CH617853A5 - Process for the preparation of a tumour polypeptide antigen - Google Patents

Description

The present invention relates to a method for producing a polypeptide antigen that is monospecific and is present in a wide variety of human cancers of different locations.

Many attempts have been made to demonstrate the presence of tumor-reactive antibodies in sera obtained from animals given preparations of human cancer tumors. If such demonstrations showed that they were consistently reproducible, it would show the presence of important antigens in human cancer tissue that are not present in normal tissues, and would lead to a better understanding of the nature of the neoplastic process. For a better understanding of cancer, human cancer tissue has been studied to find out the existence of monospecific cancer-related antigen and, most importantly, to find a reproducible way to isolate and purify such antigen.

In the presence of antigens associated with colon and digestive adenocarcinomas, Gold et al., J. expt. Med., 121 (1965), pages 439 to 462 and J. expt. Med. 122 (1965), pages 467 to 487. In these reports, reference is made to earlier work by B. Björklund, which shows the existence of human cancer-related antigen, which is common in the majority of all known malignant tumors of epithelial origin, see boarding school. Arch. Allergy and Appi. Immunol. 1966, 8, page 179 and boarding school. Arch. Allergy and Appi. Immunol. 1958, 12, page 241. In U.S. Patent 3,663,684 to Freedman et al. a carcinoma-embryonic antigen is described which shows so-called «CEA activity». However, this antigen is completely different from the antigen of the invention, which will be shown in detail below.

A practical and commercially usable isolation of antigen itself and its combination with various

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carcinomatous diseases have not been achieved. So far, it has not been possible to isolate and label human tumor antigen using practical and reproducible methods, or the presence of the antigen in the blood of people suffering from hidden or open cancer, with the help of a diagnostic test that is suitable for investigation in is suitable on a large scale to prove or even to make it credible.

In order to take full advantage of the presence of antibodies specific for tumor antigen in animal antisera as a diagnostic agent, a test must be developed that shows the presence of the tumor antigen in the patient's blood. Previously proposed methods have proven to be ineffective in diagnosing cancer.

The inventive method for producing a tumor polypeptide antigen (CAPA) is defined in claim 1.

In the characterizing isoelectric precipitation by converting the polypeptide molecules into the form of zwitterions, the basic groups present in the polypeptide molecule are saturated with the acidic groups in a type of salt formation.

When eluting the gel in stage (d) of claim 1, a fraction with a molecular weight of 10 • 106 to 20 • 106 is obtained, which is further processed in stage (e). This material only appears to have a molecular weight in the range given, since it actually consists of an agglomerate of smaller molecules which are connected to one another by ion attraction and hydrophobic interaction, hydrogen bonds, etc. This causes the fraction obtained from the gel filtration to react like a material having a molecular weight in the range from 10 to 20 • 106, while the actual molecules of the material have a considerably lower molecular weight, in any case lower than about 50,000. When the material eluted from the gel filtration is subjected to isoelectric precipitation and then pH gradient elution in the manner indicated, the molecular forces are released and result in a fraction, the molecules with a weight in the range of approximately 20,000 to 27,000 to have.

In the description, the expression “tumor polypeptide antigen” is abbreviated as “CAPA”.

The CAPA can be used a) to diagnose the presence of cancer by indicating the presence of circulating CAPA and b) to produce antibodies that are monospecific to such CAPA.

A material that has CAPA activity is isolated and purified by the method by homogenizing malignant tissues from autopsies and collecting carcinoma tissues of various types and locations to obtain tumor accumulation. A source material that can be used instead is tissue from human placenta or cultures of malignant cells in vitro (for details on such cultures, see B. Björklund, V. Björklund and I. Hdlöf, J. Nat. Cancer Inst., 26, pages 533 to 545 (1961) and "Antigenicity of Pooled Human Malignant and Normal Tissues by Cytoimmunological Technique. III. Distribution of Tumor Antigen"), normal tissues are also collected in parallel with the malignant tissues, and a useful sequence of procedures leading to the isolation of a pure antigen leads to achieve, a new test procedure was developed, which by the attached Fïg. 1 is explained. For example, the application of biochemical separation methods was guided by a sensitive immunological test method that allows the discovery of less than one ng (nanogram) antigen. The greatest value of the test method as passed through

Fig. 1 is explained based on their selectivity. Differences in antigen between the extracts to be compared were made clear against a background of unimportant similarities, the experimental set-up serving as a feedback control of a number of chemical methods that were varied so as to form tumor fractions

1. maximum antigen reactivity with antitumor sera absorbed by normal antigens, and

2. minimal antigen reactivity with antitumor sera absorbed by tumor antigens. Tumor fractions of minimal reactivity with antinormal sera absorbed by normal or tumor antigens were also created for additional control purposes.

Cancer tissue and normal tissue are mixed in the frozen state with water at a temperature of about 0 ° C and homogenized in such a way that they do not generate excess frictional heat that would cause the CAPA in question to be inactivated. The temperature of the suspension should not be allowed to rise above about 0 ° C.

The resulting cold mixture of liquid homogenate and solids is mixed with an organic solvent capable of dissolving lipids, such as neutral fats, such as acetone or ethyl ether, at a temperature at about 0 ° C, the solvent treatment including removal of Lipoid substances for the purpose and leads to increased exposure of antigenic groups. The solids are separated from the mixture, conveniently by centrifugation, and the aqueous layer and the solvent layer are discarded. The temperature should not exceed + 4 ° C.

The solids recovered are lyophilized and the lyophilized tissue powder is ground, for example in a stainless steel ball mill, at a low temperature, preferably below about 0 ° C and suitably at a temperature below about -70 ° C. The grinding results in a fine one gray-brown powder.

If desired, the resulting powder is extracted with a saline solution, such as a saline solution, at a neutral pH at a low temperature, conveniently at about 0 ° C, discarding the top layer after centrifugation. The resulting sediment can be resuspended in cold water, after which the sediment is collected and lyophilized. The resulting powder can be stored in closed bottles at around 4 ° C for years.

When the above polypeptide-containing tumor powder and the normal powder are extracted with H20 at an alkaline pH, the extracts become cloudy when NaCl is added at a neutral or somewhat alkaline pH. This cloudiness is mainly due to the presence of contaminants of a nucleic acid character. After centrifuging the extracts treated with saline, all activity remains in the clear liquid above. This is precipitated isoelectrically at an acidic pH, advantageously around 4.8, and the resulting precipitates are dissolved in an aqueous phosphate-buffered solution with a pH of around 7.0.

The solutions resulting from the above process are filtered with a suitable molecular sieve gel in pearl form, which allows a fractionation of compounds with a molecular weight range of 10 • 106 to 20 • 106, especially about 15 • 106, the filtering gel with a buffer of about neutral pH eluted and the molecular weight fraction from 10 • 10® to 20 • 106, especially from about 15 • 106, is obtained. The gel or molecular sieve can be selected from polyacrylamide gels, cross-linked dextran gels, agar and agarose gels as well as equivalent gels or molecular5

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seven are selected, such as Bio-Gel A-50 m [trade name for an agarose gel from Bio-Rad Laboratories, Munich, 100 to 200 mesh, reject limit (Daltons) 50-10®, fractionation range (Daltons) IO5 to 50-10®, approximate agarose content 2%] or Sepharose 2 B (trade name for an agarose gel from Pharmacia Fine Chem., Uppsala, Sweden, 60 to 300 microns, beads, fractionation range 10s to 20 • 10®, approximate agarose content 2%). The gel is equilibrated 1:10 with a suitable buffer solution at a pH of about 7.0, such as Sörensen buffer (1/15 molar buffer solution, based on the sodium and potassium phosphates). The type of molecular sieve or gel used for this gel filtration is not critical and the only requirement in this regard is that it should have the ability to fractionate compounds in the molecular weight range mentioned above. Another gel that can be used for the invention is Bio-Gel A-150 m (Bio-Rad Laboratories, 100 to 200 mesh, cut-off limit (Daltons) 150 ■ 10®, fractionation range (Daltons) 10® to> 150 • 106). For further details of the gel filtration method, reference is made to H. Determann, “Gel Chromatography”, Springer-Verlag, Berlin-Heidelberg-New York, 1967. The eluent used for the filtration is a buffer solution with a pH of about 7, and in the outlet the antigen activity is found in the fraction containing this molecular weight range and this fraction is obtained.

The settled active fractions from the above gel filtration are dissolved in a suitable buffer with a pH of about 7.0, such as a Sörensen buffer diluted to 1:10, and the resulting solutions are filtered through a column which has a weak ion exchange molecular sieve gel filled with weak ion exchange properties such as a polyacrylamide gel such as Bio-Gel P-2 (as defined above), the column being equilibrated with a similar buffer with a pH of about 7.0. The fraction showing antigen activity is found as the first fraction of the solution leaving the column and this fraction is obtained.

The fraction obtained in the chromatography on Bio-Gel P-2 as above is subjected to a special process which was developed for the purpose of further cleaning the CAPA. In principle, this process comprises the following stages:

The fraction is subjected to isoelectric precipitation to form hermaphrodite ions by adding acid until the polypeptides show a minimum solubility.

The precipitate obtained is mixed with a suitable gel, which is selected by considering the protein mixture on hand. The resulting mixture of precipitate and gel is applied as a layer on a column containing an identical gel of the same isoelectric pH, i. H. the pH value, which is isoelectric for the proteins and has precipitated them in the previous stage. The column is then eluted with a gradual increase or decrease in pH. In the eluate, the fraction or fractions obtained are those which contain the desired protein or proteins. Although the method of the invention is not intended to be bound by any particular theory, it is believed that the separation of types of molecules in the protein mixture can be explained in the following manner:

The precipitated proteins are exposed to an ion flow that brings one protein after another into solution. The time required to solubilize the various proteins depends on the ionic strength, pH, temperature and flow rate. Since the partition coefficient of each dissolved protein is less than that of the ion gradient, the protein migrates into the gel faster than the gradient. As a result, the protein precipitates again and becomes stationary until it is redissolved by the incoming ion front. This process is repeated countless times in succession, thus separating the types of molecules that differ only slightly from one another.

Bio-Gel P-2 is a cross-linked polyacrylamide (100 to 200 mesh) with a molecular weight cutoff of about 2000, but this cutoff is not critical. Other useful gels are cross-linked dextran gels, such as Sepha-dex G-25, which has a molecular weight cutoff of about 25,000, but this cutoff is also not critical. These gels are molecular sieves and are commonly used in pearl form. Any such molecular sieve gel with molecular weight cutoffs of at least about 2 to 3000 can be used satisfactorily. With regard to the molecular sieve gels which are suitable for gel filtration in connection with a gradient elution according to the invention, reference is made to Gellotte, Fractionation of Proteins, Peptides and Amino Acids by Gel Filtration, in "New Bio-chemical Separations", Van Nostrand, London / New York, 1964.

The normal fraction and the CAPA fraction obtained in the above chromatography are subjected to precipitation of the active substance at a pH of about 5, whereby the sediment is recovered and in water with a pH of about 8.5 is dissolved. The pH of the clear solutions is then adjusted with formic acid, suitably to about 5.0, and this treatment leads to precipitation. Each precipitate is mixed with a slurry of a similar gel as above which has been equilibrated with HCOOH-HCOONH4 to a pH of about 5.0. Each of these mixtures is applied to a gel column equilibrated as above.

A pH gradient was used to elute the precipitates, in this case with a gradually decreasing pH. A suitable buffer system is HCOOH-HCOONH4 and NH4HC03-NH3. The antigen activity is found in the outlet in the pH range from the initial pH to about 3.

The run-out fraction obtained in the above-mentioned pH range contains the desired CAPA, which can be obtained by freeze-drying. In order to determine the molecular weight of the CAPA of this run-out fraction, the fraction can be subjected to a filtration on a gel column equilibrated with HCOOH-HCOONH4 at a pH of about 3.0, the gel being selected from the list of those mentioned above and bio- Gel P-30 (trade name for a polyacrylamide gel from Bio-Rad Laboratories, Munich, 100 to 200 mesh, exclusion limit (Daltons) 40,000, fractionation range (Daltons) 2500 to 40,000) and Sephadex G-75 or G-50 (trade name for Dextran gels from Pharmacia Fine Chemicals, Uppsala, Sweden, 40 to 120 and 50 to 150 microns, fractionation range 3 to 70,000 and 1 to 30,000) are particularly suitable gels for carrying out these filtrations. The eluted fractions of CAPA with a wavelength at the spectrophotometric absorption maximum in the range from approximately 229 to approximately 233 nm and a molecular weight from approximately 20,000 to approximately 27,000 are obtained.

The CAPA can be obtained from the active fraction after filtration by freeze-drying and stored for a long time in a vacuum, in the cold and in the dark.

The isolated material consists of a polypeptide based on a single peptide chain, as shown by treatment with performic acid. The CAPA shows a basic reaction and is soluble at a pH in the range from about 1 to 3.5. The unprotected polypeptide irreversibly denatures at neutral pH values, namely at pH values that

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exceed about 4.5. The antigen is hygroscopic and turns yellow, inactivated and greasy when it absorbs moisture.

The purified CAPA contains a fluorescent group, and this group is subject to activation at a wavelength of approximately 288 nm while emitting light at a wavelength of approximately 350 nm. The fluorescence is proportional to the amount of CAPA and can be used to determine the presence of polypeptide during the process for its isolation.

The CAPA shows a spectrophotometric peak absorption at a wavelength in the range from about 229 to about 233 nm. Furthermore, it shows a strong tendency to aggregate and cannot be filtered on normal filter media which are used for sterilization. The CAPA has been demonstrated to come from the cancer cell walls, since antibodies raised by injecting the CAPA into a living animal body cause complete lysis or degradation of cancer cells in vivo when such cells are exposed to the influence of such antibodies.

As shown above, the polypeptide has a molecular weight in the range of 20,000 to 27,000, more specifically about 24,000. Analyzes show that it contains no carbohydrate and spectrophotometry shows that the CAPA is free of nucleic acids. Analyzes made with amino acid analyzer with and without oxidation are in agreement with this molecular weight, which was determined by gel filtration on Bio-Gel P-30 or its equivalent.

The isolated CAPA can be used in many fields of application, such as the production of monospecific antibodies, for diagnostic purposes, for immunization procedures, such as as an active ingredient in compositions useful as immunizing agents.

A new method has been developed in connection with the production of antibodies that are monospecific to the tumor polypeptide antigen. As stated above, the unprotected CAPA irreversibly denatures at neutral pH values, which means that an injection of an acidic solution of the CAPA into a living animal body results in immediate denaturation of the CAPA when it comes into contact with the neutral body fluids. To prevent such denaturation, an oil emulsion is prepared in which an aqueous solution of the CAPA at a pH in the range of about 2 to about 3 constitutes the trapped or disperse phase that is surrounded by the oil phase. When such an oil emulsion is injected into a living animal body, the acidic aqueous solution of the CAPA is protected by the surrounding oil phase,

when it comes into contact with the neutral body fluids. In this way, the CAPA maintains its activity in vivo and can be delivered to the antibody-producing cells.

One attempt to maintain antigen activity in vitro at neutral pH is to complex CAPA with an inert protein such as human or bovine albumin. When used, a complex is formed that is soluble at neutral pH values while maintaining antigen activity in vitro.

A modified hemagglutination inhibition method has been developed with regard to cancer diagnosis. This method enables the presence of CAPA to be determined in various stages of cancer development by examining materials of interest, such as patient sera, tissues, secretions and extracts from living animal bodies, including, for example, biopsy material, surgical specimens, punctures and smears. This new modified hemagglutination inhibition method consists of the following stages:

a) preparation of a series of samples of the material to be examined by serial dilution,

b) adding a predetermined amount of antiserum containing the antibodies specific for CAPA to each sample,

c) adding a predetermined amount of CAPA on a finely divided support after incubation to each incubated sample,

d) Comparison of the resulting series of treated samples with a series of control samples of decreasing known amounts of CAPA that give inhibition and predetermined amounts of antiserum containing the antibodies, and then adding a predetermined amount of CAPA on a similar fine particle carrier to each of the Control sample and e) Determine the amount of CAPA in the material examined by comparing the sample series with the control sample series in order to find out the presence of cancer and its stage of development.

Compositions useful as an in vivo active immunizer contain the CAPA along with a pharmaceutically acceptable carrier.

The invention is illustrated by the following example.

1. Preparations Isolation of Pure Antigen

Since it was shown (Int. Arch. Allergy, 36, pages 191 to 203, (1969), "Systematic Antigenic Change in Human Carcinoma Tissues by Hemagglutination Techniques", by B. Björklund) that most cancer tissue contains the antigen, cancer tissue of different types and from different locations to get a tumor cluster. In this case, the accumulation consisted of macroscopically pure, histologically confirmed cancer tissues from the following organs (autopsy): breast, bronchi, appendix, colon, duodenum, gallbladder, kidney, larynx, liver, lung, esophagus, ovary, pancreas, prostate, rectum , Stomach, uterus. Normal tissues were collected from a number of individuals to create a cluster of isoantigens, tissue antigens and single antigens. Autopsy cases were used and the tumor diagnoses were confirmed histologically. In addition, it was checked that the tumor antigen in question was intact due to its immunological cross-reactivity with fresh tumor tissues and living tumor cells. Aseptic precautions have been taken wherever possible.

2. Preparation of the tumor polypeptide antigen

The cancerous tissues and normal autopsy tissues were separated from neighboring tissues and cut into 2 to 3 cm cubes, which were washed in 0.9% NaCl at 0 ° C until they stopped delivering blood. The washed cubes were frozen and stored at -24 ° C.

.1. Homogenization:

While frozen, the tissue cubes were mixed with 10 ml of H20 at 0 ° C. per gram of frozen tissue and homogenized in a cooled MSE-Ato mixing homogenizer with rotating knives at half speed for three periods of 1 minute. It should be ensured that the temperature of the suspension is kept slightly below 0 ° C.

The cold homogenized mixture was poured into cold 100 ml polythene bottles. 400 ml of suspension and 400 ml of ethyl ether at -20 ° C (“ether ad narcosin PH Nord” with a content of 0.002% diphenylamine from Skanska Bomulls-krutfabriken AB, Dösjebro, Sweden) were mixed together in each bottle and 120 minutes at -2 ° C in a

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International PR-2 shaker centrifuge with a motor speed of 300 rpm. The mixture was centrifuged at 1000 G at -2 ° C for 5 minutes. During this stage, the lipid layer did not mix with the tissue layer. The ethyl ether and lipid were discarded and a second extraction was performed for 60 minutes, followed by centrifugation for 5 minutes and the ether lipid layer removed. Finally, it was centrifuged at 1000 G at -2 ° C for 30 minutes and the water layer was removed even though it contained some CAPA. The remaining insoluble matter was taken, the remaining ethyl ether was removed by applying vacuum for 5 minutes.

The antigenic material was suspended in 50 ml H20 at 0 ° C, transferred to 500 ml infusion bottles and frozen at -70 ° C (dry ice and ethanol).

It was lyophilized in a Texvac type IV A lyophilizer (Textor, Bad Soden / Taunus, FRG) at controlled temperature.

.2. Separation of the solids:

The lyophilized tissue powder was ground in a stainless steel ball mill ("Cytolator", Lars Ljungberg Company, Stockholm, Sweden) at a temperature of about -70 ° C for 2 hours at 40 rpm. Grinding resulted in a fine, gray-brown powder. This powder was extracted with pre-chilled ethyl ether at -2 ° C in a PR-2 shaker at 300 rpm for 60 minutes and centrifuged at 1000 G at -2 ° C for 30 minutes. The ether layer was removed and the sediment was dried in vacuo. The resulting raw tissue powder (CTP) can be stored for years at 4 ° C in closed bottles without noticeable loss of activity.

5 g of CTP were suspended at neutral pH in 500 ml of saline containing sterile ice cubes. The suspension was homogenized in a MSE-Ato mixer at half speed for three periods of 1 minute. The temperature was kept at 0 ° C. The mixture was kept at 0 ° C for 30 minutes with slow stirring. It was centrifuged at 1000 G at 0 ° C for 40 minutes. The above liquid was thrown away.

The sediment was resuspended in 500 ml of cold distilled water and slowly stirred at 0 ° C for 30 minutes. After centrifuging at 1000 G at 0 ° C for 40 minutes, the floating layer was discarded. The remaining sediment was suspended in 25 ml of cold distilled water and frozen at -70 ° C (dry ice and ethanol). Lyophilization resulted in a washed tissue powder (WTP) that can be stored in closed bottles at 4 ° C for years.

.3. Alkaline extraction:

Aqueous extracts of CAPA and normal WTP with a pH of 9.5 were prepared and concentrated 10 times by isoelectric precipitation at pH 4.8 and dissolution of the precipitate in vio of the volume of water of pH 9.5. The resulting concentrate was stabilized by rapid heating to a temperature of 98 to 100 ° C and this temperature was maintained for 5 to 10 minutes. This heat treatment destroys enzymes that would otherwise inactivate the CAPA. The stabilized extract became cloudy when salt solution was added at pH 7.5 due to the precipitation of residual impurities of nucleic acid character. In order to precipitate such residual impurities, 8.0 ml of CAPA and normal aqueous extracts were precipitated separately by adding 0.8 ml of a 10% solution of NaCl. After centrifuging at 27,000 g at 0 ° C for 1 hour, the clear, floating liquid was at pH 4.8

(pH adjustment with 0.1 l HCl or 0.1 l HCOOH) was subjected to isoelectric precipitation. The precipitates were dissolved in 2 to 3 ml m / 150 phosphate buffer, pH 7.0.

.4. Fractionation:

The resulting solutions, derived from tumor and normal WTP, were washed with Bio-Gel A-50 m (as defined above) in cooled columns with an inner diameter of 2.50 cm and a length of 138 cm with a flow rate of 6.3 ml / cm2 / hour filtered. The gel was equilibrated with m / 150 pH 7.0 phosphate buffer with 2% n-butanol and eluted with the same buffer. 4 to 8 ml of extract totaling 50 to 100 mg of protein were used in each run.

The antigen activity was found in the middle fraction of the outlet of the tumor extract. 1.0 ml portions of this fraction, a total of 160 to 180 ml, were diluted 3 times and adjusted to various pH values in the range from 2.0 to 9.0. After 5 minutes at room temperature, the samples were transferred to cuvettes and their light scatter at 500 nm was measured at an angle of 90 °. The scatter intensities, plotted against the pH value, gave a maximum at pH 4.8, this pH value being the isoelectric point of the fraction.

The remaining part of the active middle fraction was precipitated at pH 4.8 with the aid of 0.1 HCl. Centrifugation at 3800 G at 0 ° C for 5 minutes resulted in a quantitative yield of activity.

.5. Chromatographic purification:

In order to effect a further purification of the CAPA, the sedimented active middle fractions from five Bio-Gel-A-50 m columns in m / 150 phosphate buffer solution of pH 7.0 (Sörensen buffer in 2% n-butanol) were reduced to 5 % of the original outlet volume solved. This solution, containing 130 mg protein in 8.5 ml, was passed through a Bio-Gel P-2 column (as defined above) with a ratio of diameter to height of 1/35 and with a flow rate of 35 ml / cm2 / hour filtered. The column was equilibrated with pH 7.0 m / 150 phosphate buffer (Sörensen buffer in 2% n-butanol). A layer volume of 10 ml was permitted for each mg protein.

The first fraction was active and was separated with the eluent buffer at pH 7.0 while contaminants on the gel were absorbed at the low ionic strength applied.

Precipitation according to the invention

The active substance was precipitated at pH 4.8 with 0.1 M HCOOH and centrifuged at 3800 G at 0 ° C. for 5 minutes. The sediment was dissolved in 8.5 ml H20 and the pH was adjusted to 8.5 with 0.1m NH4OH. The clear solution was reprecipitated 3 times with 0.1 M HCOOH at pH 4.8, washed in the centrifuge at the same pH and dissolved at pH 8.5. The disappearance of n-butanol was followed by gas-liquid chromatography. The finished solution was stored in ampoules, frozen and lyophilized. The resulting product was a dry, almost white powder, and this powder was stored at -24 ° C in the evacuated and sealed ampoules.

An identical procedure was followed with an appropriate extract derived from normal tissue and no activity was found.

.6. Pure representation:

An elution method with a pH gradient was developed for further purification of the product obtained in the above chromatography method. In principle, this process consists of the following stages: The active one

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The protein mixture to be purified is precipitated by an isoelectric adjustment of the pH. The precipitate is mixed with a suitable gel, such as Sephadex G-25 (as defined above) or Bio-Gel P-2 (as defined above). This mixture is applied as a layer to a column which contains an identical gel with the same isoelectric pH. The column is then subjected to elution with a pH gradient at constant flow rate and temperature. The column is eluted with a medium with a continuously or gradually increasing or decreasing pH.

In the present case, pH gradient elution was performed with 15 mg of lyophilized and salt-free CAPA powder and normal antigen powder, which had been purified according to the previous section, and in this case decreasing pH values were used. The powders were separately dissolved in H20 and the pH was adjusted to 8.5 with 0.1N NR, OH. The pH of the clear solutions was then adjusted to 5.0 with 0.1 HCOOH, resulting in precipitation. Each precipitate was mixed with 10 ml of Bio-Gel P-2 slurry which had been equilibrated with an aqueous solution prepared from 0.02m HCOOH and 0.02m HCOONH4 to produce a pH of 5.0. Each of these mixtures was applied to a silicone-treated 15 ml Bio-Gel P-2 column with an inner diameter of 16 mm and a length of 150 mm, which had been equilibrated as above. The gel was held by a small piece of glass wool.

A pH gradient was used to elute the precipitates. Using a gradient mixer (Ultrograd, LKB, Sweden) the pH was kept at about 5 for a short time to wash the precipitate and then the pH was gradually lowered to 2.8. Finally, the pH was raised to 9. The buffer system comprised 0.02m HCOOH-HCOONH4 and 0.02m NH4HCO3-NH3, and elution was carried out at room temperature. The flow rate was kept at 27.2 ml / cm 2 column cross section per hour.

The outlet from the tumor extract was checked by fluorescence spectrometry (activation at 288 nm, fluorescence emitted at 350 nm). The fraction eluted between the initial pH and a pH of about 3 was recovered for further use. After lyophilization and freeze drying, the product could be stored in tightly sealed ampoules after air removal at -24 ° C.

To label the molecular size and confirm the purity, the antigen material in the form of solutions thereof was filtered with Bio-Gel P-30 (as defined above) which was equilibrated with a buffer of 0.02m HCOOH-HCOONH4 of pH 3.0 was. The pH-eluted CAPA was dissolved in a small amount of the same buffer. The flow rate was 3.25 ml / cm2 / hour and the fractions collected were 1.24 ml. The active fractions showed a maximum spectrophotometric absorption at a wavelength of 229 to 233 nm and a fluorescence of 350 nm when at 288 nm have been activated.

In a typical experiment, 10.4 mg of CAPA dissolved in 3.0 ml of the above buffer on a siliconized Bio-Gel P-30 column (inner diameter 2.54 cm, length 50 cm) resulted in complete recovery of the substance and of activity. The elution volume was compatible with a molecular weight in the range of 22,000 to 24,000. These molecular weight values were obtained by comparing the elution volumes with those of compounds of known molecular weights, such as ribonuclease and insulin. After freeze-drying, a white hygroscopic powder was obtained, which in the

Can be stored cold and in the absence of light and oxygen. No carbohydrates and no nucleic acids were found when analyzing the product. Analyzes using an amino acid analyzer showed agreement with the molecular weight, which was determined by gel filtration. Analyzes of the amino acids show the following molar percentages, the values being accurate to ± 5%. The calculated molecular weight was 23 200 ± 2500.

Alanine

8.62

Arginine

4.57

Aspartic acid

10.39

Cysteine

0.95

Glutamic acid

17.04

Glycine

6.87

Histidine

1.00

Isoleucine

4.75

Leucine

10.49

Lysine

3.69

Methionine

1.91

Phenylalanine

2.75

Proline

3.79

Serine

7.74

Threonine

5.92

Tyrosine

3.21

Valine

6.36

This amino acid content corresponds to a theoretical nitrogen content of 16.2 to 17.8%. Determination of the total nitrogen according to Dumas gave a value of 17.0 ± 0.5%. As shown by treatment with performic acid, the polypeptide rests on a single peptide chain. In addition, if the polypeptide is not protected by complexing with albumen, it irreversibly denatures at pH values above about 5.

Production of CAPA album complex

A portion of the CAPA, as produced according to the example, is dissolved in formic acid (for example 0.05 molar, the pH should be in the range from 2 to 3). A clear solution was obtained. Albumen powder or a solution of albumen with the same pH as the above formic acid solution (200 parts by weight of albumen per part by weight of CAPA) was added to this solution, resulting in a clear solution of CAPA and albumen. The pH of the solution was then slowly increased by adding a base (for example an aqueous sodium hydroxide solution or ammonia) until the pH reached about 7.5. This resulted in a clear solution containing CAPA and albumen in the form of a complex.

This 12% CAPA / ml solution can be used directly in the diagnostic procedure described below, or the complex can be isolated in the form of a white powder by freeze-drying. The powder is stable in the cold (+ 4 ° C) for a long time.

Experiments have shown that maximum utilization of CAPA activity is obtained with an albumen to CAPA weight ratio equal to or above 200: 1.

Fresh sheep blood (1 part by volume) became sterile Alse-ver solution (1.2 part by volume) (Alsever solution is made up of 250 g glucose, 80 g sodium citrate dihydrate, 42 g NaCl and water to 101 while adjusting the pH to 6. 1 made with 10% citric acid monohydrate) added. The mixture was centrifuged and the red blood cells were resuspended once in Alsever solution and twice in a pH 6.8 buffer solution. Finally, a suspension of ros

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blood cells in the pH 6.8 buffer at a concentration of 109 cells / ml.

A volume portion of the red blood cell suspension prepared as above was added to a volume portion of pH 6.8 buffer solution containing 18 wg tannic acid per ml with stirring. The red blood cells tanned in this way were centrifuged and resuspended twice in pH 7.5 buffer. Finally, the red blood cells were suspended in buffer of 7.5 with a concentration of 109 cells per ml.

A portion of the CAPA complex was dissolved in a buffer solution of pH 7.5 and gave a concentration of 3 ju g CAPA per ml (calculated on pure CAPA). A volume of the suspension of tanned red blood cells prepared above was added dropwise at 0 ° C to a volume of the CAPA complex solution over 10 minutes. The labeled cells were centrifuged and resuspended in a pH 7.5 buffer solution containing a stabilizing amount (about 1.2 parts by volume per part by volume) of inert human serum to give a suspension containing 1.6 +108 labeled cells per ml contained. These labeled cells were used in the diagnostic procedure as shown below.

Antibody production

In view of the fact that the CAPA irreversibly denatures at neutral pH values, as shown above, it was suspected that parenteral injection of the polypeptide would not lead to the production of antibodies. Experiments carried out on rabbits confirmed this and it was found that no antibodies were produced in rabbits after injection of polypeptide-albumin complex. All hemagglutination reactions were negative.

In order to be able to convert the polypeptide into antibody-producing cells in an active state, it is necessary to add the polypeptide in the solution to pH values below about 3 (0.02m HCOOH, pH 2.8) hold and emulsify the solution in oil to form extremely small drops surrounded by a protective layer of oil. Injection of the emulsion resulted in the production of satisfactory amounts of antibodies that reacted specifically with the CAPA in hemagglutination reactions in which blood cells were labeled with CAPA.

816 micrograms of pure CAPA made from accumulated human cancer tumors were dissolved in 1.0 ml of 0.02m HCOOH pH 2.8. 1.0 ml of oil (commercially available preparation from Difco Laboratorier, Detroit, Michigan, USA, Bacto-Adjuvant, Freund) was added dropwise to the resulting solution with simultaneous emulsification. The emulsification was carried out according to Freund with the aid of an injection syringe, the mixture being drawn up and down in the syringe until a uniform white emulsion was formed with approximately the same consistency as whipped cream. A drop of this emulsion was tested with ice water to confirm that it was floating and remained intact. The resulting semi-liquid emulsion was used for subcutaneous and intramuscular injections in rabbits.

A so-called complete adjuvant was used in the first injection and a so-called incomplete adjuvant was used in the subsequent injections. After the initial bleeding of each of the five rabbits to determine O values, a total of 408 micrograms of emulsified CAPA at pH 2.8 was subcutaneously injected on each right and left palm. In addition, three injections were administered at 10-day intervals, the first intramuscularly in each hind leg and the second and third subcutaneously on both front and rear sides. The selection of the injection sites was made to the

Exploiting access areas of the regional lymph glands. In total, each rabbit received approximately 1.6 mg of CAPA in the form of the emulsion. 14 and 24 days after the last injection, blood test samples were taken and subjected to hemag-glutination analysis for the presence of specific antibodies against CAPA. A maximum of blood was drawn 2 days after the last sampling, which was obtained in the form of serum, which in turn was sterile filtered and transferred in small portions to sterile tubes. These tubes could be stored in the cold, keeping their specificity.

Dignostic procedure

Blood samples were obtained from Es-Kilstuna Sweden Central Hospital, and 42 of these blood samples were from infectious clinic patients, one from the surgical clinic, two from radiotherapy, ten from post-partum women, ten from umbilical cord blood and ten from pregnant women in the first third and six by pregnant women in the second third. In addition, blood from twenty 10-year-old children from Slottsskolan and ten samples from older healthy adults from the private clinic of Dr. Lundström worried in Eskilstuna in Sweden. From the surgical and medical clinic of Ersta Sjukhus in Stockholm, Sweden, samples were taken from 44 patients, from Akeshoys Hospital, samples from 45 patients, and from the surgical clinic from Karolinska Sjukuset in Stockholm, Sweden, blood samples from 72 patients were taken for one possible surgical intervention were provided. The blood donation department of Karolinska Sjukhuset in Stockholm, Sweden, delivered 118 samples from donors aged 20 to 67 years and with an average age of 37.2 years (standard deviation ± 11.3 years), and sera from the clinical center laboratory of this hospital 29 patients were examined in the general departments of Radiumhemmet in Stockholm, Sweden, and 12 patients in the local gynecological departments.

The total number of blood samples was 431. Regarding the 111 cases from Eskilstuna, a clinical examination and a medical report were made except for the obviously healthy patients. Regarding the Stockholm cases, detailed patient studies and medical reports were prepared for 101 cases. In the remaining cases, clinical diagnoses other than malignant tumors were on hand and there was no suspicion of a malignant disease.

Venous blood was transferred to Wassermann tubes without additives and taken to the laboratory, in some cases after the coagulum and blood cells had been removed by centrifugation.

The blood analysis was based on the indication of the presence of CAPA using a modified hemagglutination inhibition method (micro method). In principle, the following procedure was used:

25 fA of patient serum absorbed with red blood cells from the sheep were titrated in eight steps with double dilution in Linbro IS-MRS-96 dilution plates, and then 25 µl of specific antiserum was diluted to a limit (approximately 1: 2000) added to each cavity. After incubation at 22 ° C for 10 minutes, 50 (i \ a suspension of about 6 to 8 million red blood cells from sheep was added to each cavity, tanned according to the method described above and labeled with isolated CAPA linked to human albumen (about 2 to 4 (each 109 blood cells). Other finely divided carriers such as latex, bentonite and collodion can also be used instead of the red blood cells. The reaction was carried out using a ge5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

9

617 853

inclined mirror and a standard series after 4 to 24 hours of incubation at 1 to 2 ° C. The following was used for control purposes:

1. Blood cells labeled with CAPA plus antiserum;

2. blood cells labeled with CAPA without antiserum; 5

3. patient serum plus blood cells labeled with human albumen;

4. patient serum and untreated blood cells;

5. CAPA-labeled blood cells and antibodies in a row where inhibition is achieved by gradually decreasing known amounts of CAPA.

The reconstituting fluids used contained collected (i.e., many people pooled) inactivated neutral human serum for the stabilization of blood cells.

CAPA reagent was prepared from accumulated human carcinoma tissues and purified according to the above with the help of gel filtration, pH gradient elution, filtration through Bio-Gel P-30 and complex formation with Al-20 trees.

Horse anti-HeLa from November 19, 1962, absorbed by normal tissues and accumulated human serum, was used as the antiserum. The immunization was carried out with washed fragments of HeLa cells that had grown in flat glass bottles in Eagles medium containing 20% inactivated human serum. The cells were harvested with EDTA, centrifuged and washed 3 times with water. In the presence of CAPA in patient serum, the horse antibodies were neutralized, which did not lead to agglutination if the blood cells labeled with polypeptide were subsequently added. The recording was made after an increasing score from 0 to 6, with 0 indicating the absence of inhibition and 6 the maximum inhibition of agglutination. The relationship between the evaluation points and the concentration of CAPA per ml of serum is shown in Table I, which was obtained by calibrating dilutions of predetermined amounts of CAPA in neutral serum.

Table I

Approximate calibration of assessment points and CAPA amounts

Evaluation points fig CAPA / ml

0

È 0.03

1

0.04-0.08

2nd

0.09-0.12

3rd

0.13-0.24

4th

0.25-0.49

5

0.50-0.99

6

êl, 0

The recordings were made without knowing the patient's state of health. The material examined was classified according to Table II below.

Table II

Presence of CAPA in 431 sera from people of different groups according to whole evaluation points from 0 to 6, average evaluation points (M) and standard deviations (SD)

Groups whole evaluation points

0 1 2 3 4 5 6 number M ± SD

1.

primary cancer with metastases

5

4th

1

2nd

12

3.00

+

1.13

2nd

extensive cancer

4th

2nd

1

4th

8th

2nd

1 22

2.91

±

1.82

3rd

non-radically treated cancer

6

10th

15

8th

39

1.64

±

0.99

4th

untreated primary cancer

3rd

1

4th

2nd

10th

1.50

±

1.18

5.

radically removed cancer

19th

1

1

21st

0.14

±

0.19

6.

malignant disease,

not cancer

7

3rd

1

11

0.45

+

0.68

7.

non-malignant disease

118

3rd

3rd

7

1

132

0.26

±

0.22

8th.

healthy adults

(M = 71 years)

8th

2nd

10th

0.20

±

0.42

9.

Blood donor (M = 39 years)

116

2nd

118

0.02

±

0.13

10th

10 year old children

19th

1

20th

0.10

±

0.44

11.

Newborn (umbilical cord blood)

1

1

1

4th

2nd

1

10th

2.80

±

1.48

12.

pregnant women

12

2nd

2nd

16

0.38

+

0.72

13.

Women after giving birth

8th

1

1

10th

0.40

+

0.97

It can be seen from the results that the highest scores were obtained with the groups characterized by "primary cancer with metastases" (local), "extensive cancer" and "newborn" (umbilical cord blood).

Low scores were obtained for blood donors. You get a middle position in the group "primary untreated cancer" and in the group "non-radically treated cancer". Assessment points, which were roughly the same as those of the blood donors, were found in the group "Radically 65 Cancer Removed".

The mean values for CAPA in all groups were compared in pairs and are shown in Table III below.

617 853

10th

Table III

Statistical significance3 of the differences between the assessment points for the presence of CAPA

in sera from nine patient categories

Groups meaning b the difference between the category numbers

1.

primary cancer with metastases

3.00

±

1.13

0

XX XX

XXX

XXX

XXX

XXX

XXX

2nd

extensive cancer

2.91

±

1.82

XX XX

XXX

XXX

XXX

XXX

XXX

3rd

not treated radically

cancer

1.64

+

0.99

0

XXX

XX

XXX

XXX

XXX

4th

untreated primary cancer

1.50

±

1.18

XXX

X

XXX

XX

XXX

5.

radically removed cancer

0.14

+

0.19

0

0

0

0

6.

malignant disease,

but not cancer

0.45

+

0.68

0

0

0

7.

non-malignant disease

0.26

±

0.22

0

XXXe

8th.

9.

old healthy people blood donor

0.20 0.02

+ +

0.42 0.13

0

the significance tests for the differences were carried out using an approximately normal distribution variable Z, which is defined as follows:

Mt-M2

Z = -

SDt2 SD22

ni n2

if Z> 1.96, then the meaning of the difference is x, which means 0.01 <P <0.05;

if Z> 2.6, the meaning of the difference is xx, which means 0.001 <P <0.01;

if Z> 3.5, the meaning of the difference is xxx, which means P <0.001.

this group includes eleven positive tests, eight of which relate to suspected but unproven cancer.

The table above clearly shows that "primary cancer with metastases" and "extensive cancer" have significantly higher mean scores than all other groups, but with the exception of newborns (see also Table II).

For the groups “non-radically treated cancer” and “untreated primary cancer”, the average rating points are significantly higher than for all other groups, with the exception of the two mentioned above, and compared with the group “malignant disease but no cancer” which is the difference (0.01 <P <0.05).

Groups 5 to 9 show no significant mutual differences, with the exception of the essential difference between 7 and 9. This difference can be explained by the fact that among the cases there can be real, hidden cancer cases which give evaluation points between 1 and 4.

The positive indication in cord blood deserves some attention. Pieces of such tissue were extracted to determine whether the CAPA was from the placenta. Amounts of 300 mg of frozen placental tissue per ml of buffered physiological saline with a pH of 7.5 were homogenized at 0 ° C. and centrifuged at 1000 ° C. for 30 minutes at 0 ° C. in conical tubes (according to Markham). The above floating fluid was tested for the presence of CAPA using the hemagglutination method described above. To rule out the possibility that the inhibition reactions obtained were caused by non-specific inactivation of the pure CAPA applied to the blood cells, these blood cells were shaken after each recording and a standard amount of antibodies specific for CAPA was added. The antigenic inactivation could be demonstrated in each of these cases. An extract of antigen powder, which had been obtained from human cancer tissues of different origins and different localizations, was used as control material. The results show that out of 26 placentas from as many individuals all contained significant amounts of CAPA without exception. The mean (geometric) for the 26 placentas was 297 ± 8 micrograms per gram of 35 moist tissue (0.03%).

Since it is clear from the above investigation that CAPA is synthesized in the placenta, and since the genes contained in the placenta must also be present in the being from the same egg cell, it is to be appreciated that the ability or capacity Developing CAPA is a normal phenomenon. This capacity is normally suppressed because normal cells do not contain the CAPA in measurable amounts. In cancer cells, however, capacity activation occurs, which is characterized by the presence of CAPA in and around the cells. The antigens and, to some extent, functional similarity between trophoblast cells of the placenta and cancer cells and the difference with respect to other cells of the individual appear to be comparable in that cancer 50 diseases are due to a change in control of inherent properties.

A statistical analysis of the experimental results shows that there is a very important correlation between positive cancer diagnosis and increased CAPA concentration 55 (P <0.001). The same applies to the relationship between the size of the tumor mass under consideration and the increased CAPA concentration within the same age groups (P <0.001). The increased concentration of CAPA was found in connection with a large variety of different cancer localizations. Regardless of the type and location of the cancerous tumors, it has been found that they contain the CAPA, so that in view of this fact the CAPA can be used for all known types of cancer, i. H. malignant tumors of epithelial origin that appear to be common.

65

Double blind test In order to further confirm the validity of the above conclusions, a double blind test was carried out among the most strictly

11

617 853

most controlled conditions. Encrypted blood samples were supplied by various departments at Eskilstuna Central Hospital in Sweden. In this case, quantitative measurements of the CAPA content of the patient sera were carried out.

The results of this double blind test and the results of the previous single blind test are summarized in Tables IV to VIII below. Table IV shows the total number of patients examined and the number of patients diagnosed with cancer,

Table V the number of patients in the different groups together with their average age and the deviations from them,

Table VI the presence of CAPA in the sera of all patients tested,

Table VII the percentages of patients with a CAPA concentration = = 0.15 μg / ml serum, excluding groups 9, 10 and 12 of table V,

Table VIII shows the presence of CAPA in relation to tumor localizations for groups 1 to 3 in Table V. The site numbers refer to “Cancer Incidence in Sweden 1968”, to which reference is made here.

Table IV Number of patients tested

10th

exam

Number of patients

Number of patients diagnosed with cancer

15

Single blind test Double blind test

431 503

104 49

934

153

Table V

Number of patients from different groups and average ages as well as deviations from them

groups

Single blind test

Double blind test

number

Average age

±

deviation

number

Average age

+

deviation

1. Cancer with metastases

34

72

+

9

14

66

±

10th

2. untreated cancer without

Signs of metastasis and not

radically treated cancer

49

67

±

11

14

66

+

12

3. Radically removed cancer

21st

66

+

15

21st

64

+

12

4. malignant disease, but none

cancer

11

70

±

7

5

58

±

18th

5. non-malignant disease

132

61

±

22

186

46

+

21st

6. healthy adults

10th

71

±

5

46

36

±

14

7. Blood donor

118

38

±

11

-

-

-

8. 10 year old and younger

Patient

20th

10th

±

0

62

2nd

±

1.5

9. Umbilical cord blood

10th

0

±

0

20th

0

±

0

10. Women after giving birth

10th

21st

±

3rd

20th

25th

±

4th

11. pregnant women

16

26

±

4th

110

26

±

4.5

12. Cancer in situ

-

-

±

-

5

27th

±

6

Total 431 503

Table VI

Presence of CAPA in the sera of 934 patients

groups

CAPA

SO ^ SjUg / m number

(%)

CAPA ä 0.09 fig / ml number

(%)

total number

(%)

1. Cancer with metastases

9

(19)

39

(81)

48

(100)

2. untreated cancer without

Signs of metastasis and not

radically treated cancer

28

(44)

35

(56)

63

(100)

3. Radically removed cancer

36

(86)

6

(14)

42

(100)

4. malignant disease,

but not cancer

15

(94)

1

(6)

16

(100)

5. non-malignant disease

269

(85)

49

(15)

318

(100)

6. healthy adults

54

(96)

2nd

(4)

56

(100)

7. Blood donor

118

(100)

0

(0)

118

(100)

8. 10 year old and younger

Patient

79

(96)

3rd

(4)

82

(100)

9. Umbilical cord blood

21st

(70)

9

(30)

30th

(100)

10. Women after giving birth

25th

(83)

5

(17)

30th

(100)

11. pregnant women

123

(98)

3rd

(2)

126

(100)

12. Cancer in situ

4th

(80)

1

(20)

5

(100)

A total of

781 (84) 153 (16) 934 (100)

617 853

12

Table VII

Percentage of patients with a CAPA concentration of 0.15 (groups 9, 10 and 12 of Table V were excluded)

groups

%

number

1. Cancer with metastases

64

48

2. untreated cancer without

Signs of metastasis and not

radically treated cancer

21st

63

3. Radically removed cancer

10th

42

4. malignant disease, but none

cancer

0

16

5. non-malignant disease

6

318

6. healthy adults

0

56

7. Blood donor

0

118

8. 10 years and younger patients

0

82

11. pregnant women

1

126

A total of 869

Table VIII

Presence of CAPA in relation to tumor localization for groups 1 to 3

Localization

Job

1. Cancer with

2nd

untreated

3. radical

Ins

No.*

Metastases

Cancer with no total removed

Metastases

cancer

signs and

not radical

treated cancer

g 0.08

S 0.09

<

0.08-0.09

g 0.08

g 0.09

Back gums

144

1

1

2nd

Nasopharynx

146

2nd

1

3rd

esophagus

150

1

3rd

1

1

6

ventricle

151

1

2nd

1

2nd

8th

Small intestine

151

1

1

Large intestine

153

2nd

1

7

6

16

Rectum

154

4th

1

3rd

1

14

Gallbladder

155

1

1

1

3rd

liver

156

1

• 1

pancreas

157

3rd

1

2nd

6

Nose entrance etc.

160

1

1

2nd

Trachea, lungs, etc.

162

4th

2nd

5

14

Mammary glands

170

2nd

7

1

3rd

2nd

18th

Cervix uteri

229

2nd

3rd

1

6

Corpus uteri

198

1

1

1

1

1

2nd

7

Ovaries etc.

175

1

4th

1

1

2nd

9

prostate

177

2nd

3rd

3rd

1

9

Testicles

178

1

1

1

3rd

Penis etc.

179

1

1

2nd

Kidneys

180

1

3rd

1

5

bladder

181

1

2nd

3rd

1

10th

malignant melanoma

190

1

1

2nd

thyroid

194

1

1

1

2nd

5

primary tumor

of unknown origin

1

1

A total of

9

39

28

35

36

6

153

* according to "Cancer Incidence in Sweden 1968".

Careful statistical analysis of the values obtained in this double-blind sample again shows the extremely important correlation between the positive cancer diagnosis and an increased CAPA concentration (P <0.001). This double blind test also confirms the relationship between the

Size of the investigated tumor mass and the increased 65 CAPA concentration within the same age groups (P <0.001). The increased serum content of CAPA was shown in connection with about 20 different types and localizations of cancer. The double blind test is thus confirmed

13

617 853

completely the validity of the new knowledge and its practical usability.

In the introduction to this description, it was said that the antigen described in U.S. Patent 3,663,684 is clearly different from the antigen of the invention. To demonstrate the difference between the known antigen and the antigen of the invention, a detailed overview of some specific differences in the method of antigen isolation and in the properties of the antigen is given below. io

CEA

Source: colon cancer, fetus (meconium, good)

Extraction from the antigen-containing tissues by glycoprotein solvents at low pH at room temperature:

Canceling the extract isoelectric point:

not specified or not available

Absorption of UV light at 280 nm

Fluorescence:

not mentioned

Molecular weight 200,000, (can be as little as 70,000)

active at neutral pH contains sugar (glycoprotein)

CAPA

Source: All types of cancer, 15 cancer cell cultures in vitro, placenta. (Little, if any, in the fetus.)

Wash the fabric with water at neutral 20

pH value, extraction with ethyl ether (if necessary), throwing away the extract, extracting the solids at high pH (9.5), picking up the extract, temperature in the water treatment: 0 ° C.

isoelectric point:

about 5 (before the final stage)

Absorption of UV light at 230 nm

(very little at 280 nm) fluorescence:

Activation at 288 nm, emission at 350 nm molecular weight 23 200 ± 2500

at neutral pH no sugar destroys according to gas liquid chromatography, mass spectroscopy and other analyzes

CEA

no antibody inhibition against CAPA by hemagglutination does not mark red blood cells from sheep that have been treated with tannic acid is present in cancer in a concentration of a few nanograms from 100 to 200 nm per ml

Sample: expensive, time consuming, requires radioactive isotopes

CAPA

inhibits specific antibodies by hemagglutination. Such cells are present in the serum in concentrations of 0.09 to 8 μg / ml (equivalent to 90 to 8000 ng / ml), ie. H. in an amount about 20 times larger than CEA, it is light, fast, cheap, no isotopes and no complex equipment required

In summary, there are brief definitions of the tumor polypeptide antigen and the diagnostic test that can be carried out with it.

Definition of CAPA HeLa cells (derived from a 1952 cancer case) are grown in vitro. These cells are used as an anti-gene source. The cells obtained are injected, resulting in the production of special antibodies which are used to identify the antigen in tumors, serum, placenta, etc. The latter antigen must have and has an identical immunological reaction site (compared to the HeLa antigen), but it can of course be different in other respects if it is in a natural state.

Definition of the diagnostic test Antigen from HeLa cell cultures in votro are used to produce antibodies in horses. After absorption of unwanted antibodies, horse serum is used to agglutinate red blood cells labeled with antigen taken from tumors (or placenta or cell cultures from HeLa or HEp-2 or Detroit-6). This is the indicator system. If an un-45 known sample contains antigen activity, it interferes with the antibody and inhibits agglutination.

s

1 sheet of drawings

Claims (5)

617 853 1. A method for producing a tumor polypeptide antigen, in which a) homogenizing material with tumor polypeptide antigen activity at a temperature not exceeding 0 ° C. in a liquid, b) the solids are separated off from the mixture of liquid homogenate and solids obtained in (a) directly or after treatment with an organic solvent in the cold, c) the solids are extracted with an alkaline, aqueous solution, d) filtering the extract on a filtration sieve of the molecular sieve type in pearl form, which allows a fractionation of compounds with a molecular weight range of 10,000 to 20 x 106, eluting the gel and obtaining the fraction with a molecular weight of 10 x 106 to 20 x 106 and e) the fraction resulting from step (d) is subjected to the chromatography to remove impurities by means of a weak ion exchanger, then impurities are absorbed and the fraction having the antigen activity is recovered, characterized in that this fraction is subjected to isoelectric precipitation while converting the polypeptide molecules into the form of zwitterions by adding acid until the polypeptides show a minimum solubility, after which f) the resulting precipitate is mixed with a molecular sieve and the resulting mixture is then one pH gradient elution and g) reducing the pH to a pH of about 3, the fraction containing the tumor polypeptide antigen, which has an absorption peak in the spectrum at 229 to 233 nm and a molecular weight in the range of 20,000 up to 27,000 wins. 2. The method according to claim 1, characterized in that the material containing antigen is homogenized in water at a temperature of not more than 0 ° C. while still frozen, the resulting homogenate is treated with ethyl ether and / or acetone, solids are separated from the resulting mixture, this lyophilized and ground in a steel ball mill at a temperature below the freezing point of the treated material, the resulting solids extracted with an alkaline aqueous solution and, after centrifuging, the upper liquid layer quickly heated to 95 to 100 ° C and for 5 to 10 minutes to destroy the enzymes at this temperature, the liquid then treated with an aqueous sodium chloride solution to precipitate impurities, the resulting liquid on a cold column of a molecular sieve in pearl form with a fractionation range of 10,000 to 20 x 106, which is equilibrated with phosphate buffer with a pH of 7.0, is filtered and the active fraction of the eluate is obtained as a middle fraction and on a column of a weak ion exchange molecular sieve, which with phosphate buffer with a pH of about 7.0 is equilibrated, chromatographed; subjecting the liquid fraction having the antigen activity to the isoelectric precipitation while converting the polypeptide molecules into the form of zwitterions by adding acid until a pH of about 5 is reached, after which the resulting precipitate is coated with a molecular sieve with a molecular weight cutoff of at least 2000, which is equilibrated with HCOOH-HCOONH4 having a pH of about 5, applies the mixture thus obtained to a column of an identical and equilibrated gel, the column is subjected to a pH gradient elution and the pH is reduced the fraction containing the antigen, which has an absorption peak in the spectrum at 229 to 233 nm and has a molecular weight of 20,000 to 27,000, is recovered. 2nd PATENT CLAIMS 3. The method according to claim 1, characterized in that a tumor polypeptide antigen is produced which has a molecular weight of 23 200 ± 2500, the spectrum shows an absorption band at a wavelength of 229 to 233 nm and the one by treatment with performic acid proven single peptide chain. 4. Use of the tumor polypeptide antigen produced by the method according to claim 1 for the production of antibodies which are monospecific to the polypeptide antigen, characterized in that a) an aqueous solution of the antigen with a pH of less than 3 is prepared, b) prepares an oil emulsion therefrom in which the solution resulting from stage a) forms the dispersed phase which is protected by the surrounding oil phase, and c) injects this oil emulsion into live animals and thus the antigen contained in the emulsion to the antibody-producing cells transported and the antibodies produced in this way wins. 5. Use according to claim 4, characterized in that the aqueous solution in step a) is prepared at a pH below 3 using a weak acid, preferably formic acid.