CH659328A5 - METHOD FOR ISOLATING NUCLEOLAR ANTIGENS FROM HUMAN CANCER CELLS. - Google Patents

Description

The invention relates to a method for isolating antigens in the form of isolated nucleoli or nuclear extracts containing nucleoli components. The antigens are obtained by extraction from nuclei or nucleoli from human cancer cells. The invention also relates to a method for producing the corresponding antibodies, to a method for the immunological detection of cancer and to a corresponding diagnostic equipment.

Previous experimental animal studies have found the presence of nuclear or nucleolar antigens in tumors that were not found in non-tumor-infested tissues. (RK Busch et al, Cancer Res. 34,2362, 1974; Yeoman et al, Proc. Nati. Acad. Sci. USA 73,3258,1976; 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 earlier studies by the inventors, antibodies were prepared in nucleoli from rat normal and neoplastic cells by immunization of rabbits (R.K. Busch et al, supra; Busch and Busch, supra; Davis et al, supra). With the indirect immunofluorescence method, a bright nucleolar fluorescence was found in cells fixed with acetone. It was found that the immunoprecipitin bands in Ouchterlony gels formed by antisera to Novikoff Hepatoma nuclear antigens extracted from the Novikoff Hepatoma rats Nucleoli were different from the corresponding immunoprecipitin bands produced by nucleolar liver antigens and nucleolar anti-liver antisera (Busch) and Busch.

A further specific effect was achieved as a nucleolar antitumor antiserum, absorbed with nuclear liver extracts, which produced positive nucleolar fluorescence with respect to Novikoff Hepatoma ascite cells, but not with liver cells. On the other hand, nucleolar anti-liver antiserum, absorbed with nucleolar tumor extracts, produced no detectable nucleolar tumor fluorescence, but gave positive fluorescence in the liver nucleoli (Davis et al, supra).

Where differences in acetone-fixed tumor smears and normal smears from rat cells (especially after absorption of the antisera with normal liver nuclei and nucleoli) were observed by immunofluorescence analysis, attempts were made to use these antisera in nuclear rat tumor antigens by examining the corresponding tissue samples from human Tumors originated. Studies with antibody to nucleoli from rodent tumors showed that positive immunofluorescence was not found in human tumor nucleoli.

As a result, a new series of experiments has been carried out to find human nucleolar antigens. A positive immunofluorescence was then found in human tumor tissues, with antisera and antibodies to these new human nucleolar tumor preparations. In these studies, the antibodies were absorbed with placental nuclear sonicates as well as with fetal calf serum (Busch et al, 39, 3024, 1979; Davis et al. Proc. Nati. Acad. Sci. USA 76,892,1979; Smetana et al, Life Sci 25,227,1979).

The present invention arose from studies designed to use this novel human nucleolar antigen from a wide range of human neoplasms.

The following table gives an enumeration of human tumors in which a bright nucleolar Immu5

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nofluorescence was found with the antibodies to human tumor nucleoli. These studies supported the surprising and unexpected discovery that many human tumors contain a common nucleolar antigen that produces positive immunofluorescence with antisera or immunoglobulin fractions of these antisera (Busch et al, supra).

Table I

Bright nucleolar immunofluorescence in human tumors (from Busch et al, 1979)

I. Carcinomas

1st bladder, transition cell

2. Brain astrocytoma glioblastoma

3. Colon, adenocarcinoma (4)

Metastases: Liver Transplantable Carcinomas (GW-39)

4. Eccrine gland, carcinoma

5. Esophagus, flaky cell carcinoma

6. Liver, primary carcinoma

7. Lungs:

Adenocarcinoma (2)

Oat cells Flaky cells (5)

8. Melanoma, malignant, cerebral metastases

9. Prostate, adenocarcinoma (4)

10. Skin: basal cell carcinoma (2)

Scaly cell carcinoma metastases: lymph nodes

11. Stomach: adenocarcinoma

Metastases: liver metastases: lymphatic accounts

12. Thyroid carcinoma (2)

II. Sarcome

1. Myoblastoma malignant of the lip

Metastases to cervical lymphatic accounts

2. Osteogenic Sarcome (3) biopsy,

Tissue culture

3. Sarcom synovial

4. Lymphomas (4), non-Hodkins,

III. Hematological neoplasms

1. Hodkins disease (Reed Sternberg, 5)

2. Leukemia: CCL (5), hair cells (spleen)

3. Lymphoma, lymphocytic, spleen

4.Multiple myelomae (5)

5. Fungoid mycosis

6. Acute myelocytic leukemia (5)

7. Chronic myelocytic leukemia (5)

8. Acute monocytic leukemia (2)

IV. Cultures

1. Breast carcinoma

2. Colon adenocarcinoma

3. HeLa

4. HEp-2

5. Prostate, carcinoma (3)

6. Scaly cell carcinoma (3)

(The numbers in parentheses indicate the number of cases).

Results were generally obtained in the non-tumor tissues, benign tumors and inflammatory conditions, as indicated in Table II below:

Table II

Negative immunofluorescence in human tissues (After Busch et al, 1979)

I. Normal tissue

1st bubble

2. Bone marrow (hemoblastic lines 5) *

3rd chest

4. Leukocyte Blood (3)

5. Gallbladder

6. Intestine, small, crypt by Lieberkuhn

7. Intestine, big

8. Kidney

9.Liver (2)

10. Lungs (adjacent to the tumor)

11. Lymph nodes

12. Lymphocytes, normal (2)

13. Pancreas

14. Epyphysis

15. Pituitary

16. Placenta

17. Prostate

18. Skin

19. Stomach

20. Thyroid

II.

1. Breast, adenoma

2. Parathyroid, adenoma (2)

3. Prostate, hyperlasia (3)

4. Thyroid gland, adenoma (3)

Knot goiter

III. Inflammatory diseases

1. Chronic ulcer colitits

2. Glomerulonephitis

3. Granuloma and fibrosis of the lungs

4. Liver cirrhosis and hepatitis

5. Lupus profundes (mammary gland and skin)

6. Pemphigus - bullous

7. Gastric ulcer

8.Inflammatory hyperplasia lymph nodes (4)

9. Infectious mononucleosis (5)

IV. Cultures

1. Breast fibroblasts

2. Lymphocytes, PHA stimulated

(The numbers in parentheses indicate the number of cases).

These results, originally obtained by immunofluorescence, were verified by extensive immunoperoxidase procedures.

The present invention relates to the method for producing antigens defined in claim 1, the antigens thus obtained, the method for producing antibodies defined in claim 8, the antibodies thus obtained, the diagnostic method defined in claim 10 and the one defined in claim 11 diagnostic equipment.

The present invention is based on the surprising and unexpected finding that general nucleolar antigens are found in a wide range of human cancer cells, but not in human normal cells. The antigens and proteins, which may have gene control or other functions, are resistant to mitosis in a perichromosal position.

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The antigen in the cancer cells and cancer cells and very low concentrations in not

The antigens were present in a wide range of human cancerous cells or whether they were fetal antigens related to cancer, including central nervous cancer, as earlier in the comparative studies on nuclear anti-

systems, the gastrointestinal tract, the genital tract, the Novikoff heptoma gene in the rat and normal advice

the lungs, skin, hematopoietic tissue and endothelial liver cells (Yeoman et al, 1976).

crine and exocrine glands found. The malignant human cells include, for example, HeLa cells, human tumors and other tissues

Carcinomas of the prostate, other carcinomas, sarcoma and all stages for the preparation and analysis of samples from hematological neoplasms. The antigens can be derived from human tissue, blood and serum from

Nuclei or nucleoli of the human malignant cells were identified by the «Human Research Com-

be extracted. The antigens were not found in the Baylor College of Medicine, Houston, Texas, and related non-tumor-infested tissues. the connected hospitals. Samples of human

Using the method according to the invention, tumors were extracted from frozen sections of surgical

for the immunological detection of cancer and for post-test samples, biopsies or conserved cryostatic

About 1% false negative samples were obtained from the malignant cells, mainly from the department ti ve and 3% false positive results. The fai- of Pathology of the Houston Vétérans Administration Medicai see negatives are necrotic tumor tissue or from center and also from the Michigan Cancer Foundation for unknown reasons unreactive tumors. The wrong Detroit, Michigan, and the Department of Internal Medicine

There are two positive cases of preneoplastic tissues from Charles University in Prague, Czechoslovakia. These and weak positives in occasional focal regions in 20 cuts were due to the presence of nuclear anti

hyperplastic tissues. Two focal positive regions gene by indirect immunofluorescence and Immunope-

were identified as preneoplastic regions or roxidase techniques were examined.

focal neoplastic transformation in gastrointestinal inflamed tissues. Purification of the nucleolar antigen

The antigens from human cancer cells have a major 2s. The antigens were extracted by extracting the species 6 times and at least one and probably several nuclei or nucleoli with 10 mM Tris HCl / 0.1 mM PMSF /

Minor antigen species. The main species (a) has a disph value 8, purified, the volume ratio of the Cretan isoelectric points being in the range from 6.0-6.7 and in washing solution to the nuclei or nucleoli being 20: 1. The rule at approximately 6.3, determined by isoelectric extract, was first at 27,000 x g for 10 min and

Focus; the isoelectric focusing can then be centrifuged in a 30 at 100,000 x g for 16 h. Ammonium

pH gradients of pH 3-10 in polyacrylamide gel sulfate with 40% saturation were carried out to remove the; (b) has a molecular weight that uses impurities. The 40-100% ammonium

is in the range of 50,000-60,000 u, and the determination of sulfate fraction was centrifuged off and dialyzed against can be done by two-dimensional gel electrophoresis, 20 mM Tris HCl / pH 7.6. The antigens were chro-

the electrophoresis in the second dimension in 35 matographed in DE-52 cellulose columns (1x10 cm). The

Presence of SDS (sodium dodecyl sulfate) performed antigens were in the 0.15M NaCl / 0.1 mM PMSF / pH

becomes; (c) are soluble in 0.01 M Tris-HCl, pH 8 when eluting 7.6 fraction. Isoelectric focusing has been linked to the identification and purification not tightly to nuclear or nucleolar ribonucleoprotein of the antigens; (d) both nuclear and extranu use of gels are performed. These contained 4%

kleolar, but remain intranuclear or chromosomal 40 acrylamide / 8M urea / 2% ampholine (pH 3.5-10).

sociates during cell division. The antigens, pl 6.3 and 6.0, respectively, were extracted from the gels

A second antigen species could be cut as a secondary antigen species. In SDS (sodium dodecyl sulfate) gels were determined for zies, which found a pI value of approximately 6.0 each of these antigens as a major zone.

(determined by the same method as for the main antigen species) and also has a molecular weight in the range of 50,000-60,000 u in the production of HeLa cell nuclei or nucleoli. It is possible that HeLa cell nuclei or nucleoli were made by it a modified product of the main antigen, but it did separate HeLa cells from spinner culture bottles (7-8 has not been determined to be structurally related. The liters). The cells should and were in the log phase. Secondary antigen species are in a relatively lower concentration - 7-8 x 105 cells / ml. The cells were centrifuged when present as the major antigen species. so 800 x g for 8 min and formed cell platelets. The cell antigens are also present in particles of nucleolar ribonu plate in (PBS) phosphate buffered saline solution (RNP), which can be determined by ultracentrifu- (0.15 M NaCl, 0.01 M phosphate, pH 7.2 ) can be obtained by slightly yawing the tri-extracts. You will get homogenization suspended with a loose Teflon pistol, preferably as described below. That and centrifuged at 800 x g for 8 min. The cell antigen present in these particles is firmer. Pro 55 was washed a second time with PBS and the cell lines and ribonucleic acid RNA bound as the antigen plate was weighed. The cell platelets were replaced by light fractions soluble in the Tris. It is not yet clear whether homogenization in 20 volumes of reticulocyte standard - the antigens in the RNP particles are identical to the jepuffer (RSB) pH 7.4, suspended and 30 min on ice in the supernatant fraction, however your isoelectric swell. The cells were then the same at 1000 x g point and they absorb the antibodies to the 60 centrifuged for 8 min and again by light homo-antigens. Nuclear antigens, which are present in fibrils, detected in RSB buffer plus 1/20 volume of the network, apparently in the nuclear ribonucleoprotein network, are suspended in Nonidet P40 (10% in RSB). The final volume in the cancer cells by immune light micro- des Nonidet was 0.5%. The cells were diagnosed with a dounce scan. These are extranucleolar structure homogenizer with 20-60 strokes homogenized until the doors, which can represent elements from which the 65 cells were broken and the nuclei are derived from the cytoplasmic RNP particles. were exempt. The cells were then 1000 x g for 8 minutes

It remains to be determined whether the antigens are subcentrifuged, resuspended by slight homogenization.

represent that in high concentrations in 0.88 M sucrose, 0.5 mM Mg acetate (20 x weight /

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Volume) and centrifuged at 1500 x g for 20 min. The platelets obtained contained the HeLa nuclei which were used to prepare the antigen extract as described below. Nuclei from other human malignant cells were similarly obtained. For isolation of the nucleoli, the nuclear pellet, prepared as described above, was then suspended by gentle homogenization in 0.34 M sucrose, 0.5 mM Mg acetate using 2 ml sucrose for each gram of the original cells. The nuclei were then sonicated periodically in a Branson Sonifier for 10 s (at 10 s intervals). The total time was between 60-110 s. The released nucleoli were examined by microscopic examination. To make the nucleoli visible, they were pickled with Azur C (the solution consisted of 1% Azur C in 0.25 M sucrose). After sonication with the Branson Sonifier, the preparation should be free of nuclei. The fraction sonicated with the Branson Sonifier was sub-layered with three times the volume of 0.88 M sucrose (without Mg acetate) and centrifuged at 1500 x g for 20 min. The platelets obtained contained HeLa nuclei, which can be used as an immunogen.

A satisfactory cleaning was generally achieved by the method described above (Busch and Smetana, 1970) and light microscopy showed that the quality of the preparations was essentially satisfactory. However, electron microscopic analysis indicated the presence of chromatin and nuclear contaminants. The key problem with adequate cleaning of these preparations is the limited amount of Original HeLa cells in the cultures, which limits the number of re-purification steps. Nucleoli obtained from 5-10 g HeLa cell preparations, in contrast to the 0.5-1 g amounts used in previous experiments, gave enough material for sufficient purification. The conditions for the growth of HeLa cells and isolation of the placental nuclei were essentially the same as those previously described (Davis et al, 1979).

Production of HeLa-Tris extract

HeLa Tris extract was prepared by suspending the HeLa nuclei in Na C1-EDTÀ buffer 10 x weight / volume, 1 g nuclei / 10 ml buffer (buffer: 0.075 M NaCl, 0.025 M Na EDTA / pH value 8.1 mM PMSF) . Phenylmethyl sulfonyl fluoride (PMSF) was dissolved at 100 mM in isopropyl alcohol. It was added to each solution before extraction. The suspension was homogenized with a Dounce homogenizer with 20 strokes and centrifuged at 3000 x g for 5 min. The supernatant solution was separated. The above extractions were repeated two more times for the nuclear pellets. The NaCl-EDTA extract was not used in the present antigen work, so it was discarded. The nuclear platelets were suspended in 100 x weight / volume 0.01 MTris-HCl; pH 8.1mM PMSF and homogenized with a dounce homogenizer with 20 strokes, although 0.01M Tris-HCl pH 7-9 is satisfactory . The supernatant solution was separated and kept on ice. During the tri-extractions the nuclei broke and chromatin was released. The nuclear breakup was followed by microscopic examination. The platelets were resuspended in the tris buffer and the nuclei were allowed to swell on ice for 15 min. It was then treated with a 20 stroke dounce homogenizer and centrifuged at 12,000 x g for 10 min. The supernatant solution was saved. The pellets were resuspended and had a whitish, flaky appearance. It was homogenized again (dounce homogenizer, 20 strokes) and centrifuged at 27,000 x g for 30 min. The supernatant solution was separated and combined with the previous supernatants from the s Tris extracts.

The tri-extracts were then concentrated by ultrafiltration with an Amicon-Um-10 or PM-10 Diaflo membrane. Generally the volume at the beginning is approximately 50 ml and has been concentrated to 4-5 ml. The final concentration of the protein is approximately 4-5 mg / ml. The rabbit was immunized with this tri-extract.

Extracts can also be made from HeLa Nucleoli and Nuclei or Nucleoli from other human malignant cells by the method described above.

To prepare the trisimmunogen, 250 μl tris extract (4-5 mg / ml) are prepared as above with 250 (il PBS.

This is then combined with Freund's adjuvant for the nucleolar immunogen as described below.

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Immunization of rabbits with HeLa cells. Nuclear preparations for the production of antibodies

Antibodies were produced by immunizing rabbits with nuclear preparations from HeLa cells 25 as follows:

The HeLa nuclei were weighed (20-30 mg wet weight) and evenly suspended in 0.5 ml 0.01 M phosphate buffered saline, pH 7.2. They were then mixed with 0.6 ml Freund's complete adjuvant 30 (GIBCO) as follows:

The suspended nucleoli were placed in a 5 ml syringe and the Freund's adjuvant in a second syringe. An 18 gauge needle with the tip removed was connected to each syringe. The needles were then passed through a 35 I.D. 0.047 (Clay-Adams) connected. The contents of the syringes were mixed until the preparation thickened and was difficult to move through the tube.

The rabbits were shaved on their backs and injected intradermally with 6 punctures, 0.1 ml / per puncture. The 40 remaining 0.4-0.5 ml were injected, half subcutaneously (under the loose skin on the upper back) and half intramuscularly (into the soft muscle). The injections were given once a week with similar amounts of the nucleis for 3 weeks. The first bloodletting was performed 7-10 d after the third week of immunization. A rabbit ear cup (Bellco) and a vacuum pump were used to collect the blood. The blood was allowed to clot (approximately 45-50 ml) at room temperature for 3-4 hours. The serum was removed from the tube and centrifuged at 1000 g for 30 min (this sedimented free of red blood cells). The clear serum was separated and absorbed, or kept in the refrigerator until the absorption procedure was carried out. The blood clot can be stored in the refrigerator overnight. It releases 55 a few more ml of serum. The serum from each bloodletting was checked for the presence of nucleolar antibodies by an indirect immunofluorescence method.

Other, non-human hosts (e.g. goats, sheep, horses, chicks etc.) can be immunized with preparations from human 60 malignant cell nucleoli in order to raise the antisera or antibodies of the nucleolar antigens. Antisera can also be made by immunizing non-human hosts with extracts (e.g., Tris extract) from human malignant cell nuclei or nucleoli.

Absorption of antinuclear antiserum

The absorption of antinucleolar antisera was increased

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performed by absorbing rabbit antiserum with 20% normal human serum and 20% fetal calf serum (GIBCO). 20% normal human serum was added to the rabbit antiserum (4 ml / 20 ml) and incubated for 1 h at 37 ° C in a water bath with shaking. The container was removed, 20% fetal calf serum (4.8 ml / 24 ml) was added and the incubation was carried out for 1 hour at 37 ° C. with shaking in a water bath. The container was removed and incubated for 1 hour at room temperature, mixing by gentle stirring every 15 minutes. The mixture was then centrifuged at 15,000 x g for 30 min and the supernatant solution (absorbed serum) was removed and stored. The absorbed serum was converted to the immunoglobulin (Ig) form by performing the procedure described below for the (NHU ^ SOt precipitation of serum. The Ig preparation from the nucleolar antiserum which was absorbed with normal human serum and fetal calf serum was now absorbed with normal human tissue (placenta or liver) An equal volume of sonicated placental nuclear preparation in PBS 7.2 (10-15 mg protein / ml) was added to the absorbed nuclear immunoglobulin (10 ml Ig plus 10 ml sonicated nuclear preparation) and incubated with shaking in a water bath for 1 hour at 37 ° C. The mixture was then incubated for 1 hour at room temperature, mixing with gentle stirring of the bottle every 15 minutes and then centrifuging at 15,000 × g for 30 minutes was separated and the absorbed Ig precipitated again with (NHO2SO4 as described. This Ig can be used as a final antibody product or which can be further purified by chromatography with diethylaminoethyl cellulose (DEAE cellulose) as follows:

The Ig contained in the 0.01 M phosphate buffer saline, pH 7.2, is dialyzed against 0.0175 M phosphate buffer, pH 6.3 (without saline). After dialysis, it is centrifuged at 2500 x g for 20 min. The supernatant solution is placed on a DEAE column (20 mg protein per gram of cellulose, Whatman DE52). The IgG is eluted from the column with the 0.0175 M phosphate buffer. After elution, the IgG fraction is dialyzed against a 0.01 M phosphate buffered saline solution, pH 7.2.

The same procedure was carried out with control serum, which consists of preimmune serum obtained by bleeding rabbits (or other non-human hosts) before the start of immunization.

Production of rabbit immunoglobulin Ig

A saturated (NH4) 2S04 solution (760 g / 1) was prepared and an equal volume of cold saturated (NHO2SO4 was added dropwise to the antiserum with stirring. A white precipitate formed which was left to aggregate for 1 Yi-2 h , stirring with a magnetic stirrer in the cold. The precipitated antiserum was centrifuged at 3000 xg for 20 min. The supernatant solution was separated and the sediment resuspended in PBS, pH 7.2 (approximately half the volume of the original The solubilized sediment was placed in a dialysis tube and dialyzed against 100 volumes of PBS overnight in the cold with magnetic stirring. The dialysis container was placed in fresh PBS (100 x volume) the following morning and dialysis continued for 6 hours. The immunoglobulin was carefully removed from the dialysis container and centrifuged at 2500 xg for 20 min The supernatant solution was separated.

Some preferred immunological detection methods are described below.

Immunofluorescence 5 The previously described method (RK Busch et al, 1974; Hilgers et al, 1972) for immunofluorescence was used in this study as follows:

Indirect immunofluorescence method 10 150 (il of an antinucleolar antiserum (diluted 1:50) was applied to acetone-fixed HeLa cells or a fixed tissue sample (according to Hilgers et al, 1972; RK Busch et al, 1974). More than 150 | il to be used if the tissue sample covers a large part of the slide. The dilution of the antiserum (As) depends on the antibody (Ab) titer. Other dilutions can be used up to the point where As or Ab Dilutions are too dilute to admit a positive response to known positive cells (eg HeLa) 20 The slides are incubated in a humid chamber for 45-50 min at 37 ° C. The humid chamber can consist of a large petri dish in which After incubation, the antiserum was removed from the slide by careful addition of PBS, and the slides were placed in a holder and washed in PBS for 1 hour changed three times at 15.30 and 45 min. The slides were removed from the PBS and immersed in distilled or deionized water 10 times with rapid up and down movements. The 30 slides were dried with a hair dryer with cold air for 2-3 minutes, being careful not to over-dry. 150 μl of fluorine-labeled goat anti-rabbit antiserum (Hyland or Cappel), which was diluted 1:10, was placed on the slide 3S and incubated in the humid chamber for 30-35 min at room temperature. The second antibody was removed from the strip by gentle washing with PBS. The slides were then washed with PBS for 1 hour, changing the wash solution three times, at 15.30 and 40 for 45 minutes; or after the first 15 minutes of washing, they can be placed in fresh PBS and stored in the refrigerator overnight. After the last wash with PBS, the slides were immersed 10 times in deionized or distilled water, with rapid 4s up and down movements and dried with cold air from a hairdryer (2-3 min). A 1: 1 solution of glycerin and PBS was added to the cells or tissue samples and covered with a coverslip. The samples can be stored for several months if the cover slip is sealed with a sealant such as clear nail polish and is kept in the cold. The slides were then examined with a fluorescence microscope. Nucleolar fluorescence was not observed in preimmune immunoglobulin or preimmune Ig fractions SS. The other immunological techniques used were the same as those used in previous studies (Kendali, 1938; Lowry et al, 195 l; Dale and Lautner, 1969; Laurell, 1972; Wallace et al, 1974; Marashi et al, 1979) . For the analysis of the nucleo-60 lar localization of the immunofluorescence, a phase contract illumination was switched on and off for the samples during the fluorescence observation.

Immunoperoxidase Method 65 Instead of the fluorose-labeled goat anti-rabbit antiserum, the immunooxidase method can be used. For example, 150 μl peroxidase-labeled goat anti-rabbit was diluted 1:10 or 1:20

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added. Localized peroxidase activity can be demonstrated by a number of redox color systems for light or electron microscopic studies. Other enzymes can serve as labels for the indirect method and peroxidase and other enzymes can be used directly by labeling the primary antibody.

The Karnofsky incubation medium is produced as follows:

Weigh out enough diaminobenzidine (Sigma) and suspend in 0.05 M Tris-HCl, pH 7.6, so that the concentration is 0.5 mg / ml. Prepare a hydrogen peroxide solution of 0.02% (in the 0.05 M Tris-HCl buffer). Mix 0.5 mg / ml DAB and 0.02% H2O2 in equal parts in a 1: 1 ratio. (This solution was freshly prepared for use each time and was kept cold during manufacture).

200-300 jil DAB and H2O2 mixture are placed on the slide and incubated for 30 min in a moist chamber at room temperature.

After this incubation, the DAB and H2O2 mixture is removed by washing with 0.05 M Tris HCl, pH 7.6, buffer, to which 0.01 M NaCl has been added. The slides are washed twice in 0.05 M Tris HCl, pH 7.6, 7.1 M NaCl for 10 min. The slides are de-treated as indicated in steps 11-14, except that PBS is replaced with Tris-HCl. The finished slides are checked using light microscopy.

Production of the slides with the HeLa cell preparations for immunofluorescence

A stock of fixed HeLa cells was made as follows:

Active growing cells from the HeLa culture bottle were removed and washed with PBS, pH 7.2. The cells were suspended so that there were at least 1.5 x 106 cells / ml. A drop of the HeLa cell suspension was applied to a washed slide (washed with a detergent, rinsed with distilled or deionized water, cleaned with alcohol, rinsed and dried with heated air) and spread lightly and allowed to dry overnight at room temperature or in the cold. The dried cells were fixed by placing the slides in acetone at 4 ° C for 12 h. The slides were numbered using a diamond-tipped marker pen. The slides were used as positive controls for immunofluorescence.

Detection of the malignant tumor with test antibody

The present studies confirm that nucleolar antigens are present in tumor cells, but are not found in tissues without a tumor. Preliminary studies showed that bright nucleolar fluorescence was generated in cell cultures with human tumors as well as in samples from autopsy or biopsy samples using the double antibody technique (indirect immunofluorescence) and that no corresponding results were obtained with a series of non-tumor tissues (Davis et al, 1979). In later studies, more than 60 malignant tumors were studied and a variety of tissue controls were evaluated. It is of great interest that this broad series of experiments on malignant tumors of ectodermal, endodermal, and mesodermal origin show the presence of one or more general nuclear antigens (Table I).

Example I

Normal tissues - No nucleolar fluorescence was observed in 17 non-tumor tissues after incubation of the antisera or antibodies with the various fixed cell preparations. It was of particular interest that neither the skin's Malpighian layer, bone marrow cells, nor Lieberkuhn's crypts showed positive immunofluorescence in this procedure. In addition, the number of non-tumor tissues following the neoplasms was also negative, including many tissues of various types. A group of benign tumors that were evaluated, including various types of thyroid adenoma, were also negative (Table II).

Example 2

Inflammatory Affections - To determine whether an “inflammation” reaction was associated with the appearance of these antigens, studies were conducted with 8 types of inflamed tissues. In most cases, no remarkable fluorescence was observed in the nucleoli of the cells. However, sections were found in samples of ulcerative colitis and gastric ulcer that showed positive nucleolar fluorescence. In particular, two out of three sections of ulcerative colitis were negative and one showed a definite positive nucleolar fluorescence. In gastric ulcer, one of two sections showed positive nucleolar fluorescence. These results are particularly interesting in view of the known tendency of these affections to undergo a malignant change. It was of particular interest to observe both the focal positive and negative regions of these stripes in the heme toxininose stained sections. These showed that there are indeed regions in these affections which not only show mitotic figures, but also an accumulation of epithelial lining. This result suggested that these cells represent pre-neoplastic affections or carcinomas in situ. It is possible that this discovery of the fluorescent regions can assist in decisions about the surgical procedure by either local or more general resection of the affected damage.

Example 3

Contra-facts - The gastric epithelium found a fluorescent region in each cell that was not nucleolar, which appeared to represent a non-specific location of the fluorescent antibody. In a crypt of the small intestine, non-specific localization of the antibody appeared to occur in the form of aggregates. In most cases, the prefiltration of the antibodies by a 0.34 (in the millipore filter eliminated these aggregates. In a sample of breast tissue that was negative for nucleolar fluorescence, small non-specific immunofluorescent particles were generally distributed without a specific location regarding the cell morphology The diameters of these very small non-specific precipitates were 0.5 to 0.1 µm compared to the nucleolar diameters in the nuclei and nucleoli, which were 4 to 6 pm.

Example 4

Fluorescence during the phases of the cell cycle - The nucleolar fluorescence was made clearly visible in the interphase nucleoli. In the metaphase, the nucleolar fluorescence was not visible as a distinct whole, but was visible between the chromosomes and in the connecting areas between the nucleus from the

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Cryptoplasm. Inasmuch as the nucleus generally disappears during metaphase and RNA, the synthesis stops in the late prophase, so it was not surprising that the nucleolar fluorescence was not visible as a distinct entity in these cells (Tan and Lerner, 1972). However, the finding that remnants of the immunofluorescent products persist during mitosis suggested that the nucleolar substructures (unlike the nucleolar products) contain antigens that are epigenetically stable.

Example 5

Negative malignant tumors - In the series of malignant tumors, negative regions were found on the slides to varying degrees. In general, these can be associated with either necrotic or abscess parts of the neoplasm. In a sample of a brain tumor, the mass that showed no positive fluorescence was necrotic, many leukocytes were present, but there was no defined structure. Adenocarcinoma that metastasized to the brain showed no positive fluorescence, the reasons were not clear. 61 of 63 tumors examined showed a positive nucleolar fluorescence, 97% of the examined series was positive. These studies have now been expanded to include over 300 human cancer specimens, including a series of breast, prostate, lung, and haematological tumors with very similar results.

Example 6

Labeling - Direct immunochemical methods to demonstrate the antibodies include labeling the primary antibody with one or more of the following labels: a radioisotope for autoradiography such as 12SI, mI, 14C or 3H, a fluorescent dye such as fluorescein or tetramethylrhodamine for

Fluorescence microscopy, an enzyme that produces a fluorescent or colored product to determine fluorescence or light microscopy, or that produces an electron-dense product for demonstration using electron microscopy, or an electron-dense molecule such as ferritin for direct electron microscopic visualization.

Indirect immunochemical methods include labeling the second antibody or other binding pro- teins that are specific for the first antibody with a fluorescent dye, an electron-dense compound, an enzyme that produces a product that can be detected by light, fluorescence, or electron microscopic examination or a radioisotope that can be detected by auto-15 radiography.

The indirect immunochemical methods for the visualization of the antibodies are: Use of hybrid, primary or secondary antibodies or antibody fragments (F (ab ') j) in which a part of the hybrid antibody preparation is specific for the nucleolar antigens (hybrid primary antibodies) or for the primary antibody (hybrid second antibody) and part specific for a label, such as those mentioned in the previous paragraph.

Diagnostic equipment

Labeled conjugated and non-conjugated antibodies can be packaged separately in phosphorus buffered saline (PBS) or other buffered suspending agents for marketing. Suitable suspending agents are glycerin, heparin or sucrose. Suitable buffers are buffers based on barbiturates, morpholine buffers, MOPS-3- (N-morpholino) propanesulfonic acid, Hepes-N-2-35 hydroxyethylpiperazine-N-2-ethanesulfonic acid or Triscar-bonate.

B

Claims (11)

659328 1. A method of isolating antigens in the form of isolated nucleoli or nuclear extracts containing nucleoli components and which antigens have a major species and at least one minor species, the major species of the antigens having the following characteristics: Isoelectric point pl 6.0-6.7, determined by isoelectric focusing, Molecular weight = 50,000-60,000 u, soluble in 0.01 M Tris HCL ph 8, and the antigens are primarily located in nucleoli of human cancer cells, characterized in that the antigens from nuclei or nucleoli of human cancer cells are obtained by extraction with 0.01 M Tris HCl with a pH of 7-9. 2. The method according to claim 1, characterized in that the antigens are additionally subjected in succession to ammonium sulfate precipitation, DEAE column chromatography, dextran gel exclusion, cation exchange and calcium phosphate gel chromatography and isoelectric focusing. 2nd PATENT CLAIMS 3. The method according to claim 1 or 2, characterized in that as antigens nucleoli are extracted from malignant human cells from the family HeLa cells, carcinomas, sarcomas and hematological neoplasms. 4. The method according to any one of claims 1 or 2, characterized in that nucleoli are extracted from HeLa cells or human prostatic carcinoma cells as antigens. 5. The method according to any one of claims 1 or 2, characterized in that extracts of nuclei or nucleoli of malignant human cells from the family consisting of HeLa cells, carcinomas, sarcomas and hematological neoplasms are obtained as antigens. 6. The method according to claim 1 or 2, characterized in that extracts of nuclei or nucleoli from HeLa cells or human prostatic carcinoma cells are obtained as antigens. 7. Antigens obtained by the method according to any one of claims 1-6. 8. A method for producing antibodies which are specific against the antigens from nuclei or nucleoli of human cancer cells, characterized in that a host animal is immunized with an antigen according to claim 7 and the antibodies are obtained from the immunized animal. 9. Antibodies obtained by the method according to claim 8. 10. A method for the immunological detection of cancer in human tissue sections, smears and desquamated cytological preparations, characterized in that samples thereof are brought into contact with antibodies according to claim 9. 11. Diagnostic equipment for performing the method according to claim 10, characterized in that it contains antibodies according to claim 9, which are present in a buffered suspension medium or in solution and which are marked by a label which by one or more of the methods mentioned below Detection allowed: light fluorescence or electron microscopy, indirect immunochemical test methods and autoradiography.