JPS5817168B2 - Gankanlen polypeptide kogen - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention exhibits monospecificity and exists in a wide variety of human cancers in different locations.

Concerning the problem of isolating cancer-associated antigens.

Many attempts have been made to demonstrate the presence of tumor-reactive antibodies in serum obtained from animals treated with human cancer tumor specimens.

If such a demonstration were to become consistently reproducible, it would indicate the presence in human cancer tissue of important antigens not present in normal tissue, and thus the process of neoplastic production. will lead to a better understanding of the nature of

Human cancer tissues were studied with the aim of discovering the existence of monospecific cancer-associated antigens with the aim of better understanding cancer diseases and, above all, with the aim of discovering methods for reproducible isolation and purification of such antigens.

The presence of antigens associated with adenocarcinomas of the colon and digestive system has been shown by Gold et al. (J, Expt.

Med-7121 (1965), 439-462, J.
Expt.

Med, J 122 (1965), 467-487
reference).

Reference is also made in these reports to previous studies by B. J. and B. B., who demonstrated the presence of human cancer-associated antigens common to the majority of all known malignant tumors of epithelial origin (Internat, Arch. , Al
lergy and Appl, Immunoll, (196
6) See 8.179 and (1958) Rai, 241)
.

US Pat. No. 3,663,684 to Freeman et al. describes carcinoembryonic antigens that exhibit so-called "CEA activity."

However, this antigen is completely different from the antigen of the present invention, as will be shown in detail later in the specification.

Isolation of a practical and commercially useful antigen as such as well as its association with cancerous diseases of this type has not been achieved so far.

Thus, it has not been possible to isolate and characterize human cancer-associated antigens by a practical and reproducible method.

And it is also impossible to determine or even demonstrate the presence of antigens in the blood of a person suffering from latent or overt cancer disease by diagnostic means suitable for large-scale testing. Ta.

In order to fully utilize the presence of antibodies specific for tumor-associated antigens in animal antisera as diagnostic agents, test methods must be developed to demonstrate the presence of tumor antigens in the patient's blood. .

The methods proposed so far have proven insufficient when attempting to make certain diagnoses regarding the presence of cancerous diseases.

The main objective of the present invention is to provide a method for isolating and purifying cancer-associated antigens starting from human tumor tissue or other tissues containing such antigens.

Another object of the invention is to characterize such antigens after their isolation.

Yet another object of the present invention is to provide diagnostic tests based on the use or presence of such human cancer-associated antigens.

Yet another object of the present invention is to provide a method for generating antibodies specific to the cancer-associated polypeptide antigen.

Yet another object of the present invention is to provide a composition useful as an immunizing agent.

Yet another object of the invention is to provide compositions containing an antigen and a stabilizing amount of albumen in admixture therewith, and methods for making such compositions.

Yet another object of the invention is to provide a diagnostic composition containing particulate material labeled with an albumen stabilized antigen.

In the disclosure of this specification including the claims, [
The expression "cancer-associated polypeptide antigen" is abbreviated as CAPA.

Accordingly, the present invention provides, inter alia: (a) purification, isolation, characterization of CAPA and confirmation of its identification and specificity; (b) such a method for diagnosing the presence of cancer by demonstrating the presence of circulating ff1cAPA; The present invention provides a practical method for the use of CAPAs and (C) the generation of antibodies monospecific for such CAPAs.

Other objects and characteristic aspects of the invention will become apparent from the following description of the invention in general terms and by specific examples.

Substances with CAPA activity are isolated and purified by the method of the invention by homogenizing malignant tissue from autopsies, where cancerous tissue of different types and locations are brought together to form a tumor pool. Ru.

Alternative starting materials are tissue from the human placenta or cultures of malignant cells in vivo (details of such cultures can be found in B. J., Nat., Ca.
ncer In5t.

Seal, 533-545 (1961), rAntigen
City of PooledHuman Mal i
gnant and NormalTissues b
y Cytoimmunological Technique
(See ``Distribution of Tumor Antigens'').

While in the frozen state, the cancerous and normal tissues are mixed with water having a temperature of about 0° C. and homogenized in a manner that does not generate excessive frictional heat that would cause inactivation of the CAPA involved. .

The temperature of this suspension should not rise above about 0°C.

The resulting cold mixture of liquid homogenate and solid is mixed at a temperature below about 0°C with an organic solvent such as acetone or ethyl acetate capable of dissolving lipids such as neutral fats, this solvent treatment removing inter alia lipoidal material. The purpose is to increase the exposure of antigenic groups.

The solids are separated from the mixture, suitably by centrifugation, and the water and solvent layers are discarded.

This temperature should not exceed about 4°C.

The recovered solids are lyophilized and the lyophilized tissue powder is heated, for example, in a stainless steel ball mill at a low temperature, preferably below about 0°C and suitably about -70°C.
Grind at temperatures below ℃.

As a result of this grinding, a fine gray-brown powder is produced.

If desired, the resulting powder is extracted with a salt solution, such as saline, at neutral pH and at low temperature, suitably at about 0.degree. C., and the supernatant after centrifugation is discarded.

The resulting precipitate can be suspended in cold water, collected and lyophilized.

The powder obtained can be stored year after year in a closed bottle at about 4°C.

When the polypeptide-containing tumor and normal tissue powders are extracted with H2O at alkaline pH, the extract becomes cloudy upon addition of NaCt in neutral or acid-alkaline pl.

This turbidity is mainly due to the presence of impurities of nucleic acid nature.

After centrifugation of the saline-treated extract, all activity remains in the clear supernatant.

The supernatant is isoelectrically precipitated at an acidic pH suitably 4.8 and the resulting precipitate is dissolved in an aqueous phosphate buffer solution at a pH of about 7.0.

The solution obtained from the above operation was mixed with 10XIO' to 20X1
The filtration gel is eluted with a buffer of approximately neutral pH, and the filtration gel is eluted with a buffer of approximately neutral pH, and the filtration gel is eluted with a buffer of approximately neutral pH and ×106~20X106
Specifically, a MW fraction of approximately 15×10 6 is collected.

These gels or sieves include polyacrylamide gels, cross-linked dextran gels, agar and agarose gels, and equivalent gels or molecular sieves. , Removal limit (Dalton) 50X106, Fractionation range (Dalton) 10
5-50X10', approximate agarose content 2%) or Sepharose 2B (trade name of agarose gel from Pharmacia Fine Chemicals, Sweden, 60-300
micron beads, fractionation range 105-20x106, approximate agarose content 2%).

This gel is then diluted to 10% in a suitable buffer solution with a pH of about 7.0, such as Seerensen's buffer (1/1).
Equilibrated on a 15M sodium and potassium phosphate basis (buffer solution).

The type of sieve or gel used for this gel filtration is not critical and the only requirement in this regard is that it is capable of giving a fraction of compounds within the molecular weight range identified above.

Other gels useful for the purposes of the present invention are Biogel A-150
m (manufactured by Biorad Laboratories, 100-200 mesh, exclusion limit (Dalton) 150x106, fractionation range (Dalton) 106->150x106).

For further details regarding gel fractionation techniques, see "Gel Chromatography" by H. Butterman (Springer Verlag, 1967 edition).

The eluate used for this filtration is a buffer solution with a pH of about 7, and in this eluate the antigenic activity is found in the fraction containing the MW range.

Collect the fractions. The precipitated active fraction from the gel filtration is added to a suitable buffer, e.g.
:10 diluted in Soerensen's buffer and the resulting solution is dissolved in a weak ion exchanger molecular sieve type gel with weak ion exchange properties such as a polyacrylamide gel such as Biogel P-2 (defined below). ) is filtered through a column packed with

The column was equilibrated with a similar buffer at a pH of approximately 7.0.

The fraction exhibiting antigenic activity is found to be the first fraction of the solution coming out of this column, and this fraction is collected.

The fractions collected from the Biogel P-2 chromatography are subjected to a special procedure developed for further purification of CAPA.

In principle, this operation involves the following steps.

That is, a protein or a mixture of complex proteins is precipitated by adjusting the isoelectric point of pH.

The precipitate thus obtained is mixed with an appropriate gel selected considering the protein mixture present.

The resulting mixture of precipitate and gel was placed on top of the same gel-containing column with the same isoelectric point pH, i.e. the pH of the protein isoelectric point from which the protein was to be precipitated in the previous step.The column was then subjected to pH removal. Elute by increasing or decreasing gradually.

The fraction or fractions to be collected in the eluate are those containing the desired protein or proteins.

Although the method of the present invention is not bound by any particular theory, it is believed that the separation of molecular species in protein mixtures can be explained as follows.

The precipitated proteins are exposed to a stream of ions that carry the proteins one after the other in solution.

The time required to solubilize various proteins depends on ionic strength and pH.
1 depends on temperature and flow rate.

Since the dispersion constant of each dissolved protein is lower than that of the ionic gradient, the proteins migrate into the gel more quickly than the gradient.

As a result, the protein reprecipitates and remains static until redissolved by the approaching ion front.
in a state.

This process repeats itself ad infinitum, thus resulting in the separation of molecular species that differ only slightly from each other.

Biogel P-2 is a cross-linked polyacrylamide (100-200 mesh) with a molecular weight removal limit of about 2000, but this removal limit is not critical.

Other useful gels are cross-linked dextran gels such as Sephadex G-25, which contain approximately 25,000
It has a molecular weight removal limit of 0.

However, this removal limit is not critical.

These gels are of the molecular sieve type and are usually used in bead form.

Any such sieve type gel having a molecular weight removal limit of at least about 2000-3000 can be used satisfactorily.

For a review of molecular sieve type gels suitable for gel filtration combined with gradient elution according to the invention, see Gelot,
See "Fractionation of Proteins, Peptides and Amino Acids by Gel Filtration" in "New Biochemical Separations" (Pan Norstrand, 1964 edition).

Normal and CA recovered from the chromatography
The PA fraction is subjected to active substance precipitation at a pH of approximately 5.

This precipitate is collected and dissolved in water having a pH of approximately 8.5.

The pH of this clear solution is then brought to an acidic pH, suitably about 5.0, with formic acid.

This treatment produces a precipitate.

Each precipitate was treated with HCO at a pH of approximately 5.0 as described above.
Mix with a slurry of a similar gel equilibrated with OH-HCOONH4.

Each of these mixtures is equilibrated as described above and placed on top of the gel column.

For elution of this precipitate, a pH gradient with progressively decreasing pH was used in this case.

Suitable buffer systems are HCOOH-HCOONH4 and NH4HCO3-NH3.

Antigenic activity is found in the effluent solution within a pH range of about 3 from the initial l pH.

The effluent fraction collected within the pH range identified above contains the desired CAPA, which can be recovered by lyophilization.

In order to determine the molecular weight of CAPA in the effluent fraction, this fraction was treated with HCOOH-HCOON at a pH of about 3.0.
It can be filtered on a gel column equilibrated with H4.

The gel may be selected from those listed above, including Biogel P-30 (trade name for polyacrylamide gel from Bio-Rad Laboratories, 100-200 mesh);
Removal limit (Daltons) 40,000, Fractionation range (Daltons) 2,500-40,000) and Sephadex G-75 or G-50 (Pharmacia Fine Chemicals trade name for dextran gel, 40-12
0 and 50-150 microns, fractionation range is 3
~70,000 and 1-30,000) are particularly suitable gels for performing this filtration.

An eluted fraction of CAPA having a spectral absorption peak wavelength in the range of about 229 to about 233 nm and a molecular weight of about 20,000 to about 27,000 is collected.

CAPA can be recovered from this active fraction by lyophilization after filtration and can be stored for long periods of time in a cool, dark place under vacuum.

The isolated material is composed of polypeptides based on a single peptide chain, as shown by treatment with performic acid.

This CAPA exhibits a basic reaction and about 1 to 3.5
It is soluble at a pH within the range of .

This unprotected polypeptide denatures irreversibly at neutral pH, ie, at a pH above about 4.5.

This antigen is hygroscopic and when it absorbs moisture it turns yellow and becomes inactivated and becomes sticky.

Purified CAPA contains a fluorescent group that is activated at a wavelength of about 288 nm, during which it emits light at a wavelength of about 350 nm.

This fluorescence is proportional to the amount of CAPAO and can be used to determine the presence of the polypeptide during its isolation procedure.

This CAPA exhibits peak spectroscopic absorption at wavelengths within the range of about 229 to about 233 nm.

Moreover, it shows a great tendency to agglomerate and cannot be filtered on the usual filtration media used for sterilization.

It was confirmed that CAPA is derived from cancer cell walls.

The reason is that the antibodies produced by injecting CAPA into a living animal body will not completely lyse or degrade cancer cells in vivo if they are brought under the influence of such antibodies. This is because it causes

As mentioned above, this polypeptide has a molecular weight of 20,000 to 27,
More specifically, it has a molecular weight within the OOO range of about 24,000.

Analysis shows that it contains no carbohydrates, and spectrophotometric measurements show that CAPA does not contain any nucleic acids.

Analysis performed on an amino acid analyzer with or without oxidation shows agreement with the molecular weight estimated by gel filtration on Biodel P-30 or its derivatives.

Isolated CAPA can be used as an active ingredient in compositions useful as immunological agents in many uses, such as monospecific antibody production, diagnostic purposes and in immunological methods.

A novel method has been developed for producing antibodies that are monospecific for cancer-associated polypeptide antigens.

CAPA, which is not protected as described above, denatures irreversibly at neutral pH.

This means that when an acidic solution of CAPA is injected into the body of a living animal, when CAPA comes into contact with neutral body fluid, its denaturation occurs instantaneously.

Oil emulsions are manufactured to prevent such denaturation, but
In that case, an aqueous solution of CAPA with a pH in the range of about 2 to about 3 constitutes an encapsulated phase surrounded by an oil phase.

When such oil emulsions are injected into a living animal, the acidic aqueous solution of CAPA is protected by the surrounding oil phase even when it comes into contact with neutral body fluids.

In this way CAPA retains its in vivo activity and can be transported to antibody producing cells.

A method of retaining in vitro antigenic activity at neutral pH consists in complexing CAPA with an inert protein such as human or bovine albumin.

By utilizing such methods, complexes are formed that are soluble at neutral pH and retain their in vitro antigenic activity.

For cancer diagnosis, a modified hemagglutination inhibition technique has been developed.

This technique is used to diagnose various types of cancer by examining the material of interest, such as patient serum, tissues, secretions and extracts from the living animal body, including, for example, biopsy material, surgical specimens, punctures and smears. It makes it possible to measure the presence of CAPA in advanced stages.

This new and modified hemagglutination inhibition technique involves the following steps: a) producing a series of samples of material for testing by serial dilution; and b) containing each of said samples an antibody specific for CAPA. C) Adding a predetermined amount of antiserum to each of said culture samples after incubation, d) Adding a predetermined amount of CAPA supported on a microparticulate carrier to each of said culture samples; comparing a series of treated samples with progressively decreasing amounts of inhibiting CAPA and a series of control samples with antiserum containing a predetermined amount of said antibody;
in the test material by subsequently adding to each of said control samples a predetermined amount of CAPA supported on a similar microparticulate carrier; and e) comparing said series of samples with said control samples. The method comprises the steps of determining the amount of CAPA.

The present invention also provides compositions useful as in vivo active immunological agents, which compositions include a CAPA as defined herein, along with a pharmacologically acceptable carrier. .

The invention will now be described in further detail by the following non-limiting examples.

Example 1 Isolation of pure antigens has been developed since it was shown that most cancer tissues contain the antigens of the present invention (Int-Arch, Al
Cancer tissues of different types and from different sites have been collected to form tumor pools.

In this case, this pool was created from macroscopically pure histologically proven carcinomatous tissue from 75 organs (cadaver autopsy).

Namely, the organs are breast, bronchus, cecum,
These were the colon, bifatty intestine, sputum, kidneys, larynx, liver, lungs, esophagus, ovaries, pancreas, prostate, rectum, stomach, and uterus.

Normal tissue was collected from a large number of individuals to establish pools of alloantigens, tissue antigens, and individual antigens.

A cadaveric autopsy case was used and the tumor diagnosis was histologically proven.

Furthermore, we established that the relevant tumor antigens remained intact due to immunological cross-reactivity between fresh tumor tissue and living tumor cells.

Aseptic care was taken in all cases whenever possible.

Cancer tumor tissue and normal tissue from cadaver autopsy were excised cleanly and separately from adjacent tissue and 2-3 crIL
The cubes were washed with 0.9% NaCl at 0°C until blood stains disappeared.

The washed cubes were frozen and stored at -24°C.

While still in the frozen state, cubes of this tissue were mixed at 0° C. with 107 d(7) H2O/g of frozen tissue in a chilled MSE atomic homogenizer with a rotating knife. 3 periods of 1 minute each
Homogenized at half speed.

It is observed that in this connection the temperature of the suspension should be kept at about 0° C., preferably slightly below said temperature.

The cold homogenate was poured into a chilled 1000 TL polythene bottle.

At -20°C 400 rrLl of the suspension and 400 ml of ethyl ether (Narcosine containing 0.002% diphenylamine, added ether, pH neutral, from Bomlskultfabriken Aktebolag, Sweden) were mixed in each bottle. , and agitated for 120 minutes at −2° C. in an International PR centrifugal shaker at a motor speed of 300 RPM.

The mixture was centrifuged at 1000xQ for 5 minutes at -2°C.

During this stage liquid. The layers do not mix with tissue layers.

Discard the ethyl ether and lipids and repeat the second extraction at 6
Centrifugation was performed for 0 minutes, followed by centrifugation for 5 minutes, and the ether-lipid layer was removed.

Finally, centrifuge at 1000xG for 30 minutes at -2°C.
The aqueous layer was then removed, although it contained some CAPA.

The remaining insoluble material was saved. Residual ethyl ether was removed by applying vacuum for 5 minutes.

The antigenic material was suspended in 50 TLl H2O at 0°C, transferred to a 500ml injection bottle and hard frozen at -70°C (dry ice and ethanol).

Freeze-drying was carried out in a Texvaak Model IV freeze dryer under controlled temperature.

The lyophilized tissue powder was run in a stainless steel ball mill at a temperature of about -70°C for 2 hours at 40 RPM.

As a result of this grinding, a fine gray-brown powder was obtained.

This powder was mixed with ethyl ether pre-cooled to -2℃ for 30 minutes.
Extracted for 60 minutes in an ORPM PR-2 shaker,
It was then centrifuged at 1000xG for 30 minutes at -2°C.

The ether layer was removed and the precipitate was dried in vacuo.

The resulting crude tissue powder (CTP) can be stored for many years at 4°C in closed bottles without appreciable loss of activity.

5 g of CTP was neutrally suspended in 5001 rLl of saline containing sterile ice blocks.

This suspension was washed in the MSE Atomics for 3 minutes at half speed for 1 minute.
Homogenized for a period of time.

This temperature was kept at 0°C. The mixture was kept at 0° C. for 30 minutes with slow stirring.

Centrifugation was performed at 100 OXG for 40 minutes at 0°C.

The supernatant was discarded. The precipitate was resuspended in 5001 Ll of cold distilled water and slowly stirred at 0°C for 30 minutes.

After centrifugation at 100 OXG for 40 minutes at 0°C, the supernatant was discarded.

The remaining precipitate was suspended in 25T/1 cold distilled water, and -
Freeze (shell-
Freeze).

When freeze-dried, washed tissue powder (WTP) was obtained.
This can be stored year after year in a sealed bottle at 4°C.

An aqueous extract of CAPA and normal WTP at pH 9.5 was prepared, which was then subjected to isoelectric precipitation at p)I 4.8 and 10 times concentrated by dissolving 1/10 the volume of the precipitate in water at pH 9.5. Concentrated into

The resulting concentrate was quickly heated to a temperature of 98-100°C and stabilized by holding at said temperature for 5-10 minutes.

This heat treatment destroys the enzyme, otherwise the enzyme will inactivate CAPA.

The stabilized extract became cloudy upon addition of pH 7.5 saline due to precipitation of residual nucleic acid contaminants.

8.0 TL to precipitate such residual contaminants.
1 of CAPA and normal aqueous extract were separately precipitated by adding 0.8 ml of 10% NaC7 solution.

After centrifugation at 27,000×G for 1 hour at 0°C, the clear supernatant was incubated at pH 4.8 (0, IN) ICt or 0
．． Adjust the pH with 1 nncoon) and perform isoelectric precipitation.

This precipitate was dissolved in M/150 phosphate buffer 2-3 rfLl, pH 7.0.

The resulting solution, derived from tumor and normal WTP, was mixed at 6.3 ml/cr? in a cooling column with an internal diameter of 2.50 crfL and a length of 138 cfI'L. L/
It was filtered using Biogel A-50m (as defined above) at a flow rate of h.

The gel was equilibrated with M/150 phosphate buffer pH 7, Q with 2% n-butanol and eluted with the same bathophore.

In each experiment, 50 to 100 m? An extract of 4-8 rILl containing a total amount of protein was used.

The antigenic activity was found in the central fraction of the tumor extract effluent.

1.0 ml aliquots of this fraction, totaling 160-180 TLl, were diluted 3 times and adjusted to various pH values ranging from 2.0 to 9.0.

After 5 minutes at room temperature, the samples were transferred to cuvettes and their 500 nm light dispersion was measured at an angle of 90°C.

The scattering intensity plotted against pH showed the maximum value at pH 4 and 8.

This pH was the isoelectric point of this fraction.

This method is a novel technique developed for the purpose of determining the exact and necessary conditions for the purification of CAPA.

Conventional techniques for determining the isoelectric point cannot be used here due to the low concentration of the peptide.

The remaining part of this active central fraction is set to 0. INHC7
was used to precipitate at pH 4.8.

Centrifugation for 5 minutes at 3.800×G at 0° C. resulted in a quantitative yield of activity.

For further purification of CAPA, the precipitated active central fraction from five Biogel A-50 columns was buffered in M/150 phosphate buffer (2%
Soelensen's buffer in n-butanol) was dissolved to 5% of the original effluent volume.

This solution containing 130774p protein in 8.5 ml was mixed with Biogel P-
35ml/cr through 2 columns (as defined above)?
One passage was carried out at a flow rate of t/h.

This column has a pH of 7.0 M/150 phosphate buffer = (
It had been equilibrated with Zellensen's buffer in 2% n-7'tanol.

1 mL of protein each! A bed volume of 10 m1 was allowed per bed.

The first fraction was active and was removed from the gel with the pH 7.0 elution buffer, while impurities were adsorbed onto the gel at the low ionic strength used.

The active substance was precipitated with 0.1 M) (COOH, pH 4,8) and centrifuged at 3,800 G for 5 min at 0°C.

The precipitate was dissolved in 8.5 ml H2O and 0.1
The pH was adjusted to 8.5 with M NH4OH.

This clear solution was diluted three times with 0.1M HCOOH to pH 4,
reprecipitated at pH 8.8, washed in a centrifuge at the same pH and dissolved at pH 8.5.

The disappearance of n-butanol was followed by gas-liquid chromatography.

The final solution was stored frozen (shell-frozen) and lyophilized in ampoules.

The resulting product was a dry, almost white powder that was stored at -24°C in a vacuum sealed ampoule.

The same procedure was performed on extracts derived from normal tissue and no activity was found.

In order to further purify the products obtained by the chromatographic method, a pH gradient elution method was developed.

This method basically consists of the following equipment:

That is, the protein mixture to be purified with respect to its active components is precipitated by adjusting the pH to its isoelectric point.

This precipitate is mixed with a gel such as Sephadex G-25 (
(as defined above) or with Biogel P-2 (as defined above).

This mixture is loaded on top of a column containing the same gel at the same isoelectric point pH.

The column is then subjected to a pH gradient elution at constant flow rate and temperature.

That is, the column is eluted continuously or stepwise with a medium of increasing or decreasing pH.

Gradient elution in this case was performed using 15 m7 of lyophilized and salt-free CAPA powder and normal antigen powder purified as described in the previous section, in this case at pH
This was done while decreasing the

This powder was separately dissolved in H2O and its pH was adjusted to 0.1.
Adjusted to 8.5 with NNH40H.

The pH of this clear solution was then brought to 5.0 with 0.1 M (COOH) and a precipitate formed.

Each precipitate was washed with 0.02M'HCOOH and 0.02M
It was mixed with 10 ml of Biogel P-2 slurry prepared from HCOONH, pH 5, and equilibrated with an aqueous solution.

Each of these mixtures was equilibrated as described above.
It was loaded onto a 15TLl Biogel P-2 in a siliconized column with an internal diameter of 150 m and a length of 150 m.

The gel was supported by small pieces of glass wool.

A pH gradient was used to elute this precipitate.

The pH was kept at approximately 5 for a short time to wash the precipitate by means of a gradient mixer (Ultrograd, type LKB).

The pH was then gradually lowered to 2.8.

Finally, the pH was raised to 9.

This buffer system is 0.02M HCOOH-HC0O
NH4 and 0.02 M NH,HCO3-NH3, and elution was performed at room temperature.

Is the flow rate 1 cI column cross section per hour? It was kept at 27.27d per L.

The effluent from the tumor extract was analyzed by fluorescence spectroscopy (activation at 288 nm, emission fluorescence at 350 nm).
.

Fractions eluted between the initial pH and a pH of about 3 were collected for further use.

After lyophilization and freeze-drying, the product could be stored at -24° C. in sealed ampoules after removing the air.

To characterize the molecular weight and confirm its purity,
The antigenic substance in the form of a solution of 0.02M at pH 3.0
It was filtered using Biogel P-30 (as defined above) equilibrated with a buffer of HCOOH-HCOONH4.

This pH-eluted CAPA was dissolved in a small volume of the same buffer.

Is this flow rate 3.25 ml/ct? t/hr,
The fractions collected contained 1.24ml.

The active fraction exhibited a spectrophotometric absorption maximum at wavelengths of 229-233 nm and fluorescence at 350 nm when activated at 288 nm.

In a typical experiment, siliconized biogel P-
10.47n9 dissolved in 30Tll of the above buffer on a 30 column (inner diameter 2.54cIrL1 length 50m)
of CAPA resulted in complete recovery of substance and activity.

The elution volume is within the range of 22,000 to 24,000 M
It was equivalent to W.

These molecular weight numbers were obtained by comparing this elution volume to the elution volume of compounds of known MW such as bonuclease and insulin.

After freeze-drying, a white hygroscopic powder was obtained, which can be stored in the cold and under light and enzyme exclusion.

In analysis of this product, no carbohydrates and nucleic acids are found.

Analysis using an amino acid analyzer agrees well with the molecular weight estimated by gel filtration.

Amino acid analysis shows the following molar straddle, but this number is ±5%
The accuracy is as follows.

The calculated molecular weight was 23.200±2.5000 (standard deviation).

Alania 8.62 Arginine
4.57 Aspartic acid 10.39 Cysteine 0.95 Glutamic acid 17,04 Glycine 6.87 Histidine 1.00 Inleucine 4.75 0 Isine 10.49 Lysine 3,69 Methionine 1.91 Phenylalanine 2.75 Proline 3 The amino acid content of .79 serine 7.74 threonine 5.92 tyrosine 3.21 valine 6.3 corresponds to the theoretical nitrogen content of 16.2-17.8%.

Total nitrogen measurement using the Duwa method shows a value of 17.0±0.5%.

This polypeptide is based on a single peptide chain as shown by treatment with performic acid.

Furthermore, this polypeptide, when unprotected by complex formation with albumen, has a pH of about 5 or higher.
It denatures irreversibly.

Example 1a A polypeptide was produced from human placental tissue in exactly the same manner as described in Example 1 above.

Example 1b Preparation of CAPA-Albumen Complex A dose of CAPA prepared in Example 1 was added to formic acid (e.g. 0.05 M
, the pH should be within the range of 2-3).

A clear solution is obtained. Albumen powder or an albumen solution (CAP) having the same pH as the formic acid solution is added to the solution.
Addition of 200 parts by weight of Albumen gives a clear solution of CAPA and Albumen.

The pH of this solution is then gradually increased by adding a base (eg, an aqueous solution of sodium hydroxide or ammonia) until it reaches about 7.5.

This results in a clear solution containing CAPA and albumen in the form of a combrex.

This solution containing 12 ggcAPA/ml can be used in the diagnostic method described in the Direct Method.

Alternatively, this complex can be isolated in the form of a white powder by lyophilization.

This powder is stable for long periods in the cold (+4° C.).

Experiments have shown that maximum utilization of CAPA activity is obtained when the weight ratio of albumen to CAPA is equal to or greater than about 200:1.

Example 1c Tannic acid treatment and production of labeled red blood cells
2 parts by volume) (Alsavar solution contains 250 g glucose, 80 g sodium citrate dihydrate, 42 g N
aCt (prepared by adding water to 10 A) and adjusting the pH to 6.1 with 10% citric acid monohydrate).

The mixture is centrifuged and the red blood cells are resuspended once in Alserver solution and twice in pH 6.13 buffer solution.

Finally, this suspension of red blood cells is brought to a concentration of 109 cells//7711 in a pH 6.8 buffer.

One volume part of the red blood cell suspension prepared as described above was added to a solution containing about 18 μI of tannic acid/ml at pH 6.8.
Add to 1 volume part of the buffer solution with stirring.

The red blood cells thus treated with tannic acid are centrifuged and resuspended twice in a pH 7.5 buffer.

Finally, the red blood cells were placed in a pH 7.5 buffer for 1 hour.
Suspend at a concentration of 0.09 cells/ml.

A single dose of the CAPA complex prepared in Example 1b above was dissolved in a buffer solution of pH 7.5 to 1 m
3μ per liter! The concentration is 17cAPA (pure CAP
Calculated based on A standard).

1 volume part of the tannic acid-treated red blood cell suspension prepared as described above was heated at 0°C for 10 minutes to add 1 volume part of CA.
Add dropwise to PA complex solution.

The labeled cells were centrifuged and resuspended in a pH 7.5 buffer solution containing a stabilizing amount (approximately 1.2 parts by volume) of inactivated human serum at 1.6 x 108 per ITL.
A suspension containing labeled cells is generated.

These labeled cells are used in the diagnostic method of the present invention as described below.

EXAMPLE ■ Production of Antibodies As previously described herein, in view of the fact that the CAPA of the invention is irreversibly denatured at neutral pH, parenteral injection of this polypeptide does not result in antibody production. It was thought that there would not be.

Experiments conducted using rabbits confirmed this.

It was discovered that no antibody production occurred after rabbits were injected with a polypeptide complexed with ampene.

All hemagglutination reactions were negative.

In order to make it possible for this polypeptide to be transferred into antibody-producing cells in an active state, this polypeptide should be dissolved in a solution with a pH of about 3 or less (0.02 ml HCOOH, p
)12.8) and emulsify this solution in oil to produce extremely small droplets surrounded by a protective oil layer.

Injection of this emulsion resulted in a satisfactory amount of antibody production.

This reacted specifically with CAPA in a hemagglutination reaction in which red blood cells were labeled with CAPA.

Production of Immune Agent Pure CAPA816 produced from pooled human cancer tumors
, l of 0.02 M HCOOH at pH 2,8 1,0 m
It was dissolved in l.

LO ml of adjuvant oil (commercially available from Difco) was added dropwise to the resulting solution while simultaneously emulsifying.

This emulsification was accomplished using a syringe by drawing and extruding the mixture into the syringe until it formed a homogeneous white emulsion with the same consistency as whipped cream.

A drop of the emulsion was tested in ice water to confirm that it separated, floated and remained intact.

The resulting semi-liquid emulsion was used for subcutaneous and intramuscular injections in rabbits.

Immunization of 5 rabbits: In order to establish a value using the so-called complete adjuvant for the first injection and the so-called incomplete adjuvant for subsequent injections, each of the 5 rabbits was initially immunized. After blood was drawn, a total of 408 gg of emulsified CAPA (pH 2,8) was injected subcutaneously into the right and left sides of each animal.

Three further injections were given over the course of 10 days, the first being administered intramuscularly in each hind leg and the second and third subcutaneously on the flank and both dorsal sides, respectively.

The injection site was selected with the aim of utilizing the reach of local lymph vessels.

Each rabbit received a total of ~1.6~ of CAPA in emulsion form.

Blood test samples were taken 14 and 24 days after the last injection and subjected to hemagglutination analysis for the presence of specific antibodies against CAPA.

Two days after the last sample, the maximum amount of blood was removed, sterile filtered, and collected in the form of serum aliquoted into sterile tubes.

These tubes could be stored cold, during which time specificity was retained.

Example ■ Diagnostic method a) Single-blind test Blood samples were obtained from the central hospital in Eskilsna, Sweden, of which 42 were from patients in the infectious disease unit and 1 from the surgical unit. 2 from the radiotherapy room, 10 from postpartum women, 10 from cord blood, 10 from pregnant women in their third month of pregnancy, and 6 from pregnant women. The sample belonged to a pregnant woman who was six months pregnant.

Additionally, blood was obtained from 20 10 year old children and 10 samples from older healthy adults were obtained from a private clinic.

Samples from 44 patients were obtained from a surgical and medical clinic in Stockholm, Sweden.

At Ekehofs Hospital, materials were obtained from 45 patients.
Blood samples were obtained from 72 patients hospitalized for surgical treatment at the Surgical Clinic in Stockholm, Sweden.

The average age of the blood donor department in Stockholm, Sweden, was 37.2 years (SD ± 1).
1.1 from donors in the age range of 20 to 67 years)
Eighteen samples were provided.

and from the central clinical laboratory of the hospital, 29 patients undergoing treatment in the general department of Radiumhemmet, Stockholm, Sweden, and 12 patients in the gynecology department.
Sera from human patients were obtained.

The total number of blood samples was 431.

Clinical studies were carried out on 111 cases from Eskilstuna, and medical records were made, except for apparently healthy participants.

For the Stockholm case, a detailed study of patient medical records for 100 cases was carried out.

For the remainder, a clinical diagnosis other than malignant was available, and there was no indication of malignancy.

Venous blood was transferred to Wallaselman tubes without additives and then transferred to the laboratory after removal of aggregates and blood cells by centrifugal means, if any.

Blood analysis is based on demonstrating the presence of CAPA by a modified hemagglutination inhibition technique (microvolume method).

In principle, the following method was used: 25 μt of patient serum absorbed with sheep red blood cells was subjected to Lympro IS S-MR.
Titration was performed in eight 2-fold dilution steps in a C-96 dilution plate.

Then 25 μt of specific antiserum at extreme dilution (approximately 2000)
was added to each recess.

After incubation for 10 minutes at 22°C, human albumen-bound CA was tannin-treated and isolated as described in Example Ic above.
50 μt of a suspension of approximately 6-8 million sheep red blood cells labeled with PA was added to each well (red blood cells 109
(approximately 2 to 4 μg per piece).

Other particulate carriers such as latex, bentonite and collodion may be used in place of red blood cells.

The reaction was carried out with a tilted mirror and at 1-2 °C for 4 to
Recorded using a standard system after 24 hours of incubation.

The following were used for control purposes: 1. Red blood cells labeled with CAPA plus antiserum; 2. Red blood cells labeled with CAPA without antiserum; 3. Blood cells labeled with patient serum plus human albumen; 4. Patient serum and untreated blood cells; 5. A series of CAPA-labeled blood cells and antibodies (in which inhibition is provided by known, stepwise decreasing amounts of CAPA).

The dilution liquid used contained inactivated neutral human serum for stabilization of pooled (ie, combined from many individuals) blood cells.

CAPA reagent was obtained from pooled human carcinoma tissue.

It was prepared and purified by gel filtration, pH gradient elution, filtration through Biogel P-30 and complex formation with albumen as described above.

As antiserum, horse anti-Hela cells from November 19, 1962 absorbed with normal tissue and pooled human serum were used.

Immunizations were performed using washed fractions of Hela cells grown in Eagle's medium containing 20% inactivated human serum in flat vials.

The cells were harvested with EDTA, centrifuged and washed with water for 3 minutes.
It had been washed several times.

In the presence of CAPA in patient serum, neutralized horse antibodies do not aggregate when polypeptide-labeled blood cells are added.

A progressive score from 0 to 6 was made, where 0 indicates that no inhibition occurred and 6 indicates maximum aggregation inhibition.

The relationship between this score and the concentration of CAPA in 1 ml of serum can be read from Table 1 obtained by calibrating with pre-measured dilutions of CAPA in neutral blood blue.

This record was made without knowledge of the patient's health status.

The materials used in the study were divided into groups as shown in Table 2 below.

It is clear from the results that the highest scores are obtained for the four groups consisting of "-human cancer and metastasis" (local) and "extended cancer" and the "neonatal" (cord blood) group.

Low scores are obtained from blood donors.

The intermediate position is occupied by the group "untreated - human cancer" and the group "cancer not completely treated".

Scores approximately equal to those of blood donors are found in the "completely removed cancer" group.

The mean values for CAPA in all groups were compared in a setwise fashion and are shown in Table 3 below.

From the table above, it can be seen that there is a significantly higher mean score for ``single cancer that has metastasized'' as well as for ``cancer that has spread'' compared to all other groups with the exception of neonates. It is clear (see also Table 2).

For the "Not fully treated cancer" group as well as the "Untreated - human cancer" group, the mean scores were compared to the groups other than the first two listed, and the "Cancer significantly higher than other malignant diseases.

Relative to the latter, the difference is significant (0.01
<P<0.05).

Groups 5-9 show no significant differences between each other except for the significant difference between 7 and 9.

This difference can be explained by the fact that among the cases showing scores between 1 and 4, there are cases of true hidden cancer.

A positive indication in knee band blood deserves some attention.

To confirm whether CAPA originates from the placenta,
A piece of such tissue was extracted.

Frozen placentas in an amount of 300"2/ml of buffered physiological NaC7 solution at pH 7.5 were homogenized at 0°C and centrifuged in a triangular tube (Marcum) at 100°C for 30 minutes.

CAPA in this supernatant by the hemagglutination technique described above.
tested for the presence of

The resulting inhibitory reaction is added to the blood cells of pure CAP.
To eliminate the possibility that this was caused by non-specific inactivation of A, the blood cells were shaken after each recording and a standard amount of CAPA-specific antibody was added.

Antigen inactivation was not demonstrated in any case.

As control materials, extracts of antigen powders from human cancer tissues of different origins and produced from different locations were used.

The results showed that all of the 26 placentas from multiple individuals without exception contained significant amounts of CAPA.

The average value (geometric) for 26 placentas is wet tissue 1
It was 297±8 μg (0.03%) per g.

As is clear from the above studies, since the CAPA of the present invention is synthesized in the placenta, the genes contained in the placenta must also be present in individuals derived from the same egg cell. From, CA
It follows that the potential and ability to produce PA is a normal phenomenon.

This ability is generally suppressed. This is because normal cells do not contain measurable amounts of CAPA.

However, in cancer cells, activation of this ability occurs, characterized by the significant presence of CAPA within and around the cells.

There are antigenic and, to some extent, functional similarities between placental trophoblast cells and cancer cells, and differences with respect to other cells in the individual arise from regulatory changes in the properties of cancer disease. It seems that it can coexist with the viewpoint that it is something that does something.

Statistical analysis of the experimental results shows that there is a highly significant Σ correlation between the presence of a cancer diagnosis and increased CAPA concentration (p<0.001).

The same is true for the relationship between possible tumor mass size and increased CAPA concentration in the same age group.

Increased concentrations of CAPA have been discovered and associated with a wide variety of cancer sites.

It has been discovered that cancer tumors, regardless of type and location, contain CAPA.

From that point of view, the CAPA appears to be common to all known types of cancer, malignant tumors of epithelial origin.

(b) Double-blind study In order to further prove the certainty of the above conclusions, a double-blind study was carried out under the most strictly controlled conditions.

Labeled blood samples were obtained from various departments of the central hospital in Erkilstuna, Sweden.

In this case, quantitative measurements of CAPA levels in patient serum were performed.

The results of this double-blind study as well as the single-blind study described above are summarized in Tables 4-8 below.

In that table, Table 4 shows the total number of individuals examined and the number of individuals presenting with a cancer diagnosis.

Table 5 shows the number of individuals in different groups along with their average age and their deviation.

Table 6 shows the presence of CAPA in serum from all patients tested.

Table '7 is ≧0.15. 9, 10 and 12 in Table 5, showing the % of individuals with CAPA concentrations of li'/ml serum.
The group is removed.

Table 8 shows C related to tumor location for groups 1 to 3 in Table 5.
Indicates the presence of APA.

Position numbers are included herein by reference.
cerIncidence in Sweden 1
968J.

(14) A complete statistical analysis of the data obtained from this double-blind test also revealed that the presence of a cancer diagnosis and increased CAPA
It shows that there is a highly significant correlation between the concentration (
p<o, o o i ).

This double-blind test also confirms the association between possible tumor mass size and increased CAPA concentration in the same age group (P<O, C101).

This increased serum level of CAPA has been found in association with 20 different types and locations of cancer.

Thus, double-blind testing fully confirms the robustness of this new discovery and their practical utility.

It was indicated in the introductory part of this disclosure that the antigen disclosed in US Pat. No. 3,663,684 is clearly different from the antigen of the present invention.

To illustrate the differences between prior art antigens and the antigens of the present invention, a detailed review of some of the characteristic differences, both with respect to antigen isolation methods and antigen properties, is presented below.

For purposes of summary, a brief definition of the cancer-associated polypeptide antigens and diagnostic tests of the present invention is provided below.

Definition of CAPA Hela cells (derived from the 1952 cancer case) are grown in vitro.

These cells are used as a source of antigen.

When the harvested cells are injected into horses, they result in the production of specific antibodies, which are used to identify antigens in tumors, serum, placenta, etc.

This latter antigen must, and does, have the same immunologically reactive site (compared to the Hera antigen), but this is of course not present in the natural state. If they are, they can differ in other ways.

Diagnostic Test Definition Antigens from Hela cell cultures in vitro are used to induce antibodies in horses.

After absorption of unwanted antibodies, this horse serum was applied to the tumor (or placenta or spatula or HEP-2 or Detroit 6).
It is used for agglutination of antigen-labeled red blood cells extracted from cultured cells).

This is an indicator system.

If the unknown sample contains antigenic activity, it will interfere with the antibody and inhibit agglutination.

Various modifications and equivalents will be apparent to those skilled in the art and may be made to the methods and products of the invention without departing from the spirit of the invention.

The gist of the present invention and specific examples of embodiments are listed below, all of which are included in the practice of the invention as defined in Article 65-3 of the Patent Law.

1. The following steps: (a) homogenizing the CAPA-containing material in a liquid at a temperature below about 0°C; (b) separating the solid from the mixture of liquid homogenate and solids obtained from step (a); (c) extracting the solid content with an alkaline aqueous solution; (d)
The extract obtained from step (C) was
0x106, in particular on a molecular sieve type filter in the form of beads that allows the fractionation of compounds with a molecular weight range comprising about 15X10', elutes this gel and fractionates in particular from 10XIO6 to 15XL06. Approximately 15X10
(e) chromatography of the fraction obtained from step (d) on a weak ion exchanger to adsorb impurities thereon and collect the first fraction emerging from said exchanger; (f) subjecting said first fraction to isoelectric precipitation, (g
) mixing the resulting precipitate with a molecular sieve type gel and subjecting said gel to pH gradient elution; and (h) reducing the pH until a pH of about 3, in which a spectral absorption peak at 229-233 nm is detected. A method for isolating cancer-associated polypeptide antigen (CAPA)' comprising the steps of collecting a fraction containing CAPA having a wavelength and having a molecular weight within the range of about 20,000-27,000.

2. The fraction obtained from the pH gradient elution is subjected to gel filtration on a gel column and the eluted fraction of the antigen having a spectral absorption peak wavelength of 229-233 nm and a molecular weight within the range of about 20,000-27,000 2. The method according to item 1 above, wherein the image is collected.

3. The homogenate obtained from step (a) is subjected to step (b)
2.) The method according to item 1 above, wherein the method is subjected to cold organic solvent treatment before step 1.

4. The third method, wherein the solvent used is ethyl ether.
The method described in section.

5. The method according to item 1 above, wherein the homogenization operation is performed in water at a temperature of about 0° C. or lower.

6. The method of paragraph 1, wherein the solid obtained from step (b) is freeze-dried and then ground in a cold steel ball mill.

7. The method according to item 6 above, wherein the grinding is carried out at a low temperature.

8. The method according to item 6 above, wherein the pulverization is carried out at a temperature below the freezing point of the material to be treated.

9. The method of item 6, wherein the grinding is carried out at a temperature of about -70<0>C.

10. The filtration in step (d) is carried out on a gel selected from the group consisting of polyacrylamide gel, cross-linked dextran gel, agar and agarose gel and equilibrated with phosphate buffer at a pH of about 7. Said first
The method described in section.

11. In step (e) the chromatography is carried out at neutral p
2. The method of claim 1, wherein the method is carried out on a column of a weak ion exchanger of H.

12. The method according to paragraph 11, wherein the chromatography is carried out on a weak ion exchanger polyacrylamide gel in the form of a column at neutral pH.

13. The method according to paragraph 1, wherein in step (g) a gel selected from the group consisting of polyacrylamide gel, cross-linked dextran gel, agar and agarose gel is used.

14. The method according to item 2, wherein the gel filtration is performed on a gel selected from the group consisting of polyacrylamide gel and cross-linked dextran gel.

15. The method of paragraph 1, wherein the aqueous extract obtained from step (C) is subjected to rapid heating to a temperature within the range of about 95 to about 100<0>C to destroy the enzymes.

16. The method of claim 15, wherein the aqueous extract is rapidly heated to a temperature within the range of about 98-100°C and held at this temperature for about 5-10 minutes.

17. While the antigen-containing material is still frozen, it should be heated to about 0°C.
Homogenization was carried out in water at a temperature of The solid fraction is lyophilized and milled in a steel ball mill at a temperature below the freezing point of the material being processed, the resulting solid is extracted with an alkaline aqueous solution and after centrifugation the supernatant liquid is
5 to about 100°C for rapid heating to a temperature within the range of about 100°C.
The enzymes are destroyed for about 10 minutes, the heated supernatant is treated with an aqueous sodium chloride solution to precipitate contaminants, and the supernatant liquid is collected and the resulting liquid has a concentration of about 110.0
00 to at least 20 x 106 and packed on a cold column packed with a molecular sieve type gel in the form of beads equilibrated with phosphate buffer at about pH 7.0 and the eluate The central fraction is collected and the central fraction is chromatographed on a column packed with a weak ion exchange molecular sieve type gel equilibrated with phosphate buffer at approximately pH 7.0, and collecting a first fraction of the resulting liquid, subjecting said first fraction to isoelectric precipitation at a pH of about 5 and the resulting precipitate having a molecular weight removal limit of at least about 2000 and at a pH of about
5 with a molecular sieve type gel equilibrated with HCOOH-HCOONH, and place the resulting mixture on a column packed with the same gel equilibrated in the same manner.
This column was eluted with a pH gradient and the CAPA content with a spectral absorption peak wavelength of 229-233 nm and a molecular weight of 23.200±2,500 (S, D,) was eluted with decreasing pH up to approximately pH 3. CA, including collecting images.
Method for producing PA.

18,23,200 ± 2,500 (S, D, Cancer-associated polypeptide antigen (CAPA), which is based on a single peptide chain and denatures irreversibly at pH above about 5 if not protected by complex formation with the albumen.

19. The polypeptide contains the following mole percent amino acids: Alanine 8.62 Arginine 4.57 Aspartic acid 10.39 Cysteine 0.95 Glutamic acid 17.04 Glycine 6.87 Histidine 1. OO Isoleucine 4.75 0 Isine 10.49 Lysine 3.69 Methionine 1.91 Phenylalanine 2.75 7.

Su7 3.79 Serin
CAPA020 according to item 18 above, containing 7.74 threonine 5.92 tyrosine 3.21 valine 6.36;
19. The CAPA of claim 18, which contains a fluorescent group that emits fluorescence at about 350 nm when excited at a wavelength of about 288 nm.

21. The following steps: a) preparing an aqueous solution of CAPA according to paragraph 18 above at a pH of about 3 or less; (b) the solution obtained from step a) being protected by an oil phase surrounding it; C)
A method for producing monospecific antibodies with respect to CAPA, wherein the oil emulsion is injected into a living animal body, whereby the CAPA carried by the emulsion is transferred to antibody-producing cells.

22. The method according to paragraph 21, wherein in step b) Freund's adjuvant is used for oil emulsion preparation.

23. The method of claim 21, wherein the aqueous solution in step a) is prepared at a pH of about 3 or less using a weak acid as the acidifying agent.

24. The method according to item 23 above, wherein the weak acid used is formic acid.

25. In determining the presence of CAPA at various stages of cancer progression by testing materials selected from the group consisting of serum, tissues, secretions and extracts from the animal organism being tested, the following steps: a) preparing a series of samples of said material by serial dilution; b) adding to each of said samples a predetermined amount of antiserum containing antibodies specific for that antigen; and C) preparing said cultured samples after incubation. d) add a predetermined amount of stabilized (complexed) CAPA supported by a particulate carrier to each of ) compared to control samples of known progressively decreasing amounts of CAPA and predetermined amounts of said antibody-containing antiserum;
Thereafter adding a predetermined amount of CAPA supported by a microparticulate carrier to each of said control samples, and-) comparing said series of samples with said control sample; A method comprising determining the amount of CAPA in a material to indicate the presence of cancer and its stage of progression.

26. The method according to item 25, wherein the material is patient serum.

27. The antigen-containing material is tissue from a human placenta;
The method according to item 1 above.

28. The method according to item 1, wherein the antigen-containing material is human tumor tissue.

29. The method according to item 1, wherein the antigen-containing material is a culture of cancerous cells grown in vitro.

30. The method of item 25, wherein the material is a biopsy, a surgical specimen, a puncture, and a smear.

31. The method according to item 25 above, wherein the polypeptide antigen used is as described in item 18 above.

32. The method of claim 25, wherein the material of step a) is absorbed into a particulate carrier selected from the group consisting of sheep red blood cells, latex, bentonite and collodion.

33. The method according to item 25 above, wherein the blood cells used in step C) are sheep red blood cells.

34. The method of claim 25, wherein the antiserum used in step b) is derived from an animal immunized with a substance containing CAPA.

35. The method according to item 34, wherein the immunized animal is selected from the group consisting of horses and rabbits.

36. A composition useful as an immunizing agent containing CAPA as an active ingredient together with a pharmacologically acceptable carrier.

37. The composition of claim 36, wherein the CAPA-containing aqueous solution at a pH of less than about 3 comprises an encapsulated phase and an oil pore agent protected by a surrounding oil phase.

37. The composition according to item 36, wherein the oil pore agent is a Freund's adjuvant.

39, a composition comprising CAPA and a stabilizing amount of albumen in admixture therewith.

40. The composition of claim 39, wherein the ratio of albumen to CAPA is at least 200:1 by weight.

41. Mix CAPA and albumen in acid solution;
and increasing the pH of the solution to cause interaction between these components.

42 further comprising the step of lyophilizing the composition.
42. The method according to item 41.

43. A diagnostic composition comprising particulate material labeled with albumen-stabilized CAPA.

44. The diagnostic composition according to item 43, wherein the particulate material is selected from the group consisting of tannic acid-treated red blood cells, latex, bentonite, and collodion.

[Brief explanation of drawings]

The accompanying drawing is a flow sheet illustrating a method of isolating cancer-associated polypeptide antigen (CAPA) according to one aspect of the invention.

Claims (1)

[Scope of Claims] 1. The following steps: (a) homogenizing the cancerous tissue in a liquid at a temperature of about 0° C. or less; (b) combining the liquid homogenate obtained from step (a) with a solid medium; (c) extracting the solid from the mixture with an alkaline aqueous solution; (d)
The extract obtained from step (C) was
0 x 10', in particular on a molecular sieve type filter in the form of beads that allows the fractionation of compounds with a molecular weight range comprising about 15 x 10', this gel is eluted and 10 x 10' Fractionation especially about 15X
(e) chromatography of the fraction obtained from step (d) on a weak ion exchanger to adsorb impurities thereon and collecting the first fraction emerging from said exchanger; (f) subjecting said first fraction to isoelectric focusing and (g
) mixing the resulting precipitate with a molecular sieve type gel and subjecting said gel to pH gradient elution; and (h)
22 to a pH of about 3 while decreasing the pH.
It has a spectral absorption peak wavelength of 9 to 233 nm and about 2
C with a molecular weight within the range of 0,000 to 27,000
Collect the fractions containing APA. HCO'0 at a pH of about 3.0, characterized by
A polyacrylamide gel column equilibrated with H=HCOONH4 [the gel had a particle size of 100-200 mesh, an exclusion limit (Dalton) of 40,000 and a
500 to 40,000 Daltons], 20.000 to 27;
Cancer-associated polypeptide antigen (CA
PA) (However, the polypeptide is between 229 and 233
It has a spectral peak absorption at wavelengths in the nm range, is based on a single polypeptide chain, and contains aspartic acid, glutamic acid, leucine, phenylalanine and tyrosine as essential constituents, as can be demonstrated by treatment with performic acid. , which reacts monospecifically with antisera produced by Hela cells and absorbed by normal tissues and human serum, and which reacts monospecifically with antisera produced by Hela cells and absorbed by normal tissues and human serum, and which, when unprotected by a complex with albumen, have a pH above about 5. method for producing a product which irreversibly denatures in value.