is obtained by growing artificial cancer cells in a fermentation culture, extracting the disintegrated cells with a neutral buffer, separating dissolved proteins with a pK range of 1-4 from the extract, and isolating a product with a mol. wt. of 10, 0000 from the proteins. (I) and (II) can be used to produce anti-recognin anti-bodies, "target reagents" (i.e. complexes of the recognins with a carrier), and "target-attaching globulins" (i.e. products obtained by contact of a target reagent with a biological fluid followed by acid cleavage). These products can be used for diagnostic purposes, e.g. for selective staining or radioactive tracing of tumours, and for the treatment and prophylaxis of cancer.

Description

Recognine

The invention relates to a new group of compounds herein referred to as "Recognine".

Recognins are made by looking at normal or abnormal cells or tissue, e.g. tumor cells or artificial cancer cells, treated and the desired Separates products. The Recognine can be used to change their chemical Manufacture counterparts, i.e. by placing the Recog'nine or the one on a carrier brings the Recognine in contact with body fluids. This chemical Counterparts are for diagnostic and therapeutic purposes, i.e. for diagnosis and cancer treatment. The chemical counterparts resp. Chemo-reciprocal are substances that have an immunochemical-like specificity react with a Recognin in vivo or in vitro, e.g. with a quantitative one Precipitation test, with the Ouchterlony double diffusion or with immunofluorescence.

One of the recognins according to the invention is astrocytin. Astrocytin is generated from brain tumor tissue, preferably brain glioma tumor tissue. Protein fractions, which contain the astrocytin precursor are first extracted from the tissue. A preferred extraction method is to use the fabric a neutral buffer under homogenization conditions or by using others Cell and tissue fragmentation techniques to treat protein fractions, which contain the astrocytin precursor to solubilize.

At this point the astrocytin precursor is still in need of substances with large molecular weight such as e.g.

Proteins, glycoproteins, lipoproteins, nucleic acids, nucleoproteins etc. bound. The solubilized proteins are then from the resulting Tissue extract separated. The extract solution from the tissue is then clarified, to remove insoluble particles. Low molecular weight impurities are thereupon by a pre-evaporation concentration technique from the resulting Solution removed. The resulting solution is then treated to make the astrocytin precursor from other impurities to obtain a protein fraction, which has a pK range between 1 and 4. Thus, for example, the solution is on a chromatographic Given column and eluted with increasingly acidic solvents. All political groups, which elutes in the neutral or acidic range down to a pK value of 4 are discarded, and those fractions with a pK range from 1 to 4 are collected. The eluate is then treated to produce a molecular weight product of about 8000.

This is done, for example, by filtering the material first, low molecular weight substances, i.e., those with a molecular weight below 1000, and that one again filters to remove those substances with a molecular weight above 25,000 to remove. The faction with one Molecular weight between 1000 and 25,000 is then further treated, e.g. by thin layer gel (TLG) chromatography to obtain astrocytin.

Thus, astrocytin can be made by producing brain glioma tumor tissue with a neutral buffer with repeated homogenization and high-speed centrifugation extracted, from the resulting extract a fraction with a pK range of separates about 1 to 4, from this fraction the substances with a high molecular weight, i.e. up to about 230,000, and that the product astrocytin, which has a molecular weight of about 8,000, isolated.

The astrocytin product made by this process is thereby characterized that it with its specific antibody in quantitative precipitin- respectively.

Coagulin tests and Ouchterlony gel diffusion tests a single line precipitate or a single line precipitate that it forms in water and aqueous solutions soluble at an acidic or neutral pH and at an alkaline pH insoluble is that there is a wavelength of the spectrophotometric absorption peak of 280 m / u and that it finally has a molecular weight of about 8,000.

Astrocytin is also characterized by being very high Percentage of residues of glutamic acid and aspartic acid and a very high one Has ratio of these acids to histidine. A closer analysis of astrocytin is made given below.

In a similar way as described above, another recognition is called "malignin" from artificial cancer cells, i.e. cancer cells, which grow in an in vitro fermentation. Malignin has a molecular weight of about 10,000 and a similar but different amino acid residue composition versus astrocytin, i.e. high ratios of glutamic acid and aspartic acid and high ratios of these acids too Histidine. A closer analysis of malignin is given below.

Thus, malignin can be produced in such a way that one in a Fermentation culture grown artificial cancer cells with a neutral buffer extracted with repeated homogenization and high-speed centrifugation, from the resulting extract the fraction with a pK range of approximately 1 separates up to 4, from this fraction the substances with a high molecular weight, i.e. up to about 230,000, and that this gives the product having a molecular weight isolated from about 10,000.

The amount of malignin produced by artificial cell fermentation is produced, and the percentage of the total protein that is produced in the artificial cell fermentation which is malignin can be increased by getting the growth the artificial cancer cell culture in large-sized containers.

Malignin produced according to this process is characterized by that it with its specific antibody in quantitative precipitin tests and Ouchterlony gel diffusion tests a single line precipitate or a single line precipitate forms that it is in water and aqueous solutions with an acidic or neutral pH soluble and insoluble at an alkaline pH that it is a wavelength of the spectrophotometric absorption peak of 280 m / u and that it has a molecular weight of about 10,000.

Recognins are also characterized by the fact that they are able to do so are to form a complex with bromoacetylcellulose, whereby bromoacetylcellulose-Recognin is formed and that after injection into mammals the produce specific antibodies anti-recognin. The anti-recognition is specific to added the Recognin precursor in situ. For example, there is an anti-recognition, namely Anti-malignin, toxic to brain tumor cells in vitro.

Recognins like astrocytin, malignin and similar substances are considered Products useful that can be introduced into a biological system to prevent foreign reactions to reduce, for example by coating a material with a Recognin. Another example is the introduction of a Recognins to the biological System to generate the chemical counterpart. The Recognine can also be used as nutrients used to stimulate the growth of certain biological systems from which they are part of encouraging. Another possible use of the Recognine is the generation of target reagents which complex the recognizein complexes with a carrier included in order to facilitate the applicability in biological systems. So promoted E.g. the complex the physico-chemical properties of the Recognin itself. The carrier should be selected from those that have a complex with the Recognin and which are essentially biologically inert.

Any known substances with polypeptides or Proteins form a stable complex, can complex with the Recognin be suitable.

An example is a cellulosic material such as bromoacetyl cellulose. Apart from the fact that the carrier must be inert to the biological system, the carrier should be such that it supports the specific physicochemical Properties of the recognition that are useful for the purposes specified above, not changed.

The complexes of Recognin and its carrier are suitable for this; around in any biological systems with which they are brought into contact, the chemical counter +) or target or point of attack reagents pieces or to generate, separate and identify chemical reciprocals. The Recognin Carrier Complex is also suitable for the production of the chemical counterpart precursor in to stimulate any biological system into which it is introduced.

A class of chemical counterparts are the anti-recognins, i.e., anti-astrocytin and anti-malignin.

These can be produced in such a way that the Recognin in a biological system is injected. An immunologically effective dose of Recognin is used with body tissues or fluids according to known for the generation of antibodies Techniques contacted in such a way that an antibody response is induced. The Anti-Recognine can be used to manufacture materials such as diagnostic, Food and drugs for specific cells or sites in biological systems can be used, whereby the agent in complex form with the anti-recognin in the introduces biological system. The Anti-Recognine are also used to diagnose the presence of tumor cells in histological sections, using the anti-recognin in combination or conjugation with a marker substance such as dyes and supplies radioactive substances to the section. There is a staining or radioactive labeling only on the tumor cells. Another use for Anti-Recognine is increasing the yield of other usable chemical Precursor products (such as TAG products as described below) of mammals, with an immunologically effective dose of Recognin in the Injected into a mammal or into another biological system.

So far, glycoprotein complexes have been made from brain tissue and appropriate antibodies generated. Separated materials known as 10B glycoproteins known and those from the brain of Tay-Sachs disease were injected into rabbits and were made that way Antibodies made. These Tay-Sachs antibodies were used for immunofluorescence studies used by tumor-containing brains: These antibodies were only reactive normal non-tumor glia, but not tumor glia, stained.

In contrast, when the astrocytin has been generated from tumor tissue was and antibodies against astrocytin (anti-astrocytin) produced and in immunofluorescence examinations of the brain were used, then only tumor glia but not normal (non-tumor) glia stained by the anti-astrocytin. Thus, due to the original tissue and due to the nature of the antibodies as well as the specific types of stained Cells anti-Tay-Sachs antibodies and anti-astrocytin clearly different products.

Another class of chemical counterparts are target reagents, which have been complexed with their chemical counterparts. So e.g. target (the product that is obtained when astrocytin with a carrier how bromoacetyl cellulose is complexed) in contact with anti-astrocytin brought. This type of connection can thus be converted into a complex and for the production of diagnostic nutrients and medicines for specific cells or sites in biological systems are used. These connections can can also be used for cleaning processes. For example, anti-astrocytin can be produced in this way be by getting bromoacetylcellulose-astrocytin-anti-astrocytin through a hydrolytic Treatment with an acid or a proteinase enzyme as a decomplexing treatment subject. Target reagents are also useful in determining the amount of TAG products (as described below) to increase in biological systems, e.g. by placing an immunologically effective dose of target in contact with body tissue or liquids.

Further chemical counterparts are TAG reagents (e.g. on Target adherent globulins). The TAG products are manufactured using target reagents brings in contact with body fluids over varying periods of time to one To form a complex, and that the TAG is split off from it.

Two suitable embodiments are S-TAG and F-TAG.

In a method of making S-TAG (slowly on target adhering globulin = slow-target-attaching-olobulin) one proceeds in such a way that one blood serum or another body fluid with a target (i.e. bromoacetylcellulose malignin) about 2 hours or more at a low temperature, e.g., about 4 ° C and that the S-TAG from the resulting material, for example with diluted Acid, for a period of about 2 hours and at a temperature of about 37 ° C split off. The S-TAG produced according to this process is characterized by that it is soluble in aqueous, buffered solutions; that it is a single-line precipitate with its corresponding Recognin in Ouchterlony gel diffusion tests that it is not dialyzable through cellophane membranes that it is through millipore filters is retained, which molecules with a molecular weight of over 25,000 hold back that there are molecular weights in different aggregation states, determined by thin layer gel chromatography, of about 50,000 and multiples of which in the macroglobulin range and that it has a wavelength of the spectrophotometric Has absorption peaks of 280 mlu.

In a process for the production of F-TAG (quickly at the target adhering globulin = fast target attaching globulin) one proceeds in such a way that one blood serum or another target body fluid (i.e. bromoacetylcellulose malignin) about 10 minutes at low temperature, e.g. about 4OC, and that one from the resulting material F-TAG, e.g. with dilute acid, over a period of time of about 2 hours and at a temperature of about 37 ° C. That F-TAG not produced in this way is characterized by the fact that it is in aqueous, buffered solutions that it is a single line precipitate with its corresponding recognition in Ouchterlony gel diffusion tests that it is not dialyzable by cellophane membranes that it is retained by millipore filters which will retain molecules with a molecular weight of over 25,000 that it molecular weights in different states of aggregation, determined by Thin layer gel chromatography, of about 50,000 and multiples thereof in the macroglobulin range and that it has a wavelength of the spectrophotometric absorption peak of 280 m / u.

TAG products are used to treat cancerous tumors in living mammals to be discovered by determining the concentration of S-TAG and F-TAG by a known volume of the mammalian blood serum or other body fluid is generated, and by relating this concentration to amounts that have been diagnosed as indicating cancer. TAG products are also suitable for to diagnose the presence of tumor cells in histological sections. One proceeds in such a way that one TAG, which with a marking substance, e.g. a Dye or a radioactive substance, combined, is applied to the section, whereby staining or radioactive labeling occurs only on the tumor cells. It it has also been shown that TAG products act against tumor cells are cytotoxic. TAG products are also used in the manufacture of diagnostic, nutritional and drugs suitable for specific cells or sites, said agents be introduced in complex form with the TAG product.

Normal cell division in plants or animals is restricted or inhibited when the cells come to completely occupy a respective space. The mechanisms (a) by which normal cells "recognize" that they are responsible for they have filled in the available space, and (b) after which the expiry of this recognition mechanism in turn inhibits cell division, both have been unknown. It was now a group of compounds produced whose precursors have an increased concentration have when normal detection and learning occur. These connections exist with the recognition and learning in particles and cells and with the connection of the Cells in relation to each other. These connections are known as recognize. When attempting to make these compounds from normal cancer cells, it was found that they do not exist as such and that at the same time where the cancer cells have lost their ability to (a) recognize that they have filled their normal volume and / or (b) interrupt the division when it has filled its normal volume changes in their molecular structure have taken place.

There are new compounds as well as methods of making them Connections have been discovered. These new connections are known as recognize. Recognins are new compounds that have physicochemical properties those configuration properties of the cancer cells with regard to their inability to increase recognize and stop cell division, imitate. The use of the Recognine goes beyond a mere insight into the cancer mechanism, as this results in products and Procedures are made available for diagnosis, treatment and prophylaxis are suitable from cancer.

Methods have been found by which to artificially cultivate Cells can be used to produce malignins. An advantage of the The methods described here are that now malignins and new products from them can be effectively produced in virtually unlimited quantities.

The present invention transcends the field of cancer research, and it is directly applicable to any biological system which is desired to affect overall growth and metabolism. Consequently can by making the respective compound or compounds of the corresponding cell type in artificial cultures and furthermore by production of products made from these substances showed a specific influence for the first time any tissue, cell, cell organelle, sub-organelle molecule or molecule cluster practiced in any living system. Thus, for the first time, specific Dietary influences at critical points in development, specific diagnostic, prophylactic and treatment methods, as well as the construction of artificial bioelectrical systems (e.g. in tissue or organ transplants) be carried out. These artificial bio-electrical systems can be characteristic Properties of the specific recognin, malignin or their chemical counterparts normal tissue or component to which they are adjacent, and they can thus be "recognized" as "foreign", thereby causing foreign body reactions with Inclusion of rejection can be avoided.

Another aspect of the present invention is manufacture a usable, specific, antibody-like product (anti-astrocytin) to a specific brain product (astrocytin), which enables that this antibody-like product for specific complex formation with it and as specific delivery vehicle for specific points in the nervous system of all types can be used. Malignins and Astrocytin are Recognins Another aspect The present invention is the creation of new products, namely at Target adherent globulins (TAG) from biological fluids. These products are so named because they are generated by two reactions, being the first reaction is the conversion of biological fluids with a synthetic complex, which contains physicochemical configurations that mimic those of the malignins, and which is referred to as the target. The second reaction will be specific TAG cleaved from the complex. By measuring the TAG generated in this way thus becomes a quantitative indication of the biological fluids of the living Organisms receive information on whether a tumor is present in these organisms. Thus becomes according to the invention provided a diagnostic test for tumors. Da TAG products and anti-malignin physicochemically opposite or complementary to malignins they are referred to herein as chemical counterparts or chemo-reciprocal.

It was also found that two quantitatively and qualitatively different TAG products depending on how long it takes to implement the Serum with the specific target reagent allowed, and depending on the period of time for the splitting off of the product that has been converted into a complex, allowed to be produced.

After studying the quantities of these products that come from a number from various people with brain tumors and various other medical Disorders as well as those that did not have an apparent disease process, it became apparent that the quantities of these two new products, that can be generated in a given person, give an indication of whether the individual has had a brain tumor. It is thus a new serum diagnostic Test for brain tumors discovered.

The suitability of these new products, in addition to their use to diagnose from the serum and other biological fluids, the presence of brain and other tumors display is illustrated by the fact that TAG and anti-recognin compounds in histological sections of brain tumor preferably on the glial tumor cells and the surrounding tissue, which is used during surgery Surgery of the brain tumor has been removed, adhere. This preferred marker the tumor cells by TAG and Anti-Recognine is measured by standard immunofluorescence techniques demonstrated. Thus, the invention also provides a new method put to through a histological examination with a new degree of certainty to determine whether tumor cells have advanced to the corners or edges of the removed tissue are what gives an indication of the likelihood that in the brain or the other organ still has a tumor or that tumor cells are attached to the Periphery of the removed tissue are no longer present. This creates a Statement given about the possibility that the entire tumor from the brain or removed from another organ. It has also been shown that TAG and anti-malignins, the production of which has been described above, against glioma brain tumor cells, grown in vitro in tissue culture have become cytotoxic are. This high affinity for tumor cells in another medium that is in this one Case grown in tissue culture is another piece of evidence for the specific coupling potential of the new TAG products. This will make the Name "globulins adhering to the target" (target-attaching globulins) (TAG) explained, as does the properties of TAG with regard to the synthetic target product and do the tumor cells in histological sections.

The cytotoxicity of TAG and anti-recognins against tumor cells provides another, new, diagnostic test for the serum of patients who are suspected of having a tumor. Thus, for example, the serum or another body fluid of these patients with Target implemented with the formation of TAG. The TAG product will tested for cytotoxicity in tissue cultures of tumor cells. Both the concentration on TAG as well as the level of cytotoxicity exhibited by the TAG which Can be generated from the serum of a given person, not just for diagnostic purposes Purposes to be appropriate but also of value to the course of the disease preoperatively and to follow up postoperatively in a given patient. The coupling of radioactive and Dye Tracers to TAG will deliver new TAG products that can be used in vivo for diagnosis of tumors and their exact localization are suitable. Thus, the intra-arterial or intravenous injection of appropriately labeled TAG into the cerebrospinal fluid or directly in the brain tissue or its cavities for the presence of a brain tumor by radioactive means or by visualization by the coupled dye because the TAG specifically only adheres to the tumor cells. Also permitted this method enables the precise visualization of the location of the brain tumor. This represents an improvement to this in vivo diagnostic method using in Anti-astrocytin produced by rabbit blood to mark the brain tumor because of the possibility of using TAG made from human serum of foreign protein reactions. Because TAG and Anti-Recognine are chemically specific Have properties that include preferential attachment to astrocytin precursors Allow tumor cells both in vitro and in vivo, these products can both can be used therapeutically as well as diagnostically, e.g. when they are radioactive Agents, proton trapping agents or other toxic physical or chemical agents are coupled so that these toxic substances are preferred by the specificity of these compounds on tumor cells compared to neighboring ones normal cells can be localized to adhere. This selectivity is called essential, or at least viewed as an essential factor, to an effective to achieve chemical or physical therapy of tumors. One such factor has not yet been achieved. The TAG has now shown its effectiveness prefers to adhere to the tumor cells, so that there is a certain amount of these reasons Should have a prospect as a new drug.

In the serum of patients with malignant tumors, as shown in the examples one type of TAG, namely Slow-TAG (SLOW-TAG) (S-TAG) versus Schnell-TAG (FAST-TAG) (F-TAG) in relatively larger amounts from a given Serum volumes are generated than in patients without such tumors. This suggests indicate that the concentration of a naturally occurring precursor (P-TAG) for TAG is increased or that there are other factors that affect the relative in vitro production favoring S-TAG over F-TAG.

The possible relationship between the function of the actual synthetic Products Target and TAG and their predecessors and, in turn, the Functions of the postulated but not yet shown cell "antigens" and the circulating "antibodies" against which they can exist in vivo is so far has not yet been cleared up.

For example, F-TAG and S-TAG result in only one of them in an antibody-like manner or individual discrete lines of the reaction with astrocytin in the Ouchterlony gel diffusion, and injection of Target into rabbits induces an increase in the yield of TAG products from the rabbit serum after implementation with the target. The discovery that a there may be normal levels of a precursor to the circulating antibody to a cell antigen that is hidden in the non-dividing cells is, raises the question of a possible function of the pair.

It is suggested here that the TAG precursor (P-TAG) and target-like Substances exist in vivo that have a function of controlling cell proliferation and cell death. For example, during cell division a cell component exposed to serum proteins, which is usually not directly. Exposure to this cell component could result in this component is converted into a target-like substance to which the attachment of a P-TAG-like molecule from the serum could then take place, which stimulate or inhibit cell division would. Alternatively, a non-dividing cell that has been damaged or can that works incorrectly, release a target-like substance to which the attachment of the P-TAG-like molecules can be repairing. However, under certain cell conditions, the attachment of the P-TAG-like molecules can induce the destruction of the cell (e.g., synthetic anti-glioma TAG as described herein markedly cytotoxic to glioma tumor cells growing in a tissue culture). This could thus be the mirror image of a normal mechanism for controlling cell division and either for repair or removal of individual Represent cells in the body throughout the life of the organism. if the exposure of the cellular components is abnormally increased, causing abnormally large amounts are formed by target-like cell substances, which is, for example, in rapidly dividing cancer cells as in brain gliomas can then cause an increase in concentration be induced by one type of serum P-TAG versus another.

Regardless of the actual function of the precursors, the increase is the relative amount of a predominant type of TAG, Slow-TAG (SLOW-TAG) (S-TAG), the in vitro according to the methods described from the serum of patients with malignant Tumors can be prepared based on the examples below described serum diagnostic tests.

Example 1 Preparation of one containing the astrocytin precursor Crude fraction.

Human brain glioma tumor tissue removed during a surgical procedure Intervention, is removed from surface blood vessels and normal brain tissue as much as possible cut free. For a typical amount of excised tumor tissue of 11 Weigh the tissue into six 1.5 g samples and two 1.0 g samples. Every sample is treated as follows.

Each sample is placed in a neutral buffer solution by sonication or other mechanical measures are homogenized. For example, every part in 100 cc of a 0.005 M phosphate buffer solution with a pH of 7 per gram of tissue homogenized in a Waring mixer - the homogenization should be done in the cold, denaturation of proteins to prevent. E.g. the mixer is pre-cooled in a cold room from 0 to 5 0C and only about 3 minutes be put into operation for a long time.

The homogenate is then used for clarification, for example with the 80 O00 times gravity for 30 minutes in a refrigerated ultracentrifuge, centrifuged. The soluble supernatant is decanted and kept in the cold. Of the insoluble residue is rehomogenized with a further 100 cc of the neutral buffer and centrifuged as described above. The second soluble extract is made with combined with the first extract. Best results are obtained using this practice the homogenization and centrifugation is repeated until less than 50 / ug protein / ml Solution can be obtained in the supernatant product. Most fabrics will this is achieved by the fifth extraction.

The solutions obtained in this way are combined and by Evaporation and subsequent dialysis, e.g. dialysis against a .006 M phosphate buffer in the cold, concentrated to make 15 ml. The volume of this Solution is noted and an aliquot is removed for total protein analysis. The remainder is fractionated to produce a protein fraction with a pK range in between 1 and 4. The preferred method of fractionation is chromatographic Method as described below.

The solution is in a cold room (40C) on a column with DEAE cellulose (Cellex-D) with the dimensions 2.5 x 11.0 cm fractionated. The pillar is with a 0.005 M sodium phosphate buffer has been equilibrated. It will be gradual Changes in the eluting solvent with the following solvents (solutions) carried out: Solution (1): 4.04 g NaH2PO4 and 6.50 g Na2HP04 become dissolved in 15 l of distilled water (0.005 molar, pH 7); Solution (2): 8.57 g NaH2P04 are dissolved in 2480 ml of distilled water; Solution (3): 17.1 g of NaH2P04 become dissolved in 2480 ml of distilled water (0.05 molar; pH 4.7); Solution (4): 59.65 g of NaH2PO4 are dissolved in 2470 ml of distilled water (0.175 molar); solution (5): 101.6 g of NaH2P04 are dissolved in 2455 ml of distilled water (0.3 molar, pH 4.3) dissolved; Solution (6): 340.1 g of NaH2P04 are dissolved in 2465 l of distilled water (1.0 molar, pH 4.1) dissolved; Solution (7): 283.64 g of 80% phosphoric acid (H3P04) will be dissolved in 2460 ml of distilled water (1.0 molar, pH 1.0).

It becomes a nerve tissue extract with a volume of 6 to 10 ml added. The column is then passed through. This is followed by the Solution (1) and a reservoir of 300 ml of solution (1) is attached, so that this solution drips onto the column by gravity. Aliquots of 3 ml of the effluent product are collected by means of an automatic fraction collector collected. The following eluting solutions are gradually added to the following Numbers of elution tubes exchanged: Solution (2): at tube 88; the solution is placed in the column on top of the resin, then is deliberated and a reservoir of 50 ml of the solution (2) added; Solution (3): at tube 98; the solution is placed in the column on top of the resin, then overlaid and a reservoir of 75 ml of solution (3) is added; solution (4): at tube 114; the solution gets into the column on top of the resin applied, then pondered and a reservoir of 150 ml of the solution (4) attached; Solution (5): at tube 155; the solution is in the column on top of the resin applied, then considered and there is a reservoir of 125 ml of the Solution (5) added; Solution (6): at tube 187; the solution is in the column is applied to the top of the resin, then overlaid and it becomes a reservoir of 175 ml of solution (7) was added.

Elution continues to tube 260 where the elution is complete. There will be freshly made resin for each new volume of tissue extract used.

Each tube for the effluent is quantified for protein examined. The eluates in the tubes with the numbers 212 to 230 are combined and they contain the raw products from which astrocytin is made.

Regarding this raw material, referred to as Fraction 103 values have already been published [Protein Metabolism of the Nervous System, pp 555-69 (Pleum Presse, 1970); Journal of Neurosurgery, Volume 33, pp.281-286 (September 1970)], but now the splitting off of the specific product, referred to herein as astrocytin, from Fraction 10B. The raw fraction 103 can be used as a product in amounts between 0.1 and 10 0 mg / g of the original fresh Nervous system tissue from which it is obtained. Additionally to an astrocytin precursor it contains varying amounts of covalently bound Carbohydrate residues including a number of hexoses, namely glucose, galatose, Mannose; Hexosamines including glucosamine, galatosamine and mannosamine; and occasionally other sugars, e.g.

Fructose, ribose and possibly rhamnose. It contains even High molecular weight protein products, multiple lipids and nucleic acids.

Example 2 Production of purified astrocytin from the crude fraction, which contains the astrocytin precursor.

The fraction containing the astrocytin precursor will continue freed from impurities. In preferred embodiments, the material of Example 1 on Sephadex G-50 resin in a typical one length column of 40 cm, a diameter of 2.5 cm and a volume of 196 ml. The pressure applied is 40 mm Hg. The flow rate is 35 ml / h and a 0.05 molar phosphate buffer solution with a pH of 7.2 used. The first (flow through) peak contains the astrocytin precursor together with impurities, while the following peaks contain only impurities.

In the preferred embodiment, the products in the first flow-through peak concentrated on Sephadex G-15 and then on a column from Cellex-D with the same solution or the same solutions (1) to (7) as in Example 1, and the same elution steps are used as in Example 1 carried out. The product astrocytin lies as a sharp peak in the same tubes (No. 212 to 230) as before. It thus maintains its behavior in chromatography on Cellex-D without the presence of a large number of impurities.

Low molecular weight impurities can then post known techniques, e.g., by filtration through Millipore disks will. In the preferred method, the product is astrocytin from salt and other low molecular weight contaminants from filtration through millipores Pellicon discs No. 1000 with 13 mm freed. This filter keeps substances with them molecular weight greater than 1000 and allows the passage of Substances with a molecular weight of less than 1000. The product astrocytin remains on the Pellicon discs and is removed from it by washing with the solution (1) of Example 1 obtained.

Astrocytin is then obtained by making the compound with a molecular weight of about 8,000 isolated from the above solution. At a The preferred method is thin-layer gel (TLG) chromatography as follows.

A commercially available device from Boehringer is used as the device Mannheim GmbH; Pharmacia Fine Chemicals and CAMAG (Switzerland) used. The resin, namely 2.5 g Sephadex G-200 superfine, is in 85 ml 0.5 M NaCl in 0.02 M Na2HP04KH2P04 phosphate buffer, pH 6.8 (6.6 to 7. o) prepared. It will take 2 or 3 days at room temperature Occasional, gentle mixing left to swell.

(Magnetic or other stirrers should not be used.) The swollen gel is stabilized for 3 weeks at refrigerator temperature, however bacterial and fungal growth can disrupt the swollen gel. When the gel is over Should be held for longer periods then a small amount of a bacteriostatic should be used By means of (e.g.

0.02% sodium azide) can be added. 2.5 g of dry gel are added used to make two glass plates with dimensions 20 x 20 cm and a thickness of 0.5 mm to be coated. The plates are either at room temperature for 10 minutes left to dry for a long time and transferred to a humid chamber, where they are about 2 weeks can be stored long or they are immediately after a suitable pre-ins-equilibrium -Settlement used (usually during the night for a minimum of 12 hours). The main function of equilibrium is to normalize the volume ratio between the stationary and the mobile phase. In a horizontal position the pre-equilibrated plates become the substances to be detected applied with micropipettes as spots or stripes on the starting line. 10 Up to 20 ml of the 0.2 to 2% protein solution are on the edge of a microscopic Object plate (18 x 18 mm) applied and held against the gel surface. Within the solution penetrates the gel within a few seconds. All samples are first made on the coverslip and then quickly applied. If not enough Material is used, then it is difficult to separate individual stains after separation to locate. If too much material is applied, then there is no defined one Separation. The samples are diluted with buffer for easier handling and the Separation of the samples is carried out according to the descending technique, with the plate using is arranged at an angle of 220.

A flow rate of about 1 to 2 cm / h is most suitable. Marking substances (e.g. cytochrome C, hemoglobin, myoglobin or with bromophenol blue labeled albumin) are applied at various points across the plate and they serve as reference proteins for calculating the relative distance (mobility) of unknown substances. After the samples have been applied, the plates are placed in the The device is replaced and the paper tampon is gently pressed down to form a ensure good contact with the gel layer. The paper tampon must not drip. Excess moisture is wiped off. The liquid solvent in the reservoir is held constant at 1 cm from the top of the jar. The individual rounds are usually completed in 4 to 7 hours depending on the progress of the separation. In the case of colored substances the separation can be followed directly. The severed spots of protein are easily visualized by transfer them to a paper copy of the TLG plate after the chromatographic Separation has been completed and kept on the system for 48 hours pre-washed methanol + H20 + acetic acid - 90: 5: 5 stains. As a sheet of paper a 3 mm filter paper was used. A sheet of paper measuring 20 x 18 cm is placed over the gel layer and pressed (rolled) just enough that a Contact with the gel is guaranteed.

Care was taken to ensure that no air is trapped under the paper (the copy) and that the gel layer is not disturbed. The liquid phase is sucked from the gel layer through the paper, and this is removed after about 1 minute, immediately dried for 15 minutes in an oven at 60 ° C. and colored in the normal way using any routine coloring process. The coloring is done by spraying the copy paper with 0.03% diazotized sulfanilic acid in 10% sodium carbonate (Pauley's reagent). The coloring can also be carried out with a saturated solution of amido black in methanol-acetic acid (90:10 vol / vol are used). The staining time is 5 to 10 minutes. To decolorize, it is rinsed with 2 volumes of a solution of 90:10 methanol and acetic acid mixed with 1 volume of H20. It is difficult to get low background staining without extensive washing. The plates themselves can also be dried at around 600C (in an oven with air circulation), but only if the astrocytin is to be stained. For insulation purposes, the plate should only be air-dried at room temperature. Overheating can lead to cracking, but this can usually be avoided at a temperature of 50 to 60 ° C, at which a Sephadex G-200 sheet dries in 15 to 30 minutes. The dry plates are swollen in a mixture of methanol + H2O + acetic acid (75: 20: 5) for 10 minutes and stained with saturated amido black in the same solvent system for 5 hours and then washed by bathing in the same solvent for 2 hours . Then they are dried. To determine the molecular weight, the distance from the start line to the center of each zone is measured with an accuracy of 0.05 mm either directly on the print (copy) or on the densitogram. The result is expressed as the Rm value, which is defined as the ratio of the migration distance of the tested protein (dp) to that of the cytochrome C or myoglobin (dm) which is used as a reference protein. The relationship between the migration distance of the tested substance and the standard gives the formula A calibration line is obtained if the logarithm of the molecular weight of the standard samples used is plotted against Rm. The molecular weight of the unknown protein can be obtained from this straight line. To achieve the most exact values, equal parts of the protein sample solution are mixed with the standard, in this case cytochrome C, before application to the plate.

By the TLG procedure above, the product astrocytin is classified as separated spots at a distance of about 0.83 +/- 0.02 based on the Standard substance, cytochrome C, determined, which gives an approximate molecular weight of 8000 for the astrocytin results. There are several different products from separated from astrocytin on the basis of slight differences in molecular weight. Thus, three products are worn as contaminants up to this point with molecular weights of about 64,000, 148,000 and 230,000 and occasionally found a product with a molecular weight of 32,000 and removed by the TLG methods described above. The product astrocytin comes with the gel in which it is contained is sucked off in dry form, dissolved in the solution (1) and removed by centrifugation or similar measures freed from the resin.

The product astrocytin produced at this stage is soluble in distilled water, soluble at neutral and acidic pH and at insoluble in alkaline pH. It has a spectrophotometric at a wavelength of 280 m / u Absorption peak. It is a polypeptide with a molecular weight of about 8000 as stated above. The hydrolysis with 6N HCl gives the covalently linked Amino acids. The quantitative automatic investigation provides the following Average composition of amino acids: Approximate number of aspartic acid residues 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 approximate total 88 Cysteic acid, hydroxyproline, norleucine, Ammonia, isodesmosine, desmosine, hydroxylysine, lysinonorleucine and γ-aminobutyric acid are all absent in detectable amounts, but a trace of glucosamine may be present to be available.

From ii g of the starting brain tumor tissue of Example 1 are after the above methods produced approximately 3 mg of purified astrocytin.

B e i s p i e 1 2A Production of the "tumbling or vertigo" recognin Stumbling or vertigo is a genetic disorder that animals are not are able to achieve stable, coordinated motor activity, and in which due to the deficiency of certain nerve cells, to a special place to wander in the cerebellum, the cerebellum, at a particular time of development, a state of stumbling or dizziness is generated.

In electron microscopic examinations are special glial cells have been shown, which give rise to vertical, polar-like axes along which the nerve cells migrate to their new positions in the normal state. The failure of the nerve cells these glial fibers in the wobble resp.

Climbing vertigo is believed to be due to disorders in attributed to the nerve cells or the glia or both.

According to the methods of Examples 1 and 2, Recognin was obtained from the brain a mouse that moves with the wobble resp.

Dizziness, produced and compared with Recognin, made from the brain of a normal mouse. The molecular weight of the Recognin, which has been produced from all areas of the normal mouse brain was, was 8000. The molecular weight of the tumble or

Vertigo Recognins ranged from 3,600 to 5,000.

Vertigo Recognin is abnormal as it is considerably lower Molecular weight is displayed. Furthermore, the amount of Recognin that comes from the cerebellum of the tumbling or

Dizziness mouse brain is generated, compared to the normal case considerably reduced. This is shown in Table I.

Table I Recognin concentration in the mouse brain, mg / g normal tumbling or Vertigo disease cerebellum (16 day old mouse) 2.50 0.71 cortex (16 day old Mouse) 0.60 0.96 cortex (4 day old mouse) 0.29 0.53 brain stem (16 day old mouse) 0.90 1 * 64 whole brain (17 day old mouse) 1.27 1.53 whole brain (1 day old mouse) 5.00 5.86 'Table I shows that although there was a marked decrease in concentration von Recognin is present in the cerebellum of the wobbly or vertigo mouse, but in others Areas of the brain have the same or greater amounts of recognins. The recognition cannot move from other parts of the brain to the cerebellum or the slight increase in other parts of the brain of the tumbling or. Vertigo mouse may reflect a certain compensatory effect.

This pathological Recognin in the brain of the tumbling or. Vertigo mouse is related to the inability of the brain cells to migrate to the contacts make necessary to get proper placement, thereby the role of Recognine in recognition and learning in the cells is confirmed Analogously, the anti-recognize'ins' can be compared to that of the brain from tumbling or tumbling. Vertigo mice produced recognin are made and in used in a similar manner to the anti-astrocytin and anti-malignin described above will.

Example 3 Production of the malignin precursor in cultures of artificial Cancer cells.

Care is taken to ensure that the working method is sterile is applied.

All solutions (e.g. Hank's Balanced Salt (BSS), F-10 nutrient medium, serum from calves, trypsin solution) are at about 35 ° C incubated in a water bath for approximately 20 minutes or more before use.

Cells are removed from the tumor tissue and over many in vitro Generations bred using an appropriate medium, as shown below is described. The flasks to be used are filled with a sterilizing solution, e.g. 12-proponal plus amphyl or creolin solution, pre-rinsed.

In the preferred embodiment, the artificial cancer cells (i.e. cells grown in vitro for many generations) in 250 ml flasks let grow. The liquid medium in which the cells are grown is entered in pre-rinsed beakers. The cells are then moderate with 5 to 10 ml of a Hank's compensation solution BSS or a similar, other solution Washed for about 30 seconds. Avoid stirring.

All walls and surfaces are washed. The solution is made up of cells by centrifugation in the cold for 10 minutes at 3000 rpm clarified. The medium is poured into a beaker as above. It will be a minor one Amount of buffered proteinase enzyme solution added and it will rinsed quickly to avoid cell degradation. The preferred method 1 to 2 ml of trypsin solution (EDTA) are added and it only lasts for 10 seconds flushed. The trypsin solution is poured off.

A similar volume of fresh trypsin solution is added and it is incubated until it becomes apparent by microscopic observation that the cells separate from the walls of the chamber. This usually requires 5 to 10 minutes. A suitable growth medium, for example 50 ml of a solution, becomes suitable to a 7 to 10% solution of serum from calf fetuses in 100 ml of F-10 nutrient medium.

25 ml of the fresh medium with the cells are placed in a new growth chamber convicted for reproduction.

Both chambers are placed in a 35 incubator for approximately 7 days 0C given. After working this example up to this point, a Culture the artificial cancer cells in two fresh cultures approximately every 7 days divided up. This entire procedure can be performed approximately as many times as desired 7 day intervals are repeated for each growth chamber. Thus, the The number of cells growing in vitro should be doubled approximately every 7 days.

The cells are ready to produce malignin after approximately 7 days of growth can be extracted. For example, cells in the grown in individual 250 ml growth chambers.

The medium is transferred to a centrifuge tube and placed in the cold Centrifuged at 3000 rpm for 10 minutes. The medium is discarded. In the Cells remaining in the growth chamber are scraped off the chamber walls and with a neutral buffer solution in the centrifuge tubes washed into it. The cells are washed twice with a neutral buffer solution, again in centrifuged the cold at 3000 rpm and the medium is discarded.

The washed cells are suspended in 10 ml of neutral phosphate buffer, until it is necessary for the extraction of the fraction containing the crude malignin precursor, to be ready.

Example 4 Production of the crude fraction containing the malignin precursor contains.

Washed cells suspended in the neutral buffer of the example 3, are broken mechanically under conditions where denaturation of the most proteins are avoided. The preferred method is to wash the Cells treated in the cold with a sonic device for 20 seconds.

After sonication, the cell debris is 30,000 rpm for 30 minutes centrifuged and the supernatant product is decanted. 10 ml aliquots of the buffer solution are used to wash the remaining cell debris. It is sonicated and centrifuged as described above, and the supernatant products are combined.

This procedure is repeated once.

The combined supernatant is evaporated to volume from about 30 ml to about 6 to 7 ml. An aliquot becomes the Total protein analysis is taken and the rest is performed according to the methods of the Example 1 for the preparation of the astrocytin precursor fractionated.

Example 5 Preparation of the purified malignin product from the Crude fraction that contains the malignin.

The product malignin is produced according to the methods of Example 2 further isolated from impurities by astrocytin.

At the TLG stage of the preferred embodiment, the product becomes Malignin as a pronounced stain at a distance of approximately 0.91 +/- 0.02 observed on the standard cytochrome c, which has an approximate molecular weight of 10,000 for the malignin.

The malignin produced at this stage is in distilled water soluble, soluble at neutral or acidic pH and insoluble at alkaline pH. It has a spectrophotometric absorption peak of 280 m / u.

It was a polypeptide with a molecular weight of about 10,000.

In Table II are the molecular weights of malignin determined by thin layer gel chromatography. The malignin was stabilized in Fermentation cultures produced in successive generations of the cultures been. The reproducibility of the molecular weight determinations is in view the inherent limitations of TLG chromatography.

Table II Reproducibility of the molecular weight of the produced Malignin experiment no. Mol. Trial No. Mol. Trial No. Mol.

Weight

1 9500 9 10 100 17 10 180 2 8900 10 10 180 18 10 190 3 10000 11 10 180 19 10 190 4 10 050 12 10 180 20 10 180 5 10 100 13 10 180 21 10 000 6 10 000 14 10 050 22 9 500 7 10 150 15 10 180 23 10 180 8 12 500 16 10 190 the Hydrolysis with 6nHC1 reveals the covalently linked amino acids of malignin. the quantitative analysis gives the following average composition of amino acids: Approximate number of residues 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 approximate total 89 The amino acids cysteic acid, hydroxyproline, norleucine, ammonia, isodesmosine, desmosine, Hydroxylysine, lysinonorleucine and γ-aminobutyric acid are not in detectable amounts available.

A typical yield of pure malignin from twelve 250 ml reaction chambers of Example 3 total is approximately 1 mg of malignin.

The unique structures of malignin and astrocytin were made through extensive computer tests confirmed, being their compositions have been compared with practically all known protein substances.

The amino acid composition of malignin and astrocytin, the absolute and relative amounts of each amino acid component expressed as a total number the amino acid residues / mole, and the absolute and relative amounts of each amino acid component, in terms of the molecular weight of the molecule, were a template computer analysis against the largest known enumeration of protein structures in the world, viz the National Biomedical Research Foundation, Washington, D.C. At the Matrix analysis comparison with several hundred thousand proteins and protein fragments no structure was found to be identical to that of astrocytin or malignin was identical or very close to her.

The only proteins that are structurally related in any way are in Table III for comparison along with their individual amino acid compositions and molecular weights. The computer was programmed to any degree of structural similarity from several hundred thousand possibilities identified. For example, proteins with a molecular weight that is considerably suitable is larger or smaller, in computer analysis it is not. Even such proteins fewer than 85 or more than 95 residues, nor those with less than 12 or more than 15 glutamic acid residues nor those with less than 6 or more than 11 aspartic acid residues etc.

for each of the twenty amino acids concerned are found on computer analysis no fit.

Table III Comparison of the structures of astrocytin and malignin with the closest structures through a computer analysis Astro- Malignin Cyto- Ferrodoxin Acyl Carrier Bovine Pig Gonadotropin cystine chrom Luc. Alf. E. Coli neuro- neuro- releasing b5 Eq. physin physin of the hormone aspartic acid 9 9 9 10 8 7 2 3 0 threonine 5 5 6 4 6 6 2 2 0 serine 6 5 5 7 8 3 6 7 1 glutamic acid 13 13 14 13 13 14 9 9 0 proline 4 4 3 5 3 1 8 7 1 glycine 6 5 6 7 7 4 16 14 2 alanine 9 7 4 6 9 7 6 7 0 Valine 4 6 4 6 9 7 4 2 0 1/2 Cysteine 2 1 0 5 5 0 14 14 0 Methonine 1 2 1 0 0 1 1 1 0 isoleucine 2 4 4 4 4 7 2 2 0 leucine 8 8 7 10 6 5 6 7 1 tyrosine 2 3 3 3 4 1 1 1 1 phenylalanine 3 3 3 3 2 2 3 3 0 lysine 8 6 7 5 5 4 2 2 0 histidine 2 2 7 1 2 1 0 0 1 arginine 4 5 3 2 1 1 7 5 1 asparagine 0 0 0 0 1 2 3 2 0 thypophane 0 0 1 1 1 0 0 1 0 Glutamine 0 0 0 4 3 4 5 4 0 Total number of residues 88 89 87 96 97 77 97 92 10 molecular weight 8000 10 000 10 035 10 493 8509 At this "Fingerprint analysis" must therefore match or match 22 individual variables. It turns out that although some substances have one, four or five variables match, but none match or match with respect to all 22 variables. In fact, there is no better match than with 14 variables, where thus differences in 8 variables remain.

The closest substance is therefore cytochrome b5 (from humans). As can be seen from Table III, the alanine, arginine, Aspartic, aspartic acid, cysteine, glutamine, histidine, methionine, tyrosine and residual tryptophan numbers all significantly to markedly different from those of astrocytin and malignins. Since cytochrome b5 contains 7 histidine residues, while astrocytin and malignin contain only 2 residues, they cannot have the same chemical structure.

The most unexpected finding regarding the composition of the Astrocytins and Malignins is the high concentration of glutamic acid. One would expect to find only 5 or 6 such residues under 89.

Other, closest substances are the ferrodoxins from Leucaene glauca and from alfalfa. However, these substances differ markedly in terms of four or six amino acids and noteworthy with regard to two others and finally in that they have 96 and 97 residues, respectively. The closest substance is E. coli 26 acyl carrier protein, but this substance is markedly different in terms of eleven amino acids from malignin and astrocytin and it has only 77 residues.

Finally, in Table III, other brain proteins (neurophysin, from beef and pork, as well as the gonadotropin-releasing hormone), to show how much worse the consistency in the computer memory bank for the remaining several hundred thousand protein fragments.

Respiratory proteins can contain metals and / or heme components in their situ state, but contains the isolated protein fragment, e.g. for apocytochrome b5, none of them. Another microanalysis of astrocytin and malignin showed that these substances are free of iron, sulfur, phosphorus and magnesium (i.e. they are present in amounts less than 0.01%), and the spectral properties show a typical absorption at 280 m / u. After recombination of heme with apoprotein the typical absorption spectra between 400 and 450 m / u are restored.

Despite the structural uniqueness of astrocytin and malignin compared to all other proteins and protein fragments it is worth mentioning and possibly of great importance that the closest structures are those of the Respiratory proteins are. It is known that many important relationships are dated from both developmental genetic point of view as well as from the functional point of view of the type of structure represented can be drawn. When astrocytin and Malignin are new protein products whose in situ structural equivalents are respiratory or respiratory functions, and if these proteins, as will now be shown, have a relationship with the malignancy, then a possibility has been found to solve one of the greatest mysteries of Cancer - i.e. how the energetic relationships are sufficient for these insatiable, rapidly reproducing malignant cells is done.

Apart from the theoretical importance of this discovery complement the above values regarding the increase in the percentage of malignin in more malignant cells with the values given below, indicating that anti-malignin not only adheres to the cancer cell's malignant-like chemical groups, but that this adhered product is cytotoxic to the cells.

When the malignant in situ compounds in the cancer cells Respiratory proteins are preferred because the anti-malignant products of the invention are preferred attach to these in situ connections, then it is when functional respiratory Groups play a role in this pinning, easy to understand how to do this Cancer cells die off.

The therapeutic options for the anti-malignin and others Similar chemical counterparts are identified by this structural information about malignin and astrocytin, as well as the relationship shown between the amount of malignin and the degree of malignancy is greatly increased.

Example 5A This example shows that an increased yield and an increased degree of malignancy and an increased proportion of malignin can be obtained, if you have a larger volume and a larger surface during fermentation provides.

Examples 3 to 5 were repeated, but instead of the 250 cc flask 1000 cc flasks were used.

All reagent amounts were increased three-fold. The yield of the product Malignin after a 7 day growth of the inoculum was increased almost twice, by increasing the space available for the growth of the malignant cells from 250 cc to 1000 ccm has been increased. Table IV shows the total protein yield in mg and that educated Malignin as a percentage of total protein in consecutive Generations of fermentation cultures in flasks of all sizes. Under use from 250 cc flasks, the average total protein produced was 17.5 mg. Under Using 1000 cc flasks, the average total protein produced was 40.4 mg.

Surprisingly, when the rate of growth of the malignant Cells (degree of malignancy) over a 7 day growth period by disposing increased space and surface area for cell growth, the total amount of malignin produced, expressed as a percentage of the total Protein, significantly increased. The percentage of total protein that is made up of malignin was based on an average of 10.7% using 250 cc pistons to an average of 28.3% using 1000 cc pistons at a constant Growing period increased by 7 days.

The relationship between the ratio of malignin formed and the degree of malignancy (i.e. as a function of the extent of malignant cell growth in vitro over 7 days, measured as total protein formed) is shown in the figure.

Table IV Improved Yields with Successive Generations of fermentation production cultures of malignin flask size malignin total protein % Malignin ccm mg 250 0.33 6.4 5.1 250. 0.16 6.7 2.4 250 0.21 8.9 2.4 250 1.3 26.3 4.8 250 1.4 21.6 6.4 Table IV (continued) Flask Size Malignin Total Protein% Malignin ccm mg 250 2.6 17.9 14.4 250 1.8 16.4 10.7 250 1.3 13.4 9.8 250 2.0 17.8 11.3 250 2.3 18.9 12.0 250 2.1 19.4 10.8 250 1.6 13.8 11.6 250 2.2 15.1 14.6 250 4.4 21.6 20.4 250 3.3 14.0 23.2 250 2.2 23.0 9.7 250 2.1 23.2 9.0 250 2.8 22.3 12.5 250 2.4 18.9 12.7 250 2.4 24.5 9.8 mean 17.5 mg 10, 7 1000 9.8 41.3 23.6 1000 7.2 25.4 28.4 1000 5.9 24.9 23.6 1000 11.7 37.5 31.2 1000 13.3 44.8 29.8 1000 16.5 56.5 29.4 1000 9.5 41.3 22.9 1000 10.7 38.8 27.5 1000 12.5 41.6 29.9 1000 13.3 46.7 29.4 1000 11.6 45.2 25.7 Mean 40.4 mg 28.3% That Example 5A shows that the growth of artificial cancer cell cultures on a large scale Surprisingly, growth containers have an increased proportion of the produced Malignins causes, i.e. an increase in the percentage of total protein produced, which consists of malignin. Here, under a "" large-sized growth container " to be understood such, in which the ratio of container volume to the Volume of total medium with those used according to the methods of Example 3 Cells larger than about 8: 1, e.g. 7: 1 to 10: 1.

Example 5A describes a ratio of about 8: 1.

Example 6 Preparation of Target Reagents from Recognins.

Astrocytin, made as in Example 2 above, or Malignin, made as in Example 5 above, is applied with a slide to form a target reagent transferred into a complex.

In the preferred embodiment, astrocytin or malignin dissolved in a 0.15 M NaH2PO4 citrate buffer with a pH of 4.0. A bromoacetyl resin, e.g. bromoacetyl cellulose (BAC), with 1.0 to 1.5 meq.

Bromine / g cellulose, stored in the cold, is in 0.15 M NaH2PO4 puff he prepared with a pH of 7.2. The buffer is brought to a pH of 4 adjusted by pouring off the buffer solution with the pH value of 7.2 and adding 0.15 M NaH2PO3 citrate buffer with a pH of 4.0 is added. The astrocytin or Nalignin solution and the BAC solution are mixed together (ratio of BAC to Recognin 10: 1) stirred for 30 hours at room temperature and then centrifuged.

It is preferred that all sites on the BAC be available for binding available with the Recognin will. This can be like follows to be achieved. The protruding product from the immediately preceding one Stage is lyophilized and the protein content is determined to determine the amount of astrocytin or malignin that has not been complexed with the BAC. The complexed BAC astrocytin (or BAC malignin) is in a 0.1 M bicarbonate buffer resuspended with a pH of 8.9 and stirred at 4OC for 24 hours the formation of chemical bonds between the BAC and the astrocytin or malignin to allow. After the 24 hours, the suspension is centrifuged and the supernatant Product is analyzed for protein. The complexed BAC astrocytin or BAC malignin is now in 0.05 M aminoethanol - 0.1 M bicarbonate buffer with a pH of 8.9 resuspended to block any unreacted bromine. The suspension is centrifuged and the supernatant is saved, but due to the presence of aminoethanol was not analyzed. The removal of the whole, The unbound astrocytin or malignin is then removed by centrifugation and resuspension for three washing stages in 0.15 M NaCl until in a spectrophotometer at 266 m / u Absorption is no longer measured, achievedL. The BAC-astrocytin or BAC-malignin complex is now stirred in 8 M urea solution for 2 hours at 380C, centrifuged, then Washed with 8 M urea solution (usually three washes is sufficient off) until no more absorption is shown in the wash liquid at 266 m / u. The complex is then washed twice with 0.15 M NaCl to remove the urea. The complex is then passed in 0.25 M acetic acid at 37 ° C. for 2 hours to demonstrate its stability. It is centrifuged and the supernatant product is examined at 266 m / u, whereby no absorption should be present. This chemically complexed BAC-astrocytin or BAC-malignin is therefore stable and it can now be used as a reagent in the methods described below. In this form of a stable Reagent it is called synthetically Complex, the physical and chemical properties of which make the stable, mimic the cell-bound precursors of astrocytin or malignin when it is in is in a reactive potential state with the serum components.

For storage, the target reagent is centrifuged and diluted with 0.15 M NaH24 buffer with a pH of 7.2 washed neutral.

Target reagents can be made from other bromoacetyl-liganded carriers, except cellulose, e.g. bromoacetylated resins or even filter paper will.

EXAMPLE 1 7 Production of antisera to astrocytin, Malignin and Target.

Antisera to astrocytin, malignin, or target reagents can can be produced when there is an antibody reaction against this in a mammal induced. The following procedure has proven to be satisfactory for this purpose.

1 mg of Recognin (astrocytin or malignin) is taken along with Freund's standard adjuvant injected into the toe pads of male white rabbits. Then the same injection intraperitoneally one week later, again intraperitoneally 10 days and, if necessary, 3 weeks later. Specific antibodies can be used in the Blood serum from these rabbits as early as one week to 10 days after the first injection getting discovered. The same procedure is used for the target antigen, where the amount of target injected that contains 1 mg of astrocytin or malignin, determined by the Folin-Lowry protein determination.

The specific antibody to astrocytin is called anti-astrocytin designated. The specific antibody to malignin is called anti-malignin designated. Likewise, the specific antibody to target reagent will be referred to as anti-target.

These antibodies show clearly on standard Ouchterlony gel diffusion tests for antigen-antibody reactions with specific, single, sharp reaction lines, which are formed with their specific antigen.

The presence of specific antibodies in the serum can also by the quantitative standard precipitin test for antigen-antibody reactions be tested.

Good quantitative precipitin curves are obtained and can be used therefrom the micrograms of the specific antibody are calculated.

Another evidence of the presence of specific antibodies in the serum can be anti-astrocytin by absorption of the specific antibody on bromoacetylcellulose-astrocytin (BAC-astrocytin), as it was prepared above, can be obtained. The antiserum, which contains specific anti-astrocytin, can with BAC astrocytin are implemented. If the serum is passed through BAC astrocytin, then only the specific antibodies against astrocytin bind to their specific Astrocytin antigen.

Since astrocytin is covalently bound to bromoacetyl cellulose, is now the specific antibody, anti-astrocytin, bound to the BAC-astrocytin in order to BAC-Astrocytin-Anti-Astrocytin (BACA-Anti-Astrocytin). This is because of this proved to test the rest of the serum which was washed free of BAC astrocytin has been. In the standard Ouchterlony diffusion, no antibodies remain in the serum, which could react with the astrocytin. It will therefore concluded that all specific antibodies (anti-astrocytin), previously shown to be present in the serum, now that BAC astrocytin have been absorbed. Furthermore, if the anti-astrocytin of his When binding to BAC-astrocytin is released, then it is hereby contaminated by all Antibodies have been isolated free. This release of anti-astrocytin can occur in the Way can be achieved that the coupled BACA anti-astrocytin with 0.25 M acetic acid (40C, 2 hours), showing the BAC-astrocytin binding as shown above is not broken.

Another evidence of the presence of specific antibodies Anti-malignin can be found in the serum by adsorption of the specific antibody the bromoacetyl cellulose malignin (BAC malignin) prepared above. The antiserum containing the specific anti-malignin can be reacted with BAC-malignin will.

If the serum is passed over BAC malignin, then only bind the specific antibodies for the malignin to their specific antigen malignin. Since malignin is covalently bound to bromoacetyl cellulose, it is now the specific one Antibodies anti-malignin bound to the BAC-malignin to form BAC-malignin-anti-malignin (BACM anti-malignin). This is proven by doing the rest of the serum which has been washed free of BAC malignin. With the standard Ouchterlony diffusion no antibodies remain in the serum to react with the malignin. It is therefore concluded that all specific antibodies (anti-malignin), previously shown to be present in the serum from the BAC malignin have been absorbed. If the anti-malignin continues from its binding to BAC-malignin is released then it is free of all contaminating antibodies been isolated. This release of anti-malignin can be achieved by man washes the BACM-anti-malignin complex with 0.25 M acetic acid (40C, 2 hours), whereby, as shown above, the BAC-malignin bond does not break.

The antibodies to target show clearly on standard Ouchterlony gel diffusion tests for antigen-antibody reactions with specific single reaction lines that with target formed which has a line of identity with the line of reaction to anti-astrocytin or anti-malignin antisera (i.e. those that are too the injection of astrocytin or malignin itself) show. Some rabbits have been found to have received Target prior to injection Levels of anti-target in their blood. These anti-target substances result, though they specifically with the target reagent as described in tests with human sera will be reacted to form approximately equivalent amounts of the two Types of TAG, S-TAG and F-TAG, (see the following examples).

Example 8 Discovery of malignant tumors by in vitro quantitative formation of globulins adhering to the target (target-attaching globulins) (TAG) of biological fluids.

Target reagent prepared according to Example 6 is washed to to remove unbound Recognin, which may be present due to decomposition. The following procedure is satisfactory. The target reagent will Stirred with acetic acid for 2 hours at 370C, centrifuged and the supernatant product is decanted.

The optical density of the supernatant product is read off at 266 m / u. If there is any absorption then washing is repeated until none further material is solubilized. The target is then buffered in phosphate Saline solution resuspended at pH 7.2.

(Standard S-TAG and F-TAG purified from previous reactions of human serum by the procedure below, if available, used as reference standard samples in the test of the target reagent, and Total rabbit serum determined to be S-TAG and F-TAG by others Contains target preparations.) The determination of slow binding (S-TAG) is like follows carried out. Frozen serum that contains more than has been stored for a few days. The serum is carefully made from fresh produce Whole blood or other body fluid according to standard known procedures manufactured. The following procedure has been found to be satisfactory. Blood is obtained by standing for 2 hours at room temperature in a glass test tube allowed to curdle. The clots are separated from the walls with a glass stirring rod and the blood is allowed to stand for a minimum of 2 hours (or overnight) at 40C. The clots are removed from the serum by centrifugation at 20,000 rpm for 45 minutes and 40C separated. The serum is decanted into a centrifuge tube and it becomes centrifuged again for 45 minutes at 2000 rpm and 40C. The serum is decanted and a 1% solution of methiolate (1 g in 95 ml of water and 5 ml of 0.2 M bicarbonate buffer with a pH of 10) is used to an extent of 1% of the volume of the serum added.

The serum samples obtained by the above or other procedures are made in an amount of 0.2 ml for each 0.25 ml aliquots of a target suspension reagent added, which per 0.25 ml Target reagent contains 100 to 200 / ug Recognin.

A double determination is carried out. The suspension will mixed at 40C to avoid clumping. For example, a small Rubber cap that is shaken quickly, used for 1 to 2 seconds, whereupon the tubes are slightly inclined in a Thomas shaker for about 2 hours can be shaken for a long time or more. The target reagent and that bound to it Proteins are separated from the serum. A procedure that proves to be satisfactory has been proven is the following. The tubes are then heated to 40C and 2000 for 20 minutes Centrifuged rpm and the supernatant product is decanted. The lump that formed by centrifugation is three times by remixing and Shake at room temperature washed with 0.2-0.3 ml of 0.15 M saline. It is centrifuged and the supernatant products are discarded.

The protein that remains attached to the target is cleaved from it and quantified. For example, 0.2 ml of 0.25 M acetic acid is added and the suspension is shaken in a 37C incubator for 1 to 2 hours. the Tubes are centrifuged at 2000 rpm and 40C for 30 minutes. The protruding product is carefully decanted to avoid particle carryover and the optical density of the supernatant product is read off at 280 m / u. The value The optical density is divided by a factor of 1.46, which gives the micrograms per ml of serum protein (S-TAG) can be obtained. Double determinations should not be anymore than 5%. Any other method used to determine the Protein content can be used, e.g.

the Folin-Lowry determination. However, the standards must be specified be the range of control and tumor values from S-TAG minus F-TAG concentration to determine.

The determination of the quick attachment (F-TAG) is carried out as follows. Frozen serum should not be used that will be longer than stored for a few days. The serum is carefully made from fresh produce Whole blood or other body fluid prepared using standard procedures. The procedure given in this example for the preparation of serum is satisfactory.

Serum samples prepared by the above or other methods are left to stand at 40C for 10 minutes less than the total time left in contact with the above target reagent via the S-TAG serum determinations (e.g. 1 hour 50 minutes if a "2 hour S-TAG determination was made." became). This procedure is the same as the temperature history of the S-TAG and F-TAG regulations.

There are 0.2 ml samples of the temperature equilibrium added serum to 0.25 ml aliquots of the target suspension reagent which contains 100 to 200 / ug Recognin per 0.25 ml of target reagent. It a double determination is carried out. The suspension is then at .40C approximately Blended for 10 minutes in a manner to avoid pellet formation. For example, a small rubber cap that is shaken quickly can take 1 to 2 seconds can be used for a long time, after which the tubes, which are arranged at a slight angle, are placed in a Thomas shaker Shaken for about 10 minutes. The target reagent and that bound to it Proteins are separated from the serum. A procedure that proves to be satisfactory has been proven is the following. The tubes are then run at 2000 rpm and 40C 20 Centrifuge for minutes and decant the supernatant. The through the lump formed by centrifugation is washed three times by adding 0.2 to Remix 0.3 ml of 0.15 M saline at room temperature and is shaken. It is then centrifuged and the supernatant products are discarded.

The protein that remains attached to the target is cleaved from it and quantified. The above in this example used to determine the S-TAG concentration procedure described is satisfactory. Any other procedure can also be used which is suitable for determining the protein content, e.g. the Folin-Lowry determination. However, the standards must be specified in such a way that they cover the area of control and record tumor values of the S-TAG minus F-TAG concentration.

The final results are expressed as / µg TAG / ml serum and they are equal to S-TAG minus F-TAG. The TAG values in patients without a brain tumor and other control samples currently range from zero (or a negative number) up to 135 / µg / ml serum. For all results with more than 1010 μg / ml one is repeated Determination displayed. Some other tumors, in particular, can give high TAG values when secondary (metastatic) tumors are present in the brain. The TAG values of patients with brain tumor- currently range from 136 to 500 / ug or more / ml Serum. At the first blind'1 "blind" test of 50 blood samples by the Procedures of this example using one made from astrocytine and bromoacetyl cellulose Target reagents, 11 of 11 brain tumors and 28 of 32 normals were correctly identified.

One of the four assumed normals (i.e. a control person without Brain tumor) had had a cancer of the thyroid gland, obviously some Had been successfully treated years earlier. The three remaining normals were People aged 60 to 70 who had poor health and who may have had an unrecognized cancer. Of the rest seven specimens were three of three cases of Hodgkin disease correct identified. One sample in the tumor area (136 to 500 / µg TAG / ml) matched one Patients with severe gliosis, and three samples in the non-tumor area (0 to 135 / ug TAG / ml) corresponded to patients who had an uncertain intercranial mass diagnosis, but no tumor, an osteosarcoma (a non-brain tumor) and a melanotic one Have had sarcoma (a non-brain tumor).

Following the procedures of this example, a subsequent one was made Investigation using a target reagent made from malignin malignant brain tumors and all normals.

Another followed by the procedures in this example Examination ranged the total number of human serum samples examined from 50 on 114. The suitability of these products and procedures is shown in Table V, which gives the results of these tests.

EXAMPLE 9 Diagnosing tumor cells by immunofluorescence.

The compounds have been shown to be anti-astrocytin, anti-malignin and S-TAG preferentially attach to tumor cells. This specificity allows the Use of these compounds for diagnosing tumor cells in histological sections, by adding dyes or radioactive substances with anti-astrocytin, anti-malignin or S-TAG combined. Standard marking techniques can be used for this purpose will. A suitable procedure using S-TAG is, for example, as follows.

A procedure which has been found to be satisfactory is a modified St.Marie method.

Samples of human brain tumor are frozen and 5 / u thick Sections are cut out. These will in a damp Containers stored at -700C for 4 to 8 weeks before staining. The conjugate can be a standard antiserum e.g. goat anti-rabbit conjugate. The conjugate is made by known techniques with fluorescent or other marking substances marked. Fluorescine-labeled goat anti-rabbit conjugate as commercially available can be used for this purpose. The fluorescence technique was a Standard technique used in which a 1: 200 to 1: 400 solution of TAG approx. 30 Minutes or more has been incubated on the tumor slice. Then was washed, to remove unattached TAG. Three washes with phosphate buffered saline have proven to be satisfactory. Then there is a conjugate inhibition with a fluorescine-labeled conjugate followed by washes performed on what this is followed by a microscopic examination. Normal cells and their processes are not shown both in the tumor sections and in the control sections of stained normal non-tumor brains. Fluorescence is bright in tumor glial cells and their processes.

EXAMPLE 10 Description of the cytotoxic effects of anti-astrocytin, Anti-malignin and S-TAG against tumor cells growing in tissue culture.

Standard tests can be used to determine cytotoxicity will. In general, the number of cells in a solid cell chamber is the is usually arranged to contain about 100 living cells, counted. These cells are then treated with the agent to be tested and the number the cells that are still alive are counted.

In a standard cytotoxicity test that obtained according to Example 8 S-TAG solution versus cells in tissue culture obtained from a patient using a 3rd to 4th degree glioblastoma that was of glial origin were well characterized, S-TAG revealed the death of all cells in the cell chamber even at high dilutions of 1: 100 and 1: 1000, which is only 0.2 and 0.02 / ug S-TAG / ml Solution corresponds. Similar results are obtained with high dilutions of anti-astrocytin and anti-malignin.

Both the specificity shown in Example 9 and that in Example 10 shown cytotoxicity are for the therapeutic possibilities of anti-astrocytin, Anti-malignin and S-TAG highly relevant for human brain tumors. This therapeutic Applications come to the practical diagnostic capabilities of both of these Appearances for tumor tissue that has been removed during surgery, the however, it requires diagnosis by histological examination, as described herein has already been shown.

Table V Normal malignancies Other malignancies Other malignancies Indeterminate brain tumors tumors, brain tumors, no cerebraldia primary secondary appearances. Secondary cerebral gnosis Serum Serum Serum Serum Serum Serum Serum Serum DAY DAY DAY DAY DAY DAY DAY DAY µg / ml µg / ml µg / ml µg / ml µg / ml µg / ml µg / ml µg / ml 124 19 54 65 459 270 364 1658 113 55 27 113 397 257 315 1449 105 51 41 130 236 188 44214 139 130 82 21 79 137 205 28814 20910 127 44 27 61 298 157 7510 38 127 21 123 397 18411 100 31 0 14 241 2711 125 0 14 20 241 11012 30 125 62 41 217 19215 250 118 38 34 147 39 89 93 93 127 3631 996 21 48 185 4 132 0 20 253 31 2703 120 82 253 42 7 16 20 565 34 58 20 55 277 76 24 113 0 137 48 62 72 7813 85 89 138 89 650 160 + This designation includes normal, non-tumor-related medical and surgical Disorders; 1-very sick; undiagnosed; 2-extra-intercranial brain mass, undiagnosed; 3-cut out gliosis; 4 malignant melanoma; 5 osteosarcoma; 6 brain cyst fluid kit; 7-adenocarcinoma of the colon; 8 gastrectomy; 9-headache; 10 emphysema; 11-polymyalgia; 12 colon cancer; 13 convulsions; 14-prostate cancer, secondary manifestations in the bones; 15-clinically normal, 18 months earlier than this abnormal serum TAG was obtained: now there is a severe headache as well as a loss of taste and loss of taste Odor developed.

B e i 5 p i e 1 11 Hydrolytic cleavage of the Recognine.

A solution from Recognin, in this case either Astrocytin or Malignin, with a pH between 1 and 2, is left in the cold. To 7 to 14 days, TLG chromatography shows that the molecular weight of the product has been decreased by about 200. If the solution is left to stand for a long time, then more units with a molecular weight of about 200 are every 7 split off up to 10 days. Thus, the molecular weight of astrocytin becomes 8,000 and the molecular weight of malignin of 10,000 in each case consecutively decreased by approximately 200 units.

The physicochemical properties of astrocytine are determined by everyone Maintain product down to a molecular weight of about 4000. the physicochemical properties of malignin are determined by each product up to one Maintained molecular weight down from about 5000. This is done through Ouchterlony gel diffusion tests shown against anti-astrocytin and anti-malignin.

This cleavage can also be carried out enzymatically, for example with trypsin or other proteinases, with similar results.

The molecular weight of these compounds by hydrolytic Cleavage of the Recognine may have been established approximately by the following Formulas can be defined: For products with the physicochemical properties of Astrocytin; 400 + 200 x = Y.

For products with the physicochemical properties of malignin; 5000 + 200 x = In this, Y means the molecular weight of the product and x is an integer from 0 to 19.

B e i s p i e 1 12 Manufacture of an artificial tissue or organ with recognition.

A tube made of plastic, metal, or some other suitable rigid Rigid wall material turns into a highly concentrated (i.e. 10 mg / ml) viscous Recognin solution, in this case either astrocytin or malignin, is immersed or impregnated with it until all surfaces are completely coated with the Recognin are. Alternatively, the Recognin solution can also go through and around the pipe Pressure can be applied until all surfaces are completely coated. The pipe is then dried or lyophilized in air or in a vacuum. The coating process is repeated several times to create multiple molecular layers of the Recognin coating build up.

The tube is now ready to be inserted into a cavity or tissue to become the or the astrocytin- or malignin-like precursor in the neighboring Contains tissue or fluid from a living mammal.

This artificial tissue or organ can be used to a reaction with foreign substances that would take place without a Recognin coating, to minimize or eliminate.

Artificial tissues or organs with other geometric shapes can be used can be produced in the same way.

The figure shows that the increased piston size approximately tripled that Proportional amount of the malignin product leads.

The figure also shows the relationship between malignin and the malignancy. The broken line represents an ideal linear relationship. This The relationship shown is certainly not trivial, since the ratio of the existing Malignin increases when the cells are less restricted in their growth (pathological) will. The normal in situ recognition function is available, as previously stated, related to contact inhibition of cell growth. The more pathological that As the malignant cells grow, the less contact inhibition takes place instead, and all the more, malignin becomes the predominant protein.

Claims (37)

P a t e n t a n s p r ü c h e process for the production of astrocytin, characterized in that one brain glioma tumor tissue with repeated disruption of the tissue is extracted with a neutral buffer to dissolve protein fractions or to solubilize, from the resulting extract of the dissolved or solubilized Proteins the fraction with a pK range of about 1 to 4 and that one a product with a molecular weight of about 8,000 was isolated therefrom. 2. The method according to claim 1, characterized in that the Step of separating the fraction with a pK range of about 1 to 4 in the manner carries out that the extract of the dissolved or solubilized proteins in there is a chromatographic column and eluted with increasing acidic solvents. 3. The method according to claim 2, characterized in that the Isolation is carried out in such a way that the eluate is filtered to remove a fraction to obtain the astrocytin and that you get the astrocytin from it by a Separate thin layer gel chromatography. 4. Astrocytin, characterized in that it is obtained in the manner It has been established that brain glioma tumor tissue can be broken down repeatedly extracted with a neutral buffer to dissolve or solubilize protein fractions, from the resulting extract of the dissolved or solubilized proteins the The fraction with a pK range of about 1 to 4 is separated off and a product is obtained therefrom isolated with a molecular weight of about 8,000. 5. Astrocytin, characterized in that it has its specific Antibodies in quantitative precipitin resp. Coagulin tests and Ouchterlony gel diffusion tests a single line precipitate or a single line precipitate that it forms in water and aqueous solutions soluble at an acidic or neutral pH and soluble at an alkaline pH insoluble is that there is a wavelength of the spectrophotometric absorption peak of 280 m / u and that it has a molecular weight of about 8,000. 6. astrocytin according to claim 4 or 5, characterized in that it has approximately the following amino acid composition: Approximate number of residues 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 Approximate total 88 being the amino acids Cysteic acid, hydroxyproline, norleucine, ammonia, isodesmosine, desmosine, hydroxylysine, Lysinonorleucine and γ-aminobutyric acid are not present in detectable amounts. 7. astrocytin according to any one of claims 4 to 6, characterized in that that it is able to do so with bromoacetyl cellulose to form bromoacetyl cellulose astrocytin to form a complex and, after injection into mammals, the specific antibodies Form anti-astrocytin, the anti-astrocytin being used in vitro against brain tumor cells is toxic and gives fluorescence from glioma cells when coupled with Fluorescein is performed. 8. A process for the production of malignin, characterized in that that artificial cancer cells that have grown in a fermentation culture with a neutral buffer, repeatedly disrupting the tissue, to remove the protein fractions to dissolve or to solubilize, extracted, from the resulting extract the The fraction with a pK range of about 1 to 4 is separated off and the product is obtained therefrom with a molecular weight of about 10,000 wins. 9. The method according to claim 8, characterized in that the Step of separating the fraction with a pK range of about 1 to 4 in the manner carries out that the extract of the dissolved or solubilized proteins in there is a chromatographic column and eluted with increasing acidic solvents. 10. The method according to claim 9, characterized in that the Isolation is carried out in such a way that the eluate is filtered to remove a fraction to get that Contains astrocytine, and that one contains astrocytine separated therefrom by thin layer gel chromatography. Malignin, characterized in that it is produced in the manner it has been known that one can make artificial cancer cells that are grown in a fermentation culture are, with a neutral buffer, with repeated disruption of the tissues to get the To dissolve or solubilize protein fractions, extracted, from the resulting Extract separates the fraction with a pK range of about 1 to 4 and that one the product with a molecular weight of about 10,000 is obtained therefrom. 12. Malignin, characterized in that it is specific with its Antibodies in quantitative precipitin resp. Coagulin tests and Ouchterlony gel diffusion tests a single line precipitate or a single line precipitate that it forms in water and aqueous solutions soluble at an acidic or neutral pH and soluble at an alkaline pH insoluble is that there is a wavelength of the spectrophotometric absorption peak of 280 m / u and that it has a molecular weight of about 10,000. 13. Malignin according to claim 11 or 12, characterized in that it has approximately the following amino acid composition: Approximate number of residues Aspartic acid 9 threonine 5 serine 5 glutamic acid 13 proline 4 glycine 6 - alanine 9 valine 709816/1199 6 1/2 cysteine 1 Methonine 2 isoleucine 4 leucine 8 tyrosine 3 phenylalanine 3 lysine 6 histidine 2 arginine, 5 approximate total 89 where the amino acids cysteic acid, hydroxyproline, norleucine, ammonia, isodesmosine, Desmosine, hydroxylysine, lysinonorleucine and γ-aminobutyric acid in measurable quantities are not present. 14. Malignin according to one of claims 11 to 13, characterized in that that it is able to do so with bromoacetyl cellulose to form bromoacetyl cellulose malignin to form a complex and, after injection into mammals, the specific antibodies Anti-malignin form, the anti-malignin being used in vitro against brain tumor cells is toxic and gives fluorescence from glioma cells when coupled with Fluorescein is performed. 15. Process for the preparation of globulin slowly adhering to the target (S-TAG), characterized in that blood serum or another body fluid with bromoacetylcellulose astrocytin or bromoacetylcellulose malignin about 2 Hours or more at low temperature to avoid decomposition reactions, and that the resulting material is treated with dilute acid to to split off S-TAG. 16. Globulin slowly adhering to the target (S-TAG), characterized in that that it was made in such a way that one could obtain blood serum or some other body fluid with bromoacetylcellulose astrocytin or bromoacetylcellulose malignin about 2 Hours or more at low temperature to avoid decomposition reactions, and that the resulting material is treated with dilute acid to to split off S-TAG. 17. Globulin slowly adhering to the target (S-TAG), characterized in that that it is soluble in aqueous buffered solutions that it is with astrocytin or Malignin in Ouchterlony gel diffusion tests a single line precipitate or a Single line precipitate forms that it cannot be dialyzed through cellophane membranes is that it is retained by millipore filters, which molecules with a Molecular weights in excess of 25,000 withhold that there are molecular weights in different States of aggregation as determined by thin layer gel chromatography of approx 50,000 and multiples thereof in the macroglobulin range, and that it has a wavelength of the spectrophotometric absorption peak of 280 m / u. 18. Process for the preparation of globulin rapidly adhering to the target (F-TAG), characterized in that one uses blood serum or another body fluid with bromoacetylcellulose astrocytin or bromoacetylcellulose malignin about 10 Minutes at low temperature to avoid decomposition reactions and treating the resulting material with dilute acid to produce it F-TAG split off. 19. Globulin (F-TAG) adhering quickly to the target, characterized in that that it was made in such a way that one could obtain blood serum or some other body fluid with bromoacetylcellulose astrocytin or bromoacetylcellulose malignin about 10 Minutes at low temperature to avoid decomposition reactions and treating the resulting material with dilute acid to produce it F-TAG split off. 20. Globulin (F-TAG) adhering quickly to the target, characterized in that that it is soluble in aqueous buffered solutions that it is with astrocytin or Malignin in Ouchterlony gel diffusion tests a single line precipitate or a Single line precipitate forms that it cannot be dialyzed through cellophane membranes is that it is retained by millipore filters, which molecules with a Molecular weights in excess of 25,000 withhold that there are molecular weights in different States of aggregation as determined by thin layer gel chromatography of approx 50,000 and multiples thereof in the macroglobulin range, and that it has a wavelength of the spectrophotometric absorption peak of 280 m / u. 21. Methods for the detection of cancerous tumors in living mammals, characterized in that the concentration of S-TAG and F-TAG is determined, by a known volume of the blood serum of the mammal or another Body fluid has been produced. 22. Product, characterized in that it is the physicochemical Has properties of astrocytin and that it has a molecular weight that is approximately by the formula 4000 + 200X = Y is defined where Y is the molecular weight of the product and X is an integer from 0-19. 23. Product, characterized in that it is the physicochemical Has properties of malignin and that it has a molecular weight that is approximately is defined by the formula 5000 + 200 X = Y, where Y is the molecular weight of the Product and X is an integer from 0 to 19. 24. A method for the production of anti-astrocytin, characterized in that that an antibody response to astrocytin is induced in a mammal. 25. Process for the production of bromoacetylcellulose-Recognin, thereby characterized in that Recognin is mixed with bromoacetyl cellulose in a suitable manner, that a complex is formed, one being the Recognin from the group Astrocytin and malignin. 26. Process for the production of bromoacetylcellulose-Recognin-Anti-Recognin, characterized in that anti-recognin with bromoacetylcellulose-recognin in suitable way that a complex is formed, reacted, wherein the Recognin selects from the group astrocytin and malignin. 27. A method for the production of anti-recognin, characterized in that that one bromoacetylcellulose-Recognin-Anti-Recognin through a hydrolytic treatment decomplexed with an acid or a proteinase enzyme, whereby the Recognin selects from the group astrocytin and malignin. 28. A method for the production of anti-malignin, characterized in, that an antibody response to malignin is induced in a mammal. 29. Method of diagnosing the presence of glial tumor cells in histological sections, characterized in that a substance from the Group fluorescein combined or conjugated TAG and fluorescein combined or anti-recognin conjugated applied to the incision, with a fluorescence occurs only with the glial tumor cells and where you get the Recognin from the group Selects astrocytin and malignin. 30.Methods for increasing the yield of TAG products from mammals, characterized in that one receives an immunologically effective dose of a substance from the group Recognine, Anti-Recognine and transferred into a complex with carriers Recognine injected into the mammal. 31. - Recognine, characterized in that they are able to to produce anti-recognins after injection into mammals, the anti-recognins are toxic to tumor cells in vitro and when coupling in tumor cells give fluorescence with fluorescein. 32. A process for the production of malignin, characterized in that that one has the malignin precursor in a fermentation culture of artificial cancer cells produces that a crude fraction containing the malignin precursor is produced, that a purified malignin product is obtained from the crude fraction containing malignin produces and that the fermentation culture of the artificial cancer cells in large dimensions Grows growth containers. 33. A method for the production of Recognines, characterized in that that one normal or diseased tissue with a neutral buffer under repeated Breaking up the tissue to dissolve or solubilize the protein fractions, extracted, from the resulting extract of the dissolved or solubilized Proteins the fraction with a pK range of about 1 to 4 and that one a product with a molecular weight of about 3,000 to 25,000 was isolated therefrom. 34. The method according to claim 33, characterized in that the Step of separating the fraction with a pK range of about 1 to 4 in the manner carries out that the extract of the dissolved or solubilized proteins in there is a chromatographic column and eluted with increasing acidic solvents. 35. The method according to claim 34, characterized in that the Isolation is carried out in such a way that the eluate is filtered to remove a fraction to obtain the Recognin and that the Recognin therefrom by thin-layer gel chromatography separates. 36. Recognin, characterized in that it is produced in the manner has been that you can normal or diseased tissue with a neutral buffer under repeated disruption of the tissue to dissolve or add protein fractions solubilize, extract, from the resulting extract of the dissolved or solubilized proteins separates the fraction with a pK range of about 1 to 4 and that a product with a molecular weight of about 3,000 to 25,000 isolated. 37. Recognin, characterized in that it has its specific Antibodies in quantitative precipitin resp. Kcagulin tests and Ouchterlony gel diffusion tests a single line precipitate or a single line precipitate that it forms in water and aqueous solutions soluble at an acidic or neutral pH and soluble at an alkaline pH insoluble is that there is a wavelength of the spectrophotometric absorption peak of 280 m and that it has a molecular weight of about 3,000 to 25,000.