JPS6018011B2 - Detection method for cancer-related polypeptide antigens - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the problem of detecting cancer-associated antigens that exhibit monospecificity and are present in a wide variety of humans at different locations.

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

If such a demonstration were to become consistently reproducible, it would indicate the presence in human tissue of important antigens that are not present in normal tissue, and thus the process of neoplastic production. will lead to a better understanding of the nature of Human rope tissues were studied with the aim of discovering the existence of monospecific furrow-associated antigens with the aim of better understanding bran diseases, and above all with the aim of discovering a reproducible single-nada purification method for such antigens. The presence of antigens associated with the silk intestine and the axilla of the digestive system has been shown by Gold et al. (J.

Ex-Med. , 121 (1965) ~ 439 ~ 46
2.J. Expt. Med. , 122 (1965) also 4
67-487). Reference is also made in these reports to earlier studies by BB Jorkl plate d showing the presence of human cancer-associated antigens common to most of all known malignant sitz baths of epithelial origin (car ntemat.~ch. Mountain 1er and Appl.lmmu blood 1., (i966) 8
~179 and ($19) 12 '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 this specification. Isolation of a practical and commercially useful antigen itself as well as its association with various birch diseases 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 people with latent or established cephalopathy by diagnostic means suitable for large-scale testing. Ta. In order to fully utilize the presence of antibodies specific to seed coagulation-related antigens in animal antiserum as a diagnostic agent, a test method that indicates the presence of tumor antigens in the patient's blood must be developed. Must not be.

The methods proposed so far have proven insufficient when attempting to make certain diagnoses regarding the presence of lotus disease. The main objective of the present invention is to provide a method for detecting Ren-associated antigens starting from human pupil tissue or other tissues containing such antigens.

In the disclosure of the present specification including the claims, “
The expression ``foot-associated polypeptide antigen'' is abbreviated as 庺CAPA. 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 CAPN properties are purified in a single machine by homogenizing fenestra tissue from dissections according to the method of the present invention, in which various types and ranks of rational microorganisms are collected and prepared from fly samples. ...is formed.

Alternative starting materials are ~ tissue from the human placenta or cultures of malignant cells in vivo (for details of such cultures see B. B. Jorklund et al., J. Nat. Can
cerlnst. 26-533-545 (1961)
~rAntihokonicityofPooled Hun
Ton Malignant and NonhaI
TiSS female sby C〆oimm dish ologiCa
ITechnique m, Distribution of seed field antigens).
While in the frozen state, cancer and normal tissue are mixed separately 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. A bound cold mixture of liquid homogenate and solid is heated at about 0° C. with an organic solvent such as acetone or ethyl acetate capable of dissolving substances such as neutral fats.
Mixing at the following temperatures, this solvent treatment is aimed inter alia at removing lipoidal substances and increasing 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 The powder is milled, for example, in a stainless steel ball mill at a low temperature, preferably below about 0°C and suitably below about 170 qo.

As a result of this grinding, a fine gray-brown powder is produced. If desired, the powder obtained is extracted with a salt solution, for example saline, on a neutral axis at a low temperature, suitably about 0 DEG C., and the supernatant after centrifugation is discarded. The resulting precipitate can be suspended in cold water, collected and lyophilized. The resulting powder can be stored for many years in a sealed bottle at about 4°C. When the polybebutide-containing pupil bran and normal tissue powder are extracted with alkaline water, the extract is neutral or slightly alkaline and becomes cloudy when NaCI is added.

This turbidity is mainly due to the presence of impurities that have the properties of nuclear brew. All activity remains in the clear supernatant even after the saline-treated extract is drained. The supernatant solution is precipitated at an acidic pH, suitably about 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 divided into 10 × 1 to 20
The filtration gel is filtered through a suitable molecular sieve-type gel in the form of beads that allows the fractionation of compounds with a MW range that includes approximately 1 × 1 and especially about 15 × 1, and the filtration gel is eluted with a buffer of approximately neutral pH. A MW fraction of 10 x 1 to 20 x 1, especially about 15 x 1, is then collected. Or molecular sieves such as Biogel A-5
(West German Biorad. Laboratory agarose gel Fujishina name ~ 100-200 mesh, removal limit (Dalton)
50 x 1 pi, fractionation range (Dalton) 1 to 50 x 1 pi,
(approximately agarose content 2%) or Sepharose (trade name of agarose gel from Pharmacia Fine Chemicals, Sweden, beads of 60-300 microns, dispensing box range 1-20 x 1, approximate agarose content 2%)
You can choose from. The gel is then equilibrated with a suitable buffer solution at a pH of about 7.0, such as 1:10 diluted Soelensen's buffer (1/15M sodium and potassium phosphate based 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-15h (Piolado. Laboratories, 100-200 mesh, exclusion limit (Dalton) 15
0×1pi fractionation range (Dalton) 1pi~>150×1pi)
It is. For further details regarding gel fractionation techniques, see Gel Chromatography Volume 1 by H. Determan (Springer Farark, 1967 edition). solution and in this eluate, antigenic activity is found in the fractions containing the MW range. The fractions are collected. The active fraction precipitated from the gel filtration is solution in a suitable buffer such as 1:10 diluted 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).

The column was equilibrated with a similar buffer at a pH of approximately 7.0. It was discovered that the fraction exhibiting antigenic activity was the first fraction of the solution coming out of this column.
Collect this fraction. The fractions recovered from the chromatography of the Biogel P-2 are subjected to a special procedure developed for further purification of CAPA.

In principle, this operation involves the following steps: a protein or a mixture of complex proteins is precipitated by isotropic adjustment of the plaques. The mixture of precipitate and gel is mixed at the same isosagi point p.
It is placed on top of the same gel-containing column with a pH of H, which is the protein isoelectric point at which the protein was to be precipitated in the previous step.
The column is then subjected to elution while gradually raising or lowering the plaque. 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 flow of ions that connect them one after the other in solution.

The time required for solubilization of different proteins depends on the ionic strength, temperature, and flow rate.The dispersion constant of each dissolved protein is lower than that of the ionic gradient, so the protein gels more quickly than the gradient. As a result,
The protein reprecipitates and remains in a static (steady state) state until redissolved by an approaching ion front. 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 about 2500
It has a molecular weight removal limit of 0. However, this removal limit is not critical, Q These gels are of the molecular sieve type and are usually used in bead form. at least about 2
Any such sieve type gel with a molecular weight removal limit of QOQ~3000 can be satisfactorily used. For a review of molecular sieve-type gels suitable for gel filtration in combination with gradient elution according to the invention, see also Gelot, R.W., Biochem. Separations' Protein-Veptide and Amino Acid Fractions by Gel Filtration
Norstrand, 1964 edition). Normal and CAPA recovered from the chromatography
The fractions are subjected to active substance precipitation at a time point of about 5. This precipitate is collected and dissolved in a solution having a pH of about 8.5.The pH of this clear solution is then acidified with formic acid, suitably to a pH of about 5.
. Set to 0.

This process produces precipitates. Each precipitate is mixed with a slurry of a similar gel equilibrated with HCOOH-HCOONH4 in approximately 5. Place it on top of the small column that has been prepared. For the evacuation of this precipitate, a pH gradient with a progressively decreasing pH was used in this case. A suitable buffer system is also HCOQ8-HCGQN84.
OpubiNH4HCQ31N Cooperative.

No antigenic activity is found in the current solution within a pH range of about 3 from the initial pH. The apocalyptic fraction collected within the pH range identified above contains the desired CAPA, which can be recovered by lyophilization.

This fraction can also be run for several weeks on a gel column equilibrated with HCOOH-HCOONH4 at a pH of about 30 for the purpose of determining the sperm content of CAPA in the effluent fraction. This gel tends to be selected from those listed in Funaki.
Removal limit (Dalton) 40,000 Fraction range (Daltoshi) 2,500-40,000) and Cephadex G
Monolithic or G-5Q (Pharmacia. The product name for Huain Chemica Ikezu's dextran gel is 4Q to 12■ and 50 to 15Q. The micron fractionation range is 3 to 7, respectively.
08 000 and X~309 000) are also particularly suitable gels for carrying out this filtration. have a spectral absorption peak wavelength within the range of about 229 to about 23 molar and about 20
,000 to about 278,000
Minatode branch garden is collected. CAPA can be recovered from this active fraction by lyophilization after filtration.

And it can be stored for a long time in a cold store under vacuum. Treatment with performic acid has also shown that single peptides can be made from single peptides, making them more complex than polypeptides. This CAPA exhibits a basic reaction and is soluble in a range of about 1 to 3 centimeters. This unprotected polypeptide denatures irreversibly at neutral pH, i.e., above about 4.5. The purified CAPA contains photogenic groups that are activated at skin wavelengths and emit light at approximately 35 arm wavelengths.

This fluorescence is proportional to the abundance of CAPA.
- Can be used to determine the presence of poIJ peptides during their isolation procedures. This CAPA is about 229 to about 2
It exhibits spectral peak absorption at wavelengths within the range of 3 m.
Furthermore, it showed a large amount of agglomeration and could not be filtered on the usual filtration media used for sterilization.It was confirmed that CAPA was derived from the cancer cell wall.
The reason for this is that when vCAP is injected into a living animal, the antibodies that are generated can cause the complete destruction of cancer cells in the body when cancer cells are placed under the influence of such antibodies. As mentioned above, this polypeptide has a molecular weight within the range of 2QO to 2QOO, more specifically about 2$000.

Analysis shows that it does not contain any carbohydrates, and spectrophotometric measurements show that CAPA does not contain any nucleic acids. Analyzes carried out on an amino acid analyzer with or without oxidation show agreement with the molecular weight estimated by gel filtration on a Biodil Point 30 or equivalent. Uses: For example, monospecific antibody production, diagnostic purposes, and immunological methods may also be used as active ingredients in compositions useful as immunological agents. New methods have been developed for specific antibody production.

Even with protection as described above, CAPA denatures irreversibly under neutral pH conditions. 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. To prevent such denaturation, oil emulsions are prepared in which CAPA at a pH within the range of about 2 to about 3 is used.
An aqueous solution of 100 ml of water constitutes the encapsulated phase surrounded by the oil coffin. 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 in neutral p-laws consists in conjugating CAPA with an inert protein such as human or bovine albumin.

Utilizing such a method, a complex is formed that is lacquer-like at neutral pH and retains its in vitro antigenic activity. For rope diagnosis, a modified hemagglutination inhibition technique has been developed.

This technique investigates the peculiarities of species by examining materials of interest such as patient serum, tissues, secretions and extracts from living animal bodies, including for example biopsy material, surgical specimens, punctures and smears. This new modified hemagglutination inhibition technique makes it possible to measure the presence of CAPA at advanced stages.
adding to each of said samples a predetermined amount of an antiserum containing antibodies specific for CAPA; c. After incubation, each of said cultured samples is provided with CAP supported by microgranular socles;
adding A in a predetermined amount; d adding the resulting series of treated samples to an antibody containing progressively decreasing amounts of inhibiting CAPA and a predetermined amount of said antibody; comparing a series of control samples with serum, then adding to each of said control samples a predetermined amount of CAPA supported on the same microparticulate carrier; and e. The method comprises the steps of determining the amount of CAPA in the test material by comparison with a sample.

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

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

Example 1 Isolation of Pure Antigen Since it was shown that most short tissues contain the antigen of the invention (lnt.Arch.AIlergy3
6, 191-203 (1969)) Cancer tissues of various types and from various sites are collected to form a pseudopool.

In this case, the pool was created from macroscopically pure, histologically proven brachylum tissue from the following organs (cadaver dissection): In other words, the organs include the breast, bronchial tubes,
Cecum, colon, duodenum, sputum, kidney, larynx, liver,
These were the lungs, esophagus, ovaries, pigs, 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 tumor field diagnosis was explained histologically. Furthermore, due to immunological cross-reactivity between fresh tumor tissue and living Hitotou cells, we established that the relevant Tanaba antigens remained intact. Aseptic care was taken in all cases whenever possible. The lesion tissue and normal tissue from the cadaver dissection were excised separately from the adjacent tissue and
Cut into ~3 arc cubes, which were washed with 0.9% CI at 0°C until free of blood stains.

The washed cubes were frozen and stored at -24qo. While still frozen, cubes of this tissue were mixed at a low ratio of 0° C. per frozen tissue and incubated for 1 min in a chilled MSE atomic homogenizer with a rotating knife. Homogenization was performed at "half speed" for three periods each. It is observed that in this connection the temperature of the suspension should be kept at about 0° C., preferably slightly below said temperature. This cold homogenate was cooled to l
Pour into oo0' polythene bottle.

-20 qo of 400 ml suspension and 400 ml of ethyl ether (Narcosine-added ale containing 0.002% diphenylamine from Akcebolag, Bomlskultfavrikken, Sweden) in each tank. The mixture was mixed for 12 minutes at -2°C in an International PR centrifugal shaker at a motor speed of 30 plates PM.
I was at a loss. During this stage the liquid layer does not mix with the tissue layer. The ether and lipids were discarded and a second extraction was performed for 60 minutes, followed by centrifugation for 5 minutes and removal of the ether-lipid layer. Finally 1000×G
was centrifuged for 30 min at -2°C and the aqueous layer was removed, although it contained some CAPA. Any remaining insoluble material was saved. Residual ethyl ether was removed under vacuum for 5 min. The antigenic material was suspended at a ratio of 50° C. to 500° C.
The mixture was transferred to an infusion bottle and frozen hard at -70°C (dry ice and ethanol).

Freeze-drying was carried out in a Texvak model W freeze dryer under controlled temperature.

The lyophilized tissue powder was processed in a stainless steel ball mill at a temperature of about -70° C. for 2 hours at 4 rudder PM.

As a result of this grinding, a fine gray-brown powder was obtained. Powder this powder in 30 plates of ethyl ether pre-cooled to -2°C.
The mixture was extracted in a PR-2 monkey apparatus for 60 minutes and then extracted for 3 times at 1000 x G at -2°C. The ether layer was removed and the precipitate was dried in vacuo. The resulting crude tissue powder was (CTP) can be stored for years at 4°C in a sealed bottle without appreciable loss of activity. CT
A sample of P. acetate was suspended in 500' saline solution containing sterile ice blocks at neutral pH.

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. This mixture was kept at 0° C. for 30 minutes while slowly stirring. 1
Centrifugation was performed at 00xG for 40 minutes at 0°C.

The top liquid was discarded. The precipitate was resuspended in 500°C cold distilled water and slowly stirred at 0°C for 3 minutes.

After centrifugation at 1000×G for 40 minutes at 0° C., the supernatant was discarded.

The remaining precipitate was suspended in 25°C of cold distilled water. It was then frozen (Shell M Freeze) with C (dry ice and ethanol). Wash tissue powder (WTP) when freeze-dried
was obtained, which can be stored for years in a sealed bottle at 4°C. CAPA and normal WTP at fence 9.5
An aqueous extract was prepared, which was precipitated uniformly in a bar 4.8, and the precipitate was concentrated by dissolving it in a 1'10 volume of water.

The resulting concentrate was stabilized by immediately heating to a temperature of ˜100° C. and holding at said temperature for 5 to 10 minutes. This heat treatment destroys the enzyme, otherwise the enzyme will inactivate CAPA. The stabilized extract became cloudy upon addition of pH 745 saline due to precipitation of residual nucleic acid contaminants. To precipitate such residual contaminants, the 8.0' CAPA and normal aqueous extracts were separately precipitated by adding 0.8' of 10% NaCI solution. 0℃ at 27G000×G
After 1 hour in Liaoxin, the clear supernatant liquid was adjusted to pH 4.
8 (pH with 0.1N HCI or 0.1N HCOOH)
) The isonoise point was precipitated.

This precipitate was dissolved in 2-3 ml of M 150 phosphate buffer, pH 7.0. The solution obtained here, originating from pupil moat and normal WTP, was divided into 2.50 moth inner diameter and 1
6 in a cooling column with a length of 1.

The mixture was filtered using a Biogel A-5 gelatin (as defined above) at a flow rate of 3/3.

The gel was equilibrated with M/150 phosphate buffer pH 7.0 with 2% n-butanol and eluted with the same buffer.
Extracts of 4 to 8 secretions containing total female protein were used. The antigenic activity was found in the central fraction of the effluent of the pupil extract.

A total of 160-180 fractions of 1.0 were diluted 3 times and adjusted to gutter feed values ranging from 2.0 to 9.0. After 5 minutes at room temperature, transfer the samples to a cuvette and reduce the optical dispersion of those 50 samples to 9
Measurements were taken at an angle of 0 and 0.

The scattering intensity plotted against stiffness showed a maximum value at pH 4.8. This bait was the isomer 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 isophyte points cannot be used here due to the low concentration of peptides. The remainder of this active central fraction was precipitated using 0.1N HCI 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 Piogel A-50 columns was buffered in M/150 phosphate buffer (2%
Seerensen's buffer in n-butanol) to 5% of the initial effluent volume.

This solution containing 130 o of protein in 85 [
It was filtered through a Biogel P-2 column (as defined above) with a diameter/height ratio of 1/35 at a flow rate of 35 M/h/h.

This column has a pH of 7.0 M/15 phosphate buffer (
Seerensen's buffer in 2% n-butyl alcohol). A pet volume of 10 w' was allowed for each bar of protein. The first fraction is active, pH 7.0
elution buffer from the gel, while impurities were adsorbed onto the gel at the low ionic strength used.

The active substance was precipitated with 0.1 M HCOOH at pH 4.8 and centrifuged at 3.80° C. for 5 minutes. The precipitate was dissolved on day 20 of 8.5' and 0.1 MN
The pH was adjusted to 8.5 within a day. Add this clear solution to 0.1
Reprecipitated three times with MHCOOH at pH 4.8, 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 by freezing (shell-freezing) and lyophilizing in ampoules. The resulting product was a dry, almost white powder that was stored at -24 qo in a vacuum sealed ampoule.
The same procedure was performed on corresponding extracts derived from normal tissue, but no activity was found.

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

The method essentially consists of the following steps: That is, the protein mixture to be purified with respect to its active constituents is precipitated by adjusting the fence to the isomensity point. This precipitate is mixed with a suitable gel such as Sephadex G-25 (as defined above) or Biogel P-2 (as defined above). This mixture is loaded onto the top of a column containing the same gel with the same point axis. The column is then subjected to boat gradient elution at constant flow rate and temperature. That is, the column is eluted continuously or stepwise with increasing or decreasing bait media. Gradient elution in this case was performed using 15 kg of lyophilized and salt-free CAPN powder and normal antigen powder purified as described in the previous section, in this case reducing the mother.

This powder was dissolved separately on day 20 and its pH was adjusted to 0.1.
I adjusted it to 85 in N and N days. The pH of this clear solution was then brought to 5.0 with 0.1M HCOOH and a precipitate formed. Each precipitation is 0.0 water level COO day and 0.0
Biogel P-2 slurry 10' equilibrated with an aqueous solution adjusted to pH 5.0 from 2M HCOONH4.
mixed with. Each of these mixtures was loaded onto a 15 skin Biogel P-2 of a siliconized column having an internal diameter of 16 columns and a length of 15 yards, equilibrated as described above. The gel was supported by small pieces of glass wool. A mother gradient was used to elute this precipitate.

The pH was kept at about 5 for a short time to wash the precipitate by means of a gradient mixer (U1trogQd, LK matrix). The shoe was then gradually lowered to 2.8. Finally, the pH was raised to 9. This buffer system is 0.02M HCOOH
It contained COONH4 and 0.02M COONH3, and elution was performed at room temperature.

The flow rate was maintained at 27.2 centimeters per column section per hour.
The effluent from the tumor extract was analyzed by fluorescence spectroscopy (activation at 2*m, emission fluorescence at 35m).

Fractions eluted between the first axis and a pH of approximately 3 were collected for further use. After lyophilization and freeze drying, the product could be stored at -24oo 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 0.02 MH at pH 3.0
Filtering was performed using Piogel P-30 (as defined above) equilibrated with a buffer of COOH and COONH4.

This pH-eluted CAPA was dissolved in a small volume of the same buffer. The flow rate was 3.25 M/swim/hr and the fractions collected contained 1.24 secretions. The active fraction is 22
Shows maximum absorption on a spectrophotometer at a wavelength of 9 to 23 square meters,
When activated with 2 kaede blood, 35 skin blood showed fluorescence. In a typical experiment, 10.4 μm of CAPA dissolved in 3.0 μm of the above buffer on a siliconized Biogel P-30 column (2.54 μm ID, 50 μm length) was used to analyze the substance and activity. A complete recovery occurred.

The elution volume is ~1,000 MW within the range of 24,000
It was equivalent to These molecular weight numbers were obtained by comparing this elution solution to the elution volume of compounds of known MW such as ribonuclease 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 mole percentages, which are accurate to within 5%. The calculated molecular weight is 23,200 So2,500 (
standard deviation). alanine
862 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 1,9
1 Phenylalanine 2.75 Proline
3.79 serine
7.74 Threonine
5.92 Tyrosine
3.21 Valin 6,3
6 This amino acid content is the theoretical nitrogen content of 16.2~
This corresponds to 17.8%.

Total nitrogen measurement by Dumas 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, if not protected by complex formation with albumen, irreversibly denatures at a pH above about 5. Example 1 Polypeptides were produced from human placental tissue in exactly the same manner as described in Example 1 above.

Example lb CAPA-Albumen Complex Production Example 1
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 per part by weight of AI gives a clear solution of CAPA and albumen. The pH of this solution is then gradually increased by adding a base (e.g. an aqueous solution of sodium hydroxide or ammonia) until it reaches approximately 7.5.
A clear solution containing A and albumen in the form of a complex results. This solution containing 12A/CAPA/ can be used directly in the diagnostic method described below.

Alternatively, the complex can be made into a single product in the form of a white powder by lyophilization. This powder is stable for long periods in the cold (1400 ml). Experiments have shown that the best use of CAP stability is Alpmen's CAP.
It has been shown that this is obtained when the weight ratio to A is equal to or greater than about 200:1.

Example lc Tannic acid treatment and preparation of standard red blood cell cells Fresh sheep blood (1 part by volume) is mixed with sterile Alsvar solution (1 part by volume).
．． 2 parts by volume) (Alserva solution contains 250 g of glucose, 80 g of sodium citrate dihydrate, 42 g of N
(prepared by adding water to aCI for 10 minutes and adjusting the pH to 6.1 with 10% citric acid monohydrate).

The mixture was centrifuged and the red blood cells were placed once in Alserver solution and twice in a pH 6.8 buffer solution.
Resuspend twice. Finally, this suspension of red blood cells is fed to feed 6.
．． The concentration should be 1 cell in 8 ml of buffer. Red blood cell suspension 1 produced as described above
Contains approximately 18 parts by volume of tannic acid/I pH 6
．． Add to 1 volume part of the buffer solution in step 8.

The red blood cells thus treated with tannic acid are centrifuged and resuspended twice in pH 7.5 buffer. Finally, the pH of this red blood cell? ．． Suspend the cells at a concentration of 1 cell/cell in 5 ml of buffer. C produced in Example lb above
An aliquot of the APA complex is dissolved in a pH 7.5 buffer solution to a concentration of 3 m/m of CAPA (calculated on a pure CAPA basis). One volume part of the tannic acid-treated red blood cell suspension prepared as described above is added dropwise to one volume part of the CAPA complex solution at 0° C. over a period of 10 minutes. The labeled cells were centrifuged and resuspended in a pH 7.5 buffer solution containing a stabilizing amount (1.2 parts by volume) of inactivated human serum at 1.6 x 1 part per volume. A suspension containing p labeled cells is generated. These labeled cells are used in the diagnostic method of the present invention as described below. EXAMPLE 2 Production of Antibodies As already mentioned herein, parenteral injection of this polypeptide does not result in antibody production, given the fact that the CAPA of the invention denatures irreversibly at neutral pH. It was thought that it would be.

Experiments conducted using rabbits confirmed this. It was discovered that no antibody production occurred after rabbits were injected with voriveptide complexed with Alpmen.
All hemagglutination reactions were negative. In order to enable this polypeptide to be transferred into antibody-producing cells in an active state, this polypeptide is maintained in a solution with a pH of about 3 or less (0.02 h, HCOOH, pH 2.8), and this solution It is necessary to emulsify it 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. Manufacture of immune agent Pure CAPA 816 produced from pooled human pupil was mixed with 0.02M HCO at pH 2.8.
Dissolved in 1.0M of OH.

A 1.0 soil adjuvant oil (commercially available from Difco) was added dropwise to the resulting solution with simultaneous emulsification. This emulsification was accomplished using a syringe by drawing and extruding the mixture into the syringe until it formed a homogeneous white emulsion having the same consistency as whipped cream. A drop of the previous floating L agent was tested in ice water and it was confirmed that it separated, floated, and remained intact. The resulting semi-liquid emulsion was used for subcutaneous and intramuscular injections in demons. Immunization of 5 rabbits To establish a zero value, a so-called combined adjuvant was used for the first injection, and a so-called incombinated adjuvant was used for subsequent injections.
After first drawing blood from each rabbit, a total of 4
Female muta emulsified CAPA (pH 2.8) was injected subcutaneously into the right and left sides of each.

Three further injections were then given over the course of 10 days, the first being administered intramuscularly in each hind leg and the second and third being administered subcutaneously bilaterally and dorsally, respectively.

The injection site was selected with the aim of utilizing the reach of local lymph vessels. Each rabbit received a total of approximately 1.6 doses of CAPA in emulsion form. Blood test samples were taken 14 and 24 days after the last injection and were administered to CAPA.
was subjected to hemagglutination analysis for the presence of specific antibodies against. Two days after the last sample was taken, the maximum amount of blood was taken;
This was filtered through bran bracium and collected in the form of serum, which was transferred in small portions into sterile tubes. These tubes could be stored cold, during which time specificity was retained. Example m Detection method a Single-blind method Blood samples were obtained from the central hospital in Jäskilsna, Sweden, of which 42 were from patients in the infectious disease unit and 1 from the surgical unit. , 2 from the radiotherapy room, 1 from a postpartum woman, and 1 from a pregnant woman in her 3rd month of pregnancy. Six of the samples were from pregnant women who were six months pregnant.

Additionally, blood was obtained from 20 10-year-old children and one solid sample from an elderly healthy adult was obtained from a private clinic. Samples from 44 patients were obtained from a surgical and medical clinic in Stockholm, Sweden.
Materials were obtained from 45 patients at Ekechosov Hospital.
Blood samples were obtained from the Karolinska Sudjukhuset surgical clinic in Stockholm, Sweden, from 72 patients admitted for surgical treatment. The blood donation department at Karolinska Sjukvset in Stockholm, Sweden, has an average age of 37.2 years (SD
118 samples from donors ranging in age from 20 to 67 years were provided. The hospital's central clinical laboratory obtained serum from 29 patients undergoing treatment in the general department and 12 patients in the gynecology department of Radiumhemmet, Stockholm, Sweden. The total number of blood samples was 431. Clinical studies were carried out on 111 cases from Eskilsutna, and medical records were made, except for apparently healthy participants. For the Stockholm case, a detailed study of patient medical records was carried out for 101 cases. For the remaining cases, clinical diagnoses other than malignant were available, and malignancy was not suspected. Venous blood was transferred to Watsselmann tubes without additives and in some cases transferred to the laboratory after removal of aggregates and blood cells by centrifugal means.

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

In principle, the following method was used: 25 ml of patient serum absorbed with sheep red blood cells was added to Linbro IS-M old C.
-96 dilution plate, it was added dropwise in 8 steps of 2-fold dilution.

Then 25 molecules of specific antiserum at extreme dilution (approximately 1:2000) were added to each well. 2. After incubation for 1 powder at 0.0, the above example l
About 600 to 800, labeled with human albumen-bound CAPA, tanned and monomerized as shown in c.
Fifty volumes of a suspension of ten thousand sheep red blood cells were added to each well (approximately 2-4 volumes per 10 needles of red blood cells).

Other particulate carriers such as latex, bentonite and collodion may be used in place of red blood cells. The reaction was recorded with a tilted mirror and using a standard system after incubation for 4-2 hours at 1-2°C. The following were used for control purposes: I red blood cells plus antiserum labeled with CAPA, 2 CAP without antiserum
Red blood cells labeled in A, 3. Blood cells labeled with patient serum plus human albumen, 4. Patient serum and untreated blood cells, 5. A series of CAPN-labeled blood cells and antibodies (
In this case inhibition is given by a known amount of CAPA in decreasing steps).

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

The CAPbe reagent is produced from pooled human lumbar tissue.
As described above, gel filtration, mottled gradient melting, biogel P-13
It was purified by filtration through 0 and complex formation with albumen.

As antiserum, horse anti-hela cells from November 19, 1962 absorbed with normal tissue and pooled human serum were used. Immune is placed in a flat glass pin for 20 minutes.
It was performed using a refined fraction of Hela cells produced in Eagle's medium containing % inactivated human serum. The cells were harvested with EDTA and purified three times with water. In the presence of CAPA in the patient's serum, neutralized horse antibodies do not agglutinate even when voripeptide-confirmed 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 serum 1 kite can be read from Table 1 obtained by diluting previously determined amounts of CAPA in neutral serum. Table 1 Scores and CAPA Samesho scores 〃TACAPA/I0 Mi0.03 1 0. M1 0.08 2 0.091 0.12 3 0.13- 0.24 4 0.25- 0.49 5 0.50- 0.99 6 >1.0 This record provides information on the patient's health status. Created without knowledge.

The materials used in the study were divided into groups as shown in Table 2 below. Table 2. C in 431 sera from different populations with an overall score of 0 to 6 and a mean score of 0 to 6 (SD).
Existence of APA12. Pregnant woman 12 2 2
16 0.38±0.7213. Postpartum woman 8
1 1 10 0.40 ± 0.97 This result indicates that the highest scores are obtained for the groups consisting of “primary cancer and metastases” (local) and “extended cancer” and the “newborn” (cord blood) group. is obvious.

Low scores are obtained from blood donors.

The intermediate position is occupied by the group of "untreated primary cancer" and the group of "cancer not completely treated". Scores approximately equal to those of blood donors are found in the group of "completely removed rubs". The mean values for CAPA in all groups were compared in a setwise fashion and are shown in Table 3 below.

Table 3 Linear significance of differences between scores for the presence of CAPA in serum of nine solid categories a) Significance of differences between category numbers b) a) Significance of differences test is defined as This is done using an approximately normally distributed variable Z.

b) If Z>1.96, the difference is only significant, which means 0.01<P<0.05. If Z>2.6, the significance of the difference is xx, which is <0.001<P
Means <0.01. If Z>3.5, the significance of the difference is significant, which means P<0.001. c.
) This group includes 11 positive tests, of which 8
The case concerns suspected but proven cancer. From the table above, there is a significantly higher mean score for 'metastasized primary carcinoma' for 'cancer that has expanded rapidly' compared to all other groups with the exception of neonates. It is clear that there are (
see also Table 2).

For the "not completely treated Ren" group as well as the "untreated primary cancer" group, the mean scores were higher than those for all other groups than the first two listed, and for the "untreated primary cancer" group. significantly higher than other malignant diseases. Relative to this latter difference, the difference is significant (0.01 P x 0.0
5).

Groups 5-9 show no reciprocal significant differences except for the significant difference between 7 and 9. This difference was hidden in the cases showing scores between 1 and 4. This can be explained by the fact that there may be cases of cancer.A positive indication in the band blood deserves the attention of some crabs.

To confirm whether CAPA originates from the placenta ~
A piece of such tissue was extracted. Homogenize the frozen placenta of 9 crabs of 300 to 300 g in buffered physiological NaCI solution at pH 7-5.
In a triangular tube at 0°C for 0 minutes (Marcum)
Centrifuged. The presence of CAPA in this supernatant was tested by the hemagglutination technique described above. To eliminate the possibility that the resulting inhibitory reaction was caused by nonspecific inactivation of pure CAPA added to the blood cells, the blood cells were shaken and the CAPA added after each recording. A standard amount of antibody specific for was added. Antigen inactivation was not demonstrated in any case. As a control material, extracts of antigen powders produced from different origins of human bran tissue and from different locations were used. The result is 2 from a large number of individuals.
All mirror flea placentas without exception contain significant amounts of CAP.
It was shown that even if A is contained. The average value (geometric) for 26 placentas is ~297 soils per night of green tissue 8
Since the CAPA of the present invention is synthesized in the placenta, as is clear from the above study, the gene contained in the placenta is also derived from the same egg bream gun. It follows that the possibility and ability to produce tCAPA is a normal phenomenon since it must also be present in a mature individual.

Generally, this ability is suppressed. The reason is that normal cells do not contain measurable CAPA. However, in cancer cells, activation of this ability occurs, characterized by the significant presence of CAPA within and around the cells. The antigenic and to some extent functional similarities between placental trophoblast cells and cancer cells and their differences with respect to other cells in the individual are due to regulatory changes in the unique nature of bran diseases. It seems that it can coexist with the perspective of being a thing. Statistical analysis of experimental results
It shows that there is a highly significant correlation between the presence of a cancer diagnosis and increased CAPN fly frequency (QQQ weight).

The same is true for the relationship between possible gutter pseudomass size and increased CAPA concentration in the same age group. Increased concentrations of CAPA have been found for a wide variety of cancer sites. It was discovered that Nukagoshi pseudo, regardless of type and location, contains CAPA. From that point of view ~ the QQ that seems to be common to all known types of turbidity, i.e. malignant seeds of epithelial origin. A double-blind study was also conducted under strictly controlled conditions.

Blood samples with symbols were obtained from various departments of the central hospital in Erkilssna, Sütten. In this case, quantitative measurements of CAPA levels in patient serum were performed. The results of this double-blind test and the single test method described above are shown in Table 4 below.
8 vines are summarized.

In that table, Table 4 shows the total number of individuals examined and the number of individuals exhibiting groove 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 shows the percentage of individuals with serum CAPA concentrations of 0.15 and 0.15, but groups 9, 10 and 12 in Table 5 have been removed. Indicates the presence of CAPA in relation to the location of the tumor to the group.The location number is from Cancer Inc., which is incorporated herein by reference.
Table 4 Table 5 Number of individuals and average age of different groups and their deviations Table 6
Presence of CAPA in serum from 934 individuals Table 70.
% of individuals showing CAPA concentrations of 15, 9 and 10 in Table 5
and group 12 are excluded.

Table 8 Presence of CAPA related to seed wound location in groups 1-3 1.
Cancer with metastasis 2. 3. Untreated cancer without metastatic symptoms and completely treated cancer. completely removed cancer×) ancerIncidence in Swe
denl968”.

A complete statistical analysis of the data obtained from this double-blind study again shows that there is a highly significant correlation between the presence of a nodal diagnosis and increased CAP cardiac concentrations (
Pku0.001). This single-blind test also determined the size of the possible lower back wound mass and increased C in the same age group.
It also confirms the association between AP~concentration (P<0.00
1).

Increased serum levels of CAPA have been found to be associated with 2q-specific cancers of different types and locations. 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. 3,663-4 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, below is a detailed search for some of the property differences, both with respect to antigen single method and antigen properties. For purposes of summary, a brief definition of the cancer-associated polypeptide antigens and diagnostic tests of the present invention is provided below. CAPA Nr Sardine Hela cells (derived from a 1952 case of cancer) are grown in vitro.

These cells are used as a source of antigen. When the harvested cells are injected into a horse ~ specific antibodies are produced ~ this is imitated by ants ~
Blood tests are also used to identify antigens in placentas, etc. This latter antigen must have, and indeed does have, the same immunologically reactive site (compared to the hematopoietic antigen), although this of course does not exist in its natural state. If they are, they can differ in other ways. Diagnosis of the River Mystery Using antigens from Hela cell cultures in vitro to produce antibodies in horses It is used for the agglutination of red blood cells labeled with antigens extracted from cultured cells of HEP-2 or HEP-2 or Detroit 6.

This is an icicator type. If the unknown sample contains antigenic activity ~ it will interfere with the antibodies and inhibit winterization. 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 implementation of the invention as set forth in Patent Law No. 6 Haskan-3.

1 Homogenizing the CAPA-containing material in a liquid at a temperature below about 0° C. to extracting the solid from the mixture of the liquid homogenate and the solid component obtained through the following steps: Separate ''c) Extract the solid content with an alkaline aqueous solution, 'd
} The extract obtained in step 'c' was divided into 10 × 1 to 20
The gel is filtered on a bead-shaped molecular sieve type filtering gel that makes it possible to fractionate compounds with a molecular weight range that includes 1 × 1 and especially total i5 × 1.
About 15 x 1 fractions are collected, and the fractions obtained from step [e] and [d] are subjected to chromatography on a weak ion exchanger. The impurities are adsorbed and the first fraction coming out of the exchanger is collected and the first fraction is subjected to isoelectric focusing precipitation (g
) Mixing the resulting precipitate with a molecular sieve type gel and subjecting said gel to pH gradient elution.
229 in it up to a pH of about 3 while decreasing H.
It has a spectral absorption peak wavelength of ~23m and about 20,
CAP having a molecular weight within the range of 000 to 277,000
A single method for cancer associated polypeptide antigen (CAPA) comprising steps of collecting fractions containing A.

2. The fractions obtained from the pH gradient elution were subjected to gel filtration on a gel column and the antigen having a spectral absorption peak wavelength of 9 to 2 m and a molecular weight within the range of about 20,000 to 27,000 was obtained. 2. The method according to item 1 above, wherein the eluted fraction is collected.

3 The homogenate obtained from step 'a) ~ step {b
} The method described in item 1 above is subjected to organic solvent treatment while cold.

4. The solvent used is ethyl ether.
The method described in section.

5. The method recited in Paragraph 1 above, wherein the homogenization operation is carried out in water at a temperature of about 0°C or lower.

6. The method according to paragraph 1, wherein the solid obtained from step (b) is freeze-dried and then ground in a 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 water point of the material to be treated.

9. The method of claim 6, wherein the grinding is carried out at a temperature of about 17°C.

10 Steps of filtration are carried out on gels selected from the group consisting of polyacrylamide gels, cross-linked dictran gels, agar and agarose gels and equilibrated with about 7 ml of phosphate buffer. The method described in paragraph 1.

11. The method according to paragraph 1, wherein in step 'e the chromatography is carried out on a column of a neutral matrix weak ion exchanger.

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. Polyacrylamide gel, cross-linked dextran gel, agar and agar in step (g).

2. The method according to claim 1, wherein a gel selected from the group consisting of: 14. The method of item 2, wherein the gel heating is performed on a gel selected from the group consisting of polyacrylamide gel and cross-linked dextran gel.

15. The method of claim 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 qo to destroy the enzymes.

16. The method according to page 13, wherein the aqueous extract is rapidly heated to a temperature within the range of about 98 to 100 qo and maintained at this temperature for about 5 to 10 minutes.

17 While the antigen-containing material is still in a frozen state, heat it to approximately 0°C.
The resulting homogenate was homogenized in water at a temperature of The solid fraction is lyophilized and milled in a steel pole mill at a temperature below the freezing point of the material being processed, the solid obtained is extracted with an alkaline aqueous solution and after centrifugation the supernatant liquid is reduced to about 90%
Approximately 5 pm to rapid heating to a temperature within the range of 5 to about 100 pm
~ IQ minutes to destroy the enzymes, the heated supernatant solution was treated with an aqueous sodium chloride solution to precipitate contaminants, and the supernatant fluid was recovered and the resulting liquid was heated to about 10,000
0 to at least 20×1 and a pH of about
The central fraction of the eluate was collected by filtration through a lined column filled with bead-shaped molecular sieve type gel equilibrated with a phosphate buffer of pH 7.0, and the central fraction was adjusted to a pH of about 7.0. chromatography on a column packed with a weak ion exchanger molecular sieve type gel equilibrated with 0.0 phosphate buffer, and a first fraction of the liquid coming out of this column is collected and The fraction of about p
H5 and the resulting precipitate has a molecular weight removal limit of at least about 2,000 and has a molecular weight removal limit of about K5.
The mixture was mixed with a molecular sieve type gel equilibrated at an OOH/COON ratio, the resulting mixture was placed on a column packed with the same gel equilibrated in the same way, and the column was subjected to pH gradient elution. and while decreasing the pH to about pH 3.
Collecting a CAPA-containing fraction having a spectral absorption peak wavelength of 229-2 longitudinal skin eluted up to and a molecular weight of 23,200 ± 2,500 (S.D.),
Method for producing CAPA.

18 23 has a molecular weight of 200 ± 2,500 (S.D.) and exhibits a spectral peak absorption at wavelengths within the range of about 22 m to about 23 m and as shown by treatment with performic acid. Bran-associated polypeptide antigen (CAPA) is based on a single peptide chain and denatures irreversibly at pH above about 5 if not protected by complex formation with albumen.

19. Volibeptide contains the following mole % amino acids: 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
379 se IJn
7474 Threonine
592 Tyrosine
3421 Barin 63
20 to the CAP of the above la true description containing 6
The next step, namely, a) adding a fluorescent group to the skin as described in the first flame containing fluorescent groups that emit fluorescence of about 35 m when excited at a wavelength of about 2 m; Said first
preparing an aqueous solution of CAPA as described on page 8; b) step a);
c) injecting said oil emulsion into a living animal body, whereby the emulsion A method for producing a monospecific antibody with respect to CAPA, wherein the retained CAPA is transferred to an antibody-producing cell.

22. The method of No. 21, wherein in step b) Freund's adjuvant is used for oil emulsion production.

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 described on page 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 examined, the following steps, namely a. 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) dissolving said cultured samples after incubation. to each a predetermined amount of stabilized (complexed) CAPA supported by a particulate carrier, and d) the resulting series of treated samples were stabilized (complexed) Comparison with control samples of the antibody-containing antiserum of children scheduled with progressively decreasing amounts of CAPA.
said live animal body by subsequently adding to each of said control samples a predetermined amount of CAPA supported by a microparticulate carrier, and c) comparing said series of samples with said control samples. A method comprising determining the presence of CAPA in material to indicate the presence of cancer and its stage of progression.

26. The method according to page 28, 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 a human tumor pseudotissue.

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

30. The method according to page 28, wherein the material is a biopsy material, a surgical specimen, a puncture and a smear.

31. The method described on page 25, wherein the polypeptide antigen used is as described on page 18.

32. The method of page 29, 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 page 28 above, wherein the blood cells used in step c) are sheep red blood cells.

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

35. The method of the Third Heishin Yokyo, 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 page 38, wherein the composition comprises an oil emulsion in which an aqueous CAPA-containing solution of axis lower than about 3 constitutes an encapsulated phase and is protected by a surrounding oil phase.

．． 38. The oil emulsion is based on Freund's adjuvant - the composition described on page 36 above.

39. A composition comprising CAPA and stabilizing crab albumen in a mixture therewith.

40. The composition according to item 39, wherein the ratio of albumen to CAPA' is at least 200:1 in Dokoji.

41. A method of making a CAPA-albumen composition comprising mixing CAPA and albumen in a brewing solution and increasing the pH of the solution to cause interaction between these components.

42. The method according to item 41, further comprising the step of freeze-drying the paper product.

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

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

Claims (1)

[Claims] 1 CAPA (Cancer Associated Polypeptide Antigen) at various stages of cancer progression by examining materials selected from the group consisting of serum, tissues, secretions and extracts from the animal organism to be assayed. 0 pH HCOOH-
A gel column equilibrated with HCOONH_4 (the gel has a particle size of 100-200 mesh, an exclusion limit (Dalton) of 40,000 and an exclusion limit (Dalton) of 2,500-40,0
Cancer-associated polypeptide antigens having a molecular weight of 20,000 to 27,000 when filtered on a polyacrylamide gel with a fractionation range (daltons) of 0.00 (the polypeptide is within the range of 229 to 233 nm). It has a spectral peak absorption at the wavelength of
It contains phenylalanine and tyrosine as essential components and reacts monospecifically with antiserum produced by Hela cells and absorbed by normal tissues and human serum, the antigen being protected by a complex with albumen. (which otherwise denatures irreversibly at a pH above about 5) involves the following steps: (a) preparing a series of samples of said material by serial dilution; and (b) preparing said material by serial dilution; (c) adding to each of the samples a predetermined amount of antiserum containing antibodies specific for said antigen; (c) after incubation, each of said cultured samples has a predetermined amount of stabilized antiserum (supported by a particulate carrier); complex) CA
(d) stabilized (complexed) known progressively decreasing amounts of CAPA and a predetermined amount of said antibody-containing antiserum to inhibit the resulting series of treated samples; and a control sample, after which each of said control samples was given a predetermined amount of CAP supported by a microscopic carrier.
A and (e) determining the amount of CAPA in said live animal material by comparing said series of samples with said control sample.