FI60567B - PREPARATION FOR ANALYSIS OF DIAGNOSTIC AV AVERAGE OVER FOERFARANDE FOER DESS FRAMSTAELLNING - Google Patents

Description

[mΓβΙ Μ1ν MONTHLY PUBLICATION / ne /; '? «Tj IB] <11> UTLÄGGNINGSSKRIFT bUbt t ^ (45) Patent granted 10 02 1932 Patent« eddelat (51) Kv.ik? /Int.ci.3 C 07 G 7/00. A 61 K 39/00, -; G 01 N 33/16 FINLAND —FINLAND (21) P »t * nttlh« k * mu »- Puuntamaknlnc 2099/73 (22) HakamtopUv · - AiwOknlnpdif 29-06.73 ^ (23) Alkuplhrt — Gllti | h« t * J * | 29-06-73 (41) Interpreters | utklMksl - Bllvlt offuntllg 11-01-7 ^ _ '. (44) Date of issue and date of issue. - ","

Patent and Registration Office of the Government of the United States of America 30.10.81 (32) (33) (31) Pyydetty «uoikous -Bajlrd prloriut 10-07-72 USA (US) 270273 (71) AB Bonnierföretagen, Torsgatan 21 , S-105 UU Stockholm, Sweden-Sweden (SE) (72) Knut Bertil Björklund, Bromma, Sweden-Sweden (SE) (7 ^ +) Oy Jalo Ant-Wuorinen Ab (5 ^ +) Preparation and method for the diagnosis of cancer to prepare it - Preparations for the diagnosis of carcinogens and for the preparation of framställning

Repeated attempts have been made to demonstrate the presence of tumor-reactive antibodies in sera obtained from animals administered human cancer tumor preparations. If such experiments were always reproducible, this would indicate the presence of important antigens in human cancer tissue that are not present in normal tissues, and could thus lead to a better understanding of the nature of the neoplastic process. In order to better understand cancer, human cancer tissue has been studied to detect the presence of a monospecific antigen present in cancer tissue and, above all, an attempt has been made to provide a reproducible method for isolating and purifying this antigen.

of colon and gastrointestinal tract adenocarcinoma antigens have shown the presence of the Gold, et al: J. Expt. Med. 121 (1965) 439-462 and J. expt. Med. 122, (1965) 467-! 487. These publications refer to B. Björklund's previous work, which has shown the existence of an antigen present in the context of human cancer 1 which is common to the majority of all ί i ... ... j -2- 60567 of known malignancies of epithelial origin, Internat. Arch. Allergy and Appi. Immunol. 1966, 8, 179 and Internat. Arch. Allergy and Appi. Immunol. 1958, 12, 24-1.

U.S. Patent No. 5,665,684 discloses a carcinoembryonic antigen having so-called "CEA activity". However, this antigen is completely different from the antigen used in the preparation of the present invention, which is a polypeptide antigen present in cancer tissue, i.e. CAPA, and to demonstrate this difference, a combination of certain concrete differences in both antigen isolation method and antigen properties is presented below. !

CEA CAPA

Source: colon cancer Source: all types of cancer, fetal (amniotic fluid, intestinal) cell lines in vitro, placenta.

(in the fetus little if at all)

Extraction from antigen-containing tissues- Tissue washing with water in neutral glycoprotein solvent at pH, extraction with ethyl ether at low pH at room temperature (possibly), the extract is discarded, solid

In the patient: the extract is recovered by extraction of the substances at high pH

(9.5), the extract is recovered, temperature in water treatment: £ 0 ° C

Isoelectric point: not visible Isoelectric point: about 5 defective or absent (before the final stage) UV light absorption at 280 nm UV light absorption at 250 nm (very low at 280 nm)

Fluorescence: not mentioned Fluorescence: activation at 288 nm. Radiation at 550 nm

Molecular weight 200,000 (can be -Molecular weight 25,000 ± 2,500 as low as 70,000)

Active at neutral pH Destroys at neutral pH

Contains sugar (Glycoprotein) No sugar according to gas-liquid chromatographic and dash spectrographic analyzes

Does not inhibit CAPA antibodies- Inhibits the nucleation of its specific antibody in a hemagglutination test in a hemagglutination test

Does not cause tanning of these blood cells by labeling tannin red blood cells -3- 60567

CEA CAPA

Occurs in cancers at levels ranging from 0.09 nanograms to 100-200 na- 9 yg / ml (equivalent to 90-8000 ng / ml) per mg per ml, i.e. at concentrations approximately 20 times higher than CEA .

Test procedure: expensive, time- Easy, fast, inexpensive, non-isotopic, requires radioactive pellets, does not require expensive furniture isotopes A practical and commercially viable method for isolating antigen per se has not yet been established and the antigen has not been linked to various cancers. established. Thus, to date, it has not been possible to isolate and characterize an antigen present in human cancer by practical and reproducible methods, nor has it been possible to determine or even detect the presence of the antigen in the blood of individuals with latent or established cancers by a diagnostic test suitable for large-scale research.

In order to make diagnostic use of the presence of antibodies specific for a tumor antigen in animal antisera, a test must be developed to demonstrate the presence of the tumor antigen in the patient's blood. Methods known to date have not been shown to be effective in making a definite diagnosis of cancer.

It is thus an object of the invention to provide a preparation for the diagnosis of cancer comprising an antigen present in human cancer tissue mixed with a stabilizing amount of an inert protein such as albumin, and a process for the preparation of such a preparation, and the features of the invention are set out in the appended claims.

As can be seen from the statistical studies in the description below, cancerous tumors, regardless of type and location, contain CAPA, and this CAPA is common to all known types of cancer.

M »60567

The substance with CAPA activity is isolated and purified by homogenizing malignant tissues obtained from autopsies, whereby carcinoma tissues of different types and from different sites are collected into a tumor pool ("pool"). Examples of the types of cancer studied include the colon, rectum, lung, esophagus, stomach, pancreas, kidney, bladder, breast, uterus,! ovarian, liver cancer, leukemia. Alternatively as starting materials. tissue from human placenta or malignant cell cultures can be used in vitro (for more information on such cultures, see B. <

Björklund, V. Björklund and I. Hedlöf, J. Nat. Cancer. Instr. 26: 1961, "Antigenicity of» Pooled Human Malignant and Normal Tissues hy Cytoimmunological Technique. III. Distribution of Tumor Antigen "). Normal tissues are also collected simultaneously with malignant tissues. Figure 1 shows a new test method for isolating a useful pure antigen. The application of biochemical separation methods was thus guided by a sensitive immunoassay method capable of detecting amounts of antigen of less than 1 ng (nano-gram). The main merit of the assay technique of Figure 1 is its selectivity. Differences in comparative extracts in antigenic activity were determined against a background of negligible similarities, with the assay system acting as a feedback control in a series of chemical methods that varied to obtain 1. tumor fraction with maximal antigenic reactivity with anti-tumor sera absorbed by normal antigens; 2. a tumor fraction with minimal antigenic reactivity with anti-tumor sera absorbed by tumor antigens. For additional control, tumor fractions with minimal reactivity with normal or anti-normal sera absorbed by tumor antigens were also prepared.

The carcinoma tissue and normal tissue, mixed frozen, are mixed separately with water at a temperature of about 0 ° C and homogenized without generating too much frictional heat that would cause inactivation of the relevant CAPA. The temperature of the suspension must 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, for example acetone or - 5 - 60567 ethyl ether, at a temperature below about 0 ° C, the solvent treatment of which is to remove e.g. lipids and make antigenic groups more susceptible to treatment. The solids are separated from the mixture, suitably by centrifugation, and the aqueous and solvent layers are discarded. The temperature must not rise above approx. + 4 ° C.

The recovered solids are lyophilized and the lyophilized tissue powder is ground, e.g., in a stainless steel ball mill, at a low temperature, preferably below about 0 ° C and suitably below about -70 ° C. Grinding gives a fine, greyish-brown powder.

If desired, the resulting powder can be extracted with a saline solution, such as physiological saline, at neutral pH, at a low temperature, suitably about 0 ° C, and centrifuged, after which the liquid on top of the precipitate is discarded. The slurry thus obtained can be resuspended in cold water, whereupon the precipitate is collected and lyophilized. The resulting powder can be stored for many years in closed bottles at about 4 ° C.

When the above-mentioned polypeptide-containing tumor and normal powders are extracted with water at an alkaline pH, the extracts become cloudy when NaCl is added to them at a neutral or slightly alkaline pH. This turbidity is mainly due to the presence of nucleic acid type impurities. After centrifugation of the brine-treated extracts, all activity remains in the clear liquid on top of the precipitate. This liquid can be isoelectrically precipitated at an acidic pH, suitably at a pH of about 0.8, and the resulting precipitates are dissolved in a phosphate buffered aqueous solution having a pH of about 7-0.

The solutions obtained by the procedure described above are filtered through a suitable beaded molecular sieve-type gel with which g can be fractionated from compounds with a molecular weight of 10 * 10 to 20 * 10 ^, in particular about 15 * 10 ^, whereby the filter gel is eluted with a buffer of approximately pH. neutral, and the molecular weight fraction 10-10 ^ - 20 * 10 ^, in particular about 15 * 10 ^ - is recovered. The gel or molecular sieve may be a polyacrylamide gel, a cross-linked dextran gel, an agar or agarose gel or the like, a gel or a molecular sieve such as Bio-Gel A ~ 50m (trade name for an agarose gel manufactured by Bio-Rad Laboratories, Munich, Germany-BRD; 100- 200 mesh (0.1 ^ 9-0.074 mm) separation limit 30 * 10 ^ daltons, fractionation range 10 ^ 90-106 daltons, approximate agarose content 2%) or Sepharose 2B (trade name for agarose gel manufactured by Pharmacia Fine Chemicals, Uppsala, Sweden; 60-300 microns, beads, fractionation - ~ 6 '60567 5 6 range 10 - 20-10, approximate agarose content 2%), and

the gel is equilibrated with a suitable buffer solution with a pH of about 7.0, such as Sörensen buffer (1/15 N buffer solution

based on sodium and potassium phosphates), diluted 1:10. The type of sieve or gel used for this gel filtration is not critical, and the only requirement in this regard is that it must be able to effect the fractionation of compounds in the above-mentioned molecular weight range. Another gel useful for the purpose of the invention is Bio-Gel A-150m (Bio-Rad Laboratories; 100-200 mesh, separation limit 1 ^ 0-10 ^ daltons, fractionation range 10 ^ -> 150-10 ^ daltons). For more information on gel filtration techniques, see H. Determann's "Gelchromatographie", Springer. Verlag, Berlin,

Heidelberg, New York 1967 · The eluent used for filtration is a buffer solution with a pH of about 7, and the antigen in the eluate is in the fraction containing said molecular weight range which is recovered.

The precipitated active fractions from the gel filtration described above are dissolved in a suitable buffer having a pH of about 7.0, such as Sörensen's buffer, diluted 1:10, and the resulting solutions are filtered through a column packed with a weak molecular sieve-type ion exchange gel with weak ion exchange properties, for example, with a polyacrylamide gel such as Bio-Gel P-2 (defined below), and the column is equilibrated with a similar buffer having a pH of about 7.0. The fraction with antigen activity is the fraction of the solution that leaves the column first, and this fraction is recovered.

The fraction recovered from the Bio-Gel P-2 chromatograph described above is then treated for further purification of CAPA. In principle, this treatment method consists of the following steps: a mixture of proteins or conjugated proteins is precipitated by adjusting the pH to an isoelectric point. The precipitate thus obtained is mixed with a suitable gel selected according to the protein in question. The mixture of precipitate and gel thus obtained is deposited on the top of a column containing the same gel at the same isoelectric pH, i.e. at the pH isoelectric for precipitating proteins in the previous step. The column is then eluted gradually, increasing or decreasing the pH. The fraction or fractions containing the desired protein or proteins are recovered from the eluate. Although it is not intended to bind this method to any particular theory, it is considered likely that the separation of the pxOtein mixture is different. its molecular lines can be explained as follows: - 7 - 60567

The doped proteins are exposed to ion current, which causes one protein after another to dissolve. The time required to solubilize the various proteins depends on the ionic strength, pH, temperature and flow rate. Because the partition coefficient of each dissolved protein is lower than the partition coefficient of the ion gradient, the protein moves faster in the gel than the ion gradient. As a result, the protein reprecipitates and remains immobile until it is redissolved by the approaching ionic front. This process is repeated countless times, causing only slightly different molecular species to separate from each other.

Bio-Gel P-2 is a crosslinked polyacrylamide (100-200 mesh) with a molecular weight cut-off of about 2000, but this cut-off is not critical. Other useful gels include ris-linked dextran gels, such as Sephadex G-25, which has a molecular weight cut-off of about 25,000, but even this cut-off is not critical. These gels are of the molecular sieve type and are usually used as beads. Any such molecular sieve type gel having a molecular weight cut-off of at least about 2000-3000 can be satisfactorily used. Molecular sieve type gels suitable for gel filtration in this gradient elution are described. in Gellotte's article "Fractionation of Proteins, Peptides and Amino Acids by Gel Filtration", "New Biochemical Separations", Van Nostrand, London / New York, 1964.

The normal and CAPA fractions recovered from the above chromatography are precipitated with the active substance at a pH ii of about 5, the precipitate being collected and dissolved in water having a pH of about 8.5. The pH of the clear solutions is then suitably adjusted to about 5.0 with formic acid. resulting in precipitation. Each precipitate is mixed with a gel slurry similar to the above and equilibrated with HCOOH-HCOONH 2 R a at a pH of about 5.0. The click mixture is then transferred evenly as before

to the top of the weighted gel column. I

A pH gradient, in this case a progressively decreasing pH, is used to elute the precipitates. A suitable buffer system is HCOOH-HCOONH4 or NH4HCO2H-NH2. Antigen activity is present in the eluate between the initial pH and a pH of about 3.

The eluate fraction j recovered in the above pH range contains the desired CAPA, which can be recovered by freeze-drying. To determine the molecular weight of CAPA in said eluate fraction, fraction i can be filtered on a gel column equilibrated with HCOOH-HCOONH® r11a at a pH of about 3.0, the gel being one of the above-mentioned gels. Bio-Gel P-30 (trade name for polyacrylamide gel manufactured by Bio-Rad Laboratories, Munich, Germany-BRD; 100-200 mesh, separation limit 40,000 daltons, fractional range 2500-40000 daltons) and Sephadex G-75 or G-50 ( trade name for dextran gels manufactured by Pharmacia Fine Chemicals, Uppsala, Sweden; 40-120 and 50-150 microns, fractionation ranges 3-70,000 and 1-30,000, respectively) are particularly suitable gels for this filtration. Eluted CAPA fractions with a spectrophotometric absorption peak in the wavelength range of about 229 to about 233 nm and a molecular weight; 20,000-27,000, will be recovered.

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

The isolated substance consists of a polypeptide based on a simple! peptide chain, which can be demonstrated by the formic acid concept; telyllä. CAPA reacts basic and is soluble at a pH between about; 1-3.5- The unprotected polypeptide is irreversibly denatured at neutral pH, i. When the pH exceeds about 4.5, the antigen is hygroscopic, turns yellow, inactivates, and becomes a paste when wetted.

Purified CAPA contains a fluorescent group that is activated at a wavelength of about 288 nm and emits light at a wavelength of about 350 nm. Fluorescence is proportional to the amount of CAPA, so it can be used to determine the presence of a polypeptide during its isolation step.

CAPA has a spectrophotometric absorption peak in the wavelength range of about 229-233 nm. It has a strong tendency to aggregate and cannot be filtered by the normal filtration means used in sterilization. CAPA has been shown to originate from the walls of cancer cells because antibodies developed by injecting CAPA into the body of a living animal cause complete lysis of cancer cells, i.e., lysis, in vivo when these cells are exposed to these antibodies.

As mentioned above, the polypeptide has a molecular weight between 20,000 and 27,000, more specifically about 24,000. Analyzes show that it does not contain carbohydrates, and spectrophotornetry shows that it does not contain nucleic acids. Analyzes performed with an amino acid analyzer, both with and without oxidation, confirm the molecular weight determined by filtration on Bio-Gel P-30 or an equivalent gel.

Isolated CAPA can be used for many purposes, such as the preparation of monospecific antibodies, for diagnostic purposes, immunization methods, and as an active ingredient in mixtures that can be used as immunizing agents.

Antigen activity in vitro at neutral pH is maintained by complexing CAPA with an inert protein, such as human or bovine albumin, to form a complex that is soluble in the neutral pH range while maintaining antigen activity in vitro.

A modified hemagglutination inhibition technique has been developed for cancer diagnosis. This technique can be used to determine the presence of CAPA at various stages of cancer development by examining, for example, a patient's serum, tissues, secretions, and extracts from live animals, including, for example, autopsy and surgical specimens, punctures, and smear preparations. This modified hemagglutination inhibition technique comprises a method comprising the steps of: a) preparing a series of samples of a test substance as a dilution series; b) adding a predetermined amount of anti-serum containing CAPA-specific antibodies to each sample; c) after incubation, a predetermined amount of CAPA is added to each incubated sample, using a finely divided vehicle; d) comparing the set of treated samples thus obtained with a set of control samples having decreasing, known inhibitory amounts of CAPA and predetermined amounts of antiserum containing said antibodies, and then adding to each sample a predetermined amount of CAPA using the same fine carrier ; and e) comparing said set of samples and said set of reference samples to determine the amount of CAPA in the test substance and to detect cancer and its stage of development.

The term "living animals" in this specification also means humans.

The invention is further illustrated by the following examples. Example Λ- ·

Isolation of pure antigen

Since it has been shown (Int. Arch. Allergy 56; 191-205 (1969), "Systematic Antigenic Change in Human Carcinoma Tissues by Hemagglutination Techniques", liter. B. Björklund), that most of the cancerous tissue contains antigen, cancer types of different types and from different sites were collected into a tumor pool. In this case, the pool consisted of macroscopically pure, histologically verified cancerous tissues from the following organs (necropsy): breast, bronchus, caecum, colon, duodenum, gallbladder, kidney, larynx, liver, lung, esophagus, esophagus, esophagus prostate, anus, stomach, mother. Normal tissues were collected from numerous individuals to form a pool of isoantigen, tissue antigen, and individual antigen. Autopsy cases were used and tumor diagnoses were determined histologically. In addition, immunological cross-reactivity with fresh tumor tissues and living tumor cells confirmed that the relevant tumor antigen was intact. Asepsis was maintained whenever possible. Cancer and normal tissues from autopsies were cut clean of neighboring tissue and cut into 2-5 cm cubes, which were washed in 0.9% NaCl at 0 ° C until no more blood color left. The washed cubes were frozen and preserved! at -24- ° C.

While still frozen, the tissue cubes were mixed with 10 ml at 0 ° C for each gram of frozen tissue, and homogenized in a refrigerated MSE Ato-Mix mix homogenizer (Measuring and Scientific Equipment Ltd, London, England).

with rotating blades, "at half speed," for three periods of one minute each. In this connection, it should be noted that the temperature of the suspension must be kept at about 0 ° C, preferably slightly below it.

- 11 - 60567

The cold homogenate was poured into cold 1000 ml polyethylene bottles, 4-00 ml of suspension and 400 ml of ethyl ether at -20 ° C (Aether ad narcosin pH Nord. "Incl. 0.002% diphenylamine, Skanska

Bomullskrutfabriken AB, Dösjebro, Sweden) was mixed in each flask and stirred at -2 ° C for 120 minutes in an International PR-2 centrifuge shaker (International Equipment, Needham Ltd, Mags, USA) with an engine. ,. ,. _,,. _ rotating at 300 rpm. The mixture was centrifuged at 1000 x G at -2 ° C for 5 minutes. During this step, the lipid layer does not mix with the tissue layer. The ethyl ether and lipid layer were discarded, and extracted a second time for 60 minutes, centrifuged for 5 minutes, and the ether-lipid layer removed. Finally, centrifugation was performed at 1000 x G at -2 ° C for 30 minutes, and the aqueous layer was removed even though it contained some CAPA. The remaining insolubles were recovered. The remaining ethyl ether was removed under reduced pressure for 5 minutes.

The antigen-containing material was suspended in 50 ml of HpO at 0 ° C, transferred to 500 ml infusion flasks and frozen at -70 ° C with dry ice and ethanol.

The lyophilisome was performed in a Texvac Type IV A lyophilizer (Textor, Bad Soden, Taunus, Germany-BRD) at a controlled temperature.

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 1 hour at a rate of 4-0 rpm. Grinding gave a fine, gray-brown powder. This powder was extracted with precooled ethyl ether at -2 ° C in a PR-2 shaker at 300 rpm for 60 minutes and centrifuged at 1000 x G at -2 ° C for 30 minutes. The ether layer was removed and the sediment was dried in vacuo. The resulting crude tissue powder (CTP) can be stored for years at 4- ° C in sealed bottles without significant reduction in activity.

5 g of CTP was suspended at pH neutral in 500 ml of saline containing sterile ice cubes. The suspension was homogenized in 'MSE Ato-mixes at "half speed" for three periods of one minute each. The temperature was maintained at 0 ° C. The mixture was kept at 0 ° C for 30 minutes while stirring slowly. Centrifuge at 1000 x G at 0 ° C for 4-0 minutes. The liquid on top of the precipitate was discarded.

Cod imeniti was resuspended in 500 ml of cold, distilled water and slowly stirred at 0 ° C for 30 minutes. Centrifuged at 1000 x G at 0 ° C for 40 minutes and the supernatant was discarded. The remaining sediment was suspended in 25 ml of cold distilled water and frozen at -70 ° C (dry ice and 1212 x 60567 ethanol). Lyophilization gave a washed tissue powder (WTP) that can be stored for years in sealed bottles at 4 ° C.

Aqueous extracts were prepared from CAPA and normal WTP at pH 9.5 and concentrated 10-fold by isoelectric precipitation; at pH 4.8 and dissolving the precipitate at pH 9.5 in one tenth of the volume of water used. The resulting concentrated solution was stabilized by rapidly heating to 98-100 ° C and maintaining this temperature for 5-10 minutes. This heat treatment destroys enzymes that would otherwise inactivate CAPA. The stabilized extract became cloudy when saline at pH 7.5 was added due to the precipitation of the remaining nucleic acid type impurities. To precipitate these residual impurities, 8.0 ml of CAPA and normal aqueous extracts were precipitated separately by adding 0.8 ml of 10% NaCl solution. Centrifuged at 27,000 x G at 0 ° C for 1 hour, after which the clear supernatants were precipitated isoelectrically at pH 4.8 (pH adjusted with 0.1 N HCl or 0.1 N HCl H). The precipitates were dissolved in 2-5 ml of 1 M / 150 phosphate buffer, pH 7.0.

The solutions thus obtained from tumor and normal WTP were filtered on cooled Bio-Gel A-50m (defined on page 5) columns with an internal diameter of 2.50 cm and a length of 138 cm at a flow rate of 6.3 ml / cm /B. The gel was equilibrated with M / 150 phosphate buffer, pH 7.0, 2% n-butanol, and eluted with the same buffer. 4-8 ml of extract containing a total of 50-100 mg of protein was used in each run.

Antigen activity was found in the middle fraction of the tumor extract.

A 1.0 ml sample volume of this fraction, a total of 160-180 ml, was diluted three times and adjusted to different pH values between 2.0 and 9.0.

After 5 minutes at room temperature, the samples were transferred to cuvettes and their light scattering at 500 nm was measured at a 90 ° angle. The scattering intensities were plotted as a function of pH, with a maximum observed at pH 4.8, which is the isoelectric point of the fraction. This procedure represents a new technique developed to determine the precise and necessary conditions for the purification of CAPA. Conventional technique for determining the isoelectric point cannot be used due to the low peptide content.

The remainder of the active middle fraction was precipitated at pH 4.8 with 0.1 N HCl. Centrifuged at 3800 x G at 0 ° C for 5 minutes to give a quantitative yield of activity.

To purify the CAPA edo! Icon, sedimented, active middle fractions from five Bio-Gel A-50m columns were dissolved in M / I5O phosphate buffer solution at pH. 7.0 (Sorcnsen'Ln buffer 2% - u - 60567 in n-butanol), to 5% of the initial eluate volume. This solution containing 130 mg of protein in 8.5 ml was filtered through a Bio-Gel P-2 column (as defined above) with a diameter to height ratio of 1/35 at a flow rate of 35 / cm / h. The column was equilibrated with M / 150 phosphate buffer pH 7.0 (Sorensen's buffer in 2% n-butanol). A layer volume of 10 ml was allowed for each mg of protein.

The first fraction was active and eluted with elution buffer pH 7.0, while the impurities were absorbed at the low ionic concentration used in the gel. The active substance was precipitated at pH 4.8 with 0.1 M HClOH and centrifuged at 3800 x G at 0 ° C for 5 minutes. The sediment was dissolved in 8.5 no water and the pH was adjusted to 8.5 with 0.1 M NH 4 OH. The clear solution was reprecipitated three times with 0.1 M HClOH at pH 4.8, washed in a centrifuge at the same pH and dissolved at pH 8.5. The disappearance of N-butanol was monitored by gas-liquid chromatography. The final solution was stored in ampoules, frozen and lyophilized. The product obtained was a dry, almost white powder and this powder was stored at -24 ° C in evacuated and sealed ampoules.

An identical procedure was applied to the extract from normal tissue, and no activity was observed.

To further purify the product obtained from the chromatography described above, a pH gradient elution method was developed. This method basically consists of the following steps. The protein mixture to be purified relative to its active component is precipitated by adjusting the pH to isoelectric. 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 deposited on the top of a column containing the same gel at the same isoelectric pH. The column is then eluted with a pH gradient keeping the flow rate and temperature constant. The column is thus eluted with a medium whose pH increases or decreases continuously or stepwise.

In the present case, the gradient elution was performed with 15 mg of lyophilized, unsalted CAPA powder and normal antigen powder, as previously described, using a decreasing pH. The powders were separately dissolved in traction, adjusting the pH to 8.5 with 0.1 N NH 4 OH. The clear solutions are then added to the lot to a pH of 5.0 to 0.1-11. Both precipitates were mixed with 10 ml of a Rio-Gel P-2 slurry equilibrated with an aqueous solution prepared from 0.02 M HClOH and 0.02 M HClOH to pH 5.0.

<#r .-, 4-60567 • Both mixtures were applied to the top of a 15 ml silicone-treated Bio-Gel P-2 column with an inner diameter of 16 mm and a length of 150 mm, equilibrated as described above. The gel was supported by a small amount of glass wool.

A pH gradient was used to elute the precipitates. Using a gradient mixer (Ultrograd, LKB, Sweden), the pH was maintained at about 5 for a short time to wash the precipitate, after which the pH was gradually lowered to 2.8. Finally, the pH was raised to 9 · The buffer system was 0.02-M HCOOH-HCOONH4 and 0.02-M NH2HCO2H-NH4, and elution was performed at room temperature. The flow rate was maintained at 27.1 ml per cm of column cross-section per hour.

The tumor extract eluate was analyzed by fluorescence spectrometry (activated at 238 nm, fluorescence at 550 nm). The fraction between the initial pH and about pH 3 was recovered for further use. The product was lyophilized, lyophilized, and stored in sealed, evacuated ampoules at -24 ° C.

To characterize the molecular size and ensure purity, the antigenic agent solution was filtered through Bio-Gel P-30 (defined above) equilibrated with 0.02 M HCOOH-HCOONH 2, pH 3.0 buffer. The pH-eluted CAPA was dissolved in a small amount of the same buffer.

o

The flow rate was 3.25 ml / cm / h and the collected fractions were 1.24 ml. The active fractions had a spectrophotometric absorption maximum at 229-233 nm and fluorescence at 350 nm, activated at 283 others.

In a typical run, 10.4 mg of CAPA dissolved in 3.0 ml of the above buffer was recovered from the siliconized Bio-Gel • P-30 column (inner diameter 2.54 cm, length 50 cm) activity perfectly. The elution volume ensures a molecular weight range of 22000-24000. These molecular weight numbers were obtained by comparing the elution volumes to the elution volumes of compounds of known molecular weight, such as ribonuclease and insulin. After freeze-drying, a white, hygroscopic powder is obtained which is kept in a cold, oxygen-free and dark state. No carbohydrates or nucleic acids were found during product analysis. Analyzes with an amino acid analyzer gave results consistent with the molecular weights determined by gel filtration. Amino acid analyzes gave the following percentages with an accuracy of ± 5% · The calculated molecular weight was 232ΟΟ ± 25ΟΟ (standard deviation).

_ 15 _ 60567

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

Merioin 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 16.2-17.8% · The determination of total nitrogen according to Dumas refers to a value of 17.0 + 0.5% · • The peracid acid treatment shows that this polypeptide is based on a single peptide chain. In addition, this polypeptide, unless protected by albumin complexation, is irreversibly denatured at pH above about 5-

In exactly the same manner as described above, the polypeptide is prepared from human placental tissue.

Example 2: Preparation of CAPA albumin comolex ..... - - * - —---------- s

CAPA prepared as described above is dissolved in formic acid (e.g., 0.05-M; pH should be between 2-3). A clear solution is obtained. To this solution is added albumin powder or albumin solution at the same pH as the above-mentioned formic acid solution (200 parts by weight of albumin per one part by weight of CAPA) to obtain a clear CAPA albumin solution. The pH of the solution is then slowly raised by the addition of a base (e.g., aqueous sodium hydroxide or ammonia) until the pH rises to about 7.5. This gives a clear solution containing CAPA and albumin in the rnuod part of the complex.

This solution containing 12 μg CAPA / ml can be used directly in the diagnostic method described below, or the complex can be isolated as a white powder by freeze-drying. The powder is stable in the cold (f4 ° C) for a long time.

60567 - 16 -

Experiments have shown that CAPA activity is maximally utilized when the weight ratio of albumin to CAPAr is equal to or greater than about 200: 1.

Example 3: I

- i

Preparation of tanned and labeled red blood cells j

Fresh sheep blood (1 volume) is added to a sterile Alsever solution (1.2 volumes) (Alsever solution is prepared from 250 g glucose, 80 g sodium citrate hydrate, <4-2 g NaCl and water to 10 liters). and the pH is adjusted to 6.1 with 10% citric acid monohydrate). The mixture is centrifuged and the red blood cells are resuspended once in Alsever's solution and twice in a pH 6.8 buffer solution. Finally, a suspension of red blood cells is prepared in a buffer having a pH of 6.8 so that the red cell concentration becomes 10 cells / ml.

One volume of the red blood cell suspension prepared as above is added with stirring to one volume of a buffer solution having a pH of 6.8 and containing about 18 ug of tannic acid / ml. The red blood cells tanned in this way are centrifuged and resuspended twice in a buffer of pH 7.5. Finally, the red blood cells are suspended in a buffer of pH 7.5 to a cell concentration of 10 cells / ml.

The CAPA complex prepared according to the example described above is dissolved in a buffer solution having a pH of 7.5 so that the CAPA concentration becomes 3 pg / ml (calculated as pure CAPA). One volume of the tanned red blood cell suspension prepared according to the previous paragraph is added dropwise at 0 ° C to one volume of the CAPA complex solution over a period of 10 minutes. The labeled cells are centrifuged and resuspended in a pH 7.5 buffer solution containing a stabilizing amount (about 1.2 v / v) of i.nert human g serum to give a suspension containing 1.6 x 10 labeled cells per ml. These labeled cells are used as explained below in the diagnostic method.

Γ VV a 17 - 60567

Example H:

Diagnostic procedure a) Simple blind test

Blood samples were obtained from Eskilstuna Central Hospital, Sweden, 4-2 of which were from patients in the infection clinic; one from a surgical clinic; two radiotherapy; 10 samples of newborn mothers; 10 umbilical cord blood and 10 women who were pregnant in the 1-trimester and 6 samples from women who were pregnant in the 2-trimester. In addition, 20 blood samples were obtained from a 10-year-old child from Slottsskolan and 10 samples from older, healthy individuals from Dr. Lundström's private clinic in Eskilstuna, Sweden. Samples were obtained from 44 patients at the Surgical and Internal Medicine Clinic of Ersta Hospital, Stockholm, Sweden, from 45 patients at Akeshov Hospital and from 72 patients admitted to the hospital for possible surgery from the surgical clinic of Karolinska Sjukhuset Hospital, Stockholm, Sweden. Karolinska Sjukhuset Hospital's Blood Donation Center, Stockholm, Sweden, provided 118 samples from donors aged 20-67 years, mean age 57 »2 years (SD ± 11.5 years), and sera from 29 patients in treatment were obtained from the hospitals' central clinical laboratory. In the general wards of Radiumhemmet and in 12 patients from its gynecological wards.

The total number of blood samples was 451. The 111 cases from Eskilstuna were examined clinically and a medical report was prepared, but not from apparently healthy individuals. Detailed medical history studies were carried out on 101 cases in Stockholm. j

In other cases, there were non-malignant clinical diagnoses and no suspicion of malignancies.

Venous blood was transferred to Wasserman tubes without additive and. was sent to the laboratory, in some cases first removing the clot and blood cells by centrifugation.

The blood analysis was based on the detection of CAPA by a modified hemagglutination inhibition technique (micromethod). In principle, the following procedure was used; ; 25 μΐ of patient serum absorbed into red sheep 'blood cells was titrated in 8 steps at 2-fold dilution, (disposable as a microtiter plate) ..! T.inbro IG-MRC-96 dilution plate 3 a / a, after which 25 μl of specific antiserum at x-time dilution (about 1: 2000) was added to each! Q ..... j pit. After incubation at 22 ° C for 10 minutes, 50 μl of a suspension containing approximately 6 to 8 million red sheep blood cells labeled with CAPAr1-I-labeled human albumin bound to human albumin was added to each well as described above. has been described (about 2-4 μg / 10 μl of blood cells) Other fine carriers such as latex, bentonite and collodium can be used instead of red blood cells. The reaction result was determined by means of an inclined mirror and a standard series after incubation for 4 to 24 hours at 1-2 ° C. For control, the following were used: 1. CAPA-labeled blood cells plus antiserum; 2. CAPA-labeled blood cells without antiserum ·; · 3 · Patient serum plus human albumin-labeled blood cells; 4. Patient serum and untreated blood cells; 5- CAPA-labeled blood cells and antibodies in a series in which inhibition is achieved by gradually decreasing, known amounts of CAPA.

Diluents used to stabilize blood cells contained collected (i.e., pooled from multiple individuals), inactivated, neutral human serum. j CAPA reagent was prepared from harvested human cancer tissue and! was purified as described above using gel filtration, pH gradient elution, filtration through Bio-Gel P-30 and complexation. formation with albumin.

Horse anti-HeLa from November 19, 1962, absorbed into normal tissue, and pooled human serum were used as antisera. Immunization was performed with washed HeLa cell pellet, which cells were cultured in flat glass flasks in Eagle's medium containing 20% inactivated human serum. The cells were harvested with EDTA, centrifuged and washed three times with water.

In the presence of CAPA in patient serum, equine antibodies were neutralized, resulting in the absence of agglutination when polypeptide-labeled blood cells were added. Evaluation was performed on an ascending scale of 0-6, where 0 indicated no inhibition and 6 indicated maximal inhibition of agglutination. The ratio of the scale values to the concentration of CAPA per ml of serum is shown in Table 1, obtained by calibrating dilutions of predetermined amounts of CAPA in neutral serum.

i t {~ 19 '60567

table 1

Approximate calibration of scale and amount of CAPA Scale value CAPA, pg / ml 0 0.05 1 0.04 - 0.08 2 0.09 - 0.12 3 - 0.13 - 0.24 4 0.25 - 0.49 Δ 0.50 - 0.99 6 '> 1.0

The reading was performed without knowing the patients ’health status. The study material is grouped as shown in Table 2 below.

Table 2 CAPA concentration in the serum of 431 individuals from different groups according to a numerical scale from 0 to 6, as well as mean scale values (M) and standard deviations (SD). "Radically" in this context means that an attempt has been made to completely remove the cancerous tissue.

Groups Numeric scale Read-

_0 1-? _3_4 8 Number 6 · Μ_- SD

1. -Primary cancer with metastases 5412 12 3.00 - 1.13 2. Prevalent cancer 4214821 22 2.91 - 1.82 3. Radically untreated cancer 6 ίο 15 8 39 1.64 - 0.99 4. Untreated primary cancer 3142 10 1.50 - 1.18 5- Radically removed cancer Ί9 1 1 21 0.14 - 0.19 6. 'Malignant disease other than cancer 731 11 0.45 - 0.68 7. Non-malignant disease 118 3 3 7 1,152 0.26 - 0.22 8. Healthy adults (M - 71 years) 82 10 0.20 ± 0.42 9. Blood donors (M = 39 years.) Ί16 2,118 0.02 - 0.13 - 20 - 60567

Table 2 (cont.)

Groups: Numeric scale Read-!

0 1 2 3 4'56 maara M ± SD

10. Individuals aged 10 years 19 1 20 0.10 - 0.44 11. Newborns (umbilical cord blood) 111421 10 2.80 - 1.48 12. Pregnant women.

women 12 2 2 16 0.38 - 0.72 13 · Newborns 811 10 0.40 - 0.97

The results show that the highest scale values were obtained from the groups “primary cancer with metastases”, 1 (local) and “advanced cancer” and from the group “neonates” (umbilical cord blood).

Low scale values have been obtained from blood donors. The intermediate station has a group of "untreated primary cancer" and "radically untreated cancer." Values close to those of blood donors are obtained from the group "radically removed cancer".

CAPA means from all groups have been compared in pairs and are shown in Table 3 below.

Table 3

Statistical significance of differences in serum CAPA levels in nine experimental groups a) _ _____

Groups Significance of differences in scale values between groups compared to ^ groups __M - SD 23456 7 89 · 1. Primary cancer with metastases 3.00 - 1.13 0 xx xx kkm xxx xxx xxx xxx 2. Prevalent cancer 2.91 - 1.82 xx xx 3ίχκ xxx xxx xxx xxx | 3. Radically care! untreated cancer 1.64 - 0.99 0 xxx xx xxx xxx xxx |

4. Untreated I

primary cancer 1.50 - 1.18 xxx x xxx xx xxx 5- Radically removed cancer 0.14 - 0.19 0000 6. Malignant disease other than cancer 0.45-0.68 000 7. Non-speaking \ disease 0, 26-0.22 0 xxx; 8. Older healthy individuals 0,20 to 0,42 0 j 9- Blood donors 0,02 to 0,13 to 21 to 60567 (a) Statistical significance of differences was approximately normal | by means of a distributed variable Z defined as follows; j - · Μ2 Z = --- \ / sD * SD2 V + ^ 2 ~ b) if Z> 1.96, the meaning of the difference is x, which means 0.01 <P <0.05 "Z> 2.6 , the significance of the difference is xx, which means 0.001 <P <0.01 "Z> 5.5, the significance of the difference is xxx, P <0.001 c) This group comprises 11 positive tests, 8 of which concern suspected but not detected cancer.

The table above shows that, the groups "primary cancer with metastases" and "advanced cancer" have significantly higher mean scales compared to all other groups except neonates (see also Table 2).

In the "radical untreated cancer" and "untreated primary cancer" groups, the mean scale values are significantly higher than in all other groups except the two mentioned above and in the "non-cancerous malignancy" group, for which the difference is significant (0.01 <P <0.05) ·

There are no significant differences between groups 5-9, except between groups 7 and 9. This difference may be explained by the fact that 'genuine, latent cancers may occur among cases with a scale value between 1 and 4.

A positive indication in umbilical cord blood deserves some attention. To determine whether CAPA originated from the placenta, pieces of this tissue were extracted. Aliquots of 500 mg of frozen placental tissue per ml of buffered saline at pH 7,5 | homogenized at 0 ° C and centrifuged at 1000 x G for 50 minutes at 0 ° C in conically expanding tubes (according to Markham). j

The solutions on top of the precipitate were tested for CAPA by the hemagglutination technique described above. To rule out the possibility that the inhibition reactions obtained were due to non-specific inactivation of the added CAPA in the blood cell, said blood cells were shaken after each measurement and a standard amount of CAPA-specific antibodies was added. In no case was antigen inactivation observed. An extract of antigen powder prepared from human a * - 22 - 60567 cancer tissues from different sources and locations was used as a reference substance. The results show that 26 placeholders from an equal number of individuals all invariably contained significant amounts of CAPA. The mean (geometric) of these 26 placentas was 297 to 8 micrograms per gram of wet tissue (0.03%).

Because, as shown in the above study, the CAPA used in this invention is synthesized in the placenta, and because the genes contained in the placenta must also be contained in an individual developed from the same ovum, there is a tendency or ability to produce CAPA as a normal phenomenon. Normally, this tendency is suppressed because normal cells do not contain measurable amounts of CAPA. In cancer cells, on the other hand, there has been a tendency to activate, characterized by a significant presence of CAPAr within and around the cells. The antigenic and, in some respects, functional similarity of placental trophoblast cells and cancer cells, and their difference compared to other cells in an individual, seem to agree with the notion that cancer is an expression of a change in the control of normally occurring tendencies.

Statistical analysis of the experimental results shows that there is a fairly significant correlation between the detected cancer and the increased CAPA concentration (P <0.001). The same is true for the relationship between the magnitude of the tumor mass in question and the increased CAPA concentration in the same age groups (P <0.001). Increased CAPA levels have been found in a number of different cancer sites. Irrespective of their type and location, the cancerous tumors have: been shown to contain CAPA, which appears to be,. j that said CAPA is common to all known cancers, i.e. malignant tumors of epithelial origin. ‘(B) Double-blind study;

To further confirm the above conclusions! a double-blind study was performed, under very precise conditions. Coded blood samples were obtained from Eskilstuna j

From different departments of Central Hospital, Sweden. In this case, serum CAPA levels of patients were quantified. i This double-blind study, as well as the above explained-! The results of this simple blind study are summarized in the following Tables 1-8. The tables show the following sequence

Table 4 Total number of individuals studied and number of individuals diagnosed with cancer;

Table 6 shows the number of individuals in the different groups as well as their mean age trap as well as its deviation; . 23 - 60567

Table 6 Presence of CAPA in the sera of all patients studied;

Table 7 Percentage of subjects with CAPA levels - 0.15 ug / ml serum, excluding groups 9, 10 and 12 of Table 5;

Table 8 Presence of CAPAr by tumor location in groups 1-3 of Table 5 "Site nr" refers to "Cancer Incidence in Sweden 1968".

Table 4-

Number of individuals studied

Study Number of individuals The number of individuals diagnosed with cancer

Simple blind 4-31 '104-

Double blind 503 4-9 934- 155 "2U_ 60567

Table 5

The number of individuals in different groups as well as the mean age and its deviation

RvhrlMf Simple Two nkur or nen! y blind research blind research <aus

Number- Medium + Deviation- Number- Medium + Deviation number age v. Ma amount il <ä v. Cream! 1. Cancer with metastases 3 4 72 + 9 14 66 + 10; 2. Untreated cancer! without symptoms of metastasis and rad i kaaa

Incest untreated j cancer 2 + 9 67 + 11 1 / + 66 + 12 j

3. Radically. J

removed cancer 21 66 + 15 21 64 + 12 4. Malignant disease other than cancer '11 70 + 7 5 5β + 1β 5. Non-malignant | assisted illness 132 61 + 22 1β6 46 + 21 6. Healthy adults 10 71 + 5 46 36 + 14 7. Blood donors 11Ö 30 + 11 - - Ö. 10-year-olds and! the younger! individuals 20 10 + 0 62 2 + 1.5 9. Umbilical cord blood 10 0 + 0 20 0 + 0! 10. Newborns- | thank you 10 21 + 3 20 25 + 4; 11. Pregnant women 16 26 + 4 pleasure 26 + 4.5 12. Cancer in situ - - 5 27 + 6 ί

Total 431 503 i j i t • 25 '60567

Table 6 CAPA concentration in sera from 934 individuals

Groups CAPA CAPA Total <0.00 pg / ml —0.09 pg / ml Number (%) ____ Number (%) Number (%) _ 1. Cancer with metastasis 9 (19) 39 (Oil) 46 (100) 2. Untreated cancer without metastatic symptoms and radically untreated cancer 2Ö (44) 35 (56) 63 (100) 3. Radically removed cancer 36 (66) 6 (14) 42 (100) 4. Malignant disease other than cancer 15 (94) 1 (6 ) 16 (100) - 5 · Non-malignant disease '269 (65) 49 (15) 31Ö (100) 6. Healthy adults .54 (96) 2 (4) 56 (100) 7 ·. Blood donors II6 (100) 0 (0) llö (100) Ö. Individuals 10 years of age and younger 79 (96) 3 (4) 62 (100) 9. Umbilical cord blood 21 (70) 9 (30) 30 (100) 10. Newborns 25 (63) 5 (17) 30 (100) 11. Pregnant women 123 (96) 3 (2) 126 (100) 12. Cancer in situ 4 (00) 1 (20) 5 (100)

Total 761 (64) 153 (16) 934 (100) '26 ”6 0 5 6 7

Table 7 '

The percentage of individuals with a CAPA concentration of ^ 0.15 · Groups 9, 10, and 12 in Table 5 are omitted.

_____, _____] ___

Groups% Number 1. Cancer with metastases 64 48 2. Untreated cancer without symptoms of metastasis and radically untreated cancer 21 63 3. Radically eliminated cancer 10 42 41 Malignant disease other than cancer 0 16 5. Non-malignant disease 6 31Ö 6. Healthy adults 0 56 7. Blood donors 0 11Ö 6. Individuals 10 years of age and younger 0 82 11. Pregnant women 1 126

A total of 869 i i i i i i i 27 ’60567

Table β CAPA concentration by tumor location in groups 1-3

Location Site 1.Cancer meta- 2.Untreated 3 • Radical Co-

Nox) with stasis cancer without removable metastatic cancer symptoms and radically untreated _cancer____ gQ.oe —O.09 gQ.oe ^ 0.09 £ 0.06 SO.09__

Gingivae mandib. 144__ 1____1_2

Nasopharynx_11 + 6 ___ 2_1_ · _2__

Oesophagus_150__1__2_1_1 _; __ 6

Ventricle_151_1__2_1_2__2__β

Intestinum tenui 152_1_______1__

Colon_153__2_1_7 6__16

Rectum_1J34_1 + 1 ___ 2__1__11 +.

Ves «Fellea, etc. 155 _; _ 1_1_1____3

Hepar_156 __1______1

Pancreas__157 _2_____1_2_6

Vestibul. nasi, etc .__ l60 _ 1 <1__2

Trachea, wedding, etc. 162__1 + __ 2__2__5__14!

Mammae_170 _2__7___1_3 3_2_1β_ j

Cervix uteri_229_2__2__1__6_ j

Corpus uteri_196__1_1 _1__1__1_2__7 '

Ovarii, etc, __ 175_1__1 + __ 1_1__2__9 j

Prostate e__177_2__2_3____2_ j

Test__176__1 _1 1 _3 j

Penis, etc, _179 _____ 1_1__2

Renes ___ 1Ö0_1 ____ 3 1 5 i

Ves. urinaria, etc. l6l_ 1 g 3_ ^ 1 10

Malignant melancma 190____1 1 2

Thyroid 194 1 1 1 2 5 <

Primary tumor, unknown \ origin ___ 1___ 1! ”” ”‘ 1

Total 9 39 26 35 36 6 153 x) According to "Cancer Incidence in Sweden 1966". | ! • i - 28 - 60567 Statistical analysis of the results of this double-blind study also shows a fairly significant correlation between cancer diagnosis and increased CAPA concentration (P <0.001). This double-blind study also confirms the association between the magnitude of the tumor mass in question and the increased CAPA concentration in the same age groups (P <0.001). Elevated serum CAPA levels have been found in approximately 20 different cancer types and locations. A double-blind study thus confirms the full-! these new findings and their practical applicability. '

The following is a combination of brief definitions of the polypeptide antigen and diagnostic method of the invention. I. Definition of the CAPA

HeLa cells (from a case of uterine cancer in 1952) are cultured in vitro. These cells are used as a source of antigen.

The resulting cells are injected into horses to develop a specific antibody that is used to recognize the antigen in tumors, serum, placenta, etc. The latter antigen must have and have an identical immunological response site (as HeLa antigen) although it may naturally be different in other respects. in the wild.

Definition of the diagnostic procedure

In vitro antigen from HeLa cell cultures is used to develop an antibody in horses. Following absorption of unwanted antibodies, equine serum is used to agglutinate antigen-labeled erythrocytes from tumors (or placenta or HeLa or HEp-2 or Detroit-6 cultures). This is an indicator system. If an unknown sample contains antigenic activity, it acts on the antibody and inhibits agglutination.

m '29 "60567

Figure 1.

Purification of tumor-associated antigen

Flow diagram with branching showing the general principle of the purification procedure

_ __/Normal

\ Tissues I

_ _ v Separation ^ _ procedure _iL: _s.

Immunological comparison __ 'i', _

Is there a difference between tumor and normal _ tissue? , n - ~ ....... 1 = ---- „. Is the difference tumor plus? _ -No --- <*> - On - 1 _ \ L-_

No-Is the difference optimal? -On -> t- _ _ '. * _jd. ..._

Modify and__The procedure is repeated_ | _ N ~ ~ [introduced ____

No —- <Is CAPA clean> -On --- End

The procedure is continued -: --------— --— ... -. - Τ '

MB

Claims (18)

1. Preparations for use in the diagnosis of cancers, characterized in that it contains as an active ingredient a human peptide antigen (CAPA) present with a molecular weight of 20,000 - 27,000 daltons when filtered on an equilibrated gel column with a HCOOH-HCOONH 2 at a pH of about 3.0, the gel being a polyacrylamide gel having a grain size of 100-200 mesh (0.149-0.074 mm), an exclusion limit of 40,000 daltons and a fractionation range of 2500 - 40,000 daltons, the polypeptide having a peak spectrophotometric absorption within the wavelength range of 229-233 nm, and based on a single polypeptide chain, contains aspartic acid, glutamic acid, leuoin, phenylalanine and tyrosine as essential building blocks, producing antibodies that react with antibodies that produce antibodies. and which in an isolated state is irreversibly denatured at pH above 5, wherein the polypeptide antigen is isolated by a) CAPA-containing material being homogenized in a liquid at a temperature rature 34 6 0 5 6 7 of not more than 0 ° C, and optionally treated with organic solvents, such as ethyl ether or acetone, in cooling, b) the solid constituents from the mixture of liquid homogeneous and solid constituents from step a) are separated, c ) said solid constituents are extracted with an alkaline aqueous solution; d) the extracts from step c) are filtered on a bead-shaped molecular sieve filtration gel capable of fractionating compounds having a molecular weight range of 10 * 10® - 20 * 10®, especially about 15 * 10®, the gel is eluted and the 10 * 10 - 20 * 10 fraction, especially the fraction about 15 * 10®, is recovered, e) the fraction from step d) is chromatographed on a weak molecular sieve ion exchanger to absorb the contaminants therein , and the first fraction leaving said ion exchanger is recovered; f) said first fraction is isoelectric precipitated; g) said precipitate is mixed with a molecular sieve type gel and the mixture of precipitate and gel is pH gradient eluted on a gel column, h) by lowering the pH down to pH 3, the fraction containing CAPA is collected and which fraction is optionally gel-filtered on a column and the antigen fraction recovered having a spectrophotometric peak absorption path length of 229-233 nm and a molecular weight of 20,000. - 27,000, together with a polypeptide denaturation preventing amount of an inert protein, preferably albumin, the weight ratio of inert protein to CAPA being at least 200: 1. 2. Preparation according to claim 1, characterized in that the polypeptide contains the following amino acids with the following mole percentages: 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 35 6 0 5 6 7 Serine 7.74 Treonin 5.92 Tyrosine 3.21 Valine 6.36 Preparation according to claim 1, characterized in that the polypeptide contains a fluorescent group emitting fluorescent light at 350 nm upon activation at a wavelength of 288 nm. Process for the preparation of a preparation according to claim 1, containing polypeptide antigen (CAPA) present in human cancer tissue, characterized in that a) CAPA-containing material is homogenized in a liquid at a temperature not exceeding 0 ° C, and optionally treated with (b) the solid constituents from the mixture of liquid homogeneous and solid constituents from step a) are separated; c) said solid constituents are extracted with an alkaline aqueous solution; d) the extracts from step c) is filtered on a bead-shaped molecular sieve type gel capable of fractionating compounds having a 6 6 6 molecular weight range of 10 * 10 - 20 * 10, especially about 15 * 10, the gel eluted and 10 * 10 - 20 * 10 the fraction, especially the fraction of about 15 * 10 **, is recovered; f) said first fraction is isoelectric precipitated; g) said precipitate is mixed down a molecular sieve gel and the mixture of precipitate and gel is pH gradient eluted on a gel column, the fraction containing CAPA and which fraction is optionally gel-filtered on a column and the antigen fraction is recovered which has a spectrophotometric peak absorption wavelength of 229-233 nm and a molecular weight of 20,000 - 27,000, and then CAPA is mixed with a polypeptide denaturation prevented amount of an inert protein, preferably albumin, so that the weight ratio protein to CAPA is at least 200: 1. 5. A process according to claim 4, characterized in that the homogenization a) is carried out in water. 6. A process according to claim 4, characterized in that the solid components obtained from step b) are lyophilized and then milled in a cooling ball mill in a refrigerator. 36 6 0 5 6 7 Process according to claim 6, characterized in that the milling takes place at a temperature of -70 ° C. 8. A method according to claim 4, characterized in that during step d) the filtration is carried out on a potassium column containing polyacrylic gel, crosslinked dextran gel, agar or agarose gel and equilibrated with a phosphate buffer at pH 7. 9. A method according to claim 4, characterized in that during step e) the chromatography is carried out at neutral pH, e.g. on a column equilibrated with phosphate buffer at pH 7. Process according to claim 9, characterized in that the chromatography is carried out on a polyacrylamide gel column. Process according to claim 4, characterized in that the isoelectric precipitation f) occurs at pH 5. 12. A process according to claim 4, characterized in that during step g) a polyacrylamide gel, cross-linked dextrangel, agar or agarose gel is used. Method according to claim 12, characterized in that the gel has a molecular weight exclusion limit of at least 2000 and is equilibrated with HCOOH-HCOONH 2 at pH 5, and the pH gradient elution takes place in a column containing an identical gel equilibrated in the same manner. . 14. A process according to claim 4, characterized in that water extract from step c) is rapidly heated to a temperature in the range 95-100 ° C to destroy enzymes. Process according to claim 14, characterized in that the water extract is heated to a temperature in the range 98-100 ° C and poured at said temperature for 5-10 minutes. Process according to claim 15, characterized in that the heated aqueous extract is treated with a sodium chloride aqueous solution for precipitation of contaminants. 17. A process according to claim 4, characterized in that in acidic solution, CAPA and albumin are mixed and the pH of the solution is raised to effect the interaction between the components. 18. A process according to claim 17, characterized in that the preparation is lyophilized.