NL8007128A - DETECTION OF HUMAN CANCER CELLS WITH ANTIBODIES FOR ANTIGENS FROM THE NUCLEOLI OF HUMAN CANCER CELLS. - Google Patents

Description

S 5773-1 Β. · ^ - - * Ρ ·· & c

Detection of human cancer cells with antibodies to antigens from the nucleoli of human cancer cells.

The invention relates to nucleolar antigens (antigens from nuclear bodies), which are found in a wide range of human cancers and do not occur in corresponding non-tumor tissues, as well as antibodies and antisera specific for this (these) nucleolar antigen (s) for diagnostic purposes.

Previous animal findings have indicated the presence of nuclear and nucleolar antigens in tumors, which do not exist in non-tumor tissues (RK Busch et al., Cancer Res. 34, 2362, 1974; Yeoman et al., Proc. Natl. Acad Sci. USA 73 , 3258, 1976; 3258, 1976; Busch and Busch, Tumori 63, 347, 1977; Davis et al. Cancer Res. 38, 1906, 1978; Marashi et al., Cancer Res. 39, 59, 1979). In these previous studies of the present inventors, antibodies were prepared for nucleoli of normal and rat neoplastic cells by immunization of rabbits (R.K.

Busch et al., 1-6; Busch and Busch, l.c .; Davis et al., L.c.). Clear nucleolar fluorescence in the acetone-fixed cells was demonstrated using the indirect immunofluorescence method. It was also found that the immunoprecipitin bands in Ouchterlony gels formed with antisera for Novikoff hepatoma nucleolar antigens. extracted from nucleikins of Novikoff hepatoma in the rat, different from the corresponding immunoprecipitin bands obtained with nucleolar antigens from the liver and nucleolar anti-liver antisera (Busch and Busch, 1.c.).

A further specificity was demonstrated by the fact that anti-tumor * nucleolar antiserum, which was absorbed with extracts of liver nuclei, gave positive nucleolar fluorescence in ascites cells of Novikoff hepatoma, but not in liver cells. Conversely, anti-liver nucleolar antiserum absorbed with nucleolar tumor extracts did not provide detectable nucleolar fluorescence with tumor, but positive fluorescence in liver nucleoli (Davis et al., L.c.).

Since immunofluorescence analysis indicated that differences could be observed in acetone-fixed tumor smears and smears from normal rat cells (especially after absorption of the antisera with normal liver nuclei and nucleoli), this antisera was attempted on nucleolar antigens to be used in rat tumors to examine corresponding tissue samples obtained from human tumors. Testing with antibodies to rodent tumor nucleoli showed that positive immunofluorescence 8007128 I - 2 - t 1 was not found in human tumor nucleoli. In connection with this, the present inventors started a new series of tests to find human nucleolar antigens. Positive immunofluorescence was found in human tumor tissues with antisera and antibodies for these novel nucleic oil preparations from human tumors. In this study, the antibodies were absorbed with nuclear placental sonicates, as well as with fetal calf serum (Busch et al., Cancer Res. 39, 3024, 1979; Davis et al., Proc. Natl. Acad. Sci. USA 76, 892, 1979; Smetana et al. Life Sci. 25, 227, 1979).

The invention is the result of research intended to use these novel human nucleoar antigens to detect a wide range of human novelizations (neoplasms).

Below, Table A summarizes the human tumors, where clear nucleolar immunoflorescence was found with the antibodies on human tumor nucleoli. The results of these studies confirmed the surprising finding that many human tumors contain a common nucleolar antigen that shows positive immunofluorescence with antisera or immunoglobulin fractions of such antisera (Busch et al., L.c.).

20 Table a

Clear nucleolar immunoflorescence in human tumors (Uit; Busch et al, 1979) X Carcinomas 25 1. Bladder, transitional cell 2. Brain astrocytoma gliobastoma 3. Spine, adenocarcinoma (4) 3Q metastasis: liver transplantable carcinoma (GW-39) 4. Exocrine gland, carcinoma 5. Esophagus, carcinoma of platelet-like cells 6. Liver, primary carcinoma 22 7. Lung; adenocarcinoma (2) "oat" cell (2) platelet-like cells (5) 8. Melanoma, malignant, cerebral metastases 8007128 «* * l - 3 - 9. Prostate, adenocarcinoma (4)« Μ 10. Skin: carcinoma of basal cells ( 2) platelet cell carcinoma (7) metastasis: lymph node 11. Stomach, adenocarcinoma metastasis: liver metastasis: lymph node 12. Thyroid, carcinoma (2) II Sarcomas 1. Myoblastoma, malignant, from lip metastasis' to cervical lymph node 2. Osteogenic sarcoma ( 3), biopsy, tissue culture 3. Synovial sarcoma 4. Lymphoma (4), no Hodgkins III Hematological Neoplasms 1. Hodgkins disease (Reed Sternberg, 5) 2. Leukemia: CLL (5), hairy cells (spleen) 3. Lymph , lymphocytic, spleen 4. Multiple myeloma (5) 5. Mycosis fungoides 6. Acute myelocytic leukemia (5) 7. Chronic myelocytic leukemia (5) 8. Acute monocytic leukemia (2) IV Cultures 1. Breast carcinoma 2. Adenocarcinoma of the spine 3. HeLa 4. HEp-2 5. Prostate, carcinoma (3) 6. Carcinoma of platelet cells (3) '

Note: The numbers in parentheses indicate the number of cases.

Negative results were generally obtained in the non-tumor tissues, benign tumors and inflammatory states, as shown in B below (Busch et al., l.c.).

Table B

Negative immunofluorescence in human tissues. (From: Busch et al., 1979) ~ ~ I Normal tissue 8007128 1 1

* V

- 4 - 1. Bladder 2. Bone marrow (hemoblastic lines, (5) a ^ 3. Chest 4. Reddish gray layer in centrifuged blood (3) 5 5. Gallbladder 6. Small intestine, cavities of Liéberkuhn 7. Large intestine 8 Kidney 9. Liver (2) 10 10. Lung (adjacent to tumor) 11. Lymph node 12. Lymphocytes, normal (2) 13. Pancreas 14. Pineal gland 15 15. Pituitary 16. Placenta 17. Prostate gland 18. Skin 19. Stomach 20 20. Thyroid II Benign growing tissues 1. Breast, adenoma 2. Parathyroid adenomas (2) 3. Prostate gland, hyperlasia (3) 25 4. Thyroid, adenomas (3) nodular goiter (2) III Inflammatory diseases 1. Chronic ulcerative colitis 2 Glomerulonephitis 30 3. Granulom and fibrosis of the lung 4. Liver - cirrhosis, hepatitis 5. Lupus profundus (mammary gland and skin) 6. Buleuse pemfigus 7. Peptic ulcer 35 8. Inflammatory hyperplasia lymph nodes (4) 9. Infectious mononucleosis (5 ) IV Cultures 1. Breast fibroblasts 2. Lymphocytes, stimulated with PHA a) Numbers in brackets indicate number of cases.

8007128 1 '- + - 5 -

These results, originally obtained by immunofluorescence, have been verified and extended by immunoperoxidase methods.

The invention is based on the surprising finding that common nucleolar antigens are found in a wide variety of human cancer cells, but do not exist in normal human cells. The antigens are proteins, which may have a regulatory function on the genes or other functions and persist in a perichromosomal site throughout cell division. Important aspects of the invention are the discovery of the common nucleolar antigens found in human cancer cells, the isolation and purification of the nucleolar antigens, the formation of antisera and antibodies specific for these antigens, diagnostic test methods using antisera and antibodies specific for these antigens to detect human cancer cells, and diagnostic equipment containing antibodies or antisera or both specific for these nucleolar antigens.

The antigens have been found in a wide variety of human cancers, including cancers of the central nervous system, gastrointestinal tract, genitourinal system, lung, skin, blood-forming tissues, and endocrine and exocrine glands. As examples of malignant cells in humans can be mentioned: HeLa cells, prostate carcinom, other carcinomas, carcomas and haematological neoplasms. The antigens can be extracted from nuclei or nucleoli of malignant cells in humans. The antigens have not been found in corresponding non-tumor tissues. Using the diagnostic methods of the invention to detect malignant cells, approximately 1% false negatives and 3% false positives were detected. The false negatives represent necrotic tumor tissues or, for unknown reasons, non-reactive tumors. The false positives are 2 cases of "preneoplastic tissues" and weak positives in some of the heart regions in "hyperplastic tissue". Two focal positive areas were identified as "preneoplastic areas" or focal neoplastic transformation in inflammatory tissues in the gastrointestinal tract.

The antigens have a major species and at least one and possibly more minor antigen species. The major antigenic species from human cancer cells (a) has a clear isoelectric point of 6.0-6.7 and about 6.3, as determined by isoelectric focusing, with pH 3-10 'and polyacrylamide gel; (b) has a molecular weight of about 50,000-60,000 daltons as determined by two-dimensional gel electrophoresis with a second 8007128 -6 -dimension of SDS (sodium dodecyl sulfate); (c) is partially strongly bound asm nuclear and nucleolar RNP and partially soluble in 0.01 M Tris-HCl, pH 8; (d) and is both nucleolar and extranucleolar, but remains "intranuclear" or linked to the chromosomes during cell division.

The second antigenic residue, detected, gives a pl of about 6.0 (determined by the same method as in the main antigen species) and its molecular weight is also 50,000-60,000 daltons. It is possible that this is a modified product of the main antigen, but it has not been determined whether it is structurally related. This antigen type 10 is present in a relatively smaller concentration than the main antigen type.

Antigens are also present in nucleolar ribonucleoprotein (RNP) particles, which are obtained by ultracentrifugation of the Tris extracts described below. The antigen present in these particles is more bound to proteins and ribonucleic acid (RNA) than the antigen in the Tris soluble fraction. It is not yet clear whether the antigens in the RNF particle are identical to those in the supernatant fraction, but their isoelectric points are the same and they absorb the antibodies to the antigens. Nucleolar antigens present in fibriels, probably from the network of the nuclear ribonucleo-20 protein, are also detected in cancer cells by immune light microscopy. These are additional nucleolar structures, which can represent elements from which the RNP particles are derived.

It remains to be determined whether the antigens represent a substance which is affected in high concentrations in cancer cells and in very low concentrations in non-cancer cells or whether they are fetal antigens, as previously found in nuclear comparative studies. antigens of Novikoff hepatoma in the rat and normal liver cells in the rat (Yeoman et al., 1976).

All steps for obtaining and analyzing samples of human tissue, blood and serum from human tumors and other tissues from patients suspected of cancer had been approved by the Human Research Committee at Baylor College of Medicine, Houston, Texas and associated hospitals. COUPES. From: Human tumors were obtained from frozen sections of surgical specimens, biopsy or conserved cryostat specimens mainly from the Department of Pathology of Houston Veterans Administration Medical Center and also from the Michigan Cancer Foundation, Detroit, Michigan and from the Department of Internal Medicine of the Imperial Karei University in Prague, Czechoslovakia. These sections were analyzed for the presence of necleolar antigens 8007128

J

- 7 - V * by methods of indirect immunofluorescence and immunoperoxidase.

The purification of the antigens was performed by extracting nuclei or nucleoli 6 times with 10 mM Tris HCl / 0.1 mH PMSF / pH 8 in an amount of 20 volumes on 1 volume of nuclei or nucleoli. The extract 5 was first washed at 27,000 x for 10 minutes. and then centrifuged at 100,000 x g for 16 hours. Ammonium sulfate of 40% saturation concentration was used to remove impurities. The 40-100% ammonium sulfate fraction was collected by centrifugation and dialyzed against 20 mM Tris HCl / pH 7.6. The antigens were chromatographed on 10 DE-52 cellulose columns (1 x 10 cm). The antigens were eluted in the fraction of 0.15 M NaCl / 0.1 mM PMSF with pH 7.6. Isoelectric focusing gels were used to identify and purify the antigens. These gels contain 4% acrylamide / 8M urea / 2% ampholines (pH 3.5-10). The antigens with pi 6.3 and 6.0, respectively, were excised from the gels.

15 Op. SDS (sodium dodecyl sulfate) gels, one main spot was found for each of these antigens.

Nuclei or nucleoli of HeLa cells were prepared by collecting HeLa cells from Spinner culture bottles (7-8 liters). The cells should be and should be 5 in the log phase (7-8 x 10 cells / ml). The cells were centrifuged at 800 x g for 8 minutes to form cell tablets. These cell tablets were suspended in phosphate buffered saline (PBS) (0.15 M NaCl, 0.01 M phosphate, pH 7.2) by gentle homogenization with a loose polytetrafluoroethylene stamper and centrifuged at 800 x g for 8 minutes. The cells were washed a second time with 25 PBS and the cell tablets were weighed. The cell tablets were homogenized by gentle homogenization in 20 volumes of reticulocyte standard buffer (RSB) with pH 7.4 and swelled on ice for 30 minutes. The cells were then centrifuged at 1000 x g for 8 minutes and resuspended in RSB buffer plus 1/20 volume of the detergent Nonidet P40 (10% in RSB) by gentle homogenization. The final amount of Nonidet was 0.5 volume%. The cells were homogenized with a Dounce homogenizer with 20-60 strokes until the cells were broken and the nuclei were released and cleared of cytoplasm. The cells were then centrifuged at 1000 x g for 8 minutes, resuspended in 0.88 M sucrose with 0.5 mM Mg acetate (20 x weight volume) by gentle homogenization and centrifuged at 1500 x g for 20 minutes. The resulting material contained the HeLa nuclei and these were used to prepare the antigen extracts described below. Nuclei from other human malignant cells can be similarly obtained.

8007128 «t - 8 -

To isolate nucleoli, the core tablet obtained as described above was then suspended by gentle homogenization in 0.34 M sucrose with 0.5 mM Mg acetate using 2 ml sucrose per gram of the original cells. The cores were treated with ultra-5 sonic vibrations (with a Branson device) with 10 second bursts (and 10 seconds rest). The total time was between 60 and 110 seconds. The released nucleoli were monitored by microscopic examination. To visualize the nucleoli, they were stained with azure C (a solution of 1% azure C in 0.25 M sucrose). The preparation should be free from cores at the end of the ultrasonic treatment. The fraction treated with ultrasonic vibrations was applied three times the volume of 0.88 M sucrose solution (without Mg acetate) and centrifuged 20 times at 1500 x g. The material obtained contained the HeLa nucleoli, which can be used as an immunogen.

Usually, a satisfactory cleaning was obtained by the above method (Busch and Smetana, 1970) and light microscopy showed that the quality of these preparations was essentially satisfactory. However, electron microscopy analysis revealed the presence of chromatin and impurities from the nucleus. The main problem for efficient purification of these preparations is the limited amount of original HeLa cells in the cultures, limiting the number of renewed purification steps. Nucleoli, which were prepared from preparations of 5-10 g of HeLa cells instead of the amounts of 0.5-1 g used in previous research, provided sufficient material for adequate purification.

The conditions for culturing the HeLa cells and isolating the placenta nuclei were practically the same as those previously described (Davis et al., 1979).

A Tris extract of HeLa was prepared by suspending the HeLa nuclei in NaCl-EDTA buffer, 10 x weight / volume, i.e. 1 gram of nuclei / 10 ml buffer (buffer: 0.075 M.lIaCl, 0.025 M Na-EDTA? PH 8j ImM PMSF).

The phenylmethylsulfonyl fluoride (PMSF) is used in a concentration of 100 mM in isopropanol. It is added to each solution before extraction. The suspension was homogenized with a Dounce homogenizer using 20 strokes and centrifuged at 3000 x g for 5 minutes. The supernatant was collected. The above extractions were repeated 2 more times with the core material. The NaCl-EDTA extract was not used in the present antigen study and was therefore removed. The core material was suspended in 10 times weight / volume 0.01 M Tris-HCl; pH 8; 1 mM PMSF and homogenized with a Dounce 8007128-9 "ff (direction 20 strokes homogenized, although it is noted that 0.01 M Tris-HCl with pH 7-9 is satisfactory. The supernatant was collected and on ice During the extractions with Tris, the nuclei broke and chromatin was released The breaking of the nuclei was monitored by micros-5 copic examination The material was resuspended in the Tris buffer and the nuclei were swelled on ice for 15 minutes "Then homogenized with a 20-stroke" Dounce "apparatus and centrifuged at 12000 xg for 10 minutes. The supernatant was saved. The material was resuspended and had a whitish-fluffy appearance. It was again washed with a" Dounce " homogenized with 20 strokes and centrifuged at 27000 xg for 30 minutes.

The supernatant was collected and combined with the previous supernatants obtained from the Tris extracts.

The Tris extracts were then concentrated with a 15 Amicon UM-10 or PM-10 Diaflo membrane. Generally, the volume at the beginning was about 50 ml and concentrated to 4-5 ml. The final protein concentration is about 4-5 milligrams / ml. The rabbit can be immunized with this Tris extract.

Using the above method, one can also prepare extracts from HeLa nucleoli and from nuclei or nucleoli of other malignant cells in humans.

For the Tris immunogen, dilute 250 µl Tris extract (4-5 mg / ml prepared as above) with 250 µl PBS and combine with Freund's auxiliary liquid as described below for the nucleolar immunogen.

Antibodies were prepared by immunizing rabbits with HeLa cell nucleoli preparations as follows: the HeLa nucleoli were weighed (20-30 mg, wet weight) and suspended evenly in 0.5 ml 0.01 M phosphate buffered saline, pH 7. 2. Then, they were mixed with 0.6 ml of Freund's complete vehicle (GIBCO) as follows: the nucleucle supernatants were placed in a 5 ml syringe and the Freund's vehicle in a second syringe. A needle 1.2 mm in diameter with the tip removed was attached to each syringe. The needles were then connected to a 1.2 mm inner diameter piece of polyethylene tube (Clay-Adams). The contents of the syringes were mixed until the preparation became thick and difficult to squeeze through the tube.

The rabbit was shaved back and an injection of 0.1 ml per site was administered intradermally at 6 sites. The remaining 8007128 s - 10 - 0.4-0.5 ml was injected half subcutaneously (under the loose skin on the upper back) and half injected intramuscularly (in the thigh muscle). The injections were given once a week for 3 weeks, the same amounts of nucleoli being administered each time. The first 5 blood draws were performed 7-10 days after the third week of immunization. Hen used a rabbit ear comb (Bellco) and a vacuum pump to collect the blood. The blood (about 45-50 ml) was allowed to clot at room temperature for 3-4 hours. The serum portion was removed from the tube and centrifuged with 1000 g for 30 minutes (this causes some free red blood cells to be sedimented). The clear serum was collected and then absorbed (or kept frozen until ready for absorption). The blood clot can be cooled overnight. This will release a few more ml of serum. The serum from each blood draw was tested for the presence of antibodies to the nucleoli by the indirect immunoflorescence method.

Other non-human hosts (eg, goats, sheep, horses, chickens, etc.) can be immunized with nucleoli preparations from human malignant cells to raise the antisera or antibodies to the nucleolar antigens of the invention. Antisera 20 can also be prepared by immunizing non-human host animals with extracts (eg, Tris extract) from nuclei or nucleoli of human malignant cells.

The absorption of antinucleolar antiserum was performed by first absorbing the rabbit antiserum with 20% normal human serum and 2Q% fetal calf serum (GIBCO). 20% normal human sefum was added to the rabbit antiserum (4 ml to 20 ml) and incubated for 1 hour with shaking in a water bath kept at 37 ° C. The flask was removed from the bath, 20% fetal calf serum (4.8 ml to 24 ml) was added and incubated for 1 hour with shaking in a water bath kept at 37 ° C. The flask 30 was removed and incubated for an additional hour at room temperature, mixing by gentle swirling every 15 minutes. The mixture was then centrifuged at 15000 x g for 30 minutes and the supernatant (absorbed serum) was removed and stored. The absorbed serum was converted to the immunoglobulin volxm (Ig) using the method described below for the (NH 2) 2 SO 2 precipitation of serum.

The Ig preparation from the nucleolar antiserum, which had been absorbed with normal human serum and fetal calf serum, was now absorbed with normal human tissue (placenta or liver). An 8007128 * * ** - 11 - volume volume of sonicate (material obtained by ultrasonication) of placenta nuclei in PBs 7.2 (10-15 mg egg whey / ml) was added to the absorbed nucleolar immunoglobulin (10 ml Ig plus 10 ml core sonicate) and the mixture was incubated for 1 hour with shaking in a water bath kept at 37 ° C. Then incubation was continued for an hour at room temperature, mixing through the flask every 15 minutes - gentle to swirl; then centrifuged at 15000 x g for 30 minutes.

The supernatant was collected and the absorbed Ig was reprecipitated in the manner described with (NH 2 SO 2. This Ig can be used as the final antibody product or further purified by chromatography on diethylaminoethyl (DEAE) cellulose as follows;

The Ig, which is present in the 0.01 M phosphate buffered saline with pH 7.2, is dialyzed against 0.0175 M phosphate buffer with pH 6.3 (without salt). After dialysis, centrifugation is carried out at 2500 x g for 20 minutes. The supernatant is placed on the DEAE column (20 mg of protein per gram of cellulose, Whatman DE 52). The IgG is eluted from the column with the 0.0175 M phosphate buffer. After elution, the IgG fraction is dialyzed against the 0.01 M phosphate buffered saline pH 7.2.

The same procedure was followed for the control serum, which consisted of the pre-immune serum obtained by drawing blood from the rabbit (or other non-human host animal before the immunization had started).

Rabbit immunoglobulin Ig was prepared as follows: a saturated (NH 2 SO 2 solution (760 g / liter) was prepared and an equal volume of cold saturated (NH 2 SO 2) was added dropwise to the antiserum with stirring. one white precipitate and this precipitate was allowed to aggregate in the cold for 1 * 2-2 hours with a magnetic stirrer. The precipitated antiserum was centrifuged at 3000 xg for 20 minutes. The supernatant was removed and the material was redistilled. suspended in PBS at pH 7.2 (about half the volume of the original serum). The solubilized material was placed in a dialysis bag and dialyzed overnight against 100 volumes of PBS (with magnetic stirring). dialysis bag was placed in fresh PBS (100 times the volume) the next morning and dialysis was continued for 6 hours The immunoglobulin was carefully removed from the dialysis bag and left for 20 minutes and centrifuged at 2500 x g. The supernatant was collected.

The immunofluorescence method 8007128-12 - described in the literature (RK Busch et al., 1974; Hilgers et al., 1972) was used in this study as follows: 150 µl of antinucleolar antiserum was applied in a 1:50 dilution with acetone-fixed HeLa cells or on fixed tissue samples (according to Hilgers et al. 1972; RK Busch et al., 1974). It may be necessary to use more than 150 µl if the tissue sample covers a large portion of the slide. The dilution of the antiserum (As) depends on the titer of the antibody (Ab). Other dilutions can also be used up to the point where the As or Ab preparations are too diluted to give the positive response to known positive cells (eg HeLa. 10 Slides were run at 37 ° C for 45-50 minutes incubated in a humid space (the humid chamber may consist of a large Petri dish, into which a moist paper towel is placed.) After incubation, the antiserum was washed from the slide by carefully adding PBS and the slides were placed in a container and 1 washed in BBS for 15 hours The PBS was changed 3 times, after 15 minutes, after 30 minutes and after 45 minutes Slides were removed from PBS and dipped 10 times in distilled or deionized water with rapid up and down movements. Slides were dried with cold air from a fan or hair dryer (2-3 minutes), being careful not to over-dry 150 µl of 20 fluorescein-labeled goat anti-rabbit serum (Hyland or Capp) el) in the 1:10 dilution on the slides and incubated 30-35 at room temperature in the humid chamber. The second antibody was removed from the slide by gentle washing with PBS. The slides were then washed in PBS for 1 hour, with 3 changes, after 15 minutes, 30 minutes, and 45 minutes; instead, after the first 15 minutes of washing, they can also be placed in fresh PBS and left in a refrigerator overnight. After final washing with PBS, the slides were dipped 10 times in deionized or distilled water with rapid up and down movements and dried with cold air from a fan or hair dryer (2-3 minutes). A 1: 1 ratio of glycerol and PBS was added to the cells or tissue sample and covered with a coverslip. The sample can be stored for several months if the coverslip is sealed with a sealant, eg uncoloured nail polish, and stored cold. The slide was then examined with a fluorescent 35 microscope. Nucleolar fluorescence was not observed with pre-immune immunoglobulin or pre-immune IgG fractions. The other immunological methods used were the same as those used in previous research (Kendall, 1938; Lowry et al, 1951; Dale and Latner, 1969; Laurell, 1972; Wallace et al, 1974; Marashi et al. 1979). For analysis 8007128 # · ·· I. - --13- of the nucleolar localization of the immunofluorescence, samples ** were placed in and outside phase contrast illumination while observing the fluorescence.

Instead of fluorescently labeled goat anti-rabbit serum, the immunoperoxidase method can also be used. For example, 150 µl of peroxidase-labeled goat anti-rabbit serum was added in the 1:10 or 1:20 dilution. Local peroxidase activity can be demonstrated with a number of redox dye systems suitable for light microscopy. - or electrical microscopy. Other enzymes can serve as markers for the indirect method, and peroxidase and other enzymes can be used directly by labeling the primary antibody.

Incubation medium according to Kamofsky is prepared as follows: Sufficient diaminobenzidine (Sigma) is weighed out and suspended in 0.05 M Tris = HCl with pH 7.6, so that the concentration is 0.5 mg / ml. A hydrogen peroxide solution of 0.02% (in the 0.05 M Tris-HCl buffer) is prepared. The 0.5 mg / ml DAB and 0.02% solutions are mixed in a 1: 1 weight ratio (this mixed solution was prepared fresh each time it was used and kept cold during the preparation). 200-300 µl of the mixture of DAB and HjCl2 are placed on the slide and incubated in a humid chamber at room temperature for 30 minutes.

After this incubation, the DAB and mixture are removed by washing the slide with the 0.05 M Tris-HCl buffer of pH 7.6, to which 0.1 M NaCl has been added. The slides are then washed 2 times for 10 minutes each in 0.05 M Tris-HCl with 7.6, which is 0.1 M NaCl. The processing of the microscope slides as indicated in steps 11-14 is terminated (except that the PBS is modified iii Tris-HCl). After completion, the slide is examined by light microscopy.

Microscope slides with HeLa cells for immunofluorescence were prepared as follows: A stock of fixed HeLa cells was prepared by removing and growing actively growing cells from the HeLa culture flask with PBS pH 7.2. The cells were suspended 6 'so that at least 1.5 x 10 cells / ml were present. 1 drop of the HeLa cell suspension was placed on each washed slide (cleaned with detergent, rinsed with distilled or deionized water and cleaned with alcohol, then rinsed and dried with hot air from a hair dryer) and spread slightly and left dry at room temperature (or overnight in the cold). The dried cells were fixed by placing the slides in acetone at 4 ° C for 12 minutes. Slides were numbered with a diamond marker marker for glass with diamonds.

8007128 - 14 -

The slides were used as a positive control for inmurio fluorescence.

The present study confirms that nucleolar anti-genes are present in tumor cells, but were not found in non-tumor tissue requirement. An initial study showed that, in both cell cultures of human tumors and in samples obtained by autopsy or biopsy, clear nucleolar fluorescence was obtained by the double antibody technique (indirect immunofluorescence) and that no similar result was achieved with a series of non- tumor tissues (Davis et al., 1979). Later studies examined more than 60 malignant growths 10 and also examined a series of control tissues. It is of great importance that this wide range of malignant growths of ectodermal, endodermal and mesodermal origin exhibits the presence of one or more common nucleolar antigens (Table A).

example I

Normal tissue requirement: No nucleolar fluorescence occurred in 17 non-tumor tissues after incubation of the antisera or antibodies with the different fixed cell preparations. Of particular interest, neither the Malpighian layer of the skin, nor the bone marrow cells, nor the Lieberkuhn cavities showed positive immunofluorescence in this method.

In addition, the various non-tumor tissues adjacent to the neoplasms were also negative; below are many fabrics of different types. A number of benign growths examined, including various types of thyroid adenomas, were also negative (Table B).

Example II

25. Inflammatory Damages: To determine whether an inflammatory response was associated with the appearance of these antigens, a study was conducted with 8 types of inflammatory tissue requirement. Most of them showed no detectable fluorescence in the nucleoli of the cells examined. However, sections in ulcerative colitis and gastric ulcer samples were found, showing positive nucleolar fluorescence. In particular, two or three sections of the ulcerative colitis were negative, while one showed markedly positive nucleolar fluorescence.

In the rat ulcer tissue, one of the two sections analyzed showed positive nucleolar fluorescence. These results are particularly interesting in connection with the known tendency of these lesions to change into malignant growths. Of particular interest was to review both the focal positive and negative regions of these platelets in the hematoxylin, with eosin stained sections; these showed that there were indeed areas in these lesions not only showing mitotic figures, but also an accumulation of the epithelial layer.

This possibly indicated that these cells could form preneoplastic lesions or carcinoma in situ. It is possible that the discovery of these fluorescent regions may assist in decisions to perform surgery with local or more general resections of the affected lesions.

. example lil

Artifacts: In the epithelium of the stomach, there was a region of fluorescence in each cell which was not nucleolar and which seemed to represent a nonspecific localization of the fluorescent antibody. In a cavity of the small intestine, nonspecific localization of the antibody was found to occur in the form of aggregates; in most cases, these aggregates were removed by pre-filtration of the antibodies through a 0.45 µm pore Millipore filter. In a sample of breast tissue negative on nuclear fluorescence, generally small, not -specific immunofluorescence spots' distributed without special localizing characteristics in terms of cell morphology. The diameters of these very small, nonspecific precipitates were 0.5-0.1 µm, whereas the nucleolar diameters in the nuclei and nucleoli were 4-6 µm.

v Example IV

Fluorescence-during cell cycle phases: Nuclear fluorescence was easily visualized at the nucleoli in-interphase. In the metaphase, nucleolar fluorescence was not observed as a clear unit, but was visible between the chromosomes and in the junction regions between the "nuclei" and the cytoplasm. Since the nucleolus largely disappears during the metaphase and the synthesis of rRNA in late prophase ceases, it was surprising that the nucleolar fluorescence was not visible as a distinct unit in such cells (Tan and Lerner, 1972).

However, the finding that residues of the immunofluorescence products persist during mitosis indicates that the nucleolar sub-structures (instead of the nuclearolar products) contain the antigens that persist epigenetically.

Example V

35 Malignant growths, negatives: In the series of malignant growths negative areas were found to varying degrees across all slides.

In general, these correlated with necrotic or abscess-affected parts of the neoplasms. In a brain tumor sample, the mass, which showed no positive fluorescence, was necrotic; many leukocytes 8007128 -16-t were present, but there was no particular structure. An adenocarcinoma, which gave brain metastases, showed no positive fluorescence; the reasons for this were not clear. Since 61 of the 63 tumors examined showed positive nucleolar fluorescence, 97% 5 of the series tested were positive. These studies have now expanded to more than 300 human cancer samples, including a range of cancerous growths of the breast, prostate, lung and hematological tumors, all with very similar results.

Example VI

10 Brands; For example, direct immunochemical methods of detecting the antibodies include labeling the primary antibody with one or more of the following markers: a radioisotope for autoradiography, e.g.

125 131 14 3 - X, .1, C, or H; a fluorescent dye, e.g. fluoresceins or tetramethylrodamine for fluorescence microscopy; an enzyme which gives a fluorescent or colored product for detection by fluorescence or light microscopy or which gives a product of high electron density so that it can be detected by electron microscopy; or a molecule of high electron density, e.g., ferritin, for direct visualization by electron microscopy.

Indirect immunochemical methods include labeling the second antibody or other binding protein, which is specific for the first antibody, with a fluorescent dye, with a high electron density compound, with an enzyme, which gives a product detectable is by light, fluorescence or electron micros-25 microscopic examination or a radioisotope detectable by autoradiography.

As indirect immunochemical methods for the visualization of the antibodies, use can be made of hybrid primary or secondary antibodies or antibody fragments (F (eb ') 2) / in which part of the hybrid antibody preparation is specific for the nucleolar antigens (hybrid primary antibody ) or for the primary antibody (hybrid secondary antibody) and a portion is specific for a label, e.g., those mentioned in the above paragraphs.

Labeled conjugated or unconjugated antibodies can be individually packaged in phosphate buffered saline (PBS) or other buffered suspending agents for distribution as diagnostic kits. Glycerol, heparin and sucrose can be mentioned as suitable suspending agents. Barbital buffers, 8007128, can be mentioned as suitable buffers. Morpholine drivers, MOPS [3- (N-morpholino) propanesulfonic acid], Hepes [N-2-hydroxyethylpiperazine N-2-ethanesulfonic acid], Tris carbonate and the like.

* Λ \ r% 8007128

Claims (29)

1. A method of immunologically detecting cancer in human tissue sections, smears and exfoliative cytological preparations from humans, using antibodies raised against human malignant nucleoli or extracts from nuclei or nucleoli of such cells, characterized in that samples are contacted with these antibodies and demonstration of the localization of these antibodies in the nucleoli of malignant cells but not of normal cells. 2. A method according to claim 1, characterized in that the antibodies are raised against nucleoli isolated from one of the types of malignant human cells: HeLa cells, carcinomas, sarcomas and haematological neoplasms. Method according to claim 1 or 2, characterized in that the antibodies are raised against nucleoli isolated from HeLa cells or cells of human prostate cancer. Method according to claim 1, characterized in that the antibodies are raised against extracts of nuclei or nucleoli of malignant human cells of the type: HeLa cells, carcinomas, sarcomas and haematological neoplasms. Method according to claim 4, characterized in that the antibodies are raised against extracts of nuclei or nucleoli of HeLa cells or cells of human prostate cancer. 6. Method according to claims 1 and 4, characterized in that the antibodies are raised against extracts of nuclei or nucleoli of malignant human cells against by extraction with 0.01 H Tris HCl at pH 7-9. Method according to claim 6, characterized in that the antibodies are raised against extracts of nuclei or nucleoli of HeLa cells or human prostate cancer. 8. A method according to claims 1-7, characterized in that the localization of the antibody is demonstrated by a direct or indirect immunochemical method. Method according to claims t-8, characterized in that the immunological detection of cancer is carried out with antibodies according to claim 1, by labeling the primary antibodies with one or more of the following markers: a radioisotope, which is detectable by autoradiography, a fluorescent dye, detectable by fluorescence microscopy, 8007128 • · 1. - 19 - an enzyme, which gives a fluorescent or colored product, which is detectable by fluorescence or light microscopy, an enzyme, which gives a product with a high electron density, which is detectable by electron microscopy and a molecule with a high electron density, which is detectable by make it directly visible with electron microscopy. Method according to claim 9, characterized in that radio-. . . ...... 125 131 14 3 isotope selects from I, I,, C and H. 11. Process according to claim 9, characterized in that the fluorescent dye is selected from fluoresceins and tetramethylrodamine. 12. Method according to claim 9, characterized in that the enzyme giving a fluorescent or colored product detectable by fluorescence or light microscopy, peroxidase, beta-galactosidase, alkaline phophatase or cytochrome C, is chosen, The method of claim 9. characterized in that the enzyme-produced dense molecule detectable by direct electron microscopy is ferritin, hemocyanin, virus particles or latex spheres. 14. Method according to claims 1-8, characterized in that the immunological detection of cancer is carried out by indirect immunochemical detection of the antibodies, wherein at least one labeled second antibody or other binding protein, specific for the primary antibodies or modifications thereof, applies, 15. A method according to claim 14, characterized in that the marker is selected from fluorescent dye, a compound with a high electron density and an enzyme, which gives a product detectable by examination by light microscopy, fluorescence microscopy or electron microscopy, and a radio isotope, which is detectable by autoradiography. 16. Indirect immunochemical method of visualizing the antibodies of claim 1, characterized in that one is hybrid. uses primary or secondary antibodies and / or antibody fragments ((F (ab ')), where part of the hybrid antibody preparation is specific for the hucleolar antigens (hybrid primary antibody) or for the primary antibody (hybrid second antibody) and part specific is for a marker, including a high electron density compound for electron microscopy detection, an enzyme which gives products detectable by light, fluorescence or electron microscopy, or a radioisotope labeled compound for autoradiography 8007128 - KI ff 17. A method of immunologically detecting cancer in human tissue sections, smears and exfoliative cytological preparations using modified or fragmented antibodies raised against nucleoli of malignant human cells or extracts of nuclei or nucleoli of such cells, characterized in that samples are contacted with these modified or fragmented antibodies and the localization of the modified antibodies or antibody fragments is demonstrated by one of the following methods: light microscopy, fluorescence microscopy, electron microscopy and auto-radiography by direct and / or indirect immunochemical agents, the localization of which takes place in the nucleoli of the malignant human cells, but not in the nucleoli of normal cells. 18. Antigens related to human cancer cells, containing main and secondary species, the main species being the following. 15 has properties: an isoelectric focusing pi of about 6.0-6.7 and a molecular weight of about 50,000-60,000 daltons, soluble in 0.01 M Tris HCl at pH 8 and primarily present in the nucleoli. 19. The main antigen species according to claim 18 in practically purified form. 20. A method of preparing antibodies with specificity against antigens present in nucleoli and nuclei of human cancer cells, characterized by immunizing non-human host animals with the antigens of claim 18 and harvesting the antibodies from the immunized animal. Method according to claim .20, characterized in that antibodies are prepared with specificity against antigens which occur in nucloli of human cancer cells. 22. Method for preparing and isolating nucleolar anti-genes, characterized in that the antigens are extracted from nuclei or nucleoli of human cancer cells with 0.01 and Tris HCl with pH 7-9. Cell nucleus preparation containing Tris-HCl extracts from nuclei or nucleoli of human cancer cells. 24. Method for purifying nucleolar antigens, characterized in that they are successively purified by precipitation with ammonium sulphate, chromatography on a DEAE column, gel exclusion with Sephadex, cation exchange and gel chromatography with calcium phosphate and preparative isoelectric focus. 25. A method of purifying antibodies, characterized by 8007128 Λ '* S— - * • · .......... ................. Using antibodies obtained from a non-human host animal, in which they have been induced by immunization with a nucleolar antigen. 26. Diagnostic equipment characterized by antibodies specific against nucleolar antigens present in human cancer cells and labeled to be detectable by light, fluorescence or electron microscopy, indirect assay or autoradiography, in a buffered suspending agent or buffered solution. 27. Diagnostic equipment, characterized in that it contains unlabelled primary antibodies, which have specificity against nucleolar antigens present in human malignant cancer cells, in a buffered suspending agent or buffered solution, in addition to suitable reagents, whether or not labeled for detecting the primary antibodies in fixed human malignant cells by a method selected from: light microscopy, fluorescence microscopy, electron microscopy and autoradiography. 28. Human cancer cell related antigens containing a major and minor species, the major species having the following properties: an isoelectric focusing pi of about 6.0-6.7 and a molecular weight of about 50,000 -60,000 daltons, which is soluble in 0.01 M Tris HCl with pH 8 and is primarily located in de-nucleoli prepared according to the method of claim 22 or equivalent. The main antigen species of claim 18 in a substantially purified form prepared by the method of claim 24 or an equivalent thereof. * i 'y • · · * ·. «· 8 0 0 7 1 2 8