NO793362L - PROCEDURE FOR DETECTION OF DEPARTURED TUMOR CELLS - Google Patents

Description

The present invention relates to a method for detecting the presence of cancerous or malignant tumor cells in a cell collection in vitro or in vivo, and more particularly it relates to such a method where products are used, here called target-attaching globulins (TAG), products which are derivatives of compounds which are here called recognisers, and which are capable of preferentially attaching to cancerous or malignant tumor cells.

Recogniners are formed during the treatment of diseased cells or tissues, e.g. tumor cells or artificial cancer cells, and secretion of the desired products. The term "recognin", when used here and throughout the description, means a compound that is associated with diseased cells or tissues, which is characterized by:

(a) to be able to form a single-line precipitate

with its specific antibody by quantitative precipitation tests and Ouchterlony gel diffusion test;

(b) to be soluble in water and aqueous solutions when acid

or neutral pH; (c) being insoluble in aqueous solutions at alkaline pH; (d) having a spectrophotometric absorption peak wavelength•of 280 my;

(e) having a molecular weight of from 3,000 to 25,000; and

(f) having an amino acid residue composition in which there is a large proportion of glutamic acid and aspartic acid in relation to the total amino acid residue composition and a high ratio between these acids and histidine.

The recons can. are used to produce their chemoreciprocals, i.e. by contacting the recognines or recognines

. on a carrier, with body fluids. The chemoreciprocals are useful for diagnostic and therapeutic purposes, i.e. for the diagnosis and treatment of cancer. The chemoreciprocals are substances that

reacts with immunochemical-like specificity with a recognin in vivo in vitro, ie by quantitative precipitation test, by Ouchterlony double diffusion or by immunofluorescence.

A recognin is astrocyte. Astrocyte is formed from brain tumor tissue, preferably brain glioma tumor tissue. Protein fractions containing the astrocyte precursor are first extracted from the tissue. A preferred method for performing the extraction is to treat the tissue with a neutral buffer under homogenizing conditions or by other techniques, to disrupt the cells and tissue to solubilize protein fractions containing the astrocyte precursor.

At this point, the astrocyte precursor is still bound to many high molecular weight substances, including protein, lycoproteins, lipoproteins, nucleic acids and nucleoproteins.

The solubilized proteins are then separated from the resulting tissue extract. The extract solution from the tissue is then prepared to remove insoluble particles. The low molecular weight impurities are then removed from the resulting solution by a pre-evaporation-concentration technique. The resulting solution is then treated to cleave the astrocyte precursor from other contaminants to obtain the protein fraction having a pK range between 1 and 4. Thus, e.g. the solution placed on a chromatography column and eluted with solvents of increasing acidity. All the fractions that are eluted in the neutral or acidic range down to pK 4 are discarded, and the

fractions with a pK range of from 1 to 4 are collected. The eluate is then treated to obtain a product with a molecular weight of

about. 8000.. This will be e.g. carried out by first filtering the material to remove low molecular weight substances, i.e. those with a molecular weight below 1,000, and then filtering again to remove those with a molecular weight above 25,000. The fraction which has a molecular weight between 1000 and 25,000 is then processed further, i.e. by thin layer gel (TLG) chromatography, to obtain astrocyte.

Astrocyte can thus be prepared by extracting brain glioma tumor tissue with a neutral buffer, by repeated homogenization and high-speed centrifugation, separation, from the resulting extract, of the fraction having a pK range from 1 to 4, separation, from said fraction, of the substances that have a high molecular weight, i.e. up to approx. 230,000, bg from there isolation of the product astrocytin, which has a molecular weight of approx. 8000.

The product astrocyte prepared in accordance with this method is characterized by forming a single-line precipitate with its specific antibody in quantitative precipitation tests and Ouchterlony gel diffusion tests, by being soluble in water and aqueous solutions of acidic or neutral pH, and by being insoluble at alkaline pH, by having a spectrophotometric absorption peak wavelength of 280 my and by having a molecular weight of approx. 8000.

Astrocyte is also characterized by having a very high percentage of residues of glutamic acid and aspartic acid and a very high ratio between these acids and histidine. A further analysis of astrocyte is provided below.

In a similar manner to that described above, another recognin, called malignin, was prepared from artificial cancer cells, i.e., cancer cells grown in vitro. Malignin has a molecular weight of approx. 10,000 and a similar but different amino acid residue composition to astrocytin, i.e. large ratio of glutamic acid and aspartic acid, and high ratio between these acids and histidine. A further analysis of malignin is set out below.

Malignin can thus be prepared by extracting

artificial cancer cells grown in culture with a neutral buffer by repeated homogenization and high-speed centrifugation, separate from the resulting extract the fraction that has a. pK range of approx. 1 to 4, separate from said fraction the substances that have a high molecular weight, i.e. up to approx. 230,000,

and from there isolate the product which has a molecular weight of approx. 10,000.

The amount of malignin that is formed by yeast cell fermentation

and the percentage of total protein amount formed by artificial cell fermentation which is malignin can be increased by cultivation of artificial cancer cell culture in large growth containers.

Malignin prepared in accordance with this method is characterized by forming a single-line precipitate with its specific antibody by quantitative precipitation tests and Ouchterlony gel diffusoris tests, by being soluble in water and aqueous solutions of acidic or neutral pl-l, and insoluble at alkaline pH,

by having a spectrophotometric absorption peak wavelength of 280 my and by having a molecular weight of approx. 10,000.

Recogniner is also characterized by being able

to form complexes with bromoacetylcellulose to form bromoacetylcellulose-recognin and to form the specific anti-recognin antibodies by injection into mammals, said anti-recognin attaching specifically to the recognin precursor in situ and producing fluorescence on glioma cells by coupling with fluorescein, as described in more detail below.

An anti-recognin, anti-malignin, is toxic to brain tumor cells in vitro. Anti-recogniners are antibodies to the group of antigens here called recognizers, and they form a single-line precipitate with recognizers in quantitative precipitation tests and Ouchterlony gel diffusion tests.

Recognizens, such as astrocytin, malignin and similar substances, are useful as products that can be introduced into a biological system to reduce foreign reactions, such as by coating a material with a recognizen. A further example could be to introduce a recognizen to form the chemoreciprocals in the biological system. They can also be used nutritionally to encourage the growth of a particular biological system of which they are a part. A further utility for recognizen is in the preparation of Target reagents comprising the complexes of recognizen with a carrier to facilitate its applicability in biological systems. The complex thus promotes e.g. the physicochemical properties of recognin itself. The carrier should be chosen from those which form a complex with recogninet, and which are essentially biologically inert.

Any substance known in the industry that will form a stable complex with polypeptides or proteins may be useful for complexation with recognizen. An example is a cellulose-based material, such as the aforementioned bromoacetyl cellulose.

In addition to being inert in biological systems, the carrier should be such that it does not alter the specific physicochemical properties of the recognizen useful for the purposes stated herein.

The complexes of recognizen and its carrier are useful in the preparation, separation and identification of their chemoreciprocals in any biological system with which they are contacted. Recogniz- . the carrier complex is also useful in stimulating the production of its chemoreciprocal precursor by any biological system into which it is introduced.

The group of chemoreciprocals called anti-recognins, e.g. anti-astrocytin and anti-malignin, can be generated by injecting recognizen into a biological system. Thus, an immunologically effective dose of recognin can be brought into contact with . body tissues or fluids in a manner that induces an antibody response, in accordance with known techniques in the industry, for the production of antibodies. The anti-recognins can be used to deliver materials such as diagnostic, nutritional and therapeutic agents to specific cells or locations in a biological system, which includes introducing said agent in complexed form; together with the anti-recognit in the biological system. The anti-recognines are also useful for diagnosing the presence of tumor cells in a histological section by applying the anti-recogninet conjugated with a labeling substance such as dyes and radioactive substances to said part, whereby staining or radioactive labeling only takes place with tumor cells. Yet another application for anti-recognins is to increase the yield of other useful chemoreciprocal products (such as TAG, described below) from a mammal, which involves injecting an immunologically effective dose of recognizen into the mammal, or other biological system.

Prior to the present invention, glycoprotein complexes were prepared from brain tissue, and antibodies were formed for them. Separated materials known as 10B glycoproteins produced by Tay-Sachs' diseased brain were injected into rabbits and antibodies were formed. These Tay-Sachs<1>antibodies were used in immunofluorescence studies of brain tumor: only reactive normal non-tumor glia, not tumor glia, were stained with these antibodies.

However, when astrocyte was prepared from tumor tissue, and antibodies to astrocyte (anti-astrocytin) were prepared and used in immunofluorescence studies of brain, only tumor glia, not normal (non-tumor) glia, were stained with anti-astrocytin. Thus, by the original tissue and the nature of the antibody, and also by the specific type of cells stained, Tay-Sachs antibodies and anti-astrocyte were clearly different products.

Another group of chemoreciprocals are Target reagents complexed with their chemoreciprocals. E.g. the Target product of astrocytin complexed with a carrier such as bromoacetyl cellulose is brought into contact with anti-astrocytin. This type of compound can be complexed and used for transmission

of diagnostic, nutritional and therapeutic agents to specific

cells or locations in a biological system. These compounds can also be used in cleaning processes. E.g. anti-astrocytin can be formed by decomplexation of bromoacetyl cellulose-astrocytin-anti-astrocytin by hydrolytic treatment with an acid or a proteinase enzyme. Target reagents are also useful for increasing the amount of TAG products (described below) in a biological system, such as by contacting an immunologically effective dose of Target with body tissue. or liquids.

Additional chemoreciprocals are TAG reagents (i.e.

Target-attaching globulins). TAG products are manufactured by

contacting the Target reagents with body fluids for varying periods of time to form a complex and cleave the TAG therefrom. Two useful editions are S-TAG and F-TAG.

A method for preparing S-TAG (slow-Target-attaching globulin) comprises reacting blood serum or other body fluid with Target (eg bromoacetyl cellulose malignin) for approximately two hours or more at a low temperature, e.g. about.

4°C, and cleave the S-TAG from the resulting material, e.g. with diluted acid for approximately two hours at a temperature of approx. 37°C. The product S-TAG prepared in accordance with this method is characterized by being soluble in aqueous buffered solutions, forming a single-line precipitate with its corresponding recognin by Ouchterlony gel diffusion test, being non-dialyzable in cellophane membranes, retained with millipore- filters that retain molecules with a molecular weight above 25,000, have molecular weights in different aggregate states, as determined by thin-layer gel chromatography, of approximately 50,000, and multiples thereof in the macroglobulin range,

and having a spectrophotometer absorption peak wavelength of .280 mu.

A method for producing F-TAG (rapid-Target-attaching globulin) comprises reacting blood serum or other body fluid with Target (e.g. bromoacetyl cellulose malignin) for approximately 10 minutes at a low temperature, e.g. about. 4°C, and cleave the F-TAG from the resulting material, e.g. with for-

diluted acid for approximately two hours at a temperature of approx. 37°C.

The product F-TAG prepared in accordance with this method is characterized by being soluble in aqueous buffered solutions, forming a single-line precipitate with its corresponding recognin in Oucherlony gel diffusion tests, being non-dialyzable in cellophane membranes, retained by millipore filters that retain molecules with a molecular weight above 25,000, have molecular weights in different states of aggregation, as determined by thin-layer gel chromatography, of approximately 50,000, and multiples thereof in the macroglobulin range, and have a spectrophotometer absorption -peak wavelength of 280 my.

TAG products are useful for the detection of cancerous tumors in living mammals, by determining the concentration of S-TAG and F-TAG produced by a known volume of the mammal's blood serum or other body fluid, and comparing this concentration with amounts determined to be indicative for cancer. TAG products are also useful for providing a diagnosis for the presence of tumor cells in a histological section or other cell collection, and this includes applying TAG conjugated with a labeling substance, such as dye or radioactive substance, to said section, whereby staining or radioactive labeling only takes place with tumor cells. TAG products have also been found to be cytotoxic to tumor cells. TAG products are also useful for directing the delivery of diagnostic, nutritional and therapeutic agents to specific cells or sites by introducing said agents in complexed form with the TAG product.

The present invention consequently provides a method for detecting the presence of cancerous or malignant tumor cells in a cell collection, which comprises applying an anti-malignant TAG product attached to visible or signal-emitting agents to said cell collection, whereby said anti- malignin TAG product preferentially attaches to cancerous or malignant cells, and detect said cancerous or malignant tumor cells by means of said visible or signal-emitting means.

In a preferred embodiment, the present invention provides a method for detecting the presence of cancerous or malignant tumor cells in a cell collection, which comprises applying to said cell collection an anti-malignant TAG product, and then applying fluorescently conjugated anti-TAG thereon, whereby fluorescence takes place only in the cancerous or malignant tumor cells upon illumination.

The method according to the present invention

is applicable to both cell collections in vivo and in vitro, and also for the detection of both glial and non-glial tumor cells. It is advantageous that the TAG product used in the method according to the present invention is formed by reaction of anti-malignin serum with a malignin-based reagent, preferably bromoacetyl cellulose malignin.

It is preferred that the TAG product used in the method according to the present invention is at least partially free of substances that are little or not reactive in fluorescent detection, when applied to known cancerous cells, and that if a signal-emitting agent is used, then this is a radioisotope.

Normal cell division in plants or animals is limited or inhibited when the cells come to completely occupy a special space. The mechanisms (a) by which normal cells "recognise" that they have filled the space available to them, and. (b) whereby, the operation of this recognition mechanism in turn inhibits cell division, both have been unknown. The inventor has formed a group of compounds called recognizers, the precursors of which are increased in concentration when normal recognition and learning takes place, and which are connected with the recognition and learning of particles and cells, and with the attachment of cells to each other. When attempting to produce these compounds from normal cancer cells, it has been discovered that they are absent as such, and that changes in their molecular structure have taken place at the same time that the cancer cells have lost their ability (a) to recognize that they have filled their normal volume, and/or (b) to stop sharing when they have filled their normal volume.

Recognizers are compounds that have physiochemical properties that mimic such configurational properties of cancer cells expressed by their lack of ability to recognize and stop cell division. Recogniner is useful in providing immediate products and methods that are themselves useful in the diagnosis and treatment of cancer, and in the prevention of cancer.

Methods have also been discovered whereby artificially cultured cells can be used to produce malignin for the first time. An advantage of the methods discovered here is that malignins and new products from them can now be produced efficiently in

. truly unlimited quantities.

The present invention is applicable to any and all biological systems whereby it is desired to influence all growth and metabolism. In the production of the particular compound or compounds of the appropriate cell type in artificial culture, and the further production of products from these substances,

specific influence can thus be achieved for the first time;,

on all types of tissues, cells, cell organelles, sub-organelle molecules or molecule aggregates in any living system. Thus, specific nutritional influence at critical times in development, specific diagnostic methods, preventive and. treatment methods, and the construction of artificial bioelectrical systems (as in tissue and organ transplants) are all affected. These artificial bioelectrical systems can now be formed so that they carry the properties of the specific recognin, alignin or their chemoreciprocals in the normal tissue or a component that they will approach and thus avoid being "recognized" as "foreign",

and thus avoid the reactions to foreign substances, including expulsion.

One aspect of this invention is the production from biological fluids of two new products, Target-attaching globulins (TAG) which are called this because they are formed by two reactions, where, in the first, biological fluids are reacted with a synthetic complex containing physiochemical configurations that mimic those of the malignancies and which are called 'Target', and where it,

in the second, the specific TAG is split off from the complex, and by measuring the TAG thus formed, a quantitative indication is obtained from the biological fluids in living organisms as to whether a tumor is present in that organism, and this is consequently a diagnostic test for tumor. Because TAG products and anti-malignin are physiochemically complementary to malignins, they are called chemoreciprocals.

It has further been discovered that two quantitatively and qualitatively different TAG products can be produced, depending on the time allowed for reaction of serum with the specific Target reagent used, and depending on the time allowed for cleavage of the product which is made complex.

After examining the amounts of these products that could be formed from a number of different individuals with brain tumors and various other medical disorders, and also from those with no apparent disease, it became clear that the amounts of these two new products that could be formed in a particular. individual, was an indication of whether this individual had a brain tumour, and this is consequently a serum diagnostic test for brain tumours.

The applicability of these new products, in addition to their use in diagnosing, from serum and other biological fluids, the presence of brain tumor and other tumors, is illustrated by the demonstration that TAG and anti-recognin compounds attach preferentially to glial and non-glial tumor cells in cell collections, e.g. histological parts of e.g. brain tumor and surrounding tissue removed during brain tumor surgery. This preferential labeling by TAG and anti-recognins of tumor cells is e.g. 'demonstrated by standard immunofluorescence techniques although signal-emitting agents can also be used. A new method is thus also available to determine with a new degree of certainty when examining cell collections whether tumor cells are present in the removed tissue, and whether these tumor cells have penetrated right to the edges of the removed tissue, indicating the probability that there is still tumor remaining in the brain or other organs, or that tumor cells are absent from the periphery of the removed tissue, indicating the possibility that all tumor has been removed from the brain or other organ. Also, TAG and anti-malignins are prepared as described. found to be cytotoxic to lioma brain tumor cells grown in tissue culture in vitro. This high affinity for tumor cells in another medium, here grown in tissue culture, is further evidence of the specific binding potential of the new product TAG. Furthermore, the cytotoxicity of TAG and anti-recognins to tumor cells provides an additional new diagnostic test for the serum of patients suspected of suffering from a tumor. Thus, e.g. the serum or other body fluid from these patients is reacted with Target to form TAG and the product TAG is tested in tissue culture growth of tumor cells for cytotoxicity. Both the concentration of TAG and the degree of cytotoxicity manifested by that TAG

<:> which can be formed from a given individual's serum, can be not only diagnostic, but can also be of value in tracing the course of the disease before and after surgery on a given patient. Coupling radioactivity and dye traces to TAG provides a new TAG product such as e.g. is useful in vivo in the diagnosis of tumors and their precise location. Thus will

e.g. injection of an appropriately labeled TAG either intra-arterially or intravenously, into the cerebrospinal fluid, or directly into the brain tissue or its cavity, afford the opportunity to show by means of such radioactive agents, or by visualization of the coupled dye, the presence of a brain tumor, for it is only to tumor cells that TAG is specifically attached. Furthermore, this method allows accurate visualization of the location of the brain tumor. Similar methods can be used for other types of tumor. This can be seen as an improvement of the in vivo diagnostic method using anti-astrocytin formed in rabbit blood to label the brain tumor, due to the fact that by using TAG formed from human serum, the possibility of foreign protein reactions is avoided. Since TAG and anti-recognins have the chemical specificity that allows preferential attachment to astrocyte precursor containing tumor cells both in vitro and in vivo, these products can also be used therapeutically, as well as diagnostically, when linked, e.g. with radioactive, proto-taking agents, or other toxic physical or chemical agents, so that these toxic substances can be localized preferentially by the specificity of these compounds for attachment in tumor cells compared to their normal neighboring cells. This selectivity is universally recognized as the decisive, or at least a decisive factor,

for achieving effective chemical or physical therapy on tumors. TAG has thus shown that it is effective in preferential attachment to the tumor cells, and should be promising as a new therapeutic product for these reasons.

In the serum of patients with a malignant tumor, as will be seen from the examples below, one type of TAG, slow-TAG (S-TAG), as distinct from fast-TAG (F-TAG), can be produced in relatively large amounts from a given volume of serum than in patients without such a tumor. This suggests that one of TAG's naturally occurring precursors (P-TAG) has increased concentration or that there are other factors that favor the relative formation in vitro of S-TAG over F-TAG.

The possible relationship between the function of the relevant synthetic products Target and TAG and their precursors, and further between functions of postulated but not demonstrated cellular "antigens" and circulating "antibodies" to them that may be present in vivo, has not yet been clarified . Thus forms, for example,

in an antibody-like manner, F-TAG and S-TAG simple separate reaction

lines with astrocyte by Ouchterlony gel diffusion, and injection of Target into rabbits induces an increase in the yield of TAG products from rabbit serum after reaction with Target. The finding that there may be a normal amount of precursor-like circulating antibodies to a cell antigen hidden in the non-dividing cell raises a question as to the possible function of the pair. It is proposed here that TAG precursor (P-TAG) and Target-like substances exist in vivo as functioning in the regulation of cell proliferation and cell death. Thus, e.g. ' exposure of a cell component which is not normally directly exposed to serum proteins occurs during cell division. The exposure of this cell component could result in the component being converted into a Target-like substance to which the attachment of a P-TAG-like molecule from serum can then occur, which would stimulate cell division or inhibit it. Alternatively, a non-dividing cell that is damaged or malfunctioning may expose a Target-like substance to which the attachment of P-TAG-like molecules may be reparative. However, under certain cell conditions the attachment of P-TAG-like molecules can induce destruction of the cell (eg, anti-glioma-TAG synthetically generated as described herein is clearly cytotoxic to glioma tumor cells grown in tissue culture). This could thus represent a mirror of a normal mechanism for regulating cell division, and for either repairing or removing individual cells in the body throughout the organism's life. If the exposure of cell constituents increases abnormally so that abnormally large amounts of cell Target-like substances are formed, such as can occur with rapid division of cancer cells such as in brain gliomas, an increase in the concentration of a type of serum P- TAG relative to another.

Whatever the real function of the precursors is, the increase of the relative amount of predominantly one type of TAG, slow-TAG (S-TAG), which can be formed in vitro by the methods described here from the serum of patients with malignancy tumor, basis for the serum diagnostic test described in the examples that follow.

The following examples illustrate the invention.

Example 1

Preparation of purified astrocyte precursor-containing fraction.

Human brain glioma tumor tissue, removed by surgery, is dissected as free as possible from surface blood vessels and normal brain tissue. For a typical amount of excised tumor tissue of 11 g, the tissue is weighed in 6 aliquots of 1.5 g and 2 of 1.0 g. Each aliquot is then processed in the following manner.

Each aliquot is homogenized in neutral buffer solution by sonification or other mechanical device. Each aliquot becomes e.g. homogenized in 100 cm 3 per g tissue of 0.005M phosphate buffer solution, pH 7, in a Waring blender. The homogenization should be carried out cold to prevent denaturation of proteins. The mixer should e.g. cooled in advance in a chilled room at 0-5°C, and it should be in operation only for approx. 3 minutes.

The homogenate is then centrifuged to make it clear, e.g. at 80,000 times gravity for 30 minutes in a cooled ultracentrifuge. The soluble supernatant is decanted and stored cold. The insoluble residue is rehomogenized with

3 a further 100 cm of neutral buffer and centrifuged as before, and the second soluble extract is combined with the first. The best yields are obtained when this process of homogenization and centrifugation is repeated until less than 50 micrograms of protein.per ml of solution is obtained in the overflow liquid. For most tissues this is achieved by the fifth extraction.

The solutions thus obtained are combined and concentrated by evaporation and subsequent dialysis, such as by dialysis against 0.005 M phosphate buffer in a cold environment to form a volume of 15 ml. The volume of this solution is noted, an aliquot is taken for total protein analysis, and the remainder is fractionated to obtain the protein fraction having a pK range between 1 and 4. The preferred method of fractionation is chromatographic, as indicated in the following.

The solution is fractionated in the cold room (4°C) on a DEAE cellulose (Cellex-D) column, 2.5 x 11.0 cm, which is equilibrated with 0.005M sodium phosphate buffer. Stepwise elution-solvent changes are made with the following solvents (solutions): solution (1) 4.04 g of NaH2PO4 and 6.50 g of Na2HP04 are dissolved in 15 liters of distilled H20 (0.005 molar, pH .7);

solution (2) 8.57 g of NaH 2 PO 4 is dissolved in 2480 ml of distilled H 2 O;

solution (3) 17.1 g NaH2P04 is dissolved in 2480 ml distilled H20

(0.05 molar, pH 4.7);

solution (4) 59.65 g NaH 2 PO 4 is dissolved in 2470 ml distilled H 2 O (0.175 molar);

solution (5) 101.6 g of NaH2P04 is dissolved in 2455 ml of distilled H20

(0.3 molar, pH 4.3);

solution (6) 340.2 g NaH2P04 dissolve in 2465 ml distilled H20

(1.0 molar, pH 4.1);

solution (7) 283.64 g of 80% phosphoric acid (H3P04) is taken up in 2460 ml of distilled H20 (1.0 molar, pH 1.0).

Add extract of nervous tissue, 6 to 10 ml in volume. Let it enter the column. It is then coated with solution (1) and a container with 300 ml of solution (1) is attached so that it drips onto the column with the help of gravity. Aliquots of 3 ml

of effluent is collected using an automatic fraction collector. The subsequent elution solutions are changed step by step using the following elution tube numbers. Solution (2) at tube 88, as the solution is supplied to the column on top of the resin, and then a container with 50 ml of solution (2) is coated and attached. Solution (2) at tube 98, as the solution is supplied to the column on the resin top, and then a container with 75 ml of solution (3) is coated and attached. Solution (4) at pipe 114, as solution is supplied to the column on the resin top, and then a container with 150 ml of solution (4) is coated and attached. Solution (5) at tube 155, as solution is added to the column on the resin top, and then a container with 125 ml of solution (5) is covered with and attached. Solution (6) at tube 187, as the solution is introduced onto the column on the resin top, and then a container with 175 ml of solution (7) is coated and attached. Elution is continued until elution is complete at

tube 260 - Newly prepared resin must be used for each new volume of tissue extract. Each effluent tube is quantitatively analyzed for protein. The eluates in the tubes numbered 212 to 230 are combined, and.they contain them. unpurified products from which astrocyte will be produced.

Data on this impure material, called fraction 10B, have recently been published [Protein Metabolism of the Nervous System, pp. 555-69 (Pleum Press, 1970), Journal of Neurosurgery, vol. 33, pp. 281-286 (September 1970)], but now the separation from fraction 10B of the specific product here called astrocyte has been carried out. Unpurified fraction 10B can be produced as a product in amounts between 0.1 and 10 mg per g of original fresh nervous system tissue from which it is obtained. In addition to an astrocyte precursor, it contains varying amounts of covalent

bound carbohydrate residues included a variety of hexoses, namely glucose, galactose, mannose; hexosamines, including glucosamine, galactosamine and mannosamine; and occasionally, to others. sugars, such as fucose, ribose and perhaps rhamnose. It also contains protein products with a high molecular weight, several lipids and nucleic acids.

Example 2

Preparation of purified astrocyte from crude astrocyte precursor-containing fraction.

The astrocyte precursor-containing fraction is further isolated from contaminants. In the preferred embodiments, the material from Example 1 is chromatographed on Sephadex G^50 resin with a typical column that is 40 cm long, 2.5 cm in diameter and has a volume of 196 ml. The pressure used is 40 mm Hg, the flow rate is 35 ml per hour and the buffer is 0.05 molar phosphate buffer solution, pH 7.2. The first (flow-through) tip contains astrocyte precursor along with contaminants, while subsequent tips contain only contaminants.

In the preferred embodiment, the products at the above-mentioned first regenor flow tip are then concentrated on Sephadex G-15, then introduced onto a column of Cellex-D with the same solutions.

(1) to (7) as in Example 1, and the same elution steps as carried out in Example 1. The product astrocyte is present as a sharp

spi-s in the same tubes (number .212-230) as before, thus maintaining its behavior in Cellex-D chromatography without the presence of

a large number of pollutants.

Low molecular weight contaminants can then be removed by techniques known in the industry, such as millipore disc filtration.

In the preferred method, the product astrocyte is made free of salt and other low-molecular-weight impurities by filtration through millipore Pellicon disc No. 1000, 13 mm, which retains substances with higher molecular weights than 1000 and allows through- . leading those with molecular weights less than 1000. The product astrocytin remains on the Pellicon disc, and is recovered from there by washing with solution (1) from example 1.

Astrocyte is then obtained by isolating the compound which has a molecular weight of approx. 8000 from the above solution. In a preferred method, thin layer gel (TLG) is used. chromatography in the following manner: The apparatus used is a commercially available edition constructed by Boehringer Mannheim GmbH; Pharmacia Fine. Chemicals and CAMAG (Switzerland). The resin, 2.5 g of superfine Sephadex G-200, is prepared in 85 ml of 0.5 M NaCl in 0.02 M Na 2 HPO 4 KH 2 PO 4 phosphate buffer, pH 6.8 (6.6-7.0). It is allowed to swell for 2 or 3 days at room temperature with occasional gentle mixing (magnetic and other stirrers should not be used). The swollen gel is stabilized for 3 weeks at refrigerated temperature. However, bacterial and fungal growth can interfere with the swollen gel. If the gel is to be kept for longer periods of time, a small amount of bacteriostatic agent should be added

(sodium azide 0.02%). 2.5 g of dry gel is used to form two glass plates, 20 x 20 cm and 0.5 mm thick. The plates are either given the opportunity to dry at room temperature for 10 minutes and transferred to a humid room where they can be stored for approx. 2 weeks, or they are used immediately after appropriate pre-balancing (usually overnight and a minimum of 12 hours). The main effect of the balancing is to normalize the ratio between the stationary and mobile phase volumes. With the pre-equilibrated plates in a horizontal position, substances to be examined are applied with micropipettes as spots or as a line at the starting line. 10 to 20 ml of 0.2-2% protein solution is placed on the edge of a microscopic coverslip (18 x 18 m) and held against the gel oyer surface. In a few seconds, the solution will sink into the gel. All samples are first formed on the cover slide and then quickly applied. As sufficient material is not used, it is difficult to locate individual spots after separation. If too much material is applied, no defined separation takes place. The samples are diluted with buffer to facilitate processing, and the separation of the samples is performed by a descending technique with the plate held at an angle of 22°. A flow rate of approx. 1-2 cm/hour is most suitable. Marker substances. (such as cytochrome C, hemoglobin, myoglobin or bromophenol blue-labeled albumin) is applied at different locations across the plate to obtain a control for possible variation of flow across the plate, and also serves as a reference protein for calculation of relative distance (mobility) by unknowns. After applying the samples, the plates are put back in the apparatus and the paper wick is gently pushed

downwards to ensure good contact with the gel layer. The paper wick must not drip. Excess moisture is wiped away. The liquid solvent in the container is kept constant at 1 cm from the upper end of the vessel. The experiments are usually completed in 4 to 7 hours depending on the progress of the separation. With colored substances, the separation follows directly. The separated spots of protein are made readily visible by transferring them to a sheet of carbon paper. TLG plate after the chromatographic separation is complete, and by spotting them on pre-washed areas, methanol + 1^0 + acetic acid - 90:5:5, for 48 hours. The paper sheet is 3 mm filter paper. A sheet of paper, 20 x 18 cm, is placed over the gel layer and pressed (rolled) just enough to ensure contact with the gel. It is ensured that no air is introduced under the paper (the copy) and

that the gel layer is not disturbed. The liquid phase is sucked up from the gel layer with the paper and removed after approx. 1 minute, immediately dried in an oven at a temperature of 60°C for 15 minutes and stained in the normal way by any of the routine staining processes. Staining is performed by spraying the copy paper with 0.03% diazotized sulfanilic acid in 10% sodium carbonate (PauleyV's reagent). Staining can also be carried out with a saturated solution of amido black in methanol-acetic acid (90:10 volume/volume is used), and the staining time is 5-10 minutes. For de-staining, rinse with two volumes of the 90:10 solution of methanol and acetic acid mixed with one volume of f^O. It is difficult to achieve low background staining without very extensive washing. The plates themselves can also. dried at approx. 60°C (in an oven with air circulation), but only if astrocyte is to be stained. For icing 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 50-60°C whereby a Sephadex G-200 plate is dried in 15-20 minutes. The dry plates are allowed to swell for 10 minutes in a mixture of methanol + U^O + acetic acid (75:20:5) and stained in saturated amido black in the same solvent system for 5 hours and then washed by bathing for 2 hours in the same solvent before drying. For molecular weight-determining Iser, the distance from is measured. the starting line to the center of each zone with an accuracy of 0.05 mm either directly on the printed matter (the copy) or the densitogram. The result is expressed with the R value defined as the ratio between

m the migration distance of the tested protein (d^) and that of cytochrome C or myoglobin (dm) which is used as a reference protein:

The relationship between migration distance for tested substance to

standard is the formula (~Rm= ^ A straight calibration line is obtained by plotting the logarithm of the molecular weight of the standard used against -R . From this line the molecular weight of the uk girl . protein can be obtained. To obtain the most exact results possible, 6 equal parts are compared with protein sample solution with the standard, in this case cytochrome C, before application to the plate. In the above TLG process, the product astrocyte is observed as a separate spot at a distance of approximately 0.83 t 0.02 referred to the cytochrome C standard, and this gives an approximate molecular weight of 8000 for astrocyte. Several separate products are separated from astrocyte by this method on the basis of small differences in chemical structure and large differences in molecular weight. Thus, 3rd products have been carried as impurities up to this point, with molecular weights of approx. 64,000, 148,000 and 230,000, and a random one with a molecular weight of 32,000, have been detected and removed by the TLG methods described above. n is aspirated with the gel in which it is contained, in dry form, and dissolves in it

solution (1) and is made free of resin by centrifugation or

in any other suitable manner.

The product astrocytin which has been produced by this

step, is soluble in distilled water, soluble at neutral and acidic pH

and insoluble at alkaline pH and has a spectrophotometric absorption peak wavelength at 280 my. It is a polypeptide with a molecular weight, as indicated above, of approximately 8000. Its covalently bound amino acids are shown by hydrolysis with 6N HCl and

so quantitative automatic determination, to have the following average composition of amino acids:

Cysteine, hydroxyproline, norleucine, ammonia, isodesmosine, desmosine, hydroxylysine, lysinonorleucine and gamma-aminobutyric acid are all absent in detectable amounts, but traces of glucosmain may be present.

From 11 g of the starting brain tumor tissue in example 1,

approximately 3 mg of purified astrocyte is formed by the above methods.

Example 3

Production of "trembling" recognin

Tremor disease is a genetic disorder with which animals are unable to achieve stable coordinated motor activity, and a tremor condition is formed, due to a lack of migration of certain nerve cells to a special place in the brain, the cerebellum, at a particular time of development. In particular, it has been shown with glial cells, by electron-microscopic examinations, that they provide vertical rod-like axes along which the nerve cells migrate to their new positions in the normal state. The inability of the nerve cells to ascend along these glial fibres, in tremor disease, is thought to be due to certain disturbances in the nerve cells or in the glia or in both parts.

By following the procedures from Examples 1 and 2,

was recognin formed from mouse tremor brain, and this was compared with recognin formed from normal mouse brain. The molecular weight of recognizen formed from all areas of normal mouse brain was 8,000. The molecular weight of "tremor" recognin was 3,600 to 5,000. The "tremor" redognin is morbid, as evidenced by its much lower molecular weight. Furthermore, the amount of recognin that can is formed from the cerebellum in the tremor mouse brain greatly reduced compared to a normal one This is shown in Table I.

Table I shows that while there is a clear decrease in the concentration of recognizen in the cerebellum of tremor mice, there are equal or greater amounts of recognizen in other areas of the brain. Recogninet may not be moved from the other areas of the brain to the cerebellum, or the slight increase in other areas of tremor-brain may reflect some compensatory effect.

This pathological recognitionin in concussion is consistent with the inability of migrating brain cells to make the contacts that they need to achieve the correct location, and this confirms the role of recognitionin in recognition and learning in cells.

By analogy, the anti-recognins to recognin can be generated from mouse tremor brain and used in mice in a similar manner to the anti-astrocytin and anti-malignin applications described herein.

Example 4

Formation of malignin precursor in artificial cancer cell culture.

An extremely conscientious is generally maintained

sterile technique....

All solutions (e.g. Hank<1>'s balanced salt. (BSS), F-10 nutrient medium, fetal calf serum, trypsin solution) are incubated at approx. 35°C in a water bath for approximately 20 minutes or more before use.

Cells are removed from tumor tissue and cultured in vitro

for many generations using an appropriate medium, as described below. Pre-rinsing of beakers to be used is done with a sterilizing solution, e.g. 12-proponal plus amfyl or creolin solution.

In the preferred embodiment, the artificial cancer cells (ie cells grown in vitro for many generations) are all cultured, in 250 ml flasks. The liquid medium in which the cells are grown is discarded into the pre-rinsed beakers. The cells are then carefully washed with 5-10 ml of Hank's BSS or other similar solution for approx. 30 seconds. Agitation is avoided. All walls and surfaces are washed. The solution is prepared for cells by centrifugation in a cold environment for 10 minutes at 3000 rpm. minute. The medium is poured into a beaker as above. A small amount of buffered proteinase-enzyme solution is added and rinsed quickly to avoid digestion of the cells. In the preferred method, 1-2 ml of trypsin solution (EDTA) is added and it is rinsed for just 10 seconds. The trypsin solution is poured off.

A similar volume of fresh trypsin solution ■ is added and incubated until the cells are seen to separate from the walls of the chambers, by microscopic observation. This usually takes 5-10 minutes. A suitable growth medium is added, such as 50 ml of a solution of a 7-10% solution of fetal calf serum in

100 ml of F-10 nutrient medium.

25 ml of the fresh medium with cells is transferred to a new growth room for propagation, and the remaining 25 ml is kept in the first room for propagation. Both rooms are placed in an incubator at 35°C for approximately 7 days. At this point in the method according to this example, an artificial cancer cell culture is split into two healthy cultures approximately every 7 days..This entire procedure can be repeated as often as desired, with approximately

7-day intervals, for each growth room. Thus, the number of cells grown in vitro can be doubled approximately every 7 days.

The cells can be extracted for the formation of malignancy after approximately 7 days of growth. E.g. cells grown in each 250 ml compartment, as described above, can be recovered in the following manner.

The medium is transferred to a centrifuge tube and centrifuged

with 3000 revolutions per minute in a cold environment for 10 minutes. The medium is discarded. The cells that are back in the growth chamber are scraped

off from the chamber walls and washed into the centrifuge tubes with neutral buffer solution. The cells are washed twice with neutral buffer solution, centrifuged again at 3000 rpm. minute in a cold environment, 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 impure malignin precursor-containing fraction.

. Example 5

Preparation of impure malignin-proooper-containing fraction.

Washed cells suspended in neutral buffer from example 3 are mechanically lysed under conditions that avoid denaturation of most proteins. In the preferred method, the washed cells are treated in a cold environment with a sonic device for 20 seconds.

After treatment in the sonic device, the cell remains are centrifuged at 30,000 rpm. minute for 30 minutes, and the overflowing liquid is decanted. Aliquots of 10 ml of buffer solution are used to wash remaining cells from the chamber and these are added to the rest of the cell remains. Sonication and centrifugation are carried out as above, and the overflowing liquids are combined. The procedure is repeated once more.

The combined supernatants are pre-evaporated to reduce the volume of approximately 30 ml to approx. 6-7 ml. One aliquot is taken for total protein analysis and the rest is fractionated,

in accordance with the methods of Example 1 for astrocyte precursor.

Example 6

Production of purified malignin product from impure malignin-containing fraction.

The product malignin is further isolated from contaminants by the methods in example 2 for astrocyte.

In the TLG step of the preferred embodiment, the product malignin is observed as a discrete spot at a distance of approximately 0.91 ± 0.02 with reference to standard cytobrom C,

and this gives an approximate molecular weight of 10,000 for malignin.

The product malignin which is formed by this step is soluble in distilled water, soluble at neutral or acidic pH and insoluble at alkaline pH, and has. a spectrophotometric absorption ion peak at 280 my. It is a polypeptide with a molecular weight of approximately 10,000.

The molecular weights of malignin formed in yeast cultures stabilized in successive generations of cultures, as determined by thin-layer gel chromatography determinations, are given in Table II. The reproducibility of the molecular weight determinations is remarkable in view of the intrinsic limitations of TLG chromatography.

Malignin's covalently bound amino acids have, by hydrolysis with 6N HC1 and then quantitative determination, been shown to consist of the following average composition of amino acids: The amino acids cysteic acid, hydroxyproline, norleucine, ammonia, isodesmosine, desmosine, hydroxylysine, lysino-norleucine and gamma-aminobutyric acid are absent in detectable quantities.

A typical yield of pure malignin from 12 reaction chambers of 250 ml from example 4 is approximately 1 ml of malignin.

The unique structures of malginin and astrocytin were confirmed by extensive computer studies comparing their composition with virtually all known protein substances.

The amino acid composition in malignin and astrocyte, their content of absolute and relative amounts of each amino acid component expressed as the total number of amino acid residues per moles, and their content of absolute and relative amounts of each amino acid component expressed as the molecular weight of the molecule, were subjected to matrix computer analysis against the largest known collection of literature in the world on protein structure, that of the National Biomedical Research Foundation, Washington, D.C. The. no structural identity or even one very close to that of astrocyte or malignin was found in the matrix analysis compared to several hundred thousand proteins and protein fragments.

The only proteins that were structurally related in any way are shown in Table III with their individual amino composition and molecular weights for the composition. The computer is programmed to identify, from the several hundred thousand possibilities, and show the degree of similarity in structure. Thus, e.g. proteins with molecular weight much larger or smaller do not fit,

and also not those with less than 85 or more than 95 residues, and also not those with less than 6 or more than 11 asparagine residues, and so on for each of the two amino acids involved.

This "fingerprint" thus has as many as 22 individual ones

variable to match. Some substances will fit for 1, for 4 or for 5 variables, but none will fit for all 22. In reality, none fits better than for 14 variables, and this makes a difference for 8 variables.

The most suitable is thus cytochrome b^ (human). As shown in Table III, the residue numbers for alanine, argin, asparagine, aspartic acid, cysteine, glutamine, histidine, methionine and tyrosine and tryptophan all differ markedly to clearly from astrocyte and from malginin. Because cytochrome b,- contains 7 histidine residues, while astrocytin and malginin contain only 2, it is not possible that they. have the same chemical structure;

The most unusual thing about the composition of astrocyte and malignin is the high concentration of glutamic acid. One would expect to find only 5 or 6 remnants of 89.

Other next-closest matches are the ferrodoxins of leucaene glauca and of alfalfa respectively, but these also differ clearly in 4 and 6 amino acids respectively, and noticeably in 2 others, and by having 96 and 97 residues respectively. The next closest fit is the acyl carrier protein of E. coli 26, but this also differs clearly in 11 amino acids from malignin and astrocytin, and has only

77 residues.

Some other brain proteins (neurophysin, bovine and porcine

and gonadotropin. releasing hormone) are listed in Table III for

to illustrate how much worse the adaptation is for the remaining several hundred thousand protein fragments in the computer memory bank.

Respiratory proteins may contain metals and/or heme components in their in situ state, but the isolated protein fragment, e.g. for apocytochrome b,-, contains none. Further microanalysis of astrocyte and malignin has shown that

they are free of iron, sulphur, phosphorus and magnesium (all less than 0.01%), and the spectral properties typically show absorption at 280 my. After recombination of heme with apoprotein, they are restored

typical absorption spectra between 400 and 450 m.

Despite the unique structure of astrocyte and malignin, in contrast to other proteins and protein fragments, it is remarkable and perhaps of great importance that the respiratory proteins have the closest structures. The

is well known in the industry that many important related relationships can be drawn from the structure types that are represented, both from a developmental genetic point of view and from a functional point of view. If astrocytin and malignin are new protein products if in

where structural equivalents represent respiratory functions, and these proteins, as now shown, are linked to malignancy, then an opening has been found to solve one of the biggest problems in cancer, which is how the energy is satisfied for these

•greedy, rapidly reproducing malignant cells.

Apart from the theoretical significance of this discovery, it is meaningful when the above data for the increase in the percentage content of malignin in more malignant cells, and the data provided below showing that anti-malignin does not only attach to malignin-like chemical groups of cancer cells, but is also cytotoxic for the cells when they are attached in this way. If malignin-like in situ compounds in cancer cells are respiratory proteins, and since the anti-malignin products according to this invention attach preferentially to these in situ compounds, then, if functional respiratory groups are involved in this attachment, it is easy to understand how these result in death for the cancer cells.

The therapeutic possibilities of the anti-malignins, and other similar chemoreciprocals, are greatly enhanced by this structural information about malignin and astrocyte, and also by the demonstrated relationship between amount of malignin and degree of malignancy.

Example 7

Demonstration of increased yield, degree of malignancy and amounts of malignin by providing greater volume and surface area during fermentation.

Examples 3 to 5 were repeated using 1000 ml flasks instead of 250 ml flasks. All amounts of reagents were increased in triplicate.

The yield of the product amlignin after 7 days of growth of inoculum was increased to almost double by increasing the available space for malignin cell growth from. 250 to 1000 ml.

Table IV shows the yield of total protein in mg, and the malignin formed as a percentage of total protein for successive fermentation-culture generations in each flask size. When using flasks of 250 ml of the average formation of total protein 17.5 mg. Using 1000 ml flasks, the average formation of total protein was 40.4 mg.

It was surprising that the amount of malignant cell growth (degree of malignancy) was increased per 7 day growth period by providing a larger space and surface for cell growth, and the amount of malignin formed, as a percentage of the total protein, increased significantly. The percentage of total protein that is malignin increased from an average of 10.7% using 250 ml flasks to an average of 28.3% using 1000 ml flasks at a constant growth period of 7 days.

The relationship between the amount of malignin formed and the degree of malignancy (ie, as a function of the amount of malignant cell growth in vitro in 7 days, measured as total protein formed) is shown in Figure I.

Figure I shows that the increased flask size results

in approximately triple the amounts of formed malignin.

Figure I also shows a relationship between malignancy and malignancy. The broken line represents an ideal linear relationship. This shown relationship is clearly not trivial since - the amount of malignancy present increases as the cells become more unrestrained (pathological) in their growth. The normal in situ recognin function relates, as previously stated, to contact inhibition of cell growth. The more pathological the growth of the malignant cells, the less contact inhibition works, and the more malignin becomes the predominant protein.

Example 5A shows that growth of artificial cancer cell culture

in large growth containers unexpectedly results in an increased amount of formed malignin, that is, an increase in the percentage of the total protein that is malignin. Growth containers of large size means, as used here, containers where the ratio between container volume and volume of total medium with cells used in accordance with the methods in example 3 is greater than approx. 8:1, e.g. 7:1 to 10:1. Example 5A shows a ratio of approx. 8:1.

Example 8

Preparation of Target reagents from recognizer.

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

In the preferred embodiment, astrocyte or malignin is dissolved in 0.15 M Nal 2 PO 3 citrate buffer, pH 4.0. A bromoacetyl resin, e.g. bromoacetyl cellulose (BAC) with 1.0 to 1.5 milli-equivalents of Br per grams of cellulose, stored in a cold environment, is prepared in 0.15M NaF^PO^-buffer, pH 7.2. The buffer is transferred to pH 4 by pouring away the pH 7.2 buffer solution and adding 0.15 M Naf-^PO^-citrate buffer, pH 4.0. The astrocyte or malignin solution and the BAC solution are stirred together (10:1 ratio of BAC to recognin) for 30 hours at room temperature, and then centrifuged.

It is preferred that all sites on the BAC that are available for binding to recognizen are bound. This can be performed as follows. The supernatant from the immediately preceding step is lyophilized and the protein content is determined to show the amount of astrocyte or malignin that has not yet complexed with BAC. It formed complex of BAC-astrocyte

(or BAC-malignin) is resuspended in 0.M bicarbonate buffer,

pH 8.9, stirred for 24 hours at 4° to allow formation of chemical bonds between BAC and astrocytein or malignin.

After 24 hours, it is centrifuged, and the supernatant liquid is analyzed for protein. The BAC-astrocytin or BAC-malignin complex is now resuspended in 0.05M aminoethanol - 0.1M bicarbonate buffer,

pH 8.9, to block all unreacted bromine. The suspension is centrifuged and the supernatant is retained but not analyzed due to the presence of aminoethanol. Removal of all unbound astrocyte or malignin is then performed by centrifugation and resuspension for three washes in 0.15M NaCl until no absorbance is measured on the spectrometer at 266 µm. The BAC-astrocyte or BAC-malignin complex is now stirred into

8M urea for 2 hours at 38°C, centrifuged and then washed

(three times is usually sufficient) with 8M urea until

no absorption is shown for the washing liquids at 266 my. The complex is then washed with 0.15M NaCl twice to get rid of the urea. The complex is then stirred at 37°C in 0.25M acetic acid for 2 hours to show its stability. Centrifuge and read the supernatant at 266 my - no absorbance should be present. This chemical complex BAC-astrocytin or BAC-malignin is therefore stable and can now be used as a reagent in the methods described below. In this stable reagent form it is referred to as Target (Topographic-Antigen-like-Reagent-Tamplate) on

because it is a synthetically formed complex whose physical and chemical properties mimic the stable cell-bound precursor of astrocyte or malignin when in a potentially reactive state with serum components. For storage, the Target reagent becomes . centrifuged and washed until neutralized with 0.15M Nat^PO^ buffer, pH 7.2.

The target reagents can be prepared from bromoacetyl-liganded supports other than cellulose, such as bromo-acetylated resins or even filter paper.

Example 9

Manufacture of. antisera for astrocytin, malignin and Target

Antisera to astrocyte, malignin or Target reagents can be generated by inducing an antibody response in a mammal to them. The following procedure has been found to be satisfactory. 1 mg recognin (astrocytin or malignin). injected into the toe pads of white male rabbits with standard Freud<1>'s adjuvant,

and then the same injection is done intraperitoneally 1 week later, again intraperitoneally 10 days later, and, if necessary, 3 weeks later. Specific antibodies can be detected in the blood serum of these rabbits as early as 1 week to 10 days after the first injection. The same procedure is followed for Target antigen by injecting the same amount of Target, which contains 1 mg of astrocyte or malignin, as determined by the Folin-Lowry determination of protein.

The specific antibody to astrocyte is called anti-astrocyte. The specific antibody to malignin is called anti-malignin. In a similar way, the specific antibody is called

. to Target .for anti-Target.

These antibodies clearly show by the Standard Ouchterlony gel diffusion test antigen-antibody reactions with specific single sharp reaction lines formed with their specific antigen. The presence of specific antibodies in the serum can also . tested with the standard quantitative precipitation test for antigen-antibody reactions. Good quantitative precipitation curves are obtained and the micrograms of specific antibodies can be calculated from there.

Further evidence for the presence of specific antibodies in serum can be obtained by absorption of the specific antibody-fat anti-astrocyte on bromoacetylcellulose-astrocytin (BAC-astrocytin) prepared above. The antiserum containing specific anti-astrocytin can be reacted with BAC-astrocytin. When the serum is passed over BAC-astrocyte, only the specific antibodies for astrocyte bind to their specific antigen astrocyte. Since astrocyte is covalently bound to bromoacetylcellulose, the specific antibody, anti-astrocytin, is now bound to BAC-astrocyte to form BAC-astrocytin-anti-astrocytin (BACA-antiastrocytin). This is proven by testing the rest of the serum that has been washed free of BAC-astrocyte. With standard Ouchterlony diffusion, there are no antibodies left in the serum that will react with astrocyte. It is therefore concluded that all specific antibodies (anti-astrocytin) previously shown to be present in the serum have been absorbed on BAC-astrocytin. Furthermore, when antiastrocytin is released from its binding to BAC-astrocytin, it is thus isolated free of any contaminating antibodies. This release of anti-astrocytin can be accomplished by washing the BAC-anti-astrocytin coupled with 0.25M acetic acid (4°C, 2 hours), which, as shown above,

does not break the BAC-astrocyte bond.

Still further proof of the presence of specific antibodies in serum can be obtained by absorption of the specific anti-malignin antibody on bromoacetyl cellulose malignin (BAC-malignin) prepared above. The antiserum containing specific anti-malignin can be reacted with BAC-malignin. When the serum is passed over BAC-malignin, only the specific antibodies bind to malignin. to their specific antigen malignin. Since malignin is covalently bound to bromoacetylcellulose, the specific antibody, anti-malignin, is now bound to BAC-malignin to form BAC-malignin-anti-malignin (BACM-anti-malignin). This is proven by testing the rest of the serum which is washed free of BAC malignin. With standard Ouchterlony diffusion, there are now no antibodies left in the serum that will react with malignin. It is therefore concluded that all specific antibodies (anti-malignin) previously shown to be present in the serum have been absorbed into BAC-malignin. Furthermore, when anti-malignin is released from its binding to BAC-malignin, it is therefore isolated free of any contaminating antibodies. This release of anti-malignin can be accomplished by washing the BACM-anti-malignin complex with 0.25M acetic acid (4°C, 2 hours) which, as shown above, does not break the BAC^malignin bond.

The antibodies to Target clearly show by standard Ouchterlony gel diffusion test for antigen-antibody reactions specific single reaction lines formed with Target, showing a line of identity with the reaction line of anti-astrocytin or anti-malignin antisera (i.e. such formed for the injection of astrocyte or malignin itself). Certain rabbits, it has been noted, have levels of anti-Target in their blood prior to being injected with Target. These anti-Target substances, when they are specifically reacted with Target reagent as described in tests on human sera, lead to the formation of approximately equivalent amounts of the two types of TAG, S-TAG and F-TAG (see later examples) .

Example 10

Detection of malignant tumors by quantitative in vitro formation of Target-attaching globulins (TAG) from biological fluids.

Target reagent prepared in accordance with Example 6,

washed to remove any unbound recognin that may be present due to damage. The following procedure is satisfactory. Target reagent is stirred for 2 hours at 37°C with acetic acid, centrifuged, the supernatant liquid is decanted and the optical density of the supernatant liquid is read at 266 mp. If there is any absorption, this washing is repeated until no further material is solubilized. The target is then resuspended in phosphate-buffered saline, pH 7.2. ' (Standard S-TAG and F-TAG purified from previous reactions of human serum by the method described below may be used, if available, as reference standards for testing the Target reagent, and this may also rabbit serum as determined contains S-TAG and F-TAG in other Target formulations).

The slow-binding (S-TAG) determination is performed as follows: Frozen serum stored for more than a few days should not be used. Serum is carefully prepared from fresh whole blood or other body fluid using standard industry procedures. The following procedure has been found to be satisfactory. Blood is allowed to coagulate by leaving it for 2 hours at room temperature in a glass test tube. The solidified clumps are separated from the walls with a glass stirring rod and the blood is stored at 4°C for at least 2 hours (or overnight). The solidified clumps are separated from the serum by centrifugation at 20,000 rpm. minute at 4°C for 45 minutes. The serum is decanted into a centrifuge tube and centrifuged again at 2000 rpm. minute at 4°C for 45 minutes. 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, pH 10) is added in an amount of 1% of the serum volume.

Serum samples, prepared by the above or other methods, of 0.2 ml each are added to each of 0.25 ml aliquots of Target suspension reagent containing 100-200 micrograms of recognin per 0.25 ml Target reagent, for duplicate determinations. The suspension is mixed at 4°C so that pellet formation is avoided.

E.g. a small rapid shaker with a rubber sleeve can be used in

1-2 seconds and then, with the tubes slightly sloping, it can be shaken in a Thomas shaker for approx. 2 hours or more. The target reagent and protein bound to it are separated from the serum. One of the methods which has been found to be satisfactory is the following. The tubes are then centrifuged at 2000 rpm. minute for 20 minutes at 4°C, the supernatant liquid is decanted, the pellets formed by centrifugation are washed 3 times by re-mixing and shaking at room temperature with 0.2-0.3 ml of 0.15M. saline solution,

it is centrifuged and the supernatant is discarded.

The protein that is attached back to the Target is cleaved from there and determined quantitatively. For example, 0.2 ml of 0.25 M acetic acid is added, the suspension is shaken for 1 to 2 seconds with a rubber sleeve shaker, then in a. Thomas shaker for approx. 2 hours in an incubator at 37°C. The tubes are centrifuged at 2000 rpm per minute at 4°C for 30 minutes. The supernatant is carefully decanted to avoid transfer of particles, and the optical density of the supernatant is read at 280 mp. -The value of the optical density is divided by a factor of 1.46 to obtain results in micrograms per ml serum protein (S-TAG). Duplicate determinations should not vary by more than 5%. Any other effective method for determining protein content can be used, such as the Folin-Lowry assay, but the standards must be specified to determine the regulatory range and tumor values of S-TAG minus F-TAG concentration.

The fast-binding (F-TAG) determination is performed as follows: Frozen serum stored for more than a few days should not be used. Serum is carefully prepared from freshly obtained whole blood or other body fluid using standard methods in the industry. The procedure given above in this example for serum preparation is satisfactory.

Serum samples, prepared by the above or other methods, are stored at 4°C for 10 minutes less than the total time the S-TAG serum determinations were allowed to be in contact with the Target reagent above (e.g. 1 hour and 50 minutes if a "two-hour" S-TAG determination was performed).. This method compensates for the temperature differences of S-TAG and F-TAG determinations.

Samples of 0.2 ml of temperature-equalized serum are added to each of aliquots of 0.25 ml of Target suspension reagent containing 100-200 micrograms of recognin per 0.25 ml Target reagent, for duplicate determinations. The suspension is then mixed at 4°C for approximately 10 minutes in such a way that pellet formation is avoided. For example, a small, rapidly shaken rubber sleeve can be used for 1-2 seconds and then, stirring slightly at an angle, it can be shaken in a Thomas shaker for approximately 10 minutes. The Target reagent and protein bound to it are separated from the serum. One of the methods that has been found satisfactory is the following. The tubes are then centrifuged at 2000 rpm. minute at 4°C, the supernatant is decanted, the pellets formed by centrifugation are washed 3 times by re-mixing and shaking at room temperature with 0.2-3.0 ml of 0.15M salt solution, centrifuged and the supernatant is discarded.

The remaining protein attached to the Target is cleaved from there and determined quantitatively. The procedure described above in this example for determining S-TAG concentration is satisfactory. Any other method effective for determining protein content may be used, such as the Folin-Lowry assay, but the standards must be specified to determine the control range and the tumor values of S-TAG minus F-TAG concentration.

The final results are expressed as TAG micrograms per ml serum, and is equal to S-TAG minus F-TAG. TAG values in non-brain tumor patients and other controls often range from zero (or a negative number) to 140 micrograms per ml of serum. TAG values in brain tumor patients are often in the range from 141 to 500 micrograms per ml of serum. In the first "blind" examination of 50 blood samples according to the methods of this example, using Target reagent prepared from astrocyte and bromoacetylcellulose, 11 of 11 brain tumors and 28 of 32 normal patients were correctly identified. One of the 4

putatively normal controls (ie, non-brain tumor controls) showed

turned out to have cancer of the thyroid gland, which had apparently been successfully treated a few years earlier. The 3 remaining normal controls were individuals aged 60-70 who were in poor health, possibly with undiagnosed cancer. Of the remaining 7 samples, 3 out of 3 cases of Hodgkin's disease were correctly identified, Sn sample in the tumor area (141-500 yg TAG/ml) corresponded to a patient with a severe gliosis, and 3 samples in the non-tumor area.

(0-140 yg TAG/ml) corresponded to patients with respectively a

intracranial mass diagnosis uncertain but non-tumor, and osteosarcoma (non-brain tumor) and a melanotic sarcoma (non-brain tumor).

A subsequent examination according to the methods of this example using Target reagent prepared from malignin and bromoacetyl cellulose correctly identified three of three malignant brain tumors and all normal controls. A still further investigation carried out according to the methods of this example extended the total number of human serum samples tested from 50 to 114. The applicability of these products and methods is shown in Table V which shows the results of these tests.

Includes normal controls, non-tumor medical and

surgical interference.

I - very ill, undiagnosed; 2. - extra brain intracranial mass, undiagnosed; 3 - marked gliosis; 4 - malignant melanoma, 5 - osteosarcoma; 6 - brain-bladder fluid; 7 - adenocarcinoma of the colon; 8 - gastrectomy; 9 - headache; 10 - emphysema,

II - polymyalgia; 12 - colon cancer, 13 - convulsions;

14 - prostrate cancer, secondary to bone; 15- clinically "normal",

18 months earlier, when this morbid serum TAG was.achieved: riy developed severe headaches, loss of taste and smell.

Example 11

Diagnosis of tumor cells by immunofluorescence.

The compounds anti-astrocytin, anti-malignin and S-TAG

have shown that they preferentially attach to tumor cells. This specificity makes it possible to use these compounds to diagnose tumor cells in histological sections by conjugating dyes or radioactive substances to anti-astrocytin, anti-malignin or S-TAG. Standard marking techniques can be used.

A procedure for using S-TAG is as follows.

A method that has been found to be satisfactory is a modified St. Marie process. Human brain tumor samples are

frozen and then parts are cut out with a thickness of

5 microns. These are stored in a moist container at minus 70°C

for 4 to 8 weeks before spotting. The conjugate may be a standard antiserum such as goat anti-rabbit conjugate. The conjugate is labeled by techniques known in the industry with a fluorescent or other labeling substance. Fluorescently labeled goat anti-rabbit conjugate that is commercially available can be used. A fluorescence technique was used which was a standard whereby a 1:200 to 1:400 solution of TAG is incubated for approx. 30 minutes or more on the tumor section, followed by washing to remove unattached TAG.

Three washings with phosphate-buffered saline have been found to be satisfactory. Conjugate incubation with fluorescein-labeled conjugate followed by washing is then performed, followed by microscopic examination. Normal cells and their processes fail with respect to staining both for tumor sections and in control sections of normal non-tumor brain. Fluorescence is clearly present in tumor-glial cells and their processes.

Example 12

Detection of malignant non-brain cells with fluorescent signal from TAG.

The use of TAG products coupled with a signal emitter, such as a dye or a radioactive label, . to detect cancer cells, is described here. In this example, detection of malignant non-brain cells is described.

As described in example 10, human serum is used.

In the determination of TAG, after the anti-malignant antibody was bound to the immobilized antigen and unbound serum protein was washed away, the antibody was cleaved from the bond with 0.25M acetic acid at 37°C for 2 hours and the Target reagent separated from there by centrifugation. The TAG-antibody solution was quantitated by its absorbance at 280 μm. The TAG solutions were stored at 20°C, then thawed and combined, brought to pH 7 by titration with 6N NaOH, dialyzed against phosphate-buffered saline at pH 7, filtered and concentrated on millifiber Pellicon 1000 membranes, centrifuged to clear insoluble protein and the immunoglobulin complexes were concentrated and freed from immunologically inactive compounds by Cellex D and blue Sepharose CL6B (Pharmacia) chromatography. This human anti-malignin antibody reacts with anti-human gamma-globulin by Ouchterlony double-diffusion. When TAG is used with fluorescent agent conjugated to anti-human gamma- . globulin by standard double-layer Coon's immunofluorescence, it stains malignant glia, breast carcinoma, ovarian carcinoma, colon adenocarcinoma and other types of cancer cells in post-operative and biopsy tissue sections, and also in human saliva,

bronchial lavage fluids, pleural effusion fluids, gastric aspirate and bladder urine. The concentration of protein in TAG that gives clear fluorescence when the controls are negative is 1 to 10 ug per share.

The production of "purified" TAG was accomplished by reacting sera from patients with a variety of cancers with bromoacetylcellulose malignin by methods previously described (Example 10). The antibody bound by this reaction was cleaved with 0.25M acetic acid, quantitated by measurement at the O.D. 280 using a conversion factor of 1.46 for gamma globulin, frozen and stored at 20°C. This antibody was found to contain immunoglobulin as determined by anti-human gamma globulin antiserum specific for gamma chains (BioRad Laboratories, Inc.) and by anti-FAB and anti-Fe fragments (Miles Laboratories). It also reacts with rabbit anti-human albumin (BioRad Laboratories).

It was found that while 10 to 50 micrograms of protein TAG

is required to form specific immunofluorescent staining

of cells containing malignin, only 1 to 10 micrograms of purified protein TAG is required for this specific staining in all sections, and in a few cases less than one microgram has been found

to be sufficient.

It was found that the most active preparation of purified TAG is that which is eluted with the highest ionic strength, ie from 0.15M to 1.5M. Any promotion method that takes advantage of this fact is useful. Three preferred methods are given below.

Method I - Fractionation of TAG by chromatography with DEAE

cellulose (Cellex D, BioRad Laboratories) was first used by stepwise elution with increasing ionic strength and decreasing pH, the same sequence of eluents as indicated in Example I for preparation of impure astrocyte precursor-containing fraction. Good separation of the protein mass into three fractions was achieved, peak I obtained with solution 1 (see example 1) and peak II obtained with solution 6 and solution 7. Ouchterlony double diffusion showed

that TAG at tip I still contains noticeably protein with albumin mobility> and even though tip II contained most of the albumin, it could be noticeably detected with IgG. Rechromatography of peak I gave a progressively pure IgG until, after it.

7, chromatography, essentially no albumin (less than 3%) could be detected by Ouchterlony gel diffusion, by

of which 5 to 10 micrograms of human albumin was detectable with rabbit anti-human albumin. The IgG thus obtained was prone to denaturation and loss of immunological reactivity after a few days' rest at 0-5°C.

Method II - A second fractionation of TAG was performed by chromatography on Sepharose CL-6B (Pharmacia, Inc.) by

start with low-molarity buffer (0.005M phosphate) and proceed in two steps with 0.15M and 1.5M to elute the rest of the protein. As with Cellex D, one pass was found to be insufficient for separation, and recycling slowly improved the product.

Again, the most active fraction vis-a-vis anti-malignin antibody was in the 1.5M fraction.

Method III - Chromatography with Sepharose CL-6B closest to the glass fritted disk and Cellex D layer over the Sepharose proved to be the most satisfactory method.

The graphic representation in figure 2 shows the fractions obtained by chromatography of TAG using method III.

After the first eluate of 200 ml, sub-fractions of 50 ml or less were collected. The protein content in each

eluate was determined by the optical density at 280 mu with an equal factor of 1.46 based on gamma-globulin used to convert to micrograms for recovery calculation. The absolute amount of protein must be corrected for those fractions in which albumin is noticeable. The points at which the stepwise solvent changes were made are shown by arrows. The sub-fractions are denoted by Roman numerals I to VIII.

The solutions corresponding to the letters A-F by the arrows,

was the following:

A - 0.01M TRIS (pH.7.2)

B - 0.05M TRIS with 0.1M NaCl (pH 7.2)

C - PBS, 0.11M NaCl (pH 7.2)

D - PBS, 0.16 5M NaCl (pH 7.2)

E - PBS, 0.33M NaCl (pH 7.2)

F - 0.05M TRIS, 1.5M NaCl (pH 7.2).

The following table shows the recoveries from each fraction, a semi-quantitative determination in each of gamma-globulin and albumin in each, and also the activity of each fraction in the immunofluorescent staining of cancer cells.

(The plus sign means reaction, zero no reaction and plus/minus reaction in some cases).

Figure 3 is a photographic representation of the reaction line between anti-human gamma globulin specific for gamma chains for each of fractions I and II from above (left. and below in the photograph).

Figures 3a and 3b are photographs showing the application

of TAG (fraction VIII from above) to stain malignant non-brain cells. Figure 4a is a staining of bronchogenic carcinoma cells

in the bronchial washing fluids of a patient. Fibur 4b is a staining of lymphoma cells in the pleural fluid of a patient. Non-cancerous cells do not fluoresce. TAB (1 to 10 pg in

0.1 ml phosphate-buffered saline (PBS)) is applied to the surface of packed cells on a glass slide incubated 30 minutes,

washed three times with PBS and then coated with fluorescein-conjugated anti-human IgG diluted until non-malignant control tissue gives essentially no fluorescence. The cells are visualized with a Zeiss fluorescence microscope using a tungsten lamp and filters BG 23, BG 12 and 500.

Example 13

Detection of cancer cells with radioisotope signals from TAG.

In this example, the possibility of attaching a radioactive label to the TAG is demonstrated. Second, the injection into animals of this radiolabeled TAG has been performed and shown to be safe and effective. Third, the radiolabeled TAG was localized preferentially in cancer tissue when compared with normal tissue, showing that the specificity previously demonstrated in vitro on the preference for cancer cells promoted by the use of specific anti-malignin TAG products has been confirmed in vivo .

9 9 m

Herkesetting of TAG with 99m technetium (Tc)

Marking procedure

1. Two preparations of TAG were used, here designated TAG-1

and TAG-2. TAG-1 and TAG-2 (concentrations of each 0.4 mg/0.5 ml) were added to separate sterile test tubes.

2. 0.1 ml was added to each test tube. a stannous chloride solution (10 mg SnCl 2 -2H 2 O in 100 ml of 0.01N HCl). The sample glasses were mixed for 3-4 minutes. 99m 3. 0.1 ml (6mCi) of Tc pertechnate (sodium salt) was added and mixed for 2-3 minutes.

Procedure for determining the marking effect

9 9m 9 9m Samples of Tc-TAG-1 and Tc-TAG-2 were tested for labeling effect by downflow paper chromatography using Watman No. 1 paper with 85% methanol as solvent. A similar study was performed with sodium pertechnetate- 9 9 mTc acting as a control After 2 hours, the papers were removed from the chromatography tank and divided into 2 sections: (1) 1 cm from the outlet; (2) the remaining papers up to the solvent front. Each section was then counted in a gamma wave spark counter and its content of radioactivity was determined (cpm).

Approximately 50 lambdas were plated for each paper strip.

Procedure for antigen-antibody reaction

A portion of the labeled solution was also plated on an Ouchterlony gel plate to determine its ability to react with malignin in the antigen-antibody reaction. After a period of 3 hours, the resulting sharp reaction lines were removed from the gel and their content of radioactivity was measured. An equal part of the gel not involved in the reaction was also removed and its content of radioactivity was also measured as background.

Results

Branding effect

Conclusions

The following•conclusions were reached with regard to

the applied quality control tests:

9 9m 1. Tc-pertechnetate was reduced by tin (II) chloride to a more efficient oxidation state (+4+5). 2. The reduced pertechnetate formed marks with both the TAG-1 and TAG-2 preparations. 3. 99mTc-TAG-2 was tested for its ability to

retain their activity, and were found to retain the ability to react immunologically.

The use of radiolabeled TAG in vivo to detect cancer cells

Wistar rats were injected intracerebrally with cisglioma tumor cells previously screened in rats and in tissue culture. The rats were observed for the first signs of tumor growth, such as weakness, tremors or unsteadiness. These symptoms first appeared 7 to 10 days after injection, and rapidly growing tumors resulted in death within 3 to 4 days for many animals, and one week for all. As soon as symptoms appeared, the animals were injected with labeled TAG intravenously into the tail vein, and then the animals were anesthetized after varying times, the brain was removed, the tumor was cut free of normal brain, and the radioactivity in each cut sample was compared.

9 9m

Preliminary Tc-TAG trial

Samples of tumor and of normal brain were counted overnight in the gamma wave counter. All samples and standards were first and foremost corrected for simplification to the average number for the first sample in the sequence.

Conclusion

The preferential localization of radioactivity in tumor, compared to normal tissue, is shown above.

Example 14

Demonstration that anti-astrocytin, anti-malignin and S-TAG are cytotoxic for tumor cell growth in tissue culture.

Standard tests can be used to determine cytotoxicity. Usually, the number of cells in a fixed counting room, usually arranged to contain approx. 100 live cells, counted. These cells are then treated with the agent being tested,

and the number of cells still alive is counted.

In a standard test of cytotoxicity of S-TAG solution obtained in accordance with the methods of Example 10, against cells in tissue cultures derived from a patient with a glioblastoma grade III-IV, well characterized as of glial origin, S-TAG produced death for all cells in the counting room even in a high dilution of

1:100 and 1:1000, representing as little as 0.2 and 0.02 µg of S-TAG per ml solution. Similar results have been obtained with high dilutions of anti-astrocytin and anti-malignin.

Both the specificity shown in example 11 and the cytotoxicity shown in example 12 are very relevant to the therapeutic possibilities of anti-astrocytin, anti-malignin and S-TAG for brain tumors in humans. While these therapeutic applications lie in the future, the practical diagnostic potential of both of these phenomena for tumor tissue removed by surgery but requiring diagnosis by histology has already been demonstrated here.

Example 15

Hydrolytic cleavage of recognizer.

A solution of recognin, in this case either astrocyte or malignin, at a pH between 1 and 2 is stored in a cold environment. After 7 to 14 days, TLG chromatography shows that the product has been reduced in molecular weight by approximately 200. When the solution is allowed to stand longer, additional units with approximately 200 in molecular weight are split off every 7 to 10 days. Thus, for astrocyte the molecular weight is reduced from 8,000, and for malignin it is reduced from 10,000, in each case by units of approximately 200 sequentially.

The physiochemical specificities of astrocyte are maintained for each product down to a molecular weight of approximately 4000. The physiochemical specificities of malignin are maintained for each product down to a molecular weight of approximately 5000. This has been shown with the Ouchterlony gel diffusion test against respectively anti- astrocyte and anti-malignin.

This cleavage can also be carried out enzymatically, such as with trypsin and other proteinases, with similar results.

The molecular weights of these compounds produced by hydrolytic cleavage of recogniner can be approximately defined with the following formulas:

For products with the physiochemical specificities of astrocyte:

For products with the physiochemical specificities of malignin:

where y is the molecular weight of the product and x is an integer from 0 to 19.

Example 16

Production of artificial tissue or organ with recognizer.

A rigid-walled tube of plastic, metal or other suitable rigid material is dipped in. in or impregnated with a highly concentrated (ie 10 mg/ml) viscous solution of recognizen, in this case either astrocyte or malignin, until all surfaces are completely coated with recognizen. Alternatively, recognin solution is passed through and around the pipe under pressure until all surfaces are completely coated. The tube is then dried in air or in a vacuum, or lyophilized. The coating process is repeated several times to build up several molecular layers of recognizen coating.

The tube is now ready to be placed in a cavity or in a tissue containing astrocyte- or malignin-like precursors 1 nearby tissue or fluid in a living mammal. This artificial tissue or organ can be used to minimize or eliminate reactions that foreign substances without recognin coating would incite.

Artificial tissues or organs of other geometries can be produced in a similar way.

Claims (10)

1. Method for detecting the presence of cancerous or malignant tumor cells in a collection of cells, which comprises applying an anti-malignant TAG product attached to visible or signal-emitting means to said cell collection whereby said anti-malignant TAG product is preferentially attached to cancerous or malignant cells, and detection of said cancerous or malignant tumor cells by means of said visible or signal-emitting means. 2. Method for detecting the presence of cancerous or malignant tumor cells in a cell collection according to claim 1, which comprises applying to said cell collection an anti-malignant TAG product, and then applying fluorescein-conjugated anti-TAG thereon, whereby the Fluorescence takes place only in the cancerous or malignant tumor cells upon illumination. 3. Method according to claim 1 or 2, whereby the cancerous tumor cells are glial tumor cells. 4. Method according to claim 1 or 2, whereby the cancerous tumor cells are non-glial tumor cells. 5. A method according to any one of claims 1 to 4, wherein the TAG product is formed by reacting anti-malignin serum with a malignin-based reagent. 6. Method according to claim 5, whereby the malignin-based reagent is bromoacetylcellulose malignin. 7. Method according to claim 2, whereby the TAG product has been at least partially freed from substances which are little or non-reactive by fluorescent detection when applied to known cancerous cells. 8. Method according to claim 1, whereby the anti-malignin TAG product is attached to a signal emitting agent. 9. Method according to claim 8, whereby the TAG product is directly attached to the signal emitting agent. 10. Method according to claim 8, whereby the TAG product is indirectly attached to the signal emitting means.