CH639102A5 - Process for the preparation of recognins, their complexes, of antirecognins and their complexes and use of the complex prepared - Google Patents

Description

The invention relates to a method for producing recognins and a use of the complex produced.

The recognizers can be used to make their chemical counterparts, i.e. by bringing the recognizers or the recognizers on a carrier into contact with body fluids. These chemical counterparts are for diagnostic and therapeutic purposes, i.e. for the diagnosis and treatment of cancer. The chemical counterparts or chemo-reciprocals are substances that react with an immunochemical-like specificity with a recognin in vivo or in vitro, e.g. with a quantitative precipitation test, with Ouchterlony double diffusion or with immunofluorescence.

The method according to the invention for the production of recognins is characterized by the characterizing part of claim 1.

According to the present invention, these recognins are produced by repeatedly breaking open normal or diseased tissue or cells in the presence of a neutral buffer substance in order to extract protein fractions. The fractions obtained from this are then separated chromatographically, which necessitates a chemical process for isolating a protein fraction with a pK range from 1 to 4. This is achieved by chemical and enzymatic hydrolysis of the protein fraction, which results when acidic solvents are increasingly used in the chromatographic column in the elution process. The recognin is isolated therefrom as a fraction with a molecular weight of 3000 to 25,000.

One of the recognizers according to the invention is astrocytin. Astrocytin is produced from brain tumor tissue, preferably brain glioma tumor tissue. Protein fractions containing the astrocytin precursor are first extracted from the tissue. A preferred extraction method is to treat the tissue with a neutral buffer under homogenization conditions or by using other cell and tissue disruption techniques to solubilize protein fractions containing the astrocytin precursor.

At this point, the astrocytin precursor is still attached to large molecular weight substances such as Proteins, glycoproteins, lipoproteins, nucleic acids, nucleoproteins etc. bound. The solubilized proteins are then separated from the resulting tissue extract. The tissue extract solution is then clarified to remove insoluble particles. Low molecular weight contaminants are then removed from the resulting solution by a pre-evaporation concentration technique. The resulting solution is then treated to separate the astrocytin precursor from other contaminants to obtain a protein fraction which has a pK range between 1 and 4. Thus e.g. the solution was placed on a chromatographic column and eluted with increasingly acidic solvents. All fractions that elute down to a pK of 4 in the neutral or acidic range are discarded, and those fractions with a pK range of 1 to 4 are collected. The eluate is then treated to obtain a product with a molecular weight of approximately 8,000. This happens e.g. by first filtering the material to identify low molecular weight substances, i.e. remove those with a molecular weight below 1000, and filter again to remove those substances with a molecular weight above 25,000. The fraction with a molecular weight between 1000 and 25,000 is then further processed, e.g. by thin layer gel (TLG) chromatography to obtain astrocytin.

Thus, astrocytin can be prepared by extracting brain glioma tumor tissue with a neutral buffer with repeated homogenization and high-speed centrifugation, from which a fraction with a pK range of about 1 to 4 is separated from the resulting extract, from which fraction the substances with a high molecular weight, ie up to about 230,000, and that the product astrocytin, which has a molecular weight of about 8,000, is isolated therefrom.

The astrocytin product produced by this process is characterized in that it forms a single-line precipitate or a single-line precipitate with its specific antibody in quantitative precipitin or coagulin tests and Ouchterlony gel diffusion tests, that it forms in water and aqueous solutions is soluble with an acidic or neutral pH value and insoluble with an alkaline pH value that it has a wavelength of the spectrophotometric absorption peak of 280 mji. and that it finally has a molecular weight of about 8000.

Astrocytin is also characterized in that it has a very high percentage of residues of glutamic acid and aspartic acid and a very high ratio of these acids to histidine. A more detailed analysis of astrocytin is given below.

In a similar manner to that described above, another recognin, referred to as "malignin", is made from artificial cancer cells, i.e. Cancer cells that grow during in vitro fermentation. Malignin has a molecular weight of about 10,000 and a similar but different amino acid residue composition to the astrocytin, i.e. high ratios of glutamic acid and aspartic acid and high ratios of these acids to histine. A more detailed analysis of malignin is given below.

Thus, malignin can be prepared by extracting artificial cancer cells grown in a fermentation culture with a neutral buffer with repeated homogenization and high-speed centrifugation, from which the fraction with a pK range of approximately 1 to 4 is separated, from this fraction the Substances with a high molecular weight, ie up to about 230,000, and that the product with a molecular weight of about 10,000 is isolated therefrom.

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

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

Recognins are further characterized in that they are capable of complexing with bromoacetyl cellulose to form bromoacetyl cellulose recognin and that they spe5 after injection into mammals

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make specific antibody anti-recognin. The anti-recognin is specifically added to the recognizer precursor in situ. For example, an anti-recognin, namely anti-malignin, toxic to brain tumor cells in vitro.

Recognins such as astrocytin, malignin and similar substances are useful as products that can be introduced into a biological system to reduce foreign reactions, for example by coating a material with a recognizer. Another example is the introduction of a recognition to create the chemical counterpart in the biological system. Recognins can also be used as nutrients to encourage the growth of certain biological systems of which they are a part. Another possible application of the Recognine is the generation of target reagents, or target or target point reagents, which contain the Recognin complexes with a carrier, in order to facilitate the applicability in biological systems. For example, the complex the physico-chemical properties of the recognin itself. The carrier should be selected from those which form a complex with the recognin and which are essentially biologically inert.

Any known substances that form a stable complex with polypeptides or proteins can be suitable for complex formation with the Recognin. An example is a cellulosic material, e.g. Bromoacetyl cellulose. Apart from the fact that the carrier must be inert to the biological system, the carrier should be designed so that it does not change the specific physico-chemical properties of the recognin, which are useful for the purposes stated above.

The complexes from Recognin and its carrier are suitable for generating, separating and identifying the chemical counterparts or chemical reciprocals in any biological system with which they are brought into contact. The recognizer-carrier complex is also suitable for stimulating the production of the chemical counterpart precursor in any biological system into which it is introduced.

A class of chemical counterparts are the anti-recognizers, i.e. Anti-astrocytin and anti-malignin. These can be made by injecting the recognin into a biological system. An immunologically effective dose of the recognin is contacted with body tissues or fluids according to techniques known to produce antibodies in such a manner that an antibody response is induced. The anti-recognins can be used for the production of materials such as diagnostic, food and medicinal products for specific cells or sites in biological systems, the agent being introduced into the biological system in complex form with the anti-recognin. The anti-recognins are also suitable for the diagnosis of the presence of tumor cells in histological sections, the anti-recognizing being added to the section in combination or conjugation with a labeling substance such as dyes and radioactive substances. In this case, staining or radioactive labeling occurs only in the tumor cells. Another use for anti-recognin is to increase the yield of other usable chemical precursors (such as TAG products, as described below) of mammals, giving an immunologically effective dose of the recognin to the mammal or other biological system injected.

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

In contrast, when the astrocytin was generated from tumor tissue and antibodies to astrocytin (anti-astrocytin) were made and used in brain immunofluorescence studies, only tumor glia, but not normal (non-tumor) glia by the anti Colored astrocytin. Thus, anti-Tay-Sachs antibodies and anti-astrocytin are clearly different products due to the original tissues and the nature of the antibodies as well as the specific types of stained cells.

Another class of chemical counterparts are target reagents that have been complexed with their chemical counterparts. For example, Target (the product obtained when complexing astrocytin with a carrier such as bromoacetyl cellulose) is brought into contact with anti-astrocytin. This type of compound can thus be converted into a complex and used for the production of diagnostic nutrients and pharmaceuticals for specific cells or sites in biological systems. These compounds can also be used for cleaning processes. For example, Anti-astrocytin can be prepared by subjecting bromo-acetylcellulose-astrocytin-anti-astrocytin to a de-complexing treatment by hydrolytic treatment with an acid or a proteinase enzyme. Target reagents are also suitable for increasing the amount of TAG products (as described below) in biological systems, e.g. by bringing an immunologically effective dose of target into contact with body tissue or fluids.

Other chemical counterparts are TAG reagents (e.g. globulins adhering to the target). The TAG products are made by bringing target reagents into contact with body fluids over varying periods of time to form a complex and by cleaving the TAG from them.

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

One method of making S-TAG (globulin slowly adhering to the target = slow target attaching globulin) is done by taking blood serum or other body fluid with a target (ie bromoacetyl cellulose malignin) for about 2 hours or more at low temperature, e.g. at about 4 ° C, and that the S-TAG is cleaved from the resulting material, for example with dilute acid, over a period of about 2 hours and at a temperature of about 37 ° C. The S-TAG produced by this process is characterized in that it is soluble in aqueous, buffered solutions, that it forms a single-line precipitate with its corresponding recognition in Ouchterlony gel diffusion tests, that it cannot be dialysed through cellophane membranes, that it is retained by millipore filters which retain molecules with a molecular weight of more than 25,000, that it has molecular weights in different aggregation states, determined by thin-layer gel chromatography, of approximately 50,000 and multiples thereof in the macroglobulin range and that it has a wavelength of the spectrophotometric absorption peak of 280 mji.

In a process for producing F-TAG (fast

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Globulin adhering to the target (fast-target-attaching globin) is carried out in such a way that blood serum or another body fluid with target (i.e. bromoacetyl cellulose malignin) is used for about 10 minutes at low temperature, e.g. about 4 ° C, and that the resulting material F-TAG, e.g. with dilute acid, over a period of about 2 hours and at a temperature of about 37 ° C. The F-TAG produced by this method is characterized in that it is soluble in aqueous, buffered solutions, that it forms a single cell precipitate with its corresponding recognition in Ouchterlony gel diffusion tests, that it is not dialysable through cellophane membranes, that it is retained by millipore filters, which retain molecules with a molecular weight of more than 25,000, that it has molecular weights in different aggregation states, determined by thin-layer gel chromatography, of approximately 50,000 and multiples thereof in the macroglobulin range and that it has a spectral wavelength -photometric absorption peaks of 280 mji.

TAG products are useful for detecting cancer tumors in living mammals by determining the concentration of S-TAG and F-TAG produced by a known volume of mammalian blood serum or other body fluid and by measuring this concentration relates to amounts that have been determined to indicate cancer. TAG products are also suitable for diagnosing the presence of tumor cells in histological sections. The procedure is to use TAG that contains a labeling substance, e.g. a dye or a radioactive substance is combined, applied to the cut, whereby staining or radioactive labeling occurs only in the tumor cells. It has also been shown that TAG products are cytotoxic to tumor cells. TAG products are also suitable for the production of diagnostic, nutritional and medicinal products for specific cells or sites, these agents being introduced in complex form with the TAG product.

The normal cell division in plants or animals is restricted or inhibited when the cells come to occupy a particular space completely. The mechanisms (a) by which normal cells "recognize"

that they have filled in the space available to them, and (b) that the course of this recognition mechanism in turn inhibits cell division are both unknown. A group of compounds has now been prepared, the precursors of which have an increased concentration when normal recognition and learning occur. These connections are related to the recognition and learning in particles and cells and with the connection of the cells to each other. These connections are known as recognize. When attempting to make these compounds from normal cancer cells, they were found not to be present as such and at the same time that the cancer cells lost their ability to (a) recognize that they had filled their normal volume, and / or (b) interrupt the division when they have filled their normal volume, there have been changes in their molecular structure.

New compounds and methods of making these compounds have been discovered. These new connections are known as recognize. Recognins are new compounds that have physicochemical properties that mimic those configuration properties of cancer cells in terms of their inability to recognize and stop cell division. The Verwen639102

The recognition of the recognizer goes beyond a mere insight into the cancer mechanism, since it provides products and processes that are suitable for the diagnosis, treatment and prophylaxis of cancer.

Methods have been discovered by which artificially cultured cells can be used to produce malignants. An advantage of the methods described herein is that malignins and new products derived therefrom can now be effectively produced in virtually unlimited quantities.

The present invention goes beyond the field of cancer research and is directly applicable to any biological system where it is desired to affect overall growth and metabolism. Thus, by producing the respective compound or compounds of the corresponding cell type in artificial cultures and furthermore by producing products from these substances, it is possible for the first time to specifically influence any tissue, cells, cell organelles, sub-organic molecules or molecular clusters in any living system be exercised. Thus, for the first time, specific nutritional influences can be carried out at critical times in development, specific diagnostic, prophylactic and treatment methods as well as the construction of artificial bioelectric systems (e.g. in tissue or organ transplants). These artificial bioelectrical systems can carry the characteristic properties of the specific recognin, malignin or their chemical counterparts of normal tissue or the component to which they are adjacent and can thus be "recognized" as "foreign", thereby causing foreign body reactions including rejection can be avoided.

Another aspect of the present invention is the manufacture of a utilizable, specific, antibody-like product (anti-astrocytin) versus a specific brain product (astrocytin), which enables this antibody-like product to form complex with it and as a specific delivery vehicle for specific points in the body Nervous system of all types can be used. Malignins and astrocytin are recognized.

Another aspect of the present invention is the production of new products, namely globulin adhering to target (TAG) from biological liquids. These products are so named because they are produced by two reactions, the first reaction being the reaction 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. In the second reaction, the specific TAG is split off from the complex. By measuring the TAG generated in this way, a quantitative indication of the biological fluids of the living organisms is obtained whether a tumor is present in these organisms. A diagnostic test for tumors is thus provided according to the invention. Since TAG products and anti-malignin are physicochemically opposite or complementary to malignins,

they are referred to herein as chemical counterparts or chemo-ziprocal.

It was also found that two quantitatively and qualitatively different TAG products depending on the time allowed for the reaction of the serum with the special target reagent and depending on the time taken for the product to be split off has been converted into a complex, can be manufactured.

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After examining the amounts of these products made from a number of different people with brain tumors and various other medical disorders, as well as those who did not have an apparent disease process, it became apparent that the amounts of these two new products developed in give a given person an indication of whether the person had a brain tumor. A new serum diagnostic test for brain tumors has thus been discovered.

The suitability of these new products, in addition to their use in diagnosis from serum and other biological fluids, to indicate the presence of brain and other tumors, is illustrated by the fact that TAG and anti-recognition compounds are preferred in histological sections of brain tumor adhere to the glial tumor cells and the surrounding tissue that was removed during surgery of the brain tumor. This preferred labeling of the tumor cells by TAG and anti-recognizers is demonstrated by standard immunofluorescence techniques. Thus, the invention also provides a new method for determining whether tumor cells have advanced to the corners or edges of the removed tissue by means of a histological examination with a new degree of security, which gives an indication of the probability that in the There is still a tumor in the brain or other organ or that tumor cells on the periphery of the removed tissue are no longer present. This provides information about the possibility that the entire tumor has been removed from the brain or another organ. It has also been shown that TAG and anti-malignins, the production of which has been described above, are cytotoxic to glioma brain tumor cells which have been grown in vitro in a tissue culture. This high affinity for tumor cells in a different medium, which in this case have been grown in a tissue culture, is further evidence of the specific coupling potential of the new TAG products. This explains the name “target-attaching globulins” (TAG) (TAG), as do the properties of TAG with regard to the synthetic target product and the tumor cells in histological sections.

The cytotoxicity of TAG and anti-recognizers against tumor cells provides another, new diagnostic test for the serum of patients who are suspected of having cancer. Thus e.g. reacted the serum or other body fluid of these patients with target to form TAG. The TAG product is tested for tissue cytotoxicity in tumor cell cultures. Both the concentration of TAG and the level of cytotoxicity shown by the TAG, which can be generated from the serum of a given person, may not only be useful for diagnostic purposes, but may also be of value to the course of the disease to track preoperatively and postoperatively in a given patient. The coupling of radioactive and dye tracers to TAG provides new TAG products that are suitable in vivo for the diagnosis of tumors and their exact localization. Thus, intra-arterial or intravenous injection of appropriately labeled TAG into the cerebrospinal fluid or directly into the brain tissue or its cavities allows the presence of a brain tumor to be indicated by radioactive means or by visualization through the coupled dye, since the TAG specifically adheres only to the tumor cells. This method also enables the precise location of the brain tumor to be visualized. This represents an improvement to this in vivo diagnostic method using anti-astrocytin generated in rabbit blood to label brain tumor because the use of TAG generated from human serum avoids the possibility of foreign protein reactions. Since TAG and Anti-Recognine have chemically specific properties that allow preferential attachment to tumor cells containing astrocytin precursors both in vitro and in vivo, these products can be used both therapeutically and diagnostically if they e.g. are coupled with radioactive agents, proton capturing agents or other toxic physical or chemical agents, so that these toxic substances can preferably be localized by the specificity of these compounds to adhere to tumor cells compared to the neighboring normal cells. This selectivity is considered essential or at least an essential factor in order to achieve an effective chemical or physical therapy of 20 tumors. Such a factor has not yet been reached. The TAG has now shown its effectiveness in adhering preferentially to the tumor cells, so that for these reasons it should have a certain prospect as a new drug.

25 As can be seen from the examples, a type of TAG can be found in the serum of patients with malignant tumors, namely slow-TAG (SLOW-TAG) (S-TAG) versus rapid-TAG (FAST-TAG) (F-TAG ) are produced in relatively larger amounts from a given serum volume 30 than in patients without such tumors. This indicates that the concentration of a naturally occurring precursor (P-TAG) for TAG is increased or that there are other factors that favor the relative in vitro production of S-TAG compared to F-TAG. 35 The possible relationship between the function of the actual synthetic products Target and TAG and their precursors and, in turn, the functions of the postulated but not yet shown cell "antigens" and the circulating "antibodies" against which they may exist in vivo 40 is has not yet been cleared up. For example, F-TAG and S-TAG in antibody-like manner, discrete lines of reaction with astrocytin in ouchterlony gel diffusion, and injection of target into rabbits induces an increase in the yield of TAG products from rabbit serum after the reaction with the target. The discovery that there may be a normal level of precursor similar to the circulating antibody to a cell antigen that is hidden in the non-dividing cells raises the question of a possible function of the pair. It is proposed here that the TAG precursor (P-TAG) and target-like substances exist in vivo, which have a function of controlling cell proliferation and cell death. For example, during cell division, a cell component is exposed to the serum proteins, which is normally not the case. Exposure of this cell component could result in this component being converted into a target-like substance to which the attachment of a P-TAG-like molecule 60 from the serum could then take place, which would stimulate or inhibit cell division. Alternatively, a non-dividing cell that has been damaged or is malfunctioning can 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 cell destruction (e.g., synthetic, as described herein, anti-glioma TAG is versus glioma

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morocytes that grow in a tissue culture, pronounced cytotoxic). This could thus be a reflection of a normal mechanism for controlling cell division and either repairing or removing individual cells in the body throughout the life of the organism. If the exposure of the cell components is abnormally increased, so that abnormally large amounts of target-like cell substances are formed, e.g. with rapidly dividing cancer cells, such as in brain gliomas, an increase in the concentration of one type of serum P-TAG over another can be induced.

Regardless of the actual function of the precursors, the increase in the relative amount of a predominant type of TAG is slow-TAG (SLOW-TAG) (S-TAG), which can be produced in vitro from the serum of patients with malignant tumors using the methods described , the basis for the serum diagnostic tests described in the following examples.

example 1

Preparation of a crude fraction containing the astrocytin precursor.

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

Each sample is homogenized in a neutral buffer solution by sonication or other mechanical measures. For example, homogenize each part in 100 cc of a 0.005 M phosphate buffer solution with a pH of 7 per gram of tissue in a Waring mixer. Homogenization should take place in the cold to prevent denaturation of the proteins. For example, the mixer is pre-cooled in a cold room from 0 to 5 ° C and only started for about 3 minutes.

The homogenate is then centrifuged for clarification, for example with 80,000-fold gravity for 30 minutes in a cooled ultracentrifuge. The soluble supernatant is decanted and kept in the cold. The insoluble residue is rehomogenized with a further 100 cc of the neutral buffer and centrifuged as described above. The second soluble extract is combined with the first extract. Best results are obtained when this homogenization and centrifugation procedure is repeated until less than 50% protein / ml solution is obtained in the supernatant. For most tissues, this is achieved through the fifth extraction.

The solutions obtained in this way are combined and evaporated and followed by dialysis, e.g. Dialysis against a 0.006 M phosphate buffer in the cold, concentrated to obtain a volume of 15 ml. The volume of this solution is noted and an aliquot is removed for analysis of the total protein. The rest is fractionated to obtain a protein fraction with a pK range between 1 and 4. The preferred fractionation method is a chromatographic method as described below.

The solution is fractionated in a cold room (4 ° C) on a column with DEAE cellulose (Cellex-D) with the dimensions 2.5 x 11.0 cm. The column was equilibrated with a 0.005 M sodium phosphate buffer. Gradual changes in the elution solution are

carried out with the following solvents (solutions):

Solution (1): 4.04 g NaH2P04 and 6.50 g Na2HPÛ4 are dissolved in 151 distilled water (0.005 molar, pH 7); Solution (2): 8.57 g of NaH2P04 are dissolved in 2480 ml of distilled water;

Solution (3): 17.1 g of NaH2PÛ4 are dissolved in 2480 ml of distilled water (0.05 molar, pH 4.7);

Solution (4): 59.65 g of NaH2P04 are dissolved in 2470 ml of distilled water (0.175 molar);

Solution (5): 101.6 g NaH: P04 are distilled in 2455 ml

Water (0.3 molar, pH 4.3) dissolved;

Solution (6): 340.1 g NaH2PÛ4 are distilled in 2465 ml

Water (1.0 molar, pH 4.1) dissolved;

Solution (7): 283.64 g of 80% phosphoric acid (H3PO4) are dissolved in 2460 ml of distilled water (1.0 molar, pH 1.0).

A nerve tissue extract with a volume of 6 to 10 ml is added. The column is then run through. This is followed by the solution (1) and a reservoir of 30 ml of the solution (1) is attached so that this solution drips onto the column by gravity. Aliquots of 3 ml of the effluent are collected using an automatic fraction collector. The following elution solutions are gradually replaced with the following numbers of the elution tubes:

Solution (2): at tube 88; the solution is placed in the column on top of the resin, then it is considered and a reservoir of 50 ml of solution (2) is added; Solution (3): for tube 98; the solution is applied to the column on top of the resin, then pondered and a reservoir of 75 ml of solution (3) is added; Solution (4): at tube 114; the solution is applied to the column on top of the resin, then covered and a reservoir of 150 ml of solution (4) is added;

Solution (5): for tube 155; the solution is applied to the column on top of the resin, then considered and a reservoir of 125 ml of solution (5) is added;

Solution (6): for tube 187; the solution is applied to the column on top of the resin, then pondered and a reservoir of 175 ml of solution (7) is added.

Elution is continued to tube 260 where the elution is complete. Freshly made resin is used for each new volume of tissue extract. Each tube for the effluent is examined quantitatively for protein. The eluates in the tubes with the numbers 212 to 230 are combined and they contain the raw products from which astrocytin is produced.

Values have already been published with regard to this raw material, which is referred to as fraction 10B [Protein Metabolism of the Nervous System, pp. 555-69 (Pleum Presse, 1970); Journal of Neurosurgery, Volume 33, pp. 281-286 (September 1970)], but the specific product, referred to herein as astrocytin, has now also been cleaved from fraction 10B. The crude fraction 10B can be produced as a product in amounts between 0.1 and 10 mg / g of the original fresh nervous system tissue from which it was obtained. In addition to an astrocytin precursor, it contains varying amounts of covalently linked carbohydrate

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most 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 also contains high molecular weight protein products, several lipids and nucleic acids.

Example 2

Preparation of purified astrocytin from the crude fraction containing the astrocytin precursor.

The fraction containing the astrocytin precursor is still freed of impurities. In preferred embodiments, the material of Example 1 is chromatographed on Sephadex G-50 resin in a typical column with a length of 40 cm, a diameter of 2.5 cm and a volume of 196 ml. The pressure used 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 is used as the buffer. The first (flow) peak contains the astrocytin precursor along with impurities, while the subsequent peaks only contain impurities.

In the preferred embodiment, the products in the first flow peak are then concentrated on Sephadex Gl 5 and then dispensed onto a column of Cellex-D with the same solution or solutions (1) to (7) as in Example 1, and the same elution steps as in Example 1 are carried out. The product astrocytin is present as a sharp peak in the same tubes (No. 212 to 230) as before. It therefore maintains its behavior when chromatographed on Cellex-D without the presence of a large number of contaminants.

Low molecular weight contaminants can then be prepared by known techniques, e.g. be removed by filtration through Millipore discs. In the preferred method, the astrocytin product is freed from salt and other low molecular weight contaminants by filtration through Millipore Pellicon disks No. 1000 with 13 mm. This filter retains substances with a molecular weight of more 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 therefrom by washing with the solution (1) of Example 1 won.

Astrocytin is then obtained by isolating the compound having a molecular weight of about 8,000 from the above solution. In a preferred method, thin layer gel (TLG) chromatography is used as follows.

A commercially available device from Boehringer Mannheim GmbH; Pharmacia Fine Chemicals and CAMAG (Switzerland) used. The resin, namely 2.5 g Sephadex G-200 super fine, is prepared in 85 ml 0.5 M NaCl in 0.02 M Na: HPOjKHzPO-i phosphate buffer, pH 6.8 (6.6 to 7.0) . Allow to swell for 2 or 3 days at room temperature with occasional, gentle mixing. (Magnetic stirrers or other stirrers should not be used.) The swollen gel is stabilized for 3 weeks at refrigerator temperature, although bacterial and fungal growth can disrupt the swollen gel. If the gel is to be kept for long periods of time, a small amount of a bacteriostatic agent (e.g. 0.02% sodium azide) should be added. 2.5 g dry gel are used to coat two glass plates measuring 20 x 20 cm and a thickness of 0.5 mm. The plates are either left to dry at room temperature for 10 minutes and placed in a moist

Chamber transferred where they can be stored for about 2 weeks, or used immediately after appropriate pre-equilibrium (usually during the night for a minimum of 12 hours). The main function of balancing is the normalization of the volume ratio between the stationary and the mobile phase. When the plates are in pre-equilibrium, the substances to be detected are applied to the starting line using micropipettes as spots or strips. 10 to 20 ml of the 0.2 to 2% protein solution are applied to the edge of a microscopic object plate (18 x 18 mm) and held against the gel surface. The solution penetrates the gel within a few seconds. All samples are first made on the coverslips and then quickly applied. If insufficient material is used, it is difficult to locate individual spots after separation. If too much material is applied, there is no defined separation. The samples are diluted with buffer for easy handling and the samples are separated using the descending technique, the plate being arranged at an angle of 22 °. A flow rate of about 1 to 2 cm / h is most suitable. Labeling substances (e.g. cytochrome C, hemoglobin, myoglobin or albumin marked with bromophenol blue) are applied to the plate at various points and serve as reference proteins for calculating the relative distance (mobility) of the unknown substances. After the samples have been applied, the plates in the device are replaced and the paper tampon is pressed down slightly to 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 kept constant at 1 cm from the top of the vessel. The individual runs are usually completed in 4 to 7 hours depending on the progress of the separation. The separation can be followed directly for colored substances. The separated spots of the protein are easily visualized by transferring them to a paper sheet copy of the TLG plate after the chromatographic separation has been completed and by prewashing methanol + H2O + acetic acid - 90: 5 on the system for 48 hours: 5 stains. A 3 mm filter paper is used as the paper sheet. A sheet of paper measuring 20 - 18 cm is placed over the gel layer and just pressed on (rolled) enough to ensure contact with the gel. Care was taken to ensure that no air is trapped under the paper (copy) and that the gel layer is not disturbed. The liquid phase is aspirated from the gel layer through the paper, and this is removed after about 1 minute, immediately dried in an oven at 60 ° C. for 15 minutes and colored in a normal manner by any routine dyeing method. 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 amidose black in methanol-acetic acid (90:10 vol / vol are used). The dyeing time is 5 to 10 minutes. To decolorize, rinse with 2 vol. Of a solution of 90:10 methanol and acetic acid, mixed with 1 vol. H2O. It is difficult to get a low background color without washing extensively. The plates themselves can also be dried at around 60 ° C (in an oven with air circulation), but only if the astrocytin is to be stained. For insulation purposes, the plate should only be exposed to air at room temperature

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25th

30th

35

40

45

50

55

60

65

9

639102

be dried. Overheating can cause cracking, but this can usually be avoided at a temperature of 50 to 60 ° C, at which a Sephadex G-200 plate dries in 15 to 30 minutes. The dry plates are left to swell 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 being in the same solvent for 2 hours to be bathed. Then they are dried. To determine the molecular weight, the distance from the starting line to the middle 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 an Rm value, which is defined as the ratio of the migration distance of the protein tested (dp) to that of the cytochrome C or myoglobin (dm) used as the reference protein. The relationship of the migration distance of the tested substance to the standard gives the formula

(-R _ dP

v m - •

Table (continued)

Aspartic acid

Threonine

Serine

Glutamic acid proline

Approximate number of remnants

9

5

6 13

4th

Glycine

6

Alanine

9

Valine

4th

1/2 cysteine

2nd

Methionine

1

Isoleucine

2nd

Leucine

8th

Tyrosine

2nd

Phenylalanine

3rd

Lysine

8th

Histidine

2nd

Arginine

4th

approximate total 88

15

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. In order to achieve the most exact values, equal parts of the protein sample solution are mixed with the standard, in this case cytochrome C, before being applied to the plate. The above TLG procedure detects the product astrocytin as a separate spot at a distance of approximately 0.83 ± 0.02, based on the standard substance cytochrome C, which results in an approximate molecular weight of 8000 for the astrocytin. Several different products are separated from the astrocytin based on small differences in molecular weight. Thus, three products that have been carried as contaminants up to this point, with a molecular weight of approximately 64,000, 148,000 and 230,000, and occasionally a product with a molecular weight of 32,000, have been identified and removed by the TLG methods described above . The product astrocytin is suctioned off in dry form with the gel in which it is contained, dissolved in the solution (1) and the resin is removed by centrifugation or similar measures.

The product astrocytin, which has been produced in this step, is soluble in distilled water, soluble at neutral and acidic pH and insoluble at alkaline pH. It has a spectrophotometric absorption peak at a wavelength of 280 m | i. It is a polypeptide with a molecular weight of approximately 8000 as indicated above. Hydrolysis with 6n HCl indicates the covalently linked amino acids. The quantitative automatic analysis provides the following average amino acid composition:

25th

30th

Cysteic acid, hydroxyproline, norleucine, ammonia, iso-desmosine, desmosine, hydroxylysine, lysine non-leucine, and y-aminobutyric acid are all not present in detectable amounts, but there may be a trace of glucosamine.

Approximately 3 mg of purified astrocytin are produced from 11 g of the original brain tumor tissue of Example 1 using the above methods.

Example 2A

Production of the "wobble or dizziness" recognition The wobble or dizziness disease is a genetic disorder in which animals are unable to achieve stable, coordinated, motor activity and in which due to the inability of certain nerve cells to a special place in the cerebellum, the cerebellum, to migrate during a special development period, a state of wobbling or dizziness is generated. In electron microscopic examinations, special glial cells have been shown, which result in vertical, pole-like axes, along which the nerve cells migrate to their new positions in the normal state. The deficiency of the nerve cells, these glial fibers in the wobble or Climbing vertigo is thought to be due to disorders in the nerve cells or glia, or both.

Recognin was prepared using the methods of Examples 1 and 2 from the brain of a mouse with dizziness and dizziness, and compared with Recognin made from the brain of a normal mouse. The molecular weight of the recognizer, which was made from all areas of the normal mouse brain, was 8,000. The molecular weight of the wobble or dizziness recognizer was 3600 to 5000. The wobble or dizziness recognizer is abnormal, as evidenced by that lower molecular weight is displayed. Furthermore, the amount of recognition that is generated from the cerebellum of the tumbling or vertigo mouse brain is significantly reduced compared to the normal case. This is shown in Table I.

Table I

Recognin concentration in the mouse brain, mg / g

40

45

50

60

normal

Tumbling or dizziness

65 cerebellum (16 day old mouse) 2.50 0.71

Bark (16 day old mouse) 0.60 0.96

Bark (4 day old mouse) 0.29 0.53

Brain stem (16 day old mouse) 0.90 1.64

639 102

Table II (continued)

normal

Wobble or

Dizziness-

illness

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 is a marked decrease in the level of recognin in the cerebellum of the tumbling or vertigo mouse, the amounts of recognizers in other areas of the brain are the same or greater. Recognin cannot move from the other parts of the brain into the cerebellum, or the slight increase in other parts of the brain of the tumbling or vertigo mouse can reflect a certain compensatory effect.

This pathological recognin in the brain of the wobble or Dizziness mouse is related to the inability of the brain cells to migrate to make the contacts necessary for proper placement, thereby confirming the role of the recognizer in recognizing and learning in the cells.

The anti-recognins can be produced analogously to the recognin produced by the brain of tumbling or vertigo mice and can be used in a similar manner to the anti-astrocytin and anti-malignin described above.

Example 3

Production of the malignin precursor in cultures of artificial cancer cells.

Care is taken to ensure that a sterile working method is used.

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

Cells are removed from the tumor tissue and grown in vitro over many generations using an appropriate medium as described below. The pistons to be used are cleaned with a sterilization solution, e.g. 12-propanol plus amphyl or creolin solution, pre-rinsed.

In the preferred embodiment, the artificial cancer cells (i.e. cells that have grown in vitro over many generations) are grown in 250 ml flasks. The liquid medium in which the cells are grown is placed in pre-rinsed beakers. The cells are then washed gently with 5 to 10 ml of Hank's BSS leveling solution or similar solution for about 30 seconds. Avoid stirring. All walls and surfaces are washed. The solution is clarified by cells by cold centrifugation over a period of 10 minutes at 3000 rpm. The medium is poured into a beaker as above. A small amount of buffered proteinase enzyme solution is added and rinsed quickly to avoid cell degradation. In the preferred method, 1-2 ml of trypsin solution (EDTA) is added and rinsed for only 10 seconds. The trypsin solution is poured off.

A similar volume of fresh trypsin solution is added and incubated until microscopic observation shows that the cells separate from the walls of the chamber. This usually takes 5 to 10 minutes. A suitable growth medium, e.g. 50 ml of a solution of a 7- to 10% solution of veal serum in 100 ml of F-10 nutrient medium are added.

25 ml of the fresh medium with the cells are transferred to a new growth chamber for reproduction. Both chambers are placed in a 35 ° C incubator for approximately 7 days. Following the operation of this example up to this point, a culture of artificial cancer cells is divided into two fresh cultures approximately every 7 days. This entire procedure can be repeated as often as desired at approximately 7 day intervals for each growth chamber. Thus, the number of cells growing in vitro can be doubled approximately every 7 days.

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

The medium is transferred to a centrifuge tube and centrifuged in the cold at 3000 rpm for 10 minutes. The medium is discarded. The cells remaining in the growth chamber are scraped off the chamber walls and washed into the centrifuge tubes with a neutral buffer solution. The cells are washed twice with a neutral buffer solution, centrifuged again in the cold at 3000 rpm and the medium is discarded. The washed cells are suspended in 10 ml of neutral phosphate buffer until they are ready for extraction of the fraction containing the crude malignin precursor.

Example 4

Preparation of the crude fraction containing the malignin precursor.

Washed cells suspended in the neutral buffer of Example 3 are broken mechanically under conditions that avoid denaturing most proteins. In the preferred method, the washed cells are sonicated in the cold for 20 seconds.

After sonication, the cell debris is centrifuged at 30,000 rpm for 30 minutes and the supernatant is decanted. 10 ml aliquots of the buffer solution are used to wash the remaining cell residues. It is sonicated and centrifuged as described above and the supernatants are combined. This process is repeated once.

The combined supernatant is pre-evaporated to reduce the volume from approximately 30 ml to approximately 6-7 ml. An aliquot is removed for total protein analysis and the rest is fractionated according to the methods of Example 1 for the preparation of the astrocytin precursor.

Example 5

Production of the purified malignin product from the raw fraction that contains the malignin.

The product malignin is further isolated from impurities by the methods of Example 2 for the production of astrocytin.

At the TLG stage of the preferred embodiment, the product malignin is observed as a distinct stain at a distance of approximately 0.91 +/- 0.02 based on standard cytochrome C, which is an approximate molecular weight of 10,000 for that gives malignin.

The malignin produced in this stage is soluble in distilled water, soluble at neutral or acidic pH

10th

s

10th

15

20th

25th

30th

35

40

45

50

55

60

65

11

639102

and insoluble at alkaline pH. It has a spectrophotometric absorption peak of 280 mu. It was a polypeptide with a molecular weight of approximately 10,000.

Table II shows the molecular weights of malignin as determined by thin layer gel chromatography. The malignin had been produced in stabilized fermentation cultures in successive generations of the cultures. The reproducibility of molecular weight determinations is remarkable given the inherent limitations of TLG chromatography.

Tables

Reproducibility of the molecular weight of the malignin formed

attempt

Mol.

attempt

Mol.

attempt

Mol.

No.

No.

No.

1

9,500

9

10 100

17th

10180

2nd

8,900

10th

10180

18th

10190

3rd

10,000

11

10180

19th

10190

4th

10 500

12

10180

20th

10180

5

10100

13

10180

21st

10,000

6

10,000

14

10 050

22

9,500

7

10150

15

10180

23

10180

8th

12,500

16

10190

Hydrolysis with 6N HCl shows the covalently linked amino acids of malignin. The quantitative analysis shows the following average composition of amino acids:

Approximate number of remnants

Aspartic acid

9

Threonine

5

Serine

5

Glutamic acid

13

Proline

4th

Glycine

6

Alanine

9

Valine

6

1/2 cysteine

1

Methionine

2nd

Isoleucine

4th

Table (continued)

Leucine 8

Tyrosine 3

Phenylalanine 3

s Lysine 6

Histidine 2

Arginine _5

approximate total 89

The amino acids cysteic acid, hydroxyproline, nor-leucine, ammonia, isodesmosine, desmosine, hydroxylysine, lysinorleucine and y-aminobutyric acid are not present in detectable 15 quantities.

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

The unique structures of malignin and astrocytin 20 have been confirmed by extensive computer studies, their compositions being compared with practically all known protein substances.

The amino acid composition of malignin and astrocytin, the absolute and relative amounts of each 25 amino acid component, expressed as the total number of amino acid residues / mole, and the absolute and relative amounts of each amino acid component, expressed as the molecular weight of the molecule, were mapped to a template. Computer analysis against the largest known list 30 of protein structures in the world, namely the National Biomedicai Research Foundation, Washington, DC. In the matrix analysis comparison with several hundred thousand proteins and protein fragments, no structure was found which was identical to or very close to that of astrocytin or 35 malignin.

The only proteins that were structurally related in any way are listed in Table III for comparison, along with their individual amino acid compositions and molecular weights. The 40 computer was programmed to identify any degree of structural similarity from hundreds of thousands of possibilities. For example, Proteins with a molecular weight that is considerably larger or smaller are not used in computer analysis. Even proteins with 45 less than 85 or more than 95 residues, still those with less than 12 or more than 15 glutamic acid residues, still those with less than 6 or more than 11 aspartic acid residues etc. for each of the twenty amino acids in question result in computer analysis no fit.

Table III

Comparison of the structures of astrocytin and malignin with the closest structures by computer analysis

Astrocytin Malignin Cytochrome Ferredoxin Acylträger Bovine-Pig Gonadotropin b s ï m. ., E. coli neurophysin releasing neurophysin

Luc. Eq. Alf. v'y hormone

Aspartic acid

9

9

9

10th

8th

7

2nd

3rd

0

Threonine

5

5

6

4th

6

6

2nd

2nd

0

Serine

6

5

5

7

8th

3rd

6

7

1

Glutamic acid

13

13

14

13

13

14

9

9

0

Proline

4th

4th

3rd

5

3rd

1

8th

7

1

Glycine

6

6

6

7

7

4th

16

14

2nd

Alanine

9

7

4th

6

9

7

6

7

0

Valine

4th

6

4th

6

9

7

4th

2nd

0

Vi cysteine

2nd

1

0

5

5

0

14

14

0

Methionine

1

2nd

1

0

0

1

1

1

0

Isoleucine

2nd

4th

4th

4th

4th

7

2nd

2nd

0

Leucine

8th

8th

7

10th

6

5

6

7

1

639 102 12

Table HI (continued)

Astrocytin

Malignin

Cytochrome ferredoxin bs Luc. Eq.

Alf.

Acyl carrier E. coli

Bovine neurophysin

Pig neurophysin

Gonadotropin releasing

hormone

Tyrosine

2nd

3rd

3rd

3rd

4th

1

1

1

1

Phenylalanine

3rd

3rd

3rd

3rd

2nd

2nd

3rd

3rd

0

Lysine

8th

6

7

5

5

4th

2nd

2nd

0

Histidine

2nd

2nd

7

1

2nd

1

0

0

1

Arginine

4th

5

3rd

2nd

1

1

7

5

l

Asparagine

0

0

0

0

1

2nd

3rd

2nd

0

Tryptophan

0

0

1

1

1

0

0

0

1

Glutamine

0

0

0

4th

3rd

4th

5

4th

0

Total number of remnants

Molecular weight

88 8000

89 10,000

87 10 035

96 10493

97 8509

77

97

92

10th

In this “fingerprint analysis”, 22 individual variables must fit or match. It turns out that some substances match one, four or five variables, but none match or match all 22 variables. In fact, there is no better match than with 14 variables, leaving differences on 8 variables.

The closest substance is therefore cytochrome bs (human). As can be seen from Table III, the alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, histidine, methionine, tyrosine and tryptophan residual numbers all differ considerably from those of astrocytin and malignin . Since cytochrome bs 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 astrocytin and malignin is the high concentration of glutamic acid. One would expect to find only 5 or 6 such residues among 89.

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

Finally, Table III lists other brain proteins (bovine and porcine neurophysin, as well as the gonadotropin-releasing hormone) to show how much poorer the match in the computer memory was for the remaining hundreds of thousands of protein fragments.

Breathing proteins can contain metals and / or heme components in their in situ state, but the isolated protein fragment contains e.g. for apocytochrome bs, none of them. Another microanalysis of astrocytin and malignin has shown that these substances are free of iron, sulfur, phosphorus and magnesium (ie they are present in 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 ma 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 narrowest structures are those of the respiratory proteins. It is known that many important relationships can be drawn both from the developmental genetic point of view and from the functional point of view in terms of the type of structure represented 2s. If astrocytin and malignin are new protein products whose in situ structural equivalents are respiratory and respiratory functions, respectively, and if these proteins, as will now be shown, are related to malignancy, then a way has been found at 30 to be one of the greatest puzzles of the To solve cancer - ie how the energetic conditions are, which are satisfied with these insatiable, rapidly reproducing malignant cells.

Apart from the theoretical importance of this discovery, the above values for the increase in the percentage of malignin in more malignant cells complement the values given below, which indicate that anti-malignin not only adheres to malignant-like chemical groups of the cancer cell, but also 40 that this adherent product is cytotoxic to the cells. If the malignant in-situ compounds in the cancer cells are respiratory proteins because the anti-malignant products according to the invention prefer to attach to these in-situ compounds, then it is when functional respiratory groups play a role in this attachment , easy to understand how this leads to cancer cell death.

The therapeutic possibilities for the anti-malignins and other similar chemical counterparts are greatly increased by this structural information about malignin and astrocytin as well as by the shown relationship between the amount of malignin and the degree of malignancy.

Example 5A

55 This example shows that an increased yield and increased degree of malignancy as well as an increased proportion of malignin can be obtained if a larger volume and a larger surface area are provided during the fermentation.

60 Examples 3 to 5 were repeated using 1000 cc flasks instead of the 250 cc flasks. All reagent quantities were increased three times.

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 cc. Table IV shows the yield of total protein in mg and the malignin formed as a percentage of the total protein

13

639102

the following generations of fermentation cultures in flasks of all sizes. Using 250 cc flasks, the average total protein produced was 17.5 mg. Using 1000 cc flasks, the average total protein produced was 40.4 mg.

Surprisingly, when the level of malignant cell growth (degree of malignancy) was increased over a 7 day growth period by providing a larger space and surface area for cell growth, the total amount of malignin formed, expressed as a percentage of the total protein, was significantly increased. The percentage of total protein consisting of malignin was increased from an average of 10.7% when using 250 cc flasks to an average of 28.3% when using 1000 cc flasks with a constant growth period of 7 days.

The relationship between the ratio of malignant 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 produced) is shown in the figure.

Table IV

Improved yields in successive generations of malignin fermentation production cultures

Piston size

Malignin

Total protein

% Malig ccm mg 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

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

Average

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

Average

40.4 mg

28.3%

Example 5 A shows that the growth of artificial cancer cell cultures in large-sized growth containers surprisingly causes an increased ratio of the malignin produced, i.e. an increase in the percentage of total protein formed that consists of malignin. A "large-sized growth container" is to be understood here as meaning one in which the ratio of the container volume to the volume of the total medium with the cells used according to the methods of Example 3 is greater than approximately 8: 1, e.g. 7: 1 to 10: 1. Example 5 A describes a ratio of approximately 8: 1.

Example 6

Manufacture of target reagents from recognins.

Astrocytin, prepared as in Example 2 above, or malignin, prepared as in Example 5 above, is complexed with a carrier to form a target reagent.

In the preferred embodiment, astrocytin or malignin is dissolved in a 0.15 M NaH2P04 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 prepared in 0.15 M NaH2P04 buffer with a pH of 7.2. The buffer is adjusted to a pH of 4 by pouring off the buffer solution with a pH of 7.2 and by adding 0.15 M NaH2P03 citrate buffer with a pH of 4.0. The astrocytin or malignin solution and the BAC solution are stirred together (BAC to Recognin ratio 10: 1) for 30 hours at room temperature and then centrifuged.

It is preferred that all sites on the BAC that are available for binding with the recognin be bound. This can be accomplished as follows. The supernatant from the immediately preceding step 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 resuspended in a 0.1 M bicarbonate buffer with a pH of 8.9 and stirred for 24 hours at 4 ° C in order to form chemical bonds between the BAC and the Allow astrocytin or malignin. After the 24 hours, the suspension is centrifuged and the supernatant is analyzed for protein. The complexed BAC-astrocytin or BAC-malignin is now resuspended in 0.05 M aminoethanol-0.1 M bicarbonate buffer with a pH of 8.9 in order to block any unreacted bromine. The suspension is centrifuged and the supernatant is saved, but not analyzed due to the presence of aminoethanol. The removal of all unbound astrocytin or malignin is then by centrifugation and resuspending for three washing steps in 0.15 M NaCl until in a spectrophotometer at 266 ml. absorption is no longer measured. The BAC-astrocytin or BAC-malignin complex is now stirred in 8 M urea solution for 2 hours at 38 ° C., centrifuged, then washed with 8 M urea solution (usually three washes are sufficient) until none in the washing liquid at 266 mu Absorption is shown more. The complex is then washed twice with 0.15 M NaCl to remove the urea. The complex is then stirred at 37 ° C in 0.25 M acetic acid for 2 hours to demonstrate its stability. It is centrifuged and the product beyond is at 266 mji. examined, with no absorption should be present. This chemically complexed BAC astrocytin or BAC malignin is therefore stable and can now be used as a reagent in the methods described below. In this form of a sta5

10th

IS

20th

25th

30th

35

40

45

50

55

60

65

639 102

14

bile reagent, it is called a synthetically produced complex whose physical and chemical properties mimic the stable, cell-bound precursor of astrocytin or malignin when it is in a reactive potential state with the serum components. 5 For storage, the target reagent is centrifuged and washed neutral with 0.15 M NaH2P04 buffer with a pH of 7.2.

Target reagents can be derived from bromoacetyl-ligated carriers other than cellulose, e.g. bromoacetylated 10 resins or even filter paper.

Example 7

Production of antisera against astrocytin, malignin and target. 15

Antisera to astrocytin, malignin or target reagents can be made by inducing an antibody response against them in a mammal. The following procedure has proven to be satisfactory. 20th

1 mg recognin (astrocytin or malignin) is injected into the toe pads of male white rabbits along with standard Freund's adjuvant. The same injection is then carried out intraperitoneally one week later, again intraperitoneally 10 days and, if necessary, 3 weeks 25 later. Specific antibodies can be detected in the blood serum of these rabbits as early as a week to 10 days after the first injection. The same procedure is used for the target antigen, injecting that amount of target which contains 1 mg of astrocytin or 30 malignin, determined by the Folin-Lowry protein determination.

The specific antibody against astrocytin is called anti-astrocytin. The specific antibody against malignin is called anti-malignin. 35 Likewise, the specific antibody against target reagent is called anti-target.

These antibodies clearly show reactions in standard Ouchter-lony gel diffusion tests for antigen-antibody reactions with specific, single, sharp lines of reaction formed with their specific antigen.

The presence of specific antibodies in the serum can also be tested by the quantitative standard precipitate test for antigen-antibody reactions. Good quantitative precipitin curves are obtained and the micrograms of the specific antibody can be calculated from them.

Further evidence of the presence of specific antibodies in the serum can be obtained by absorbing the specific antibody anti-astrocytin on bromoacetyl cellulose-astrocytin (BAC-astrocytin) as prepared above. The antiserum, which contains specific anti-astrocytin, can be reacted with BAC-astrocytin. If the serum is passed through BAC astrocytin, then only the specific antibodies against ss astrocytin bind to their specific antigen astrocytin. Since astrocytin is covalently bound to bromoacetyl cellulose, the specific antibody, anti-astrocytin, is now bound to the BAC-astrocytin to produce BAC-astrocytin-anti-astro-cytin (BACA-anti-astrocytin). This turns 60

proven by testing the rest of the serum,

which has been washed free of BAC astrocytin. With standard Ouchterlony diffusion, no antibodies remain in the serum that could react with the astrocytin. It is therefore concluded 6s that all specific antibodies (anti-astrocytin) that were previously shown to be present in the serum have now been absorbed by the BAC-astrocytin. Furthermore, when the anti-astrocytin is released from its binding to BAC-astrocytin, it has thereby been isolated from all contaminating antibodies. This release of anti-astrocytin can be achieved by washing the coupled BACA-anti-astrocytin with 0.25 M acetic acid (4 ° C, 2 hours), as shown above, the BAC-astrocytin Bond is not broken.

Further evidence for the presence of specific antibodies in the serum can be obtained by adsorbing the specific antibody anti-malignin on the bromoacetyl cellulose malignin (BAC-malignin) prepared above. The antiserum containing the specific anti-malignin can be reacted with BAC-malignin. If the serum is passed over BAC malignin, only the specific antibodies for the malignin bind to their specific antigen malignin. Since malignin is covalently bound to bromoacetyl cellulose, the specific anti-malignant antibody is now bound to the BAC-malignin to produce BAC-malignin-anti-malignin (BACM-anti-malignin). This is proven by testing the rest of the serum that has been washed free of BAC-malignin. With the standard Ouchterlony diffusin, no antibodies remain in the serum that react with the malignin. It is therefore concluded that all specific antibodies (anti-malignin) that have previously been shown to be present in the serum have been absorbed by the BAC-malignin. Furthermore, if the anti-malignin is released from its binding to BAC-malignin, then it has been isolated free from all contaminating antibodies. This release of anti-malignin can be achieved by washing the BACM-anti-malignin complex with 0.25 M acetic acid (4 ° C, 2 hours), but as shown above, the BAC-malignin binding is not breaks up.

The antibodies to target clearly show reactions in standard Ouchterlony gel diffusion tests for antigen-antibody reactions with specific single lines of reaction formed with target which line of identity with the line of reaction against anti-astrocytin or anti-malignant antisera (i.e., those that have been formed to inject astrocytin or malignin itself). Some rabbits have been found to have anti-target levels in their blood prior to target injection. These anti-target substances, when specifically reacted with the target reagent as described in tests with human sera, result in the formation of approximately equivalent amounts of the two types of TAG, S-TAG and F-TAG, (forget the following examples).

Example 8

Discovery of malignant tumors through in vitro quantitative formation of target-attaching globulins (TAG) from biological fluids.

Target reagent prepared according to Example 6 is washed to remove unbound recognin, which may be present due to decomposition. The following procedure is satisfactory. The target reagent is stirred for 2 hours at 37 ° C with acetic acid, centrifuged and the supernatant is decanted. The optical density of the protruding product is read at 266 mjx. If there is any absorption then the washing is repeated until no more material is solubilized. The target is then resuspended in phosphate buffered saline with a pH of 7.2. (Standard S-TAG and F-TAG, cleaned from before

15

639 102

reactions of human serum by the procedure below can, if available, be used as reference standard samples in assaying the target reagent, and rabbit whole serum, which has been determined to contain S-TAG and F-TAG by other target preparations.)

The determination of the slow binding (S-TAG) is carried out as follows. Frozen serum that has been stored for more than a few days should not be used. The serum is carefully made from fresh whole blood or other body fluid according to well known standard procedures. The following procedure has been found to be satisfactory. Blood is allowed to clot in a glass test tube by standing at room temperature for 2 hours. The clots are separated from the walls with a glass stir bar and the blood is left to stand for a minimum of 2 hours (or overnight) at 4 ° C. The clots are separated from the serum by centrifugation at 20,000 rpm and 4 ° C for 45 minutes. The serum is decanted into a centrifuge tube and centrifuged again at 2000 rpm and 4 ° C for 45 minutes. The serum is decanted and a 1% solution of methiolate (1 g in 95 ml water and 5 ml 0.2 M bicarbonate buffer with a pH of 10) is added to a level of 1% of the volume of the serum.

The serum samples prepared according to the above or other procedures are each added in an amount of 0.2 ml to 0.25 ml aliquots of a target suspension reagent, which is 100 to 200 per 0.25 ml target reagent (ig Recognin contains. A double determination is carried out. The suspension is mixed at 4 ° C. in such a way that clumping is avoided. For example, a small rubber cap which is quickly shaken can be used for 1 to 2 seconds, after which the The tube can be shaken slightly inclined in a Thomas shaker for about 2 hours or more. The target reagent and the protein bound to it are separated from the serum. One procedure that has been found to be satisfactory is as follows. The tubes become then centrifuged for 20 minutes at 4 ° C. and 2000 rpm and the supernatant is decanted and the lump formed by the centrifugation is three times washed by remixing and shaking at room temperature with 0.2 to 0.3 ml 0.15 M saline. It is centrifuged and the supernatants are discarded.

The protein that remains attached to the target is split off from it and quantified. For example, 0.2 ml of 0.25 M acetic acid is added and the suspension is shaken in an incubator at 37 ° C. for 1 to 2 hours. The tubes are centrifuged for 30 minutes at 2000 rpm and 4 ° C. The supernatant is carefully decanted to avoid particle transfer and the optical density of the supernatant is read at 280 mu. The optical density value is divided by a factor of 1.46 to obtain the micrograms per ml of serum protein (S-TAG). Duplicate determinations should not vary more than 5%. Any other method suitable for determining protein content can be used, e.g. the determination according to Folin-Lowry. However, the standards must be specified to determine the range of control and tumor values from S-TAG minus F-TAG concentration.

The determination of the rapid binding (F-TAG) is carried out as follows. Frozen serum that has been stored for more than a few days should not be used.

The serum is carefully made from fresh whole blood or other body fluid using standard procedures. The procedure given in this example for serum production is satisfactory.

Serum samples made by the above or other method are left at 4 ° C for 10 minutes less than the total time that the S-TAG serum determinations have been left in contact with the above target reagent (e.g., 1 hour 50 minutes if a “2 hour” S-TAG determination was made). This procedure compensates for the temperature history of the S-TAG and F-TAG regulations.

0.2 ml samples of the temperature-balanced serum are added to 0.25 ml aliquots of the target suspension reagent, which contains 100 to 200 μg recognin per 0.25 ml target reagent. A double determination is carried out. The suspension is then mixed at 4 ° C for about 10 minutes in a manner that avoids pellet formation. For example, a small rubber cap, which is quickly shaken, is used for 1 to 2 seconds, after which the slightly inclined tubes are shaken in a Thomas shaker for about 10 minutes. The target reagent and the protein bound to it are separated from the serum. One procedure that has been found to be satisfactory is as follows. The tubes are then centrifuged at 2000 rpm and 4 ° C for 20 minutes and the supernatant is decanted. The lump formed by centrifugation is washed three times by mixing with 0.2 to 0.3 ml of 0.15 M saline solution at room temperature and shaking. It is then centrifuged and the supernatants are discarded.

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

The final results are expressed as (ig TAG / ml serum and are equal to S-TAG minus F-TAG. The TAG values in patients without brain tumor and other control samples currently range from zero (or a negative number) to 135 µg / ml serum, repeated results are indicated for all results above 100 jig / ml. Some other tumors can give high TAG values, especially if there are secondary (metastatic) tumors in the brain. The TAG values of patients with brain tumor currently range from 136 to 500 pg or more / ml serum In the first "blind" examination of 50 blood samples following the procedures of this example using a target reagent made from astrocytin and bromoacetyl cellulose, 11 out of 11 brain tumors and 28 out of 32 Correctly identified normals: One of the four assumed normals (ie a control subject without a brain tumor) had had cancer of the thyroid gland, which, apparently, some Had been treated successfully years earlier. The three remaining normals were people between the ages of 60 and 70 who were in poor health and who may have had an undetected cancer. Of the remaining seven samples, three out of three cases of Hodgkin's disease were correctly identified. A sample in the tumor area (136 to 500 (ig TAG / ml) corresponded to one

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

639102

16

Patients with severe gliosis, and three specimens removed in the stapled TAG. Three washes with phosphate

Non-tumor areas (0 to 135 [ig TAG / ml) corresponding to buffered saline have been found to be satisfactory

Patients who had an uncertain intercranial mass diagnosis. IConjugatin inhibition with but no tumor, osteosarcoma (a non-brain fluorescine-labeled conjugate, followed by washes) is then

tumor) and a melanotic sarcoma (a non-brain tumor, which was followed by a microscopic examination of the tumor). connects. Normal cells and their processes will not

Following the procedures of this example, an examination in both the tumor sections and the control follow-up was stained using a target section from normal non-tumor brains. Reagent, made from malignin, in the malignant brain. Fluorescence is bright in tumor glial cells and their protumors and all normals. io present.

Another study, performed according to the procedures of this example, extended the total number of Examples 10

examined human serum samples from 50 to 114. The proprietary description of the cytotoxic effects of anti-astro-

These products and procedures are listed in Table V cytin, anti-malignin and S-TAG versus tumor cells,

shown, which gives the results of these tests. i5 that grow in a tissue culture.

Standard tests for determining the cytotoxic

Example 9 Generally the number of

Diagnosis of tumor cells by immunofluorescence. Cells in a solid cell chamber, usually like this

The compounds have been shown to be anti-astro- arranged to contain about 100 living cells,

cytin, anti-malignin and S-TAG are preferably counted at 20 tumor. These cells are then attached to the cells to be tested. This specificity permits the use of treated agents and the number of cells still left on these compounds for diagnosis of tumor cells in histo-life is counted.

logical cuts by combining dyes or radioactive in a standard cytotoxicity test of the substances obtained according to the example with anti-astrocytin, anti-malignin or S-TAG 8 compared to cells in a tissue. Standard labeling techniques 25 can be cultured that are used by a patient with a glioblastoma. A suitable 3rd to 4th degree procedure that has been obtained as a glial down use of S-TAG is e.g. the following. were well characterized in the future, S-TAG resulted in the death

One procedure that has been found to be satisfactory for all cells in the cell chamber even in high dilution is a modified St. Marie procedure. of 1: 100 and 1: 1000, which is only 0.2 and 0.02 (ig S-TAG / ml

Samples of human brain tumor are frozen corresponding to 30 solution. Similar results are obtained with high and 5 [x thick sections are cut out. These anti-astrocytin and anti-malignin dilutions are obtained in a moist container for 4 to 8 weeks at -70 ° C.

stored before they are stained. The conjugate can have both the specificity shown in Example 9 and that shown in FIG

Standard antiserum, e.g. Goat anti-rabbit conjugate Example 10 cytotoxicity shown to be therapeutic. The conjugate is labeled by known techniques with fluo-35 possibilities of anti-astrocytin, anti-malignin and resecting or other labeling substances. S-TAG highly relevant for human brain tumors. These

Fluorescin-labeled goat-anti-rabbit conjugate, therapeutic applications come to the practical

as it is commercially available, can be used to diagnose both of these symptoms.

will. A standard for tumor tissue was added as a fluorescence technique, which

technology used, in which a 1: 200 to 1: 400 solution has been removed from 40, but which is diagnosed by histological

TAG requires about 30 minutes or more on the tumor incision, as incubated previously shown. Then it was washed so as not to be turned on.

Table V

Normal *

Malignant

Brain tumors primarily

Other malignant tumors, brain secondary symptoms

Other malignant tumors, no secondary brain symptoms

Undetermined cerebral diagnosis

serum

serum

serum

serum

serum

serum

serum

serum

DAY

DAY

DAY

DAY

DAY

DAY

DAY

DAY

µg / ml

Ug / ml p.g / ml jig / ml

Ug / ml

Hg / ml

Hg / ml jig / ml

124

19th

54

65

459

270

36 "

1658

113

55

27th

113

397

257

315

1449

105

51

41

130

236

188

44214

139

130

82

21st

79

137

205

28814

20910

127

44

27th

61

298

1577

75 '"

38

127

21st

123

397

184 "

100

31

0

14

241

27 "

125

0

14

20th

241

1101J

30th

125

62

41

217

19215

250 '

118

38

34

147

39

89

93

93

127

363 '

99 «

21st

48

185

4th

132

0

20th

253

31

2703

120

82

253

42

7

16

20th

565

17th

639 102

Table V (continued)

Normal *

Malignant

Primary brain tumors

Other malignant tumors.

Brain secondary symptoms

Other malignant tumors, no secondary brain symptoms

Undetermined cerebral diagnosis

34 76 48 85

58 24 62 89 89

20 113 72

55 0

277

137 7813

138 650 160

* This term includes normal, non-tumor related medical and surgical disorders; 1-very sick, undiagnosed; 2-extra-intercranial brain mass, undiagnosed; 3-pronounced gliosis; 4 malignant melanoma; 5-osteosarcoma; 6-brain cyst fluid; 7-adenocarcinoma of the colon; 8-guest rectomy; 9 headache; 10 emphysema; 11-polymyalgia; 12 colon cancer; 13 convulsions; 14-prostate cancer, secondary symptoms in the bones; 15 clinically normal, 18 months earlier than when this abnormal TAG serum was obtained: severe headache and loss of taste and smell have now developed.

Example 11 20

Hydrolytic cleavage of the recognition.

A solution of Recognin, in this case either astrocytin or malignin, with a pH between 1 and 2 is left in the cold. After 7 to 14 days, TLG chromatography shows that the molecular weight 2s of the product has been reduced by approximately 200. If the solution is left to stand for longer, additional units with a molecular weight of approximately 200 are split off every 7 to 10 days. Thus, the molecular weight of the astrocytin of 8,000 and the molecular weight of the 30 malignin of 10,000 are successively reduced by about 200 units in each case.

The physicochemical properties of astrocytin are maintained down to a molecular weight of approximately 4000 by each product. The physicochemical properties of the malignant are maintained down to a molecular weight of approximately 5,000 by each product. This is shown by Ouchterlony gel diffusion test against anti-astrocytin and anti-malignin.

This cleavage can also be carried out enzymatically, for example with 40 trypsin or other proteinases, to achieve similar results.

The molecular weights of these compounds, which have been prepared by hydrolytic cleavage of the recognins, can be roughly defined by the following formulas:

For products with the physicochemical properties of astrocytin; 400 + 200 X = Y.

For products with the physicochemical properties of malignin; 5000 + 200 X = Y. so

Where Y is the molecular weight of the product and X is an integer from 0 to 19.

Example 12

Production of an artificial tissue or organ with ss recognition.

A tube of plastic, metal, or other suitable rigid material with a rigid wall is immersed or impregnated in a highly concentrated (i.e., 10 mg / ml) viscous recognition solution, in this case either astrocytin or malignin, until all surfaces are completely covered with the recognition are coated. Alternatively, the Recognin solution can be passed through and around the pipe under pressure until all surfaces are completely coated. The tube is then dried in air or in vacuo or lyophilized. The coating process is repeated several times to build up multiple molecular layers of the Recognin coating.

The tube is now ready to be inserted into a cavity or tissue that contains astrocytin or malignant-like precursors in the adjacent tissue or fluid of a living mammal. This artificial tissue or organ can be used to minimize or eliminate a reaction with foreign substances that would take place without a recognition coating.

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

The figure shows that the increased flask size roughly triples the ratio of the malignin product.

The figure also shows the relationship between malignin and malignancy. The broken line represents an ideal linear relationship. The relationship shown is certainly not trivial, since the proportion of malignant present increases as the lines become less restricted (pathological) in their growth. The normal in situ recognin function, as previously indicated, is related to the inhibition of contact with cell growth. The more pathological the growth of the malignant cells, the less contact inhibition takes place and the more the malignin becomes the predominant protein.

B

1 sheet of drawings

Claims (3)

639 102 1. Process for the production of recognins, characterized in that a) normal or diseased tissue or cells are extracted with a neutral buffer with repeated disruption of the tissue or cells in order to extract protein fractions, b) a protein fraction with a pK range from 1 to 4 is separated from the resulting extract by means of a chromatographic separation method, the extracted protein fractions being placed in a chromatographic column and eluted with increasing acidic solvents, c) the recognin is isolated therefrom as a fraction with a molecular weight between 3000 and 25,000. 2. The method according to claim 1, characterized in that the tissue or cells are broken up by repeated homogenization and centrifugation. 3. The method according to claim 1, characterized in that one carries out the isolation of the recognin in such a way that the fraction separated by means of chromatographic separation is filtered and the recognin is isolated therefrom by thin-layer gel chromatography. 4. The method according to claim 1, characterized in that one uses brain glioma tumor tissue as the diseased tissue, and that one isolates from the fraction with a pK range from 1 to and with 4 a fraction with a molecular weight of approximately 8000, to be called an astrocytin Get Recognin. 5. The method according to claim 1, characterized in that artificial cancer cells are used as cells and a fraction with a molecular weight of approximately 10,000 is isolated from the fraction with the pK range from 1 to and with 4 in order to obtain a recognizable as malignin receive. 6. The method according to claim 1, characterized in that from the fraction with a pK range from 1 to and with 4 a recognin is isolated, its approximate molecular weight by the formula 4000 + 200X = Y where Y is the molecular weight of the recognin and X is an integer from 0 to 19. 7. The method according to claim 1, characterized in that from the fraction with the pK range from 1 to and with 4 a recognin is isolated, its approximate molecular weight by the formula 5000 + 200 X = Y where Y is the molecular weight of the recognin and X is an integer between 0 and 19. 8. A process for the preparation of recognition complexes, characterized in that the recognin is prepared according to one of claims 1 to 7, then complexed with at least one carrier to form a recognizer-carrier complex. 9. The method according to claim 8, for the production of recognizer-carrier-antirecognin complexes, characterized in that the resulting recognizer-carrier complex is complexed with antirecognin. 10. The method according to claim 8, characterized in that the recognin is complexed with at least one carrier which is largely biologically inert with respect to the recognin and which at least largely does not change the physical / chemical properties of the recognin, in order to form recognizer-carrier complexes , which show the physical / chemical properties of the recognition. 11. The method according to claim 10, characterized in that bromoacetyl cellulose is used as the carrier. 12. The method according to claim 8, for the production of recogn-carrier-globulin complexes, characterized in that the resulting recogn-carrier complex is brought into contact with a globulin-containing body fluid for a predetermined period of time, where complexes are formed , and then isolated. 13. The method according to claim 12, characterized in that the recognizer-carrier complex is reacted for 2 hours or more at low temperature with the body fluid, such as blood serum, and from it a recognizer-carrier globulin complex with a slowly with 20 Recognin-carrier complexes separating lcomplexing globulin. 14. The method according to claim 12, characterized in that the Recognin-carrier complex for about 10 minutes at low temperature with a body fluid 25 how blood serum reacts, and from this separates a recognizer-carrier-globulin complex with a globulin complexing rapidly with recognizer-carrier complexes. 15. A process for the production of antirecognin, characterized in that the recognition according to one of the Manufactures claims 1 to 7, thereof an immunologically effective dose in contact with body tissue or body fluid of a mammal, such that an antibody reaction sets in, and deprives the mammal of the antirecognin. 16. The method according to claim 15, characterized in that the antirecognin obtained is complexed with a recognition-carrier complex and then decomplexed again by a hydrolytic treatment with an acid or a proteinase. 17. The method according to claim 15, characterized in that the antirecognin obtained is combined with a display substance, such as with a dye or a radioactive substance. 18. A method for producing an antirecognin-Diagno-45 sticum complex, characterized in that the Recognin according to any one of claims 1 to 7, of which an immunologically effective dose is brought into contact with a mammalian body tissue or body fluid such that an antibody reaction sets in and thus deprives the mammal of antirecognin, then complexes the antirecognin with a diagnostic agent. 19. Use of the Recognin-carrier-globulin complex produced according to one of claims 12 to 14 in the in vitro cancer diagnosis. 55 20. Use according to claim 19, characterized in that in vitro in a known volume of blood or other body fluid of a mammal Recognin-carrier-globulin complexes with fast-complexing globulins and slow-complexing 60 globulins as an indicator of the presence of cancer used on living mammals. 21. Use according to claim 19, characterized in that the Recognin-carrier globulin complex together with a display substance, such as a dye 65 or a radioactive substance, a histological section is supplied, the coloring or the radioactive display only in the presence of Cancer cells. 2nd PATENT CLAIMS 3rd 639102