LU83015A1 - METHOD FOR THE DETECTION OF CANCER CELLS BY ANTIBODIES AND METHOD FOR THE PREPARATION THEREOF - Google Patents

Description

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The present invention relates to nucleolar antigens found in a wide range of human cancers and not found in corresponding non-cancerous tissues and to antibodies and antisera specific to this or these nucleolar antigens for diagnostic purposes.

Previous discoveries in experimental animals have indicated the presence in tumors of nuclear and nucleolar antigens that have not been found in * * non-cancerous tissue (RK Busch et cons *, Cancer Res. 34, 2362, 1974 ; Yeoman et cons., Proc. Natl. Acad. Sci. USA 73, 3258, 1976; Busch and Busch, Tumori (33, 347, 1977; Davis et cons., Cancer Res. 3J3, 1906, 1978; Marashi and cons ., Cancer Res. 3j3, 59, 1979) In these previous studies, antibodies were prepared for nucleoli of normal and neoplastic cells of rats by immunization of rabbits (RK Busch and cons · / supra; Busch and Busch, supra ; Davis et al., Supra) Broad nucleolar fluorescence has been demonstrated in acetone-bound cells by the indirect immunofluorescence method, and it has also been found that the immunopre-vipitin bands in Ouchterlony gels formed by antisera to hepatoma nucleolar antigens ce Novikoff nucleic extracts ols of rat Novikoff hepatoma differ from the corresponding immunoprecipitin bands produced by liver nucleolar antigens and anti-liver nucleolar antisera (Busch and Busch, supra).

In addition, specificity appeared when antitumor nucleolar antiserum absorbed by nucleic extracts ^ i ^ / /

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3 liver res produced positive nucleolar fluorescence in Novikoff hepatoma ascites cells but not in liver cells. Conversely, anti-liver nucleolar antiserum absorbed by nucleolar tumor extracts "did not produce detectable nucleolar tumor fluorescence but produced positive fluorescence in liver nucleoli (Davis et al., Supra).

Insofar as an immunofluorescence analysis indicates that differences are observable in tumor smears and smears of normal rat cells, fixed by acetone, (in particular after absorption of the antisera by nuclei and nucleoli normal liver), attempts have been made to use these rat tumor nucleolar antigen antisera in the examination of corresponding tissue samples from human tumors. Studies with antibodies to rodent tumor nucleoli have shown that positive immunofluorescence has not been found in human tumor nucleoli. In view of this, we began according to the invention a new series of experiments to find the nucleolar antigens of humans. An immu- v. Positive nofluorescence was then found in human tumor tissues with antisera and antibodies to these new human nucleolar tumor preparations.

In these studies, the antibodies are absorbed by placental nuclear products acoustically treated as well as by serum from calf bovine (Busch et cens., 39,3 02 4, 197 9; Davis et al., Proc.

Natl. Acad. Sei. USA 16, 892, 1979; Smetana et cons., Life / -, /

Sei. 25, 227, 1979). ! f / il i r 4

The present invention results from studies designed to use these new human nucleolar antigens for the detection of a wide range of human neoplasms.

Table I below presents a summary of human tumors in which broad nucleolar immunofluorescence has been found with antibodies to human tumor nucleoli. These studies have led to the surprising and unexpected discovery that a large number of human tumors contain common nucleolar antigens which show positive immunofluorescence with antisera or immunoglobulin fractions of such antisera (Busch et al., Supra).

TABLE I

LARGE NUCLEOLAR IMMUNOFLUORESCENCE IN HUMAN TUMORS. (Provenance: Busch et cons., 1979) I. Carcinomas 1. Bladder, transition cell 2. Astrocytoma glioblastoma brain 3. Colon, adenocarcinoma (4) „metastasis: liver transplantable carcinoma (GW-39) 4. Exocrine gland, carcinoma 5. Esophagus, squamous cell carcinoma 6. Liver, primary carcinoma 7. Lung: adenocarcinoma (2) oat grain cells (2) squamous cells (5) / 8. Melanoma, malignant brain matastases / - / / / 9 Prostate: adenocarcinoma (4) f 7

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"* * I __ 5 110. Skin: basal cell carcinoma (2) squamous cell carcinoma (7) metastasis: lymph node" 11. Stomach: adenocarcinoma metastasis: liver metastasis: lymph node 12. Thyroid; carcinoma (2) II. Sarcoma s 1. Malignant myoblastoma of the lip metastasis to the cervical lymph node. 2. Osteogenic sarcoma (3), biopsy, tissue culture 3. Synovial sarcoma 4. Lymphoma (4), not Hodgkin III. Hematological neoplasms 1. Hodgkin's disease (see Sternberg, 5) 2. Leukemia: CLL (5), hairy cells (spleen) 3. Lymphocytoma, spleen 4. Multiple myeloma (5) 5. Dermatomycosis 6. Acute myeloid leukemia '(5 ) 7. Chronic myeloid leukemia (5) 8. Acute monocytic leukemia (2) IV. Crops 1. Breast carcinoma 2. Colon adenocarcinoma 3. HeLa 4. HEp-2 5. Prostate carcinoma (3) 6. Squamous cell carcinoma (3)

The figures in parentheses in the table above // • t 6 represent the number of cases.

In non-cancerous tissues, benign tumors and inflammatory conditions, negative results are generally obtained as indicated in Table II below (Busch et al., Supra).

TABLE II

NEGATIVE IMMUNOFLUORESCENCE IN HUMAN TISSUES (Provenance: Busch et cons., 1979) 'I. Normal tissue 1. Bladder 2. Bone marrow (hemotlastic lines, 5) 3. Chest 4. White clot blood (3) 5. Gallbladder 6. Small intestine, Lieberkuhn crypts 7. Large intestine 8. Kidney 9. Liver (2) 10. Lung (adjacent to tumor) 11. Lymphatic gland 12. Normal lymphocytes (2) 13. Pancreas: 14. Epiphysis 15. Pituitary gland * 16. Placenta 17. Prostate 18. Skin 19. Stomach 20. Thyroid II. Benign growth tissue 1. Breast adenoma 2. Parathyroid adenomas (2) 3. Prostate hyperplasia (Ξ) /

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4. Thyroid adenomas (3) /./ nodular goiter (2) Jf i '7 III. Inflammatory diseases 1. Chronic ulcerative colitis i 2. Glomerulonephritis! 3. Granuloma and fibrosis of the lung 4. Liver: cirrhosis, hepatitis 5. Acute lupus (mammary gland and skin) 6. Bullous pemphigus 7. Gastric ulcer 8. Inflammatory hyperplasia of the lymph glands (4), 9. Infectious mononucleosis. (5) ”1 IV. Cultures 1. Breast fibroblasts 2. Lymphocytes, stimulated with ΡΗΆ.

The figures in parentheses represent the number of cases.

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

The present invention is based on the surprising and unexpected discovery that common nucleolar antigens are found in a wide range of human cancer cells but are not found in normal human cells. Antigens are proteins which may have control over genes or other functions and which are persistent through mitosis at a perichromosomal location. Important aspects of the invention are the discovery of common nucleolar antigens found in human cancer cells , isolation and purification of nucleolar antigens, production of antisera and antibodies specific for these antigens, diagnostic methods using antisera and antiV ^ '- / 1 * 8 bodies specific for these antigens to detect human cancer cells, and a diagnostic kit containing either antibodies or antisera specific to these nucleolar antigens, or both.

The antigens have been found in a wide range of human cancers including cancers of the central nervous system, gastrointestinal tract, genitourinary tract of the lung, skin, blood-forming tissue and endocrine glands and exocrines. "For example, malignant human cells include HeLay cells, carcinoma" of the prostate, felt carcinomas, sarcomas and hematological neoplasms. Antigens can be extracted from nuclei or nucleoli of human malignant cells. The antigens were not found in corresponding non-cancerous tissues. In using the diagnostic methods of the present invention to detect malignant cells, approximately 1% false negatives and 3% false positives have been detected. False negatives represent necrotic cancer tissue or non-reactive tumors for unknown reasons. False positives represent two cases of "preneoplastic tissue" and weak positives in occasional focal regions in "hyperplastic tissue". Two focal positive regions are identified as "preneoplastic regions" or as focal neoplastic transformation in inflammatory gastrointestinal tissues.

The antigens include a main species and at least one and possibly several minor antigen species.

. / The main antigen species from cancer cells - - ·· * - · -i-m'-jn .. * '· · - - - * · »*» - »······· ““ J i. ·. I "H% .ia-.tiu". J »i» »-» ι 11 —1 i '' —- * ~ 9 human lines (a) has a distinct isoelectric point pi of the order of 6.0 to 6.7 and approximately 6.3, when '' it is determined by isoelectric concentration, pH 3 to 10, polyacrylamide gel, (b) has an approximate molecular weight of 50,000 to 60,000 daltons as determined by two-dimensional gel electrophoresis with a second SDS dimension ( sodium dodecyl sulphate), (c) is partly closely linked to nuclear and nucleolar ribonucleoprotein (RNP) and partly soluble in 0.01 M Tris-HCl, pH 8, and (d) is both nucleolar and extranucleolar, but remains "intranuclear" or associated with a chromosome during cell division.

The second species of antigen that has been detected has a pI of approximately 6.0 (detected by the same methods as for the main species of antigen) and its molecular weight is also 50,000 to 60,000 daltons. It may be a modified product of the main antigen, but it has not been determined if it is structurally related. . The minor antigen species is in relatively smaller concentration than the main antigen species.

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

It remains to be determined whether the antigens represent a substance which is present 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 comparative studies on antigens of rat Novikoff hepatoma and normal rat liver cells (Yeoman et al., 1976).

All phases for obtaining and analyzing human tissue, blood and serum samples from human tumors and other tissue from suspected cancer patients are approved by the Human Research Committee of Baylor College of Medicine, Houston, Texas and affiliated hospitals. The sections of human tumors are obtained from frozen sections of surgical specimens, biopsy or specimens of pre-served cryostats, mainly from the Department of Pathology of the Houston Veterans Administration Medical Center, and also from the Michigan Cancer Foundation of Detroit , Michigan, and the Department of Internal Medicine, Charles University, Prague, Czechoslovakia. These sections are analyzed for the presence of nucleolar antigens by indirect immunofluorescence techniques and with immunoperoxidase. // i · * 11

The purification of the antigens is obtained by extraction of nuclei or nucleoli using 10 mM Tris HCl / 0.1 mM PMSF / pH 8 six times in a ratio of 20 vo-j lmes for 1 volume of nuclei or nucleoli. The extract is centrifuged first at 27,000 x g for 10 minutes and then at 100,000 x g for 16 hours. Ammonium sulfate is used at 40% saturation to remove contaminants. The ammonium sulfate fraction at 40 - 100% 1 is collected by centrifugation and dialyzed against

20 mM Tris HCl / pH 7.6. The antigens are chromatographed on DE-52 cellulose columns (1 x 10 cm). The antigens are eluted in the fraction of 0.15 M NaCl / 0.1 mM PMSF / pH

7.6. Isoelectric concentration gels are used to identify and purify the antigens. These gels contain 4% acrylamide / 8M urea / 2% ampholines (pH 3.5-10). The antigens, at pi of 6.3 and 6.0 respectively, are removed from the gels. On sodium dodecyl sulfate (SDS) gels, a main spot was found for each of these antigens.

HeLa cell nuclei or nucleoli are prepared by harvesting HeLa cells from Spinner culture bottles (7 to 8 liters). The cells can be and are in a stem phase of 7-8 x 10 cells / ml. The cells are centrifuged at 800 x g for 8 minutes to form cell pellets or pellets. The cell pellets are suspended in a phosphate-buffered saline solution (PBS) (0.15 M NaCl, 0.01 M phosphate, pH 7.2) by gentle homogenization with a pestle. Loose Teflon and centrifuged 12 to 800 xg for 8 minutes. The cells are washed a second time with PBS and the cell pellets are weighed. The cell pellets are suspended, with gentle homogenization, in 20 volumes of standard reticulocyte buffer (RSB), at a pH of 7.4, and allowed to swell for 30 minutes on ice. The cells are then centrifuged at 1000 x g for 8 minutes and resuspended, with gentle homogenization, in RSB buffer plus 1/20 of the volume of the detergent, Nonidet P40 (10% in RSB). The final volume of Nonidet is 0.5%. The cells are homogenized with a 20-60 beat Dounce homogenizer until the cells are broken and the nuclei are released and released from the cytoplasm. The cells are then centrifuged at 1000 xg for 8 minutes, resuspended, with gentle homogenization, in 0.88 M sucrose, 0.5 mM Mg acetate (20 x weight-volume) and centrifuged at 1500 xg for 20 minutes.

The resulting pellet contains the HeLa nuclei which are used to prepare the antigen extracts described below. The nuclei from other human malignant cells can be obtained in a similar way.

For nucleolus isolation, the nuclear pellet as prepared above is then suspended, with gentle homogenization, in 0.34 M of sucrose, 0.5 mM Mg acetate, using 2 ml of sucrose for each gram of original cells. The nuclei are treated acoustically (with a Branson acoustic device) with pulses of 10 seconds (and a rest of 10 seconds). The time / total is between approximately 60 to 110 seconds. The released nucleoli // * · 13 are checked by microscopic examination. To visualize the nucleoli, they are stained with Azur C (the solution consists of 1% Azur C in 0.25 M of sucrose). The preparation must be free of pits at the end of the acoustic treatment period. The fraction subjected to an acoustic treatment is placed below three times its volume of 0.88 M of sucrose (without Mg acetate) and centrifuged at 1500 x g for 20 minutes. The resulting pellet contains HeLa nucleoli which can be used as an immunogen.

Satisfactory purification is usually achieved with the above process (Busch and Smetana, 1970) and light microscopy has shown that the quality of these preparations is essentially satisfactory. However, an electron microscope analysis indicated the presence of chromatin and elements of nuclear contamination. The key problem in the proper purification of these preparations is the limited amount of original HeLa cells in the cultures, which limits the number of repurification phases. The nucleoli prepared from 5 to 10 g of HeLa cell preparations, rather than the amounts of 0.5 to 1 g used in the previous studies, provide sufficient material for proper purification. The growth conditions of HeLa cells and the isolation of placental nuclei are essentially the same as those previously reported (Davis et al., 1979).

A Tris extract of HeLa is prepared by suspending the HeLa nuclei in a NaCl-EDTA lo × weight /// buffer. / "· 14 volume, 1 g of nuclei / 10 ml of buffer (buffer: 0.075 M NaCl, 0.025 M Na EDTA / pH 8, 1 mM PMSF). Phenylmethylsulfonyl fluoride (PMSF) is produced at a concentration of 100 mM in isopropyl alcohol. It is added to each solution before extraction. The suspension is homogenized with a 20 beats Dounce homogenizer and it is centrifuged at 3000 x g for 5 minutes. The supernatant is collected. The above extractions are repeated on the nuclear pellet two more times. The NaCl-EDTA extract is not used in the present antigen treatment; therefore it is evacuated. The nuclear pellet is suspended in 10 x weight / volume of 0.01 M Tris-HCl, pH 8, 1 mM PMSF and homogenized with a Dounce homogenizer for 20 beats, although 0.01 M Tris -HCl, pH 7-9, is satisfactory. The supernatant is collected and stored on ice. During Tris extractions, the nuclei break and the chromatin is released. Nuclear fragmentation is checked by microscopic examination. The pellet is resuspended in a Tris buffer and the pits are left to "swell" for 15 minutes on ice. They are then homogenized on a Dounce homogenizer for 20 beats and centrifuged at 12,000 x g for 10 minutes. The supernatant is kept. The pellet is resuspended and has a fluffy whitish appearance. It is homogenized again in a Dounce homogenizer for 20 beats and centrifuged at 27,000 x g for 30 minutes. The supernatant is collected and combined with the previous supernatants / // from the Tris extracts. /// i Ù K »15 β ·

The Tris extracts are then concentrated using an Amicon UM-10 or PM-10 Diaflo membrane. Generally, the volume at the beginning is about 50 ml and it is concentrated to 4-5 ml. The final protein concentration is approximately 4 to 5 mg / ml. The rabbit can be immunized with this extract with Tris.

Following the above process, extracts can also be prepared from HeLa nucleoli and from nuclei or nucleoli of other human malignant cells.

For the Tris immunogen, 250 μl of Tris extract (4 to 5 mg / ml) as prepared above is diluted with 250 μl of PBS. This is combined with Freund's adjuvant as described for the nucleolar immunogen below.

Antibodies are prepared by immunizing rabbits with HeLa cell nucleolar preparations, as follows: HeLa nucleoli are weighed (20 to 30 mg, wet weight) and suspended uniformly in 0.5 ml of saline buffered - with 0.01 M phosphate, pH 7.2. They are then mixed with 0.6 ml of complete Freund's adjuvant (GIBCO), as follows: the nucleoli in suspension are placed in a 5 ml syringe and the Freund's adjuvant in a second syringe. In each syringe, an 18 gauge needle, with the tip removed # is attached. The needles are then connected by a piece of polyethylene tube, I.D. 0.047 (Clay-Adams). The contents of the syringes are mixed until the preparation becomes thickened and difficult to force through the tube. ^ ^ /

The back of the rabbit is shaved and injected intramedrically 7/16, in six places, 0.1 ml / place. The remaining 0.4 to 0.5 ml is injected, half subcutaneously (under the loose skin of the upper back) and half intramuscularly (into the thigh muscle). The injections are given once a week for 3 weeks with similar amounts of nucleoli each time. The first bleeding is done 7-10 days after the third week of immunization. A rabbit ear cup (Bellco) and a vacuum pump are used to collect blood. The blood (approximately 45 to 50 ml) is allowed to clot for 3 to 4 hours at room temperature. The serum part is removed from the tube and centrifuged at 1000 x g for 30 minutes (which sediments any free red blood cell). The clear serum is collected and is then absorbed (or kept frozen until it is ready for the absorption process). The blood clot can be refrigerated overnight. This releases a few extra ml of serum. The serum of each bloodletting is examined for the presence of nucleolar antibodies. by the indirect immunofluorescence method.

Other non-human hosts (eg, goat, sheep, horse, chicken, etc.) can be immunized with nucleolus preparations of human malignant cells in order to detect antisera or antibodies to. nucleolar antigens according to the present invention. The antisera can also be prepared by immunization of non-human host animals using extracts (for example a Tris extract) from nuclei or nucleoli of human malignant cells.

/ The absorption of antinucleolar antiserum is carried out /! / "17" by a first absorption of rabbit antiserum with 20% normal human serum and 20% fetal calf serum (GIBCO). 20% of the normal human serum is added to the rabbit antiserum (4 ml / 20 ml) and incubated for 1 hour in a stirred water bath at a temperature of 37 ° C. The bottle is removed, 20% fetal calf serum (4.8 ml / 24 ml) is added and incubated for 1 hour in a stirred water bath at a temperature of 37 ° C. The bottle is removed and incubated for an additional 1 hour at room temperature while mixing with. »Moderate brewing every 15 minutes. It is then centrifuged at a speed of 15,000 x g for 30 minutes and the supernatant is separated and stored (absorbed serum). The absorbed serum is converted into the immunoglobulin (Ig) form following the method given for the precipitation with (NH ^^ SO ^ of the serum, described below.

The Ig preparation from nucleolar antiserum, which has been absorbed by normal human serum and fetal calf serum, is now absorbed by normal human tissue (placenta or liver). An equal volume of acoustically treated placental nuclear product in PBS 7.2 (10 to 15 mg protein / ml) is added to the absorbed nucleolar immunoglobulin (10 ml of Ig plus 10 ml of nuclear acoustic treatment product) and incubated for 1 hour in a stirred aqueous bath at a temperature of 37 ° C. It is then left to incubate for an additional 1 hour at room temperature while mixing by moderate mixing of the flask every 15 minutes and then centrifuged at 15,000 x g for 30 minutes. The supernatant is collected and the absorbed Ig is reprecipitated with (NH ^ J ^ SO ^ g6zil · - // 18 * "describes me. This Ig can be used as the final antibody product or it can be further purified by chromatography with diethylaminoethyl (DEAE) cellulose, as follows:

The Ig, which is contained in the 0.01 M phosphate buffered saline solution and having a pH of 7.2, is dialyzed against a 0.0175 M phosphate buffer having a pH of 6.3 (without saline solution). After dialysis, it is centrifuged at 2500 x g for 20 minutes. The supernatant is added to the DEAE column (20 mg of protein per gram of cellulose, Whatman -. DE52). 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, having a pH of 7.2.

The same procedure is followed for the control serum which consists of preimmune serum which is obtained by bleeding from rabbits (or from another non-human host animal) before the immunization is started.

Immunoglobulin Ig this rabbit is prepared as follows

A saturated solution of (NH 4) 2 SO 4 (760 g / l) is prepared and an equal volume of cold saturated (13 4) 2 SO 4 is added dropwise to the antiserum, with stirring. A white precipitate forms and the precipitate is allowed to aggregate for 1.5 hours to 2 hours on a magnetic stirrer, when cold. The precipitated antiserum is centrifuged at 3000 x g for 20 minutes. The supernatant is removed and the pellet or pellet is resuspended in PBS, at pH 7.2 (approximately half the volume of the original serum). The solubilized pellet is placed in a dialysis tank and is dialyzed against 100 volumes of PBS, overnight, cold (with magnetic stirring).

The dialysis tank is placed in fresh PBS (100 x volume) the next morning and dialysis is continued for 6 hours.

The immunoglobulin is carefully removed from the dialysis tank and centrifuged at 2500 x g for 20 minutes. The supernatant is collected.

The previously described method (RK Busch et al., 1974? Hilgers et al., 1972) for immunofluorescence is used in this study, as follows: 150 μΐ of antinucleolar antiserum diluted to 1/50 are placed on HeLa cells fixed with acetone or on fixed tissue specimens (de Hilgers et al. 1972 7 RK Busch et al., 1974). It may be necessary to use more than 150 μΐ if the tissue specimen covers a large section of the coverslip. The dilution of the antiserum (As) depends on the antibody titer (Ab). Other dilutions can be used to the point where dilutions of As or Ab become too dilute to provide a positive response to known positive cells (e.g. HeLa). The slides are incubated in a humid chamber for 45 to 50 minutes at 37 ° C. (the humid chamber can consist of a large Petri dish to which a damp paper cloth has been added). After incubation, the antiserum is removed by washing the coverslip by the gentle addition of PBS and the coverslips are placed in a coverslip holder and washed in PBS for 1 hour. The PBS is changed three times, at 15 minutes, 30 minutes and 45 minutes. The coverslips are removed / from PBS and immersed in distilled or deionized water, ten '// 4.% 20 times, by rapid movements up and down. The strips are dried in cold air in a blower or hair dryer (2 to 3 minutes) and care is taken not to dry them excessively. 150 μΐ of goat antilapin antiserum labeled with fluorescein (Hyland or Cappel) diluted 1/10 are placed on the coverslips and left to incubate in the humid chamber for 30 to 35 minutes at room temperature. The second antibody is removed from the coverslip by gentle washing with PBS. The slides are then washed in PBS for 1 hour with three changes, at 15 minutes, 30 minutes and 45 minutes; or after the first wash at 15 minutes, they can be placed in fresh PBS and left in the refrigerator overnight. After the final wash with PBS, the slides are immersed in deionized or distilled water ten times with rapid movements up and down and dried in cold air using a blower or hair dryer (2 to 3 minutes). A solution of glycerol and PBS in a ratio of 1/1 is added to the cells or to the tissue specimen and covered with a cover slip. The specimen can be preserved for many months if the coverslip is sealed with a sealing agent, such as clear nail polish, and if kept cold. The coverslip is then examined under a fluorescence microscope. Nucleolar fluorescence is not observed with pre-immune immunoglobulin or pre-immune IgG fractions. The other immunological techniques used are the same as those used in previous studies (Kendall, 1938; Lowry et cons., 1951 /

Dale and Latner, 1969; Laurell, 1972; Wallace et al., 1974 / y / "% 21

Marashi et al., 1979). For the analysis of the nu-cleolar localization of the immunofluorescence, the samples are passed in and out of a phase contrast illumination during the observation of the fluorescence.

Instead of the fluorescein labeled goat antilapin, the immunoperoxidase method can be used. For example 150 μΐ of a 1/10 or 1/20 dilution of peroxidase-labeled goat antilapin are added. Localized peroxidase activity can be demonstrated by a number of redox dye systems for examination under an optical or electron microscope. Other enzymes can be used as tags for the indirect method and peroxidase and other enzymes can be used directly by labeling the primary antibody.

A Karnofsky incubation medium is prepared as follows:

Sufficient diaminobenzidine (Sigma) is removed and weighed so that, when suspended in 0.05 M Tris-BCl, pH 7 ', 6, the concentration is 0.5 mg / ml. A 0.02% hydrogen peroxide solution is prepared (in 0.05 M Tris-HCl buffer). The 0.5 mg / ml diaminobenzidine (DAB) and the 0.02% H 2 O 2 are mixed in equal proportions, in a ratio of 1/1 (this solution is freshly prepared each time it is used and it is stored at cold during preparation). 200 to 300 μΐ of the mixture of DAB and H 2 O 2 are added to the coverslip and the mixture is left to incubate for 30 minutes in a humid chamber at room temperature.

After this incubation, the DAB mixture is removed

/ // <· * 22 * "of H2O by washing the coverslip with 0.05 M Tris-HCl buffer, pH 7.6, to which 0.1 M NaCl was added. The coverslips are subjected to two 10-minute washes in 0.05 M Tris-HCl, pH 7.6, 0.1 m NaCl. The treatment of the coverslips is finished as indicated in phases 11 to 14 (except that the PBS has been changed to Tris HCl). The completed coverslip is examined under an optical microscope.

; Slides of HeLa cells are prepared for immunofluorescence, as follows: a reserve sample of fixed HeLa cells is prepared by separation and washing with PBS, at pH 7.2, of cells with active growth at from the HeLa culture bottle. The cells are suspended so that there are at least 1.5 x 10 cells / ml. A drop of the HeLa cell suspension is placed on each washed slide (cleaned with detergent, rinsed with distilled or deionized water, cleaned with alcohol, rinsed and dried with hot air using a hair drier), spread it out slightly and let it dry at room temperature (or in the cold overnight). The dried cells are fixed by placing the coverslips at 4 ° C in acetone for 12 minutes. The slats are numbered using a diamond-tipped glass marking pen. The coverslips are used as positive controls for immunofluorescence.

The present studies confirm that nu-cleolar antigens are present in cancer cells but are not found in non-cancerous tissue. Initial studies have shown that, both in cultures. . / 7 cell of human tumors and in specimens in pro- / f * * 23 "coming from samples either of autopsy or biopsy, a large» »nucleolar fluorescence is produced by the double technique with antibodies (indirect immunofluorescence) and that a corresponding result is not obtained with a series of non-cancerous tissues (Davis et al., 1979). In the latest studies, more than 60 malignant tumors have been studied and a wide variety of tissue controls have also been evaluated. It is of great interest that this large series of malignant tumors of ectodermal, endodermal and mesodermal origin shows the presence of one or more common nucleolar antigens (Table I).

Example 1

Normal fabrics. In 17 non-cancerous tissues, there is no nucleolar fluorescence following incubation of the antisera or antibodies with the various preparations of fixed cells. It is of particular interest that neither the Malpighi layer of the skin, nor the cells of the osseous cell, nor the Leiberkuhn crypts show positive immunofluorescence with this method. In addition, the variety of non-cancerous tissue adjacent to the neoplasms is also negative; this includes several fabrics of different types. A group of benign tumors examined, including several types of thyroid adenomas, is also negative (Table II).

Example 2 Inflammatory lesions. To find out whether an inflammatory response is related to the appearance of these antigens, studies have been done on eight types of inflammatory tissue.

/

In most of these, there is no noticeable fluorescence: // 24 "in the nucleoli of the cells studied. However, sections have been found in specimens of ulcerative colitis and gastric ulcer which show positive nucleolar fluorescence. It should be noted that two of the three sections of ulcerative cclitis are negative and only one showed a definite positive nucleo-fluorescence. In gastric ulcer, one of the two sections analyzed shows positive nucleolar fluorescence.

• These results are particularly interesting given the known propensity of these lesions to undergo a malignant change.

It is of special interest to review both the positive and negative focal regions of these lamellae in sections stained with hematoxylin-eosin, these show that there are indeed regions in these lesions which show not only mitotic forms but also a heap of epithelial coating. This discovery suggests that these cells may constitute pre-neoplastic lesions or carcinomas in situ. It is possible that the discovery of these fluorescent regions may help in the decisions to be made to proceed by surgery with either local or more general resections of the lesions concerned.

Example 3

Artifacts. In the gastric epithelium, there is a region of fluorescence in each cell which is non-nucleolar, that is to say which appears to represent a non-specific localization of the fluorescent antibody. In a crypt of the small intestine, a non-specific localization of the antibody seems to occur in the form of aggregates; in most cases, a prefilteration of the antibodies through a 0.45 µm Millipore filter removes these aggregates. In a sample of breast tissue, which is negative for nucleolar fluorescence, small, nonspecific immunofluorescent spots are distributed generally with localization characteristics not special with respect to cell morphology. The diameters of these very small non-specific precipitates are from 0.5 to 0.1 μm in comparison with the nucleolar diameters in the nuclei and nucleoli which are from 4 to 6 μτη.

Example 4

Fluorescence during phases of the cell cycle. The nucleolar fluorescence is easily observed in the nucleoli of the interphase. In metaphase nucleolar fluorescence is not observed as a separate entity but is visible between chromosomes and the junction zone between "nucleus'1 and the cytoplasm. Since the nucleolus largely disappears during metaphase and where the synthesis of rRNA ceases in the final prophase, it is not surprising that nucleolar fluorescence is not visible as a separate entity in these cells (Tan and Lerner, 1972). However, the discovery that residues immunofluorescent products persist through mitosis suggests that nucleolar substructures (rather than nucleolar products) contain antigens that are epigenetically persistent.

Example 5 Malignant tumor negatives. In the series of malignant tumors, negative regions are found in various measures across the coverslips. In general, this corresponds either to the necrotic or purulent parts of the neoplasms. In a sample // »1 26« of a brain tumor, the mass which shows no positive fluorescence is necrotic; several leukocytes are present but there is no defined structure. An adenocarcinoma, which has sent metastases to the brain, does not show positive fluorescence, * the reasons are not clear. Insofar as 61 of the 63 tumors studied exhibit positive nucleic fluorescence, 97% of the series studied are positive. These studies have now been extended widely to more than 300 specimens of human cancer, including a range of breast, prostate, lung and haematological tumors, with very similar results.

Example 5

Marking. Direct immunochemical methods for demonstrating antibodies include labeling the primary antibody with one or more of the following marks: an r; 125 131 14 dioisotope for autoradxography, such as I, I, C, or 3 H; a fluorescent dye such as fluorescein or tetramethylrhodamine for microscopic observation by fluorescence; uh enzyme which produces a fluorescent or colored product to be detected by fluorescence microscopy or by optical microscopy, or which produces an electronically dense product to be observed under an electron microscope; or an electronically dense molecule, such as ferritin, for direct electron microscopic observation.

Indirect immunochemical methods include labeling the second antibody or other protein binding specific for the first antibody with a fluorinated dye-y cent, an electronically dense compound, an enzyme that produces ÿ y ψ. ί 27 “a product detectable by examination under an optical, fluorescent or electronic” microscope, or a radioisotope detectable by autoradiography.

Indirect immunochemical methods for the observation of antibodies include the application of hybrid primary or secondary antibody or fragment (F (ab ') 2) in which part of the hybrid antibody preparation is specific for antigens nucleolar (primary antibody. hybrid) or for the primary antibody (secondary hybrid antibody), and a part is specific for a mark, such as that mentioned in the preceding paragraph.

Labeled conjugated and unconjugated antibodies can be packaged separately in phosphate buffered saline (PBS) or other buffered suspending agents for distribution as diagnostic kits. As suitable suspending agents, mention may be made of glycerin, heparin or sucrose. As suitable buffers, mention may be made of barbital buffers, morpholine buffers, MOPS-3- (N-morpholino) -propane-sulfonic acid, hepes-N-2-hydroxypiperazine-N-2 acid. -ethanesulfonic, Tris carbonate and similar substances.

It should be understood that the present invention is in no way limited to the embodiments described above and that many modifications can be made thereto without departing from the scope of this patent.

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Claims (30)

1. A method of immunological detection of cancer in sections, smears and cytological preparations exfoliated from human tissue characterized by the use of antibodies brought to light in nucleoli of malignant human cells or extracts of nuclei or nucleoli of such cells, this process comprising bringing the samples into contact with said antibodies and demonstrating the localization of the antibodies in the nucleoli of malignant cells and not of normal cells. 2. Method according to claim 1, characterized in that the antibodies are highlighted in nucleoli isolated from cells of the family of malignant human cells comprising HeLa cells, carcinomas, sarcomas and hematological neoplasms. 3. Method according to claim 1, characterized in that the antibodies are highlighted in nucleoli isolated from cells of the group comprising HeLa cells or cells of 'carcinoma of the prostate of the human being. 4. Method according to claim 1, characterized in that said antibodies are highlighted in extracts of nuclei or nucleoli of malignant human cells originating from the family comprising HeLa cells, carcinomas, sarcomas and hematological neoplasms. 5. Method according to claim 1, characterized in that the antibodies are brought to light in extracts of nuclei or nucleoli from cells of the group comprising ^ HeLa cells or carcinoma cells of the prostate of the being / // I * "'* · 29 A human. * 6. Method according to claim 1, characterized in that the antibodies are highlighted in extracts of nuclei or nucleoli of malignant human cells obtained by extraction with 0.01 M Tris HCl, at a pH of 7 to 9 . 7. Method according to claim 1, characterized in that the antibodies are brought to light in extracts of nuclei or nucleoli originating from the group comprising HeLa cells or carcinoma cells of the prostate of the human being, obtained by extraction with 0.01 M Tris HCl, at a pH of 7 to 9. 8. Method according to claim 1, characterized in that the localization of the antibody is observed by one of the direct or indirect immunochemical methods. 9. Method according to claim 1, characterized in that the immunological detection of cancer by antibodies comprises the labeling of primary antibodies by one or more of the following marks: a radiosisotope detectable by autoradiography, a fluorescent dye detectable by fluorescence microscopy , an enzyme which produces a% or colored fluorescent product detectable by fluorescence or optical microscopy, an enzyme which produces an electronically dense product detectable by electron microscopy, and an electronically dense molecule detectable by direct electron microscopic observation. 10. Method according to claim 9, characterized in that the radioisotope is chosen from the group comprising / '. f 125 131T 14. 3 f f I, I, C, and H. // / ♦ 1 30 J 11. Method according to claim 9, characterized in that the fluorescent dye is chosen from the group comprising fluorescein and tetramethylrhodamine. 12. Method according to claim 9, characterized in that the enzyme which produces a fluorescent or colored product detectable by fluorescence or optical microscopy is peroxidase, ß-galactosidase, alkaline phosphatase "or cytochrome C. 13. The method of claim 9, characterized in that the electronically dense molecule detectable by direct electron microscopic observation is ferritin, hemocyanin, viral particles or latex spheres. 14. Method according to claim 1, characterized in that the immunological detection of cancer is carried out by indirect immunochemical demonstration of the antibodies, this method comprising the application of at least one second labeled antibody and of another specific binding protein for the . primary antibodies or their modifications. 15. The method of claim 14, characterized in that the brand is chosen from the group comprising a fluorescent dye, an electronically dense compound and an enzyme which produces a product detectable by examination under an optical, fluorescence or electronic microscope, and a radioisotope detectable by autoradiography. 16. indirect immunochemical method for observing the antibodies used according to claim 1, characterized in that it comprises the application of at least one of the antibodies ι <i 31 or fragments of hybrid primary or secondary antibodies "(F (ab ') 2), part of the hybrid antibody preparation being specific for nucleolar antigens (primary antibody <hybrid) or for the primary antibody (secondary hybrid antibody), and part being specific for a brand, comprising an electronically dense compound for observation under an electron microscope, an enzyme which supplies products detectable under an optical microscope, by fluorescence or electron, or a compound labeled with a radioisotope for autoradiography. 17. A method of immunological detection of cancer in sections, smears and cytological preparations exfoliated from human tissue, characterized by the use of modified or fragmented antibodies highlighted in nucleoli of malignant human cells or extracts of nuclei or nucleoli of such cells, this method comprising bringing samples into contact with said modified or fragmented antibodies, and demonstrating the localization of the modified antibodies or antibody fragments by optical microscopy, fluorescence microscopy, electron microscopy or autoradiography at using at least one of the direct or indirect immunochemical means, the localization taking place in the nucleoli of malignant human cells and not in the nucleoli of normal cells. 18. Antigens associated with human cancer cells comprising a main species and a subsidiary species, the main species having the following characteristics: r / pi, on isoelectric concentration, of about 6.0 a / '/ // // 32 i 6 , 7, and a molecular weight of about 50,000 to about 60,000 t 'daltons,. ; * · The solubility in 0.01 M Tris HCl at pH 8, and its location in the first place in the nucleoli. 19. The main antigen species according to claim 18, in substantially purified form. 20. Process for the preparation of antibodies having a. specificity against antigens discovered in nucleoli and nuclei of human cancer cells, characterized in that it comprises the immunization of non-human host animals with the antigens according to claim 18, and the harvesting of antibodies from the immunized animals . 21. A method of preparing antibodies having specificity against the antigens discovered in nucleoli of human cancer cells, characterized in that it comprises the immunization of non-human host animals by the antigens according to claim 19 and the harvesting of antibodies from immunized animals. 22. Process for the preparation and isolation of nucleolar antigens characterized in that it comprises the extraction of antigens from nuclei or nucleoli of human cancer cells with 0.01 M Tris HCl with a pH of 7 at 9. 23. Nuclear preparation, characterized in that it comprises extracts of Tris HCl from nuclei or nucleoli originating from human cancer cells. 24. A method of purifying nucleolar antigens characterized in that it comprises their sequential purification / by precipitation with ammonium sulZate, chromatography on / / * v 33 l DEAE column. exclusion from Sephadex gel, exchange of 1 "* cations and chromatography on calcium phosphate gel, ·" and an isoelectric concentration of preparation. 25. Method for the purification of antibodies harvested from a non-human host animal induced by immunization with a nucleolar antigen. 26. Diagnostic kit, characterized in that it „comprises antibodies characterized by a specificity against nucleolar antigens found in human cancer cells, these antibodies having a mark detectable by optical, fluorescence or electronic microscopy, by methods of 'indirect examination and by autoradiography, and being in a buffered suspension agent or in solution. 27. Diagnostic kit, characterized in that it comprises unlabeled primary antibodies, characterized by specificity against nucleolar antigens found in human malignant cancer cells and found in a buffered suspending agent or in solution, and reagents labeled or unlabeled suitable for the detection of primary antibodies in human malignant cells fixed by a method chosen from the group comprising optical microscopy, fluorescence microscopy, electron microscopy and autoradiography. “28. Antigens associated with human cancer cells, characterized in that they comprise a main species and a subsidiary species, the main species having the following characteristics: a pi, on isoelectric concentration, of about 6.0 / to / / y / / - * tf. 34 l * 6.7, • a molecular weight of approximately 50,000 to approximately 60,000 daltons, • it is soluble in 0.01 M Tris HCl with a pH of 8, and it is located first in the nucleoli, when it is prepared by the process according to claim 22 or by a chemical equivalent thereof. 29. Main species of antigen as claimed. cation 18, characterized in that it is in a practically purified form, when it is prepared by the process according to claim 24 or by a chemical equivalent of the latter. 30. A method of detecting cancer cells by antibodies, as described above. Drawings: - ^ ..... plates _____15 „„ pages of which ...... A ......... guard piece ...... pr-.-Ηε of description. ......... ¾ ..... 7: „as it claims. ...... A ........ descriptive abbreviation Luxembourg, le Charles Munch? ''