JPH01112163A - Recognine and related substance - Google Patents

Abstract

PURPOSE: To diagnose and treat cancer by extracting a normal or an ill cell or organization by a neutral buffer liquid and binding lecognin that is sampled by separating a fraction of pK value 1-4 to a carrier. CONSTITUTION: Normal or ill cell or organization is extracted by a neutral buffer liquid, a fraction with pK value of 1-4 is separated from obtained solubilization protein extract, acid peptide sampled from the fraction is set as lecognin. The lecognin is bound to a carrier. The carrier is a material with cellulose as a base and is made of, for example, bromoacetylcellulose. The lecognin is bound to anti-lecognin by specifical immunity adsorption when being bound to an inactive carrier, thus determining and preparing an antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to Recognis and related substances, which are novel acidic peptides that are particularly useful in the diagnosis and treatment of malignant tumors or cancer.

Recognins are obtained by processing normal or abnormal cells or tissues (e.g. tumor cells, artificial cancer cells) and are brought into contact with body fluids, either supported on a support or without support. In particular, the chemoreciprocals (Chemoreciproca)
ls) can be obtained. These chemoresibrocals can be tested in vivo or in vitro (e.g. quantitative precipitation tests, Ouchterlony
) Antigen-antibody gel diffusion test in agar, immunofluorescence test) It is a substance that reacts with recognins with immunochemical specificity.

Recognins provided by the present invention are obtained by extracting normal or diseased cells or tissues with a neutral buffer, separating a pl (value 1 to 4 fraction) from the resulting solubilized protein extract, and collecting the recognins from this fraction. It is an acidic peptide with the following properties: (a) It is capable of forming a single linear precipitate on specific antibodies in quantitative precipitation tests and antigen-antibody gel diffusion tests in Raphthaloni-agar. I can do it.

(b) Soluble in water and aqueous solutions at acidic or neutral pH; (c) Slightly soluble in water and aqueous solutions at alkaline pH; (d) Has a spectral absorption peak wavelength of 280 Rμ; (e)
It has a molecule ff of 14,000-10,000.

0) high content of glutamic acid and aspartic acid, high ratio of glutamic acid to aspartic acid to hisdecine; (g) produces anti-recognin antibodies when injected into animals;
The antibody specifically reacts with other acidic peptides belonging to the recognin class: (h) binds to the anti-recognin antibody by specific immunoadsorption when bound to an inert carrier, thereby enabling the quantification and purification of the antibody; In addition, it enables the antibody to specifically bind to acidic peptides belonging to the recognins group or cells containing the same, both in hydro and in vitro.

Recognins specifically include various acidic peptides with the above-mentioned properties, the representative one being As)
・Locytin (AsLrocytin) and malignin (M
alignment).

Astrocytin - Astrocytin is obtained from brain tumor tissue, preferably from brain collagen tissue. First, protein components including the astrontin precursor are extracted from the tissue. In this case, a preferred extraction method is to treat the tissue with a neutral buffer and homogenization or other suitable destructive conditions. The protein fraction containing astrocytin precursors is solubilized.

At this point, the astrocytin precursor is still bound to large molecular weight substances such as proteins, glycoproteins, lipoproteins, nucleic acids, and nuclear proteins. The solubilized extract is clarified to remove insoluble particles and low molecular weight impurities are removed by evaporative concentration techniques. The remaining solution is processed to separate the astrosidin precursor from other impurities and obtain a protein fraction with a pK value of 1-4. The treatment in this case is carried out, for example, by pouring the solution into a chromatography column and eluting it with a solvent whose acidity is increased stepwise. neutral or pK
Discard the fractions eluting with acidity up to pK1
~4 fractions are collected and the collected fractions are processed to obtain a material with a molecular weight of approximately 8.000. In this case, the treatment is carried out, for example, by filtering the fraction to remove low molecular weight substances, that is, substances with a molecular weight of 1,000 or less, and then filtering again to remove substances with a molecular fi of 25,000 or more. Molecule fit, 000
~25,000 fractions are processed, for example, by thin layer gel chromatography to obtain astrocytin.

In this way, astrocytin is obtained by extracting brain nerve collagen tissue with a neutral buffer, repeating homogenization and high-speed centrifugation, and extracting the resulting extract with a K value of approximately 1 to 4.
It can be obtained by separating a fraction of , collecting a high molecular weight substance of up to about 230,000 from this separated fraction, and isolating therefrom a substance with a molecular weight of about g, ooo.

Astrocytin obtained by the above method forms a single line precipitate with its specific antibody in quantitative precipitation tests and Uftalony gel diffusion tests, is soluble in water or aqueous solutions of acidic or neutral pH, and is difficult to dissolve at alkaline pH. It is characterized by having an absorption peak wavelength of 28-μ by a spectrophotometer and a molecular weight of about g, ooo.

Astrocytin is also characterized by a very high proportion of glutamic acid and aspartic acid residues, and a very high proportion of these acids to histidine.

Malignin Neal lignin can be obtained from artificial cancer cells, i.e. cancer cells cultured in vitro, has a molecular weight of approximately 10°000, and contains amino acids similar to but different from astrocytin. It has a residue composition. That is, the ratio of glutamic acid and aspartic acid is high, and the ratio of these acids to histidine is high.

Malignin is extracted from artificial cancer cells grown in a fermentation medium by repeating homogenization and high-speed centrifugation with a neutral buffer, and a fraction with a pK value of 1 to 4 is separated from this extract. Approximately 230,0
Collect a high molecular weight substance with a molecular weight of about 1.
It can be obtained by isolating 0.000 of the substance. The amount of malignin produced in the artificial cell culture and the percentage of total protein produced in the artificial cell culture is increased by carrying out the artificial cancer cell culture in a large volume incubator.

The malignin obtained in this way forms a single line precipitate with its specific antibody in the constant M precipitation test and the Rhapthaloni gel diffusion test, is soluble in water or aqueous solutions of acidic or neutral pH, and is soluble in alkaline It is poorly soluble in pl (
The absorption peak wavelength measured by a spectrophotometer is 28h+μ,
It is characterized by having a molecular weight of about 10,000.

In addition, recognins can be complexed with bromoacetylcellulose to form bromoacetylcellulose-recognin, and when injected into mammals, anti-recognin, which is a specific antibody, can be produced. Specifically attaches to recognin precursors. For example, one antirecognin, antimalignin, is toxic to brain tumor cells in vitro.

Recognins, such as astrodin, malignin, and similar substances, are useful as substances that may be introduced into biological systems to reduce extraneous reactions such as coating materials with recognin. There are other examples of introducing recognin to generate chemoresibrocals in biological systems. Recognin may also be used to nutritionally promote the growth of specific biological systems. The utility of Recognin also lies in the ability to produce TargetL agents consisting of complexes of Recognin with carriers to promote adaptability in biological systems. And, for example, the recognin complex has the physical and chemical properties of recognin itself. The carrier should be selected from materials that form a complex with recognin and are biologically substantially inert.

Any known substance that forms a stable complex with a polypeptide or protein is useful for conjugating with recognin.

Examples include cellulosic materials such as bromoacetyl cellulose. In addition to being inert to biological systems, the carrier must not alter the unique physicochemical properties of recognin for the purposes described herein. The complex consisting of recognin and its carrier produces chemoreciprocals in the contacted biological system;
Useful for isolating and confirming. The recognin-carrier complex is also useful in facilitating the production of chemoresibrocal precursors in the biological system into which it is introduced.

The second class of chemoreciprocals are the antirecognis, namely antiastrotisin and antimalignin. These substances can be produced by injecting Recognin into biological systems. Immunologically effective doses of Recognin are calculated upon contact with body tissues or fluids to elicit an antibody response according to conventional antibody production techniques. Antirecognins can be used to administer substances such as diagnostics, nutrients, and therapeutics to specific cells or locations in a biological system by introducing a complex swelling agent with antirecognins into the biological system. . The anti-recognin can also be used to detect the presence of tumor cells in a living tissue by applying anti-recognin containing a labeling substance such as a dye and a radioactive substance to the living tissue so that the colored or radioactive label occurs only in the tumor cells. This is useful for diagnosing whether Further uses of anti-recognins include injecting anti-recognins into mammals and other biological systems.
Other useful chemoreciprocals (e.g. TAG1 below)
This is to increase output from mammals.

Traditionally, glycoprotein complexes have been produced from brain tissue to form antibodies. Tay-SachS
An isolated substance, known as IOB glycoprotein, made from the diseased brain of a man (1997) was injected into rabbits to generate antibodies. These Tysahas antibodies were used in immunofluorescence studies of tumor-bearing brains.

Only reactive normal non-tumor glia, not tumor glia, were colored by these antibodies.

In contrast, when astrocytin was produced from tumor tissue, antibodies against astrocytin (anti-astrocydin) were produced and used in brain immunofluorescence studies, and normal (non-tumor) glia Only tumor glia were colored by anti-astrocytin. It is therefore clear that, depending on the causative tissue, the nature of the antibody and the specific type of cells colored, =14- anti-Tisahas antibodies and anti-astrocytin are different substances.

Another class of chemoreciprocals are targeting agents that are complexed with their chemoreciprocals.

For example, a target (astrocytin material complexed with a carrier such as bromoacetyl-cellulose) is used to contact anti-astrocytin. Compounds of this type can be conjugated and used to administer diagnostic, nutritional, and therapeutic agents to specific cells or locations in biological systems. These compounds can also be used for purification treatments. For example, anti-astrocytin can be produced by hydrolyzing promacetylcellulose-astrocytin-anti-astrocytin with acid or proteinase enzymes. Targeting agents also serve to increase the amount of TAG substances (described below) in a biological system, such as by contacting an immunologically effective dose of the target with body tissues or fluids.

Additional chemoreciprocals are TAG agents (globulins that attach to targets). The TAG material is obtained by contacting the targeting agent with body fluids to vary the time period for complexing and degrading the TAG.

Two useful embodiments are 5-TAG and F''-TAG.

S-T A G (S L 0W-T raget
-Attaching-G 1obulin) is prepared by reacting serum or other body fluid with the target (i.e., bromoacetylcellulose-malignin) for about 2 hours or more at a low temperature, e.g., about 4°C, to remove 5-TAG from the product. cleavage, for example with dilute acid at a temperature of about 37° C. for about 2 hours. The product 5-TAG produced according to this method is soluble in aqueous buffer, forms a monolithic precipitate with its counterpart Recognin in an antigen-antibody gel diffusion test in agar, is not dialyzable on cellophane membranes,
It is retained in a Millipore filter that retains molecules with a molecular weight of 25,000 or more, and the macroglobulin range is approximately 50,000.
It is characterized by having a differentially solidifying molecular weight as determined by thin film gel chromatography of 00 and multiples thereof, and having a spectrophotometric absorption peak wavelength of 280 mμ.

F-TAG (Fast-Target-At
The method for producing F-TAG (taching-G 1 obulin) involves reacting serum or other body fluid with the target (i.e., bromoacetylcellulose-malignin) for about 10 minutes at a low temperature, e.g., 4°C, and cleaving F-TAG from the product, e.g. It consists of cleavage with dilute acid for about 2 hours at a temperature of about 37°C. Product F-TAG manufactured according to this method
Millipore is soluble in aqueous buffers, forms a single precipitate with its counterpart Recognin in an agar-in-agar antigen-antibody gel diffusion test, is not dialyzable on cellophane membranes, and retains molecules with molecular weights greater than 25,000. have a molecular weight that is retained in the filter and coagulates differently as determined by thin layer gel chromatography from the macroglobulin range to about 50,000 and multiples thereof; and
It is characterized by having a spectrophotometer absorption peak wavelength of 0 mμ.

TAG products are designed to determine the concentration of 5-TAG and 1-TAG produced by known amounts of mammalian serum or other body fluids and correlate this concentration with values established as indicative of cancer. is useful for detecting cancerous tumors in living mammals.

TAG products are also useful in diagnosing the presence of tumor cells at a tissue site, which consists of applying TAG conjugated to a labeled substance, such as a dye or a radioactive substance, to the site, thereby identifying tumor cells. Coloring or radioactive labeling occurs only in

Additionally, TAG products were found to be cytotoxic to tumor cells. TAG products are also useful for delivering diagnostic, nutritional, and therapeutic agents to specific cells or sites by introducing these agents complexed with the TAG product.

Normal cell division in plants or animals is restricted or arrested once the cells have sufficiently occupied a particular space. (a) A mechanism that “recognizes” that a normal cell has occupied a space that it could occupy; and (b)
) The mechanism by which the action of this recognition mechanism subsequently inhibits cell division was unknown so far. When normal recognition and sensing occurs, the concentration of its precursors increases and a group of compounds involved in the recognition and sensing of particles and cells that connect cells to each other are produced. Ta. These compounds are called recognins. Previous attempts to produce compounds from normal cancer cells have shown that they do not exist as such, and that cancer cells have (a) the ability and/or ability to recognize that they have occupied their normal space; (b) It was found that when it occupies its normal volume, it loses the ability to stop dividing and at the same time its molecular structure changes.

Recognins are novel compounds with physicochemical properties that mimic the morphological characteristics of cancer cells in that they are unable to recognize and stop cell division.

The use of recognins goes beyond determining the mechanism of action of cancer. This is because it provides direct products and methods useful in the diagnosis and treatment of cancer, as well as its prevention.

The present invention has advantages in the field of cancer research and is readily applicable to any biological system in which one wishes to influence growth and metabolism. Thus, by producing specific compounds or compounds of appropriate cell types artificially cultivated, and by further producing products from these materials, tissues,
Specific effects could be exerted on cells, organelles, sub-organelle molecules or molecular assemblies. Thus, specific nutritional efficacy during critical periods of development, specific diagnostic
Prophylactic and therapeutic methods, and the construction of artificial bioelectrical systems (such as in tissue or organ transplantation) can all be initially influenced. These artificial bioelectrical systems can now be made to have the properties of cognins, malignins or their chemoreciprocal cousins of the normal tissues or compositions that are adjacent to them and are therefore 'recognized' as 'foreign bodies'. , thus avoiding reactions to foreign objects that include rejection.

Yet another aspect of the invention provides TARGET-ATTACHING-GLOBU from two new product biological fluids.
The purpose is to produce L lN5 (TAG). This involves two reactions: the first reacts a biological fluid with a synthetic complex containing a physicochemical form that mimics malignins, called a target, and the second cleaves a specific TAG from the complex. It is so named by the measure of TAG that is obtained from the biological fluids of a living organism, i.e., from a tumor diagnostic test, whether or not that organism has a tumor. It is something that TAG
Since the products and antimalignins are physicochemically complementary to malignin, they are named chemoreciprocals.

Furthermore, depending on the time allowed for the reaction of the serum with the specific target reagent used and the time allowed for the cleavage of the complexed product, two quantitative It has been discovered that it is possible to produce qualitatively distinct TAG products.

After testing the quantities of these products that could be produced from a number of different people with brain tumors and various other medical disorders, as well as without any obvious signs of disease, certain individuals It turns out that these two new products that we were able to produce for patients indicate whether or not that individual has a brain tumor. Therefore, a new serologic diagnostic test for brain tumors has been discovered.

In addition to the use of these new products in diagnosing brain and other tumors from serum and other biological fluids, TAG and Recognin compounds can be used to diagnose brain tumors and surrounding tissues removed during brain tumor surgery. This is explained by the evidence that it preferentially attaches to glial tumor cells at the biological site. This preferential labeling of tumor cells by TAG and antirecognis is demonstrated by standard immunofluorescence techniques. Thus, the new method can also be used to determine whether tumor cells have penetrated to the extremities of the removed tissue, by histological examination, and whether the tumor has penetrated into the brain or other organs. The tumor cells may still be present or may have disappeared from the area around the removed tissue, indicating the possibility that all of the tumor has been removed from the brain or other organ. Furthermore, TAG and antimalignins prepared as described were found to be cytotoxic to collagen brain tumor cells grown in tissue culture in vitro.

This high affinity for tumor cells in other media, generated here in tissue culture, further demonstrates the specific binding potential of the new product TAG, with respect to the synthetic product target and histological location. TA regarding tumor cells in
As the property of G indicates, TARGET-ATTACHT
This explains the adoption of the name NG-GLOBUL IN5 (TAG). Furthermore, the cytotoxicity of TAG and anti-recognins to tumor cells provides a new diagnostic test for serum from patients suspected of having a tumor. Thus, for example, serum or other body fluids from these patients may be reacted with the target to produce TAG, and the product TAG may be cytotoxically tested for tumor cell growth in tissue culture. The concentration and degree of cytotoxicity of TAG expressed by TAG, which can be produced from the serum of a given individual, is only diagnostic.
In addition to having two advantages, it is also valuable in exploring the course of disease before and after surgery for a particular patient. Combining radioactive and stained detectors with TAG can create novel TAG products that are useful in vivo in diagnosing tumors and determining their precise location. Thus, if a suitably labeled TAG is injected either intra-arterially or intravenously into the cerebrospinal fluid of the brain, or directly into the brain tissue or its fossa, visualization of the bound dye may be performed by radioactive means or by visualization of the bound dye. This can be explained by the presence of a brain tumor. This is because TAG specifically attaches only to tumor cells. Furthermore, this method can accurately visualize the location of brain tumors.

This can be seen as an improvement on this in-vivo diagnostic method that uses anti-astrocytin made from rabbit blood to label brain tumors. This is because the possibility of foreign protein reactions can be avoided by using TAG made from human serum.

TAGs and anti-recognins have chemical properties that allow them to preferentially attach to astrontin precursors, including tumor cells, both in vitro and in vivo, such as by radioactive, proton-scavenging agents or other agents. In combination with toxic physical or chemical agents, these toxic substances can be preferentially localized due to the specialities they add to tumor cells compared to the normal cells adjacent to them. As such, these products can be used therapeutically as well as diagnostically. This selectivity is universally recognized as at least one decisive factor in achieving effective chemical or physical treatment of tumors, and as a hitherto unattained factor. . Thus, TAG has been shown to be effective in preferentially attaching to tumor cells, and for these reasons it has great promise as a novel therapeutic product.

In the serum of patients with malignant tumors, FAST-TAG (F-TAG)
One type of TAG is distinguished from 5LOW-TAG.
(S-TAG) can be produced in relatively large quantities from the serum of certain patients than in patients without such tumors. This suggests that the naturally occurring precursor of TAG (P
-TAG) or F-
This suggests that there are other factors that may cause 5-TAG to perform relatively better in vitro than T A,G.

The action of postulated but unproven cellular "antigens" and circulating "antibodies" on the actual synthetic product target and its precursors of TAG and, in turn, on those that may be present in vivo. Possible functional relationships between the two have not yet been elucidated. Thus, for example, in an antibody-like manner, F-TAG and 5-TAG produce a single, separate lineage of reactions with astrocidin in antigen-antibody gel diffusion in agar. Therefore, by injecting the target into rabbits, after reacting with the target, the yield of TAG products made from 26 rabbit serum can be increased. The discovery that there may be normal levels of circulating antibody-like precursors for cellular antigens hidden in non-dividing cells raises questions about the pair's possible actions. Here, we propose that TAG precursors (P-TAGs) and target-like substances exist within the production that act in the control of cell proliferation and cell death. Thus, for example, exposure of cellular components not normally exposed directly to serum proteins may occur during cell division. As a result of this exposure of cellular constituents, the constituents become converted into target-like substances, to which the addition of P-TAG-like molecules from the serum occurs, which can stimulate or inhibit cell division. Conceivable. Alternatively, damaged or malfunctioning non-dividing cells may expose target-like substances such that addition of P-TAG-like molecules is reversible. However, under certain cellular conditions, the addition of P(AG-like molecules) can lead to cell destruction (e.g., the synthetically produced ANT I G L r OMA-TAG described in As such, this represents a model for the normal mechanisms for the control of cell division and for the repair or removal of individual cells in the body throughout the life of the organism. Yes, when the exposure of cellular components is abnormally increased and an abnormally large amount of cellular target-like substances are formed, as occurs in rapidly dividing cancer cells such as in the case of collagen in the brain, one It may occur that the concentration of serum P-TAG of some types is increased compared to others.

Whatever the actual action of the precursor, this
The substantial increase in the amount of one major type of TAG, 5LOW-TAG (S-TAG), which can be produced in vitro from the serum of patients with malignant tumors by the described method is shown in the examples below. This is the basis for the serological diagnostic tests described.

In the Examples below, astrocidin, malignin, and leeler-recogni are shown as specific examples of recognins, and all of these are acidic peptides having the properties (a) to (h) above.

Example I Generation of a Fraction Containing Crude Astrosidin Precursors Swollen human brain collagen tissue is cut out to separate surface blood vessels and normal brain tissue as much as possible. Take a representative amount 117 of isolated swollen tissue and combine it with 6 sections of 152 and 10f!
Divide into two parts. Each part is then processed as follows.

Each portion is homogenized in a neutral buffer solution by ultrasound or other mechanical means. For example, combine each portion with 100cc of 0.005M phosphate buffer (pH 7) in a warning mixer.
/ homogenize the structure. To prevent protein denaturation, the homogenization process should be performed under cold conditions. for example,
The mixer should be pre-chilled in a cold room at 0-5°C and operated for about 3 minutes. 'Next, in order to clarify the homogeneity, centrifugation is performed, for example, in a frozen ultracentrifuge at 500 times the gravity for 30 minutes. The soluble supernatant is decanted and kept cold. The insoluble residue is homogenized again using a neutral buffer solution of 100% and centrifuged in the same manner as above to obtain 2
Combine the soluble/extract from No. 1 with that from No. 1 above. The yield is very good when homogenization and centrifugation are repeated until the protein in the supernatant is less than 50 micrograms/-solution. This means that in most organizations,
Achieved in multiple extractions.

The solutions thus obtained are combined and concentrated by thorough evaporation in subsequent dialysis. A solution of 15- is obtained by dialysis of 0.006M phosphoric acid M solution under cooling. Record the volume of this solution, use a portion for total protein analysis, and divide the remainder into P.
Obtain protein fractions K1-4. Chromatography, described below, is the preferred fractionation method.

The solution was diluted with 0.005M sodium phosphate I! in a cold room (4°C). H! DE, AP, and cellulose (C
ellex-D) column 2.5×ILOal. Elution solvent is stepwise +CZ by using the following solvent (solution): Solution 11) Na H2P O,i
Dissolve 4-042 and Na2HPO46,50F in distilled water 15,000 - (0:005 mol, PH 7):
Solution (2) Nλ) I 2P 04 Dissolve B-577 in distilled water 2480-; Solution 13] NaH2PO41
7. Dissolve IP in distilled water 2480-. (0, o
s solution, pH 4,7): Solution (4) NaH2PO459
, 659 in distilled water 247o- (0.175
mol); Solution (5) Dissolve NaH2PO410L65E in 2455 mj of distilled water (0.3 mol, pH 4.3)
; Solution (6) 1 g of NaHPO4340 in distilled water 246
Add the solution (ylso% phosphoric acid (H3PO4) zsas4F to distilled water 2460- (10 mol, PH:LO) add the nerve tissue extract 6-10-. This is passed through the column. Solution (1) is then added on top and a reservoir is attached to receive the solution (1) 300 - and allowed to fall into the column by gravity. Using an automatic fraction collection device, the eluate 3 is Collect -. Next, with the first elution number, change the elution solution and use it. Solution (
2): Convey the column solution to the top of the resin in tube 88;
Next, add 50 tnl of solution (2) from above and install the reservoir: Using tube 98, move the solution from column 2 to the "J" part of Lennon, then add 75-liter of solution (3) from above and attach the reservoir. Attach; Solution (4): With tube 114, carry the solution onto the column, on top of the resin, and add solution (4) with N.
Add s o wt from above and attach the reservoir; Solution (5): Bring the solution in tube 155 to the top of the column, on top of the resin, then add solution (5) 125xI2 from above and attach the reservoir. Install; Solution (6): Tube 187
Then, bring the solution onto the column, to the top of the resin, then add solution (7) 17 s- from above and attach the reservoir; continue elution until reaching tube 260 to complete the elution. A custom made resin is used for each volume of tissue extract. Each tube is subjected to quantitative protein analysis. Pipe 212~, 23o
Combine the eluates. This contains the crude product from which astrocytin is obtained.

In the past, data on a crude product called fraction IOB has been made public [protein
Metabolism of the Nervous System App55
5 = 69 (Pleum Press.

1970]; Nenko; Naru of Neurosurge? '
J-, Vol, 33, PP281 #286 (197
September 2003)], where we achieved the isolation of a specific product, termed astrocytin, from fraction 10B. The crude side 10B is made from 0.1 to 10 19 of product per fresh nervous system tissue 11 initially. In addition to the astrocytin precursor, it contains a number of hexoses, namely glucose, galactose, mannose; muhexose amines, including glucose, galactose and mannose amines; and occasionally other sugars, fucose, ribose and It probably contains varying amounts of covalent hydrocarbon residues, including lanose and the like. It also contains high molecular weight protein products, some lipids and nucleic acids.

Example 2 Production of purified astrocytin from crude astrocytin precursor-containing fractions.

The astrocytin precursor-containing fraction is further isolated from contaminants. In a preferred embodiment, the material obtained in Example 1 is chromatographed on Sephadex G-50 resin using a typical column (40a IC vessel x 2.5cx (diameter), xpa-volume). Working pressure is 40 o'H
g: Flow rate was 35 mg/Hr; 0.05 molar phosphate buffer solution (pH 7,2) was used as the buffer. first 7 o --
The peak of S'/L/-(flow through) contains impurities as well as astrocytin precursors, but the following peaks contain only impurities.

In a preferred embodiment, the product in the first flow-through heating above is then concentrated on Sephadex C-15 and then added to the same solution #i, (1) to (1) as in Example 1.
7) and using the same elution steps as in Example 1.
Pass through Kusu D column. The astrocytin product is present as a sharp peak in the same tubes (numbers 212-230) as above, and on Selex-D chromatography,
It behaves without the presence of multiple contaminants.

Molecular weight contaminants are removed by conventional means such as millibore disk filtration. As a preferred method,
Astrocytin product is millipore vericon disk hh'l OOOll 3m (this one is 1000
It captures substances with a larger molecular weight and allows substances with a molecular weight less than 1000 to pass through. ) to separate salts and other small molecular weight contaminants. The astrocytin product remains on the Pericone disc and is recovered by washing with solution (1) of Example 1.

Preferably, a chromatograph is then used in which astrocytin is obtained by isolating a compound having a molecular weight of about 8,000 from the above solution. The equipment used is a commercially available one designed by Botzlingen Mannheim GmbH; Pharmacia Fine Chemicals & CAMAQ (Switzerland). Sephadex G-2
00 resin (of the highest quality) 2.55'Q, 02
Phosphate buffer of 0.5 m NaCj in MNa2HPO4KH2PO4 pH 6,8 (6,6-7.0) within 85-! Prepare this. Swell at room temperature for 2 to 3 days with occasional gentle mixing. (Magnetic and other stirring means should not be used). The swollen gel is stabilized at freezing temperatures for 3 weeks. However, bacterial growth and fungal growth inhibit the swelling gel. If you want the gel to hold for a longer time, you should add a small amount of bacteriostatic agent (sodium azide 0.021.
Al+, make two glass plates with a thickness of 0.5 strands.

The plates can be dried for 10 minutes at room temperature, stored in a humid chamber for about two weeks, or used immediately after appropriate pre-equilibration. (Usually, it takes at least 12 hours at night.) The main function of equilibrium is to normalize the ratio between the static and five mobile layers. With the plate pre-equilibrated in a horizontal position, the substance to be measured is applied with a microphone, pipette as a spot or streak at the starting line. 0.
Place 10-20% of a 2-2% protein solution on the edge of the first slide (18x18) of the microscope.1. - Face the gel surface. After a few seconds, the solution soaks into the gel. All samples are first placed on cover slides and then quickly adapted. If not enough material is used, it is difficult to locate individual spots after one separation. if,
If too many substances are applied, it will not be possible to clearly separate them. Sample separation is performed by diluting the sample with a buffer for ease of handling and using a rate ramp technique at a 22° angle. A flow rate of 1-2α/hour is very suitable. Labeling substance (cytochrome C.

Hemoglobin, myoglobin or albumin (classified and identified by bromophenol blue) is applied at different positions across the plate and also functions in a matching protein and le for calculation of relative distance (mobility) of unknowns. . After applying the sample, rotate the plate into the device and
Place the wick of the vapor at the bottom of the barrel and make sufficient contact with the gel layer.

Do not immerse the vapor wick. Wipe off excess moisture. The liquid solvent in the reservoir is kept constant at ICI+ from the top of the container. The operation usually takes 4 to 7 days depending on the progress of the separation.
It will be completed in time. Separates colored substances directly. After completing the chromatographic separation, the separated protein spots were transferred to a vapor sheet replica of the TLG plate and washed with pre-washed methanol
Easily visible by staining on 20+acetic acid-90:5:5 for 48 hours. Vapor seat is 3
! It is Il+1%F paper. , a sheet of 20 x 28c11 paper is placed on top of the gel layer and rolled to ensure proper and sufficient contact with the gel, making sure that no air is trapped under the 6 paper (replica) and , being careful not to disturb the gel layer.

The liquid layer is penetrated from the gel layer with paper, removed after about 1 minute, immediately dried in an oven at 60° C. for 15 minutes, and dyed using a conventional dyeing method. Staining was carried out in 10% sodium carbonate.
．． This is accomplished by spraying the replica paper with 0.3% diazotized sulfanilic acid (Pauli Reagent). Also, methanol, -acetic acid (9,0:IOV
Dyeing can be achieved using a saturated solution of amido-brazate during the use of V/V; the dyeing time is 5 to 10 minutes.

To decolorize, wash with 2 volumes of methanol and acetic acid (90:10.) mixed with 1 volume of water.

It is difficult to achieve low background staining without multiple washings. If only astrocytin is to be stained, the plate itself may be dried (in an oven with approximately 60"cc air circulation). For isolation, the plate is air-dried at room temperature.

Overheating causes cracking, which can be avoided by using 50-60°C, a temperature that typically dries Sephadex G-200 plates in 15-30 minutes. The drying plate was prepared using methanol + H20 + acetic acid (75:20:5).
) (7) i compound for 10 minutes and swelled in the same solvent system in a saturated amic acid rack'.

It is dyed for 5 hours and then washed by soaking in the same solvent for 2 hours before drying. To determine the molecular weight, print directly on (or on) the molecular weight. The distance from the starting line to the center of each band is measured with an accuracy of 0.05 mm on the spectral cytogram. The measurement results are expressed as a width value defined as the ratio of the distance traveled by chromium C or myoglobin (used as a control protein) (d-) to the distance traveled by the test protein (dp): The relationship between the migration distance of the standard substance and the sample substance is expressed by the formula: (-1=P,).A straight calibration line is drawn by plotting the logarithm of the molecular weight of the standard substance used against Rfn. This straight line gives the molecular weight of the unknown protein. For very accurate results, mix equal parts of the protein sample solution with a standard, in this case cytochrome C, before applying it to the plate. By the above TLG method, the astrocytin product is
In contrast to standard cytochrome C, approximately 0. ．． 83 +/-0,
It is observed as individual spots at a distance of 0.02 mm, giving a molecular weight of about 8000 for astrocytin. This method separates several individual products from astrocytin based on slight differences in molecular weight. In this way, approximately 6400 molecular weight particles are transported as contaminants to this point.
Three products with 0.148000 and 230000 and often a molecular fi32000 product are found and removed in the TLG method described above. The astrocytin product, in dry form, is absorbed into the gel and dissolved in solution (1);
Separate the resin by centrifugation or other similar means.

The astrocytin product produced at this stage is soluble in distilled water, soluble at neutral, acidic pH, insoluble at alkaline pH, and has a spectrophotometer absorption peak wavelength of 2'8 Qmμ, which is similar to the above-mentioned It is a polypeptide with a molecular weight of about 8,000.The covalently bonded amino acids are 6NH
Hydrolysis with Cj followed by automated quantitative measurements shows that it has the following average composition of amino acids:

Number of residues (approx.) ・Aspanquinic acid 9 threonine, 5 serine
6 Glutamic acid 13 Prol) 4 Glycine, 6-゛l/2cysteine ・2゜Methionine ゛l-゛Isolo, Isine 2, *Isine
″ 8 Tyrosine 2 Phenylalanine 3 Lysine 8 Histidine 2 Arginine 4 Total 88 isistonic acid, hydroxyproline, norleucine, ammonia, indesmosine, desmosine.

Bitroxylidine, Risino°/Leleucine and Ga.

There are no detectable amounts of either amino acid or butyric acid, and only trace amounts of glucopmin are present.

From the starting brain tumor and cancer tissue 11g in Example 1, Example 2 reeler (REE electrop)-recognin four-fold =-.

Leela's disease is a genetic disorder in which the animal is unable to perform stable, coordinated motor muscle activity, and certain nerve cells migrate to a specific location in the brain, the cerebellum, at specific times of development. It is not possible to do so and exhibits a reeling state. Electron microscopy studies have shown that certain glial cells provide vertical rod-like axes along which new cells move to new positions under normal conditions. The inability of new neurons to climb these glial fibers in Leeler's disease is thought to be due to some disorder affecting the neurons or glia, or both.

・Glue the mouse according to the method of Examples 1 and 2.
Recognin i obtained from the brain, normal mouse brain 0OH5.
It is 000゛. 1 reeler? Licognin is unusual as indicated by its very small molecular weight;
Moreover, the comparison between the brains of reeler mice is far less likely. This is shown in Figure 1.

,・ ゛・ Recognin concentration in each Q brain of the table mouse (v9/ri) ・Normal reeler cortical layer (4 days old mouse) 0.29 0.53
Brainstem (16 day old mouse) o, 9o, 1.6
4 Whole brain (17El (7)?mouse after birth) 1.27 1
53 whole brain ° (1 day old mouse), s, oo °5
．． There was a significant decrease in Recognin concentration in the cerebellum of Harrier mice at the age of 86, indicating that there is the same amount or more Recognin in other parts of the brain. Recognin does not move from other parts of the brain to the cerebellum or
Slight increases elsewhere in the brains of Leela' mice reflect some kind of compensation.

The pathological recognizance in the brain of Leela-fus is a recognise that influences cognition within the cells.

This is related to the inability of the migrating cells to make the necessary contacts in order to ensure their role and position themselves in the proper position for recognition.

An equal amount of anti-recognin against recognin released from mouse reeler brains was prepared and used in mice in a similar manner to the use of anti-astoccytin and anti-malignin, as described above. It's fine.

Example 3.

During the production of malignin precursor in artificial cancer cell culture medium fermentation, sterilization treatment is generally carefully carried out.

, all solutions (e.g. “Bank of Balanced Salts CBS'
S), F-10 nutrient solution, fetal calf serum, trypsin solution) are incubated in a water bath at about 35° C. for about 20 minutes or more before use.

Cells are isolated from M cancer tissue and propagated in vitro for multiple generations using appropriate techniques as described below. Use a beaker that has been pre-cleaned with a sterile solution, such as 12-propanoke$ and amfil or creolin solution.

As a preferred example, artificial cancer cells (i.e., a pre-cleaned crucible are placed in one pot) and about 30
Wash gently for seconds. Avoid stirring. Clean all walls and surfaces. 3000 rPm for about 10 minutes in the cold
The solution is clarified from the cells by centrifugation. Pour the medium into one beaker as above. Add a small amount of buffered proteinase enzyme solution and wash quickly to avoid digestion of cells. A preferred method: - Add trypsin solution (EDTA) 1-2rnl' and wash for only 10 seconds. Flow out the trypsin solution. ,
Add the same volume of fresh trypsin solution and incubate until the cells are seen to detach from the chamber walls by microscopic observation. , which typically takes 5-10 minutes. suitable,
Add a 7 to 10% solution of fetal calf serum in a suitable growth hsw, such as F-10 Nutrients 100-50.

Transfer 25 ml of fresh medium with cells to a new growth chamber and propagate. Place both chambers in an incubator at 35°C.
Leave it for about 7 days. According to the previous operations in this example, the artificial cancer ii%l& is divided into two fresh media approximately every 7 days. For each growth chamber, this entire procedure is repeated as often as desired, spaced by Schift days. In this way, the number of viable cells in vitro doubles every i7 days.

After about 7 days of growth, cells are extracted for making malignin. For example, the proliferating cells on the wall of each 250-proliferation chamber described above are regenerated as follows. Transfer the medium to a centrifuge tube and centrifuge at 3000 rpm for 10 minutes in the cold. Discard the medium. Cells remaining in the growth chamber are detached from the chamber walls and flushed into the centrifuge with a neutral buffer solution. ° Cells were washed twice with neutral buffer solution and incubated again at 3000 rpm in the cold.
Centrifuge and discard the local. . For extraction of crude malignin precursor-containing fractions! When ready, wash and suspend the cells in neutral phosphate buffer.
.

, ゛Example 4 Production of fraction containing crude malignin precursor In Example-3.
The prepared and washed cells, suspended in a neutral buffer, are mechanically disrupted under conditions that avoid denaturation of most proteins. A preferred method is 1. Treat the washed cells with cold ultrasound for 20 seconds. After sonication, remove cell debris by
Centrifuge at 3000 g rpm for 0 minutes and decant the supernatant. Use 10 parts of the buffer solution to wash away remaining cell debris. Ultrasonication m-b and centrifugation are performed as described above, and the supernatants are combined. Repeat this operation again. 1.11. Combine the -3° supernatants and evaporate thoroughly to bring the volume from about 30° to about 6-7 rnt. The sample was used for total protein analysis, and the remainder was used for the above-mentioned Example 1 with 1 astrocytin precursor.
Separate according to the method described in .

Example 54. Production of Purified Malignin Product from Crude Marijuana 6 Mignin-containing Fractional Power Astrocytin 1 The malignin product is further isolated from contaminants by the following method:
, ・ ・
In the T, LG stage of a preferred embodiment, malignin-4
8 = Product is approximately 0.91 +/- compared to standard cytochrome C
It is observed as individual spots at a distance of 0.02, giving approximately 10,000 molecular ports to malignin.

The malignin product prepared at this stage is soluble in distilled water, soluble at neutral or acidic pH, insoluble at alkaline pH, and has a spectrophotometric absorption peak of 28.0 m.

It is a polypipetide with a molecular weight of approximately 10,000. - as shown by thin-layer Geluk White Madgera Buoy measurements;
The molecular weights of malignin produced in culture media that have been stabilized in successive generations of media are shown in Table H. The reproducibility of mustard mass measurement is determined by TLG chromatography rKJ, %°
This is notable from the perspective of constraints.

Table■ Reproducibility of molecular weight of malignin obtained.

Table 2 (Continued) The covalently bound amino acids of malignin are hydrolyzed with 0NHCI and then determined by constant measurements to show that they have the following average composition of amino acids.

Number of residues (approximately) Aspartic acid 9 Threonine '5 Serine
5 Glutamic acid 13 Proline
° 4 Glycine
6 alanine/9 valine
6 1/2 cysteine l methionine 2 inleucine
+40 Ishin,
8th bnn: 3・'
.

Fetanyl alanine 3 lysine °,
°6 bistidine 1 °2 arginine
There are no detectable amounts of 5, 5, 5, ammonia, isodesmosine, desmosine, human waxylysine, lysinonorleucine, and amino acid present in the 12 250-reaction chambers of Example 3. Ra°
The yield of pure malignin obtained is about lv9 malignin.

The unique structures of marimenin and astrocytin have been ascertained by exhaustive computer searches comparing their composition to virtually all known protein substances.

Marikunin and astrocytin amino acid compounds, 1
The absolute and relative amounts of each asnoic acid component, in terms of the total number of amino acid residues per mole, and each in terms of the molecular weight of the molecule. Absolute amount and relative amount of amino acid codibonent.

The most widely known literature on protein tissues in the world, that of the National Biomedical Researcher 7.7 (DC), was subjected to matrix computer analysis. . In matrix analysis comparing hundreds and thousands of proteins and protein fragments, no organization was found that was identical or even very similar to that of astrocytin or malignin, only proteins that were closely related in organization. , their individual amino compounds and molecular weights are shown in Table 3 for comparison. From a myriad of possibilities, a computer program was constructed to determine each degree of similarity in structure, for example, proteins of larger or smaller molecular weight, or less than 85 or more than 95. Claims with more residues, or less than 12 5) or 1
Does not match proteins with more than 5 glutamic acid residues, or less than 5 or more than 11 aspartic acid residues.

A fingerprint such as the one above has 22 variables to match.

It appears that some materials are compatible with one, four, or five variables, but none are compatible with all 22 variables. In fact, those that fit well on more than 14 variables (8 of which show differences).

Nari'). 'Therefore, the most closely matched masterpiece is cytochrome bs (human)! be. As seen in Table ■, its alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, histidine, methionine, tyrosine, and tryptophan residues are all markedly different from asi-locitin and malignin. be able to. Since natochrome b5 contains seven histidine molecules, astrocytin and malignin contain only two of these molecules, it is unlikely that they have the same chemical structure. The most unusual thing about the composition of astrocytin and malignin is the high concentration of glutamic acid. One would expect to find only 5-6 residues out of 89.

The next most closely matched are the ferrotoxins in Leucaene glaucsc and alfalfa, which differ in having 4 and 6 amino acids, respectively, and both have 96 residues, respectively. It differs in that it has 1 and 97 pieces. Other suitable proteins include the acyl carrier protein of Escherichia coli (E, colt)26, which has a number of residues of 11 amino acids (C), which is significantly different from that of mylignin and astrocytin; It differs in that it has only 77 i groups.

Other brain proteins (neuroficin and co-natotropin-releasing hormone in cows and pigs) have been recorded (see Table ■), but all of them are highly compatible from among the hundreds of thousands of protein fragments stored in this computer. It is a bad thing.

Respiratory proteins return to their original state and absorb metals and /°
Or it may contain a heme component, but it is isolated. state protein fragments, such as aponatochrome b
5 does not contain any metals or hemium components.According to the trace analysis results of strocytin weir and mignin, it does not contain iron, sulfur, phosphorus, or magnesium ((less than 0.01%). ), has typical absorption spectrum characteristics at 28'Omμ.As a result of forming a heme substance together with τ bobrotiine and measuring its absorption spectrum, it was found that 4
Typical absorption was observed between 00 and 450 mμ.

Although astrocytin and malignin are structurally distinct from all other proteins and protein fragments, their structures are closely related to those of respiratory proteins. It is noteworthy and perhaps very important. It is well known in the art that many important relationships can be drawn both from a developmental genetic point of view and from a functional point of view regarding its chemical structure. One of the major problems in cancer is that astrocytin and malignin are new white substances whose structural equivalents exhibit respiratory action at their location, and therefore these proteins induce other adverse effects. (
In other words, it was decided to provide light-cutting to solve the problem of how to make the energy relationship satisfactory for malignant cells that are greedy and rapidly proliferate.

Regardless of the theoretical importance of such a discovery, the above data show that the proportion of malignin increases in more malignant cells, and that anti-malignin substances only bind to malignin-like chemicals in cancer cells. However, the rhinological data showing that the bound lignin is also toxic to cancer cells are of great significance. Since the anti-malignin products of the present invention are likely to selectively bind to heme-compounds in cancer cells, if the malignin-like compound is a silent protein and therefore has a respiratory tract within the system.
It is easy to understand that if it contains a functional substance, it can kill the cancerous axon.

“The potential for therapeutic effects of antimalignins and other similar chemotherapeutic agents has already been demonstrated between the structural knowledge of malignin #, and astrocytin, and the malignancy of the amount of malignin. Significantly increased by relationships.

Example 5 Increasing the yield, degree of malignancy and proportion of malignin by increasing the volume and surface area during fermentation; 1000-flask was used instead of z50frLt flask, and the amount of all reagents was tripled. increased, Example 3~
The same operation as in 5 was performed.

``250- to 10- to 100% space for malignant cell growth
00- almost doubled the yield of malignin product after 7 days of growth from inoculation. Total protein content for each flask size! (η) The malignin produced is expressed as a percentage of the total protein content of the fermented material of successive generations, and is shown in Table 2. 25
When using a 0-flask, the average amount of total protein produced was 17.5η, 1000tn! When using a flask,
The average amount of total protein produced was 40.4.

Table ■ Improved malignin yield by culture fermentation including successive generations, Table ■ (Continued) By increasing the surface area, per 7-day growth period,
As the amount of malignant cell proliferation (degree of malignancy) increases, the amount of malignin produced, which is shown as a component of total protein, also increases significantly. During the 7-day growth period, the percentage of malignin relative to the total protein amount was 250+, from the average value xo, r% when using Lt flasks, to $ when using 10°00d flasks.
The average value increased to 28.3%.・ .

FIG. 1 shows the relationship between the proportion of malignin produced and the degree of malignancy (that is, a function of the amount of protein produced relative to the amount of malignant cell proliferation in vitro for 7 days).

The m1 diagram shows that as the flask size increases, the fraction of lignin product increases approximately three times.
. Moreover, FIG. 1 shows the relationship between malignin and the degree of malignancy. A magnetic line is a line that represents an ideal straight line.

This empirical relationship is important because as the cell proliferation becomes more difficult to prevent (pathologically), the proportion of malignin present increases. The normal function of recognin itself is related to the above-mentioned contact with cells and suppression of their proliferation.If the proliferation of malignant cells is more pathological, the suppressive effect of contact may Malignin becomes the more preferential protein and increases.

Example 5A illustrates that artificial cancer cell cultures grown in large growth vessels unexpectedly increase the proportion of malignin produced, ie, the percentage of malignin in total protein. . Large-capacity growth containers such as those used in accordance with the present invention have a ratio of container volume to total cell culture medium volume according to Example 3 that is greater than about 8=1 (e.g., the range is 7:1 to 10:1). It means that it is a container. Example 5 AIC:1 The ratio was about 8:1.

Example 6: Preparation of target reagent from recognins The malignin prepared in Example 5 was mixed with a carrier to prepare a target reagent.

In a preferred embodiment, astrocytin or malignin is first dissolved in 0.15M sodium dihydrogen phosphate-citrate buffer (p14.4.0). Bromoacetyl resin, such as cellulose I SE, A element 1. θ〜1
．． Bromoacetyl cellulose (DA
C) in 0.15M sodium dihydrogen phosphate buffer (pH
7, 2) Prepare in a refrigerator and store in a cool place. Pour the pH 7.2 buffer solution and add the above 0.15M! By adding sodium dihydrogen citrate buffer (pH 4,0),
Adjust the voice of the obtained liquid to 4. After stirring a solution of astrocytin or malignin solution and BAC (ratio of BAC to recognin 10:1) at room temperature for 30 hours,
Centrifuge.

Preferably, all sites on the BAC used to bind recognin are involved in binding. This is accomplished as follows. The supernatant obtained in the step described immediately above is lyophilized and the protein content is determined to determine the amount of astrocytin or malignin that is not bound to BAC. Complex B C-astrocytin (or B C-malignin) was suspended again in 0.1 M bicarbonate slowing solution (pH 8,9) and stirred at 4°C for 24 hours to dissolve BAC and astrocytin or malignin. forming chemical bonds between After 24 hours, the suspension is centrifuged and the supernatant is analyzed for protein. Composite BAC-
Additional 0.0 astrocytin or BAC-malignin
5M aminoethanol-0.1M hydrogen carbonate t1 line solution (
Resuspend in pH 8,9) to occlude unreacted bromine. ``This suspension is centrifuged, and the supernatant is retained as is without being subjected to analysis due to the presence of aminoethanol. It is centrifuged and the resuspension is washed three times with 0.15M sodium chloride until no absorption is observed at 266 mμ in a spectrophotometer. This B
'A.C.

8M astrocytin or DAC-malignin complex
Stir in urea at 38°C for 2 hours and centrifuge until no absorption of 266 mμ appears in the washing solution (usually 3 times).
, wash with 8M urea. This complex was washed twice with IJM (0.15M chloride) to remove urea. The complex is stirred in 0.25 M acetic acid at 37° C. for 2 hours to demonstrate its stability. After centrifugation, the resulting supernatant necessarily has no absorption at 266 mμ. Hence this chemical complex: BAC-, astrocytin or BA
C-malignin is stable and can be used as a reagent in methods such as those described below. In this stable sample type, it can be called a complex obtained by total synthesis, whose physical and chemical properties, when understood as a serum component in a potentially reactive state, are stable cell-astrocytin or It can be regarded as a binding precursor of malignin. For preservation, one sample of the target reagent is centrifuged and washed with 0.15 M +7.dihydrogen dihydrogen buffer (pH 7.2) until neutral.

Targeting reagents can even be used from bromoacetyl carriers with ligands other than cellulose, such as bromoacetylated resins or v-paper.

Example 7 Preparation of Antisera Against Astrocytin, Malignin #, and Target: Antisera against astrocytin, malignin, or target reagents are produced by inducing antibody responses against these reagents in mammals. A method satisfactory for this purpose is described below.

One gram of Recognin (astrocytin weir yohimarikunin) was injected into the heel of the foot of a white male rabbit in standard Frein along with an adjuvant, and the same injection was injected intraperitoneally after one week, then another 10 days, and if necessary for another three weeks. Perform the injection.

Specific antibodies can be detected in the serum of these rabbits as early as 1 week to 10 days after the first injection. To obtain the target antigen, 1 kg of astrocytin or malignin (measured by the poorin-lowry protein detection method)
The operation of injecting a target amount containing . A special antibody against astrocytin is called anti-Astroc7tin. Mari, anti-malig special antibody against genin = 7
(Anti-Mal 1gn1n). Similarly, a special antibody against the target reagent was prepared using anti-tar/7.7t (
Ant'i-Target).

Standard method for antigen-antibody reactions
hterlon7) It is clear by the gel diffusion test method that the specific antibodies obtained with the sonospecific antigens exhibit a single sensitive linear relationship in their reaction with the antigen.

The presence of specific antibodies in a serum can also be tested by the precipitin standard quantitative test method for antigen-antibody reactions. This test allows a good precipitin quantification curve to be obtained, from which the number of specific antibodies can be calculated.

Furthermore, the presence of special antibodies in the serum is determined by the presence of special antibodies: anti-astrocytin, as mentioned above, and bromoacetyl.

The proof can be obtained by absorption on leucellulose-astrocytin (BAC-astrocytin). That is, the antiserum containing a specific anti-astrocytin can be reacted with B"AC-astrocytin. When the serum is passed over the BAC-astrocytin,
Only specific antibodies against astrocytin bind to the specific antigen astrocytin. Astroretin is professional
Since it covalently binds to cetyl-cellulose, the specialized antibody: anti-astrocytin binds to BAC-astrocytin to produce BAC-astrocytin-anti-astrocytin (BACA-anti-astrocytin). This is demonstrated by testing serum residues separated from BAC-astrocytin by washing. No residual antibodies capable of reacting with astrocytin were found in the serum using the standard kissing antibody diffusion test. Therefore, it is clear that all the special antibodies (anti-astrocytin) whose presence was previously shown in the serum were absorbed by BAC-astrocytin. Furthermore, anti-astrocytin was combined with BAC-astrocytin by 25M acetic acid i'B.
This can be done by washing with AcA-anti-astrocytin (at 4°C for 2 hours).

In addition, the presence of specific antibodies in serum indicates that special antibodies: anti-malignin, such as bromoacetyl cellulose,
Proof can be obtained by absorption onto malignin (DAC-malignin). That is, an antiserum containing a special anti-malignin can be reacted with BAC-malignin. When serum is passed over BAC-malignin, 1 only the specific antibodies against malignin bind to the specific antigen malignin. Since malignin covalently binds to bromoacetyl-cellulose, the special antibody: anti-malignin binds to DAC-' malignin and DA
C-7 ligene-anti, lyleene (BACM-antimalignin) is produced. This is demonstrated by testing the serum residue separated from the DAC-malignin after washing. Using the standard Zaitenyu Antigen #L diffusion test method, it was found that no antibodies capable of reacting with monomalignant lignin remained in the serum. Therefore, all the special antibodies (antimalignin) whose presence in serum has been previously shown are B
It is clear that it was absorbed by AC-malignin. Further, the reaction can be carried out by combining anti-lignin with BAC-malignin (at ℃ for 2 hours).

By the standard Hyotensen S & Reima Gel diffusion test method for antigen-antibody reactions, specific antibodies against the target antigen obtained by the antiserum (such as anti-astrocytin or anti-malignin) described above ( i.e. astrocytin or malig.

It is clear that the antiserum obtained by injecting the antigen itself shows a single linear relationship in the reaction with its antigen. Of note, some rabbits retain a certain amount of anti-target prior to injecting the target. These anti-target substances produce approximately equimolar amounts (two types) of TAc, s'-Tha and F-TAG in large serum tests, especially when reacted with targeting reagents as described in (see below). (See actual flow side).

Example 8 Detection of malignant tumors by quantitative in vitro production of target-f touching globulin (TAG) from biological fluids.

The target reagent prepared according to Example 6 is washed to remove unbound recognin that may be present due to deterioration. The satisfactory method is as follows. The target reagent was stirred with acetic acid at 37°C for 2 hours, centrifuged, and the supernatant was separated by decantation.
Measure the optical density in mμ. If absorption at that wavelength l is present, repeat the washing as described above until the substance no longer dissolves. The target is then resuspended in phosphate buffered saline solution (pH 7.2),
(Refer to standard methods for testing target reagents, and if other target formulations
If it is possible to obtain whole rabbit serum that has been shown to contain C., standard 5-TAG and F-TAG purified from the reaction of large serum by the method described below can be used. .

Slow-binden f (S-TAle) by the following method
Determine: Frozen serum that has been stored for several days should not be used. Serum is carefully prepared from freshly obtained whole blood or other body fluids by conventional methods. The method that should be satisfied is as follows. The collected blood is left in a glass test tube at room temperature for 2 hours to allow clotting.

Separate the clot from the test tube wall using a glass stir bar and leave the blood at 4°C for a minimum of 2 hours (or - overnight).

Clots were separated from serum by centrifugation (2,0,000 rpm) for 45 min at 4°C, serum was decanted into centrifuge tubes, and further centrifuged for 45 min at 4°C (2,0'
OOrpm). This serum was decanted and methiolate 1
Multi-solution (95% water and 0.2M bicarbonate buffer (p
H10) 5-medium, 1g) 1% (volume) to serum
Add up to the limit of .

Serum obtained by the above method or other methods, each 0.2
4, 100 lucognin per 0,254 target reagent
~200 μl of target suspension reagent each containing 0.2
In addition to 54, there will be two test samples. The suspension is mixed at 4° C. in such a way that pellet formation is avoided. After shaking vigorously for 1-2 seconds, for example using a small rubber cap, the tube is gently decanted and shaken in a Thomas shaker for about 2 hours or more. The target reagent and its bound proteins are separated from the serum. According to the satisfactory method discovered in this invention, the tube was centrifuged at 4°C for 20 minutes (200 Qrpm), the supernatant was decanted, and then the tube was mixed with 0.15 M saline solution (0.2-0.3-
The resulting pellet is washed by performing the following operations three times: remixing with the pellets, shaking at room temperature, and centrifuging to separate the supernatant.

The remaining proteins attached to the target are then separated,
This protein is quantified. For example, 0.25M acetic acid 0.2
- and shake the suspension for 1 to 2 hours in an incubator at 37°C. Centrifuge the tube for 30 minutes at 4°C (200Orp
m) Do. The supernatant is decanted, taking care to avoid migrating particulates, and the optical density of the supernatant at 280 mμ is determined. Serum Protein (S-TAG) - To obtain the results for this rig, divide the measured optical density of 1 degree by a factor of 1.46.

There should not be a difference of more than 5 points between the two test materials.

To test the protein content, other methods can be used, such as the fluorine-lowry test, but the range of values for the concentration of the control sample and the (S-TAG)-(F-TAC,) concentration must be specified and pronounced in standard law 1ζ.

Serum should not be used. Serum is carefully prepared from freshly obtained whole blood or other body fluids by conventional methods. The method described at the beginning of this example is sufficient for preparing blood and serum.

Serum samples prepared by the above or other methods are 5-TA
Leave at 4°C for 10 minutes less than the total time the G serum measurement agent is contacted with the 7-Gelato reagent described above (e.g., if a ``2 hour'' 5-TAG measurement was performed, 1
time 50 minutes). In this method, the temperature histories of the 5-TAG and F-TAG measuring agents are balanced.

A 0.2 d sample of the temperature-equilibrated serum was tested in duplicate with 0.25 ml of Gouget's reagent (100 to 200 micrograms) of Cousat suspension reagent, containing 100 to 200 micrograms. Add to each divisor. The suspension is mixed at 4°C for approximately 10 minutes, avoiding granulation. For example, using a small rubber cap with rapid shaking for 1 to 2 seconds, then cap the tube. These can be shaken on a Thomas shaker at a slight tilt for approximately IO minutes. One is as below.Next, the tube is 200Q
Centrifuge at 4°C for 20 min at rpm and isolate the supernatant.
The granules formed by centrifugation were mixed with 0.15M saline solution at 0.2-0.
Cleavage from the 3-isomer is determined quantitatively by remixing with 3vi and shaking at room temperature. The above-mentioned method for measuring 5-TAG concentration is satisfactory.

It's worth it. . Other effective techniques for measuring protein content may be used, such as the Orin-Lowry assay, but the range of control and tumor value of 5-TAC; negative F-TAG concentration may be used. Standards must be established to determine the °“The final result is expressed as micrograms of TAG per 1 − of serum, which is
-TAG? Equivalent to inus F-TAG. TAC values in non-brain tumor patients and other controls currently range from zero (or negative number) to 135 micrograms per serum 1-. For results exceeding 1 i * s o Qmμ, anti-4'l measurements are displayed. Some other tumors may also exhibit high TAG values, especially if there are secondary (metastasized) tumors within the brain. Brain tumor patients' TAG values currently range from 136 to over 560 micrograms per serum 1-j. In an initial "blind" test of 50 blood samples performed according to the procedure of this example using Kuged's reagent prepared from astrocytin and bromoacetylcellulose,
11 out of 11 brain tumors and 28 out of 32 normal ones were correctly identified. Of the four cases that appeared to be normal (i.e., non-brain tumor), one was apparently diagnosed with thyroid cancer, which had been treated several years ago.The remaining three cases Normal people are in poor health, probably diagnosed
An individual between the ages of 60 and 70 with undiagnosed cancer. Of the remaining seven cases, three out of three cases of Hodgkins disease were correctly identified.

1 case falling within the tumor range (human G/- in 136-500μ)
applies to patients with severe gliosis, and in the non-tumor range (
The three cases falling within 6-135μ9TA (y/4) each have unclear but non-tumor head-ma, °, su(-i:
Subsequent studies were conducted in accordance with the method of the present example using reagents prepared from individuals with cancer, malignant brain tumors, and all normal individuals.

In further trials performed according to the procedure of this example, the total number of human tumor specimens tested ranged from 50 to 114. The usefulness of these products and techniques is shown in Table 5, which is intended to record the results of these tests. , anti-astrocytin, anti-malignin and 5-rh diagnostic compounds for tumor cells by A.
c; indicates that tumor cell 1 is preferentially attached. This specificity is due to the use of dyes or radioactive substances in histological sections such as anti-astrocytin, anti-malignin or s,-'.
r, Aa, these compounds can be used in the diagnosis of tumor cells. Standard labeling techniques are therefore taken into account.

The usage of 5-TAG is as follows.

This is a modification of the SLoMarie method. 9 Human brain malignant tumor specimens are frozen and cut into 5 micron thick slices. These are stored in a moist container at -70°C for 4-8 weeks before staining. The conjugate is a goat anti-rabbit conjugate. The conjugate can be a standard anti-serum such as fluorescein or other standards using goat anti-rabbit conjugates using techniques known in the art. Incubate a 1:200 to 1:400 solution of TAG on tumor sections for 30 minutes or longer, then wash to remove non-adherent TAG. Wash three times with phosphate buffered saline. Good. Bind with fluorescein-labeled conjugate, incubate, wash, and examine microscopically. Normal cells and their processes are not stained in tumor sections and normal non-tumor brain control sections. Brightly present on its protrusions.

Example 10 Experiments on the cytotoxicity of anti-astrocytin, anti-malignin t and 5-TAG against tumor cells growing in tissue culture.

Standard tests are used to measure cytotoxicity. Generally, the number of cells in a fixed calculation phase, usually prepared to contain 100 live cells, is calculated by 4° treating those cells with the reagents to be tested; Note that the number of living cells is calculated.

Glioblast cells, whose gliogenesis is well characterized, in the S-, TAG solution obtained in Example 8
In a standard test for cytotoxicity to cells in tissue culture from patients with grade 1, 5-TAG is 5-TAG per solution.
Go, 2 weir and 1 corresponding to a small amount such as 0.02μ2
High dilutions of 1:100 and 1:10° also kill all cells during the calculation phase. Similar results were obtained for anti-
Obtained in highly diluted solutions of astrocytin and anti-malignin.

The specificity # shown in Example 9 and the cytotoxicity shown in Example 10 are both anti-astrocytin and anti-
It is highly relevant to the therapeutic potential of malignin rings and 5-TAG. Their therapeutic use requires the actual diagnostic possibility of both these phenomena on surgically removed tumor tissue, in addition to the already proven histological diagnosis.

-85 = Example 1 Hydrolytic cleavage of lucognin A solution of genin, in this case astrocytin or malignin, is prepared at pH 1-2. After 7 to 14 days, thin layer chromatography shows that the product has a molecular weight reduction of about b00 - about 200 if the solution is allowed to stand longer.
The molecular weight unit cleaves in 7-10 days. Thus, for Asto Pcytin there is a decrease in molecular weight from 8°000 and for malignin from 10°000, with a subsequent decrease of about 200 units in each case. .

The physiochemical specificity of astrocytin is 400 for each product.
Maintained down to 0 molecular weight. The physiochemical specificity of malignin is maintained in each product up to a molecular weight of 5,000. This is demonstrated in gXtRA4Jt4 gel diffusion studies for anti-astrocytin and anti-malignin, respectively.

This cleavage can also be accomplished enzymatically, such as with trypsin and other proteases, with the same results. .

=86- The molecular weight of these compounds prepared by hydrolytic cleavage of recognin is approximately defined by the formula: - Product example 1 with physiochemical specificity of malignin
2. Manufacture of artificial tissues or organs with recognin.
lignin) in a highly concentrated (i.e. 10/-) solution of viscose until all surfaces are coated. Alternatively, the Recognin solution is passed under pressure into and around the tube until all surfaces are thoroughly coated. The tube is then ready to be placed into a cavity or tissue containing precursors such as astrocytin or malignin in adjacent tissues or body fluids of a living mammal. This artificial tissue or organ is used to minimize or eliminate the irritating responses of foreign materials without Recognin coating.

Other shapes of artificial tissues or organs can be similarly made.

[Brief explanation of the drawing]

FIG. 1 is a graph showing malignin production fi1 (as total protein amount (IIg) produced) in the present invention as a function of malignancy degree (as % of total protein amount). Patent applicant: Samuel Bogoch, agent: Patent attorney: Aoyama Aoyama, one personsiI! ! Map-ton birth development (%) Procedural amendment written form) 2. Name of the invention Recognins and related substances 3. Person making the amendment Relationship with the case 4 agents for the patent applicant

Claims (1)

[Scope of Claims] 1. A targeting reagent characterized in that recognin is bound to a carrier. 2. The reagent according to claim 1, wherein the carrier is a cellulose-based material. 3. The reagent according to claim 2, wherein the carrier is bromoacetylcellulose. 4. A patent in which recognin is an acidic peptide obtained by extracting normal or diseased cells or tissues with a neutral buffer, separating a fraction with a pK value of 1 to 4 from the resulting solubilized protein extract, and collecting this fraction. Claims 1 to 3
The reagent described in any of the paragraphs. 5. The reagent according to claim 4, wherein the recognin has the following properties: (a) Forms a single linear precipitate with a specific antibody in a quantitative precipitation test and an antigen-antibody gel diffusion test in Uftalony agar (b) Soluble in water and aqueous solutions at acidic or neutral pH; (c) Slightly soluble in water and aqueous solutions at alkaline pH; (d) Has a spectral absorption peak wavelength of 280 mμ (e) having a molecular weight of 4,000 to 10,000; (f) having a high content of glutamic acid and aspartic acid, and a high ratio of glutamic acid to aspartic acid to histidine; (g) having an anti-recognin antibody when injected into animals; produce,
The antibody specifically reacts with other acidic peptides belonging to the recogni family; (h) binds to the anti-recognin antibody by specific immunoadsorption when bound to an inert carrier, thereby making it possible to quantify and purify the antibody; Moreover, it enables the antibody to specifically bind to acidic peptides belonging to the recognins or cells containing the same both in vitro and in vivo. 6. The reagent according to claim 4, wherein the recognin is astrotisin having the following properties: the normal or diseased cell or tissue is a repeatedly destroyed tissue of a glial tumor tissue;
It has a molecular weight of approximately 8,000, and its amino acid composition is 9 aspartic acid, 5 threonine, 6 serine, and 1 glutamic acid.
3, proline 4, glycine 6, alanine 9, valine 4,
1/2 cysteine 2, methionine 1, isoleucine 2,
8 leucine, 2 tyrosine, 3 phenylalanine, 8 lysine, 2 histidine, 4 arginine, total 88 (detectable amounts of cysteic acid, hydroxyproline, norleucine, ammonia, isodesmosine, desmosine, hydroxylysine, lysinonorleucine, γ-amino butyric acid is absent). 7. The reagent according to claim 4, wherein the recognin is malignin having the following properties: an artificial cancer cell in which normal or diseased cells or tissues are grown in a culture medium, and has a molecular weight of about 10,000; Amino acid composition: 9 aspartic acid, 5 threonine, 5 serine, 13 glutamic acid.
, proline 4, glycine 6, alanine 7, valine 6, 1
/2 Cysteine 1, Methionine 2, Isoleucine 4, Leucine 8, Tyrosine 3, Phenylalanine 3, Lysine 6
, histidine 2, arginine 5, total 89 (no detectable amounts of cysteic acid, hydroxyproline, norleucine, ammonia, isodesmosine, desmosine, hydroxylysine, lysinonorleucine, γ-aminobutyric acid). has. 8. Recognin has the formula: 4000+200X=Y [wherein, Y represents the molecular weight of the product, and X represents 0 or an integer from 1 to 19. 5. The reagent according to claim 4, which has a molecular weight represented by: 9. Recognin has the formula: 5000+200X=Y' [where Y' is the molecular weight of the product and X is 0 or 1-19
represents an integer. 5. The reagent according to claim 4, which has a molecular weight represented by: 10. Recognin can produce anti-recognin when injected into mammals, and the anti-recognin is toxic to tumor cells in vitro and binds to fluorescein to produce fluorescence in tumor cells. The reagent according to any one of claims 4 to 10.