IE50760B1 - Detection of human cancer cells with antibodies to human cancer nucleolar antigens - Google Patents

Abstract

Specific nucleolar antigen(s) are found common to a broad range of human malignant tumor specimens and have been isolated, extracted and purified. Antibodies and antisera specific to these nucleolar antigen(s) are harvested from non-human hosts and used for detection of human cancer cells. Disclosed are (1) methods for isolating the nucleolar antigen(s), obtaining them in substantially purified form, producing the specific antibodies, obtaining them in substantially purified form, and using the antisera and antibodies induced by the nucleolar antigen(s) in diagnostic procedures for detecting human cancer cells, and (2) diagnostic kits comprising specific antibodies and antisera.

Description

This invention relates to nucleolar antigens found in a broad range of human cancers and not found in corresponding nontumor tissues and to antibodies and antisera specific to these nucleolar antigen(s) for diagnostic purposes.

Earlier findings in experimental animals have indicated the presence of nuclear and nucleolar antigens in tumors which were not found in non-tumor tissues (R. K. Busch et al, Cancer Res. 34 , 2362, 1974; Yeoman et al, Proc. Natl. Acad. Sci. OSA 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 early studies by the inventors, antibodies were prepared to nucleoli of rat normal and neoplastic cells by immunization of rabbits (R. K. Busch et al, supra; Busch and Busch, supra; Davis et al, supra). Bright nucleolar fluorescence was demonstrated in the acetone-fixed cells by the indirect immunofluorescence method. It was also found that the immunoprecipitin bands in Ouchtcrlony gels formed with antisera to Novikoff hepatoma nucleolar antigens extracted from rat Novikoff hepatoma nucleoli differed from the corresponding immunoprecipitin bands produced with liver nucleolar antigens and antiliver nucleolar antisera (Busch and Busch, supra).

Further specificity was shown when antitumor nucleolar antiserum absorbed with liver nuclear extracts produced positive nucleolar fluorescence in Novikoff hepatoma ascites cells but not in liver cells. Conversely, antiliver nucleolar antiserum absorbed with tumor nucleolar extracts did not produce detectable tumor nucleolar fluorescence but did produce positive fluorescence in liver nucleoli (Davis et al, supra).

Inasmuch as immunofluorescence analysis indicated that differences were observable in acetone-fixed tumor smears and normal rat cell smears (particularly after absorption of the antisera with normal liver nuclei and nucleoli), attempts were made to utilize these antisera to rat tumor nucleolar antigens in testing corresponding tissue samples derived frcm human tumors. Studies with antibodies to rodent tumor nucleoli showed that positive immunofluorescence was not found in human tumor nucleoli.

In view of this the present inventors began a new series of experiments to find human nucleolar antigens. Positive immunofluorescence was then found in human tumor tissues with antisera and antibodies to these new human tumor nucleolar preparations.

In these studies, the antibodies were absorbed with placental nuclear sonicates as well as fetal calf serum (Busch et al, 39, 3024, 1979; Davis et al., Proc. Natl. Acad. Sci. USA 76, 892, 1979; Smetana et al. Life Sci. 25, 227, 1979).

The present invention has resulted from studies designed to utilize these new human nucleolar antigens for the detection of a broad range of human neoplasms.

The following Table I presents a summary of the human tumors in which a bright nucleolar immunofluorescence 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 antigens which exhibits a positive immunofluorescence with antisera or immunoglobulin fractions of such antisera (Busch et al, supra.

TABLE I BRIGHT NUCLEOLAR IMMUNOFLUORESCENCE IN HUMAN TUMORS (From; Busch et al, 1979) I. Carcinomas 1. Bladder, Transitional cell 2. Brain astrocytoma glioblastoma 3. Colon, adenocarcinoma (4) metastasis: liver transplantable carcinoma (GW-39) 4. Eccrine gland, carcinoma . Esophagus, squamous cell carcinoma 6. Liver, primary carcinoma 7. Lung: adenocarcinoma (2) oat cell (2) squamous cell (5) 8. Melanoma, malignant, cerebral metastases 9. Prostate, adenocarcinoma (4) . 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. Sarcomas 1. 2. 3. 4.

Myoblastoma, malignant of lip metastasis to cervical lymph node Osteogenic sarcoma (3), biopsy, tissue culture Synovial sarcoma Lymphoma (4), non Hodgkins III Fematolocical Neoplasms 1. Hodgkins disease (Reed Sternberg, 5) 2. Leukemia: CLL (5), Hairy cell (spleen) 3. Lymphoma, lymphocytic, spleen 4. Multiple myeloma (5) . Mycosis fungoides 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 . Prostate, carcinoma (3) 6. Squamous cell carcinoma (3) ♦Numbers in parenthesis represent number of cases In the non-tumor tissues, benign tumors, and inflam matory states, negative results were generally obtained as in dicated in the following Table II (Busch et al, supra).

TABLE II NEGATIVE IMMUNOFLUORESCENCE IN HUMAN TISSUES (From.- Busch et al, 1979) I. Normal Tissue 1. Bladder 2. Bone marrow (hemoblastic lines, 5)* 3. Breast 4. Buffy coat-blood (3) . Gallbladder 6. Intestine, small, crypts Lieberkuhn 7. Intestine, large 8. Kidney 9. Liver (2) 10. Lung (adjacent to tumor) 11. Lymph node 12. Lymphocytes, normal (2) 13. Pancreas 14. Pineal gland 15. Pituitary 16. Placenta 17. Prostate gland 18. Skin 19. Stomach 20. Thyroid gland Benign Growing Tissues 1. Breast, adenoma 2. Parathyroid, adenomas (2) 3. Prostate gland, hyperlasia (3) 4. Thyroid, adenomas (3, nodular goiters (2) III. Inflammatory Diseases 1. Chronic ulcerative colitis 2. Glomerulonephitis 3. Granuloma and fibrosis of lung 4. Liver - cirrhosis, hepatitis . Lupus profundus (mammary gland and skin) 6. Pemphigus - bullous 7. Dicer, gastric 8. Inflammatory hyperplasia-lymph nodes (4) 9. Infectious mononucleosis (5) IV. Cultures 1. Breast fibroblasts 2. Lymphocytes, PHA stimulated •Numbers in parenthesis represent number of cases These results, originally obtained with immunofluorescence, have been verified and extended with immunoperoxidase methods.

The present invention is based on the surprising and unexpected discovery that common nucleolar antigens are found in a broad range of human cancer cells but are not found in normal human cells. The antigens are proteins which may have gene control or other functions and are persistent throughout mitosis in a perichromosomal location. Important aspects of the invention are discovery of the common nucleolar antigens found in human cancer cells, isolation and purification of the nucleolar antigens, production of antisera and antibodies specific to these antigens, diagnostic test methods using antisera and antibodies specific to these antigens to detect human cancer cells, and a diagnostic kit containing either antibodies or antisera or both specific to these nucleolar antigens.

The antigens have been found in a broad range of human cancers including cancers of the central nervous system, gastrointestinal tract, genitourinary tract, lung, skin, blood forming tissues and endocrine and exocrine glands. For example, the malignant human cells include HeLa cells, prostatic carcinoma, other carcinomas, sarcomas and hematological neoplasms. The antigens can be extracted from nuclei or nucleoli of human malignant cells. The antigens have not been found in corresponding nontumor tissues. In using the diagnostic methods of the present invention to detect malignant cells, approximately one percent false negatives and three percent false positives were detected.

The false negatives represent necrotic tumor tissues or nonreactive tumors for reasons unknown. The false positives represent two cases of preneoplastic tissues and weak positives in occasional focal regions in hyperplastic tissue. Two focal positive regions were identified as preneoplastic regions or focal neoplastic transformation in gastrointestinal inflammatory tissues.

The antigens have a major species and at least one and possibly more minor antigen species. The major antigen species from human cancer cells (a) has a discrete isoelectric point cf from 6.0 to 6.7 and approximately 6.3 as determined by isoelectric focusing, pH 3-10, polyacrylamide gel; (b) has an approximate molecular weight of 50,000 to 60,000 daltons as determined by two-dimensional gel electrophoresis with an SDS(sodium dodecylsulfate) second dimension; (c) is in part tightly bound to nuclear and nucleolar RHP and in part soluble in 0.01 M Tris-HCl, pH 8; (d) and is both nucleolar and extranucleolar but remains intranuclear or chromosomeassociated during cell division.

The second antigen species which has been detected has a pi of approximately 6.0 (detected by the same procedures as the major antigen species) and its molecular weight is also 50,000 to 60,000 daltons. It is possible that it represents a modified product of the major antigen, but it has not been determined whether it is structurally related.

The minor antigen species is in relatively smaller concentration than the major antigen species.

Antigens are also present in nucleolar ribonucleoprotein (RNP) particles obtained by ultracentrifugation of the Tris extracts, subsequently described. The antigen present in these particles is more tightly 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 RNP 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 fibrils, probably of the nuclear ribonucleoprotein network, are also seen in cancer cells by immune-light microscopy. These are extranucleolar structures which may represent elements from which the RNP particles are derived.

It remains to be determined whether the antigens represent a substance that is present in high concentrations in cancer cells and very low concentrations in noncancerous cells or are fetal antigens as was found earlier in the comparative studies on nuclear antigens of the rat Novikoff hepatoma and normal rat liver cells (Yeoman et al, 1976).

All steps for obtaining and analyzing samples of 7S0 human tissue, blood and serum of human tumors and other tissues of suspected cancer patients were approved by the Human Research Committee at Baylor College of Medicine, Houston, Texas and affiliated hospitals.

Sections of human tumors were obtained from frozen sections of surgical specimens, biopsy, or preserved cryostat specimens, mainly from the Department of Pathology from the Houston Veterans Administration Medical Center, and also from the Michigan Cancer Foundation in Detroit, Michigan, and the Department of Internal Medicine, Charles University in Prague, Czechoslovakia. These sections were analyzed for the presence of nucleolar antigens by indirect immunofluorescence and immunoperoxidase techniques.

The antigens are isolated from nuclei or nucleoli of human cancer cells by extracting with 0.01 M Tris HC1 at a pH of from 7 to 9. Preferably, the antigens are purified by the sequential steps of precipitation with ammonium sulphate, DEAE - cellulose column chromotography, a gel exclusion technique using gel particles of crosslinked dextran, available as SEPHADEX (Sephadex is a Trade Mark), cation exchange and calcium phosphate gel chromatography and preparative isoelectric focussing.

Typically, purification of the antigens was achieved by extraction of nuclei or nucleoli with 10 mM Tris HC1/0.1 mM PMSF/pH 8 for 6 times in a ratio of 20 volumes to one volume of nuclei or nucleoli. The extract was centrifuged first at 27,000 x g for 10 minutes and then at 100,000 x g for 16 hours. Ammonium sulfate at 40% saturation was used to remove contaminants. 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 DE-52 cell5 ulose columns (1 x 10 cm). The antigens were eluted in the 0.15M NaC1/0.1 mM PMSF/pH 7.6 fraction. Isoelectric focussing gels were used to identify and purify the antigens. These contained 4% acrylamide/8M urea/2% ampholines (pH 3.5-10). The antigens, pi 6.3 and 6.0 respectively, were cut out of the gels. On SDS (sodium dodecyl sulfate) gels, one major spot was found for each of these antigens.

Hela cell nuclei or nucleoli were prepared by collecting HeLa cells from Spinner culture bottles (7-8 liters). The cells should be and were in log phase 7-8 c x 10 cells/ml. The cells were centrifuged at 800 x g for 8 minutes to form cell pellets. The cell pellets were suspended in (PBS) phosphate buffered saline (0.15 M NaCl, 0.01 M phosphate, pH 7.2) by eentle homogenization with a loose Teflon (Teflon is a Trade Mark) pestle and centrifuged at 800 x g for 8 minutes. The cells were washed a second time with PBS and the cell pellets were weighed. The cell pellets were suspended by gentle homogenization in 20 volumes of reticulocyte standard buffer (RSB), pH 7.4 and allowed to swell for 30 minutes on ice.

The cells were then centrifuged at 1000 x g for 8 minutes and resuspended by gentle homogenization in RSB buffer plus 1/20 volume of the detergent Nonidet (Nonidet is a Trade Mark) P40 (10% in RSB). The final volume of Nonidet was 0.5%. The cells were hcstiogenized with a Dounce homogenizer 20-60 strokes until the cells were broken and the nuclei released and freed of cytoplasm. The cells were then centrifuged 1000 x g for 8 minutes, resuspended by gentle homogenization in 0.88 M sucrose, 0.5 mM Mg acetate (20 x weight-volume) and centrifuged at 1500 x g for 20 minutes. The resulting pellet contained the HeLa nuclei that were used to prepare the antigen extracts described below. Nuclei from other human malignant cells may be obtained in a similar manner.

For isolation of nucleoli, the nuclear pellet as prepared above was next suspended by gentle homogenization in 0.34 M sucrose, 0.5 mM Mg acetate using 2 ml of sucrose for each gram of original cells. The nuclei were sonicated (with a Branson (Branson is a Trade Mark) sonifier) by 10-seoond bursts (and 10 seconds rest). Total time was between 60 and 110 seconds. The nucleoli released were monitored by microscopic examination.

To visualize the nucleoli, they were stained with Azure C (the solution consists of 1% Azure C in 0.25 M sucrose). The preparation should be free of nuclei at the end of the sonication period. The sonicated fraction was underlayed with three times the volume of 0.88 M sucrose (without Mg acetate) and centrifuged 1500 x g for 20 minutes. The resulting pellet contained the HeLa nucleoli which may be used as the immunogen.

Satisfactory purification has usually resulted with the above procedure (Busch and Smetana, 1970), and light microscopy showed the quality of these preparations was essentially satisfactory. However, electron microscopic analysis indicated the presence of chromatin and nuclear contaminants. The key problem in adequate purification of these preparations is the limited amount of original HeLa cells in the cultures which limit the number of repurification steps. Nucleoli prepared from 5- to 10-g HeLa cell preparations, rather than the 0.5- to 1-g quantities used in earlier studies, provided sufficient material for adequate purification. The conditions for growing the HeLa cells and the isolation of placental nuclei were essentially the same as those reported previously (Davis et al, 1979).

HeLa Tris extract was prepared by suspending the HeLa nuclei in NaCl-EDTA buffer 10 x weight/volume, 1 g nuclei/10 ml buffer. (Buffer: 0.075 M NaCl, 0.025 M Na EDTA/pH 8, 1 mM PMSF) The phenylmethylsulfonylfluoride (PMSF) is made up at 100 mM concentration in isopropyl alcohol. It is added to each solution prior to the extraction. The suspension was homogenized with a Dounce homogenizer 20 strokes and centrifuged at 3000 x g for 5 minutes. Supernatant was collected. The above extractions were repeated on the nuclear pellet two more times. The NaCl-EDTA extract was not used in the present antigen work; therefore, it was discarded. The nuclear pellet was suspended in 10 x weight/ volume 0.01 M Tris-HCl; pH 8, 1 mM PMSF and homogenized with a Dounce homogenizer for 20 strokes, although 0.01 M Tris-HCl pH 7-9 is satisfactory. The supernatant was collected and saved on ice. During the Tris extractions, the nuclei broke and chromatin was released. The nuclear breakage was monitored by microscopic examination. The pellet was resuspended in the Tris buffer and the nuclei were allowed to swell for 15 minutes on ice. It was then Dounce homogenized for 20 strokes and centrifuged at 12,000 x g for 10 minutes. The supernatant was saved. The pellet was resuspended and had a whitish fluffy appearance. It was again Dounce homogenized for 20 strokes and centrifuged at 27,000 x g for 30 minutes. The supernatant was collected and combined with previous supernatants from the Tris extracts.

The Tris extracts were then concentrated with an Amicon UM-10 or PM-10 Diaflo (Diaflo is a Trade Mark) membrane. Generally, the volume at the beginning is around 50 ml and this was concentrated to 4-5 ml. The final concentration of protein is around 4-5 mg/ml. The rabbit may be immunized with this Tris extract.

By following the above procedure, extracts may also be prepared from HeLa nucleoli and from nuclei or nucleoli of other human malignant cells.

For the Tris immunogen, dilute 250 μΐ of Tris extract (4-5 mg/ml) as prepared above with 250 μΐ PBS.

Combine this with the Freund's adjuvant as described for the nucleolar immunogen below.

Antibodies were prepared by immunization of rabbits with HeLa cell nucleolar 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. They were then mixed with 0.6 ml Freund's complete adjuvant (GIBCO) as follows: The suspended nucleoli were placed in a 5 ml syringe and the Freund's adjuvant in a second syringe.

To each syringe an 18 gauge needle from which the tip had been removed was attached. The needles were then connected by a piece of polyethylene tubing, I.D. 0.047 (Clay-Adams).

The contents of the syringes were mixed until the preparation became thickened and was difficult to force through the tubing.

The rabbit was shaved on the back and injected intradermally at 6 sites, 0.1 ml/site. The remaining 0.4-0.5 ml was injected, half subcutaneously (under the loose skin on the upper back) and half intramuscularly (in the thigh muscle). The injections were given once a week for three weeks with similar amounts of nucleoli each time. The first bleeding was carried out 7-10 days after the third week of immunization.

A rabbit ear cup (Bellco) and a vacuum pump were used to collect the blood. The blood (approximately 45-50 ml) was allowed to clot for 3-4 hours at room temperature. The serum portion was removed from the tube and centrifuged at 1000 g for 30 minutes (this sediments any free red blood cells).

The clear serum was collected and was then absorbed (or kept frozen until ready for the absorption procedure). The blood clot may be refrigerated overnight. This releases a few additional ml of serum. The serum from each bleeding was assayed for the presence of nucleolar antibodies by the indirect immunofluorescence procedure.

Other non-human hosts (e.g. goat, sheep, horse, chicken, etc.) may be immunized with human malignant cell nucleoli preparations to elicit the antisera or antibodies to the nucleolar antigens of the present invention. Antisera may also be prepared by immunization of non-human hosts animals with extracts (e.g. tris extract) of human malignant cell nuclei or nucleoli.

Absorption of antinucleolar antiserum was accomplished by first absorbing the 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 hour in a 37°C shaking water bath. The flask was removed, 20% fetal calf serum (4.8 ml/24 ml) was added, and incubation was carried out for 1 hour in a 37°C shaking water bath. The flask was removed and incubated an additional hour at room temperature with mixing by gentle swirling every minutes. It was then centrifuged at 15,000 x g for 30 minutes, and the supernatant (absorbed serum) was removed and saved. The absorbed serum was converted to the immunoglobulin (Ig) form by following the procedure given for the (NH4)2SO4 precipitation of serum, subsequently described.

The Ig preparation from the nucleolar antiserum which had been absorbed with normal human serum and fetal calf serum was now absorbed with a normal human tissue (placenta or liver). An equal volume of placental nuclear sonicate in PBS 7.2 (10-15 mg protein/ml) was added to the absorbed nucleolar immunoglobulin (10 ml Ig plus 10 ml nuclear sonicate) and incubated for 1 hour in a 37°C shaking water bath. It was then incubated for an additional hour at room temperature with mixing by gentle swirling of the flask every 15 minutes and then centrifuged at 15,000 x g for 30 minutes. The supernatant was collected and the absorbed Ig was reprecipitated with (NH4)2SO4 as described. This Ig can be used as the final antibody product or it can be further purified by diethylaminoethyl (DEAE) cellulose chromatography as follows: The Ig which is contained in the 0.01 M phosphate buffered 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 minutes. The supernatant is added to the DEAE column (20 mg of protein per gram of cellulose, Whatman DE52). The IgG is eluted from the column with the 0.0175 M phospate 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 preimmune serum which was obtained by bleeding the rabbit (or other non-human host animal, before the immunization was started.

Rabbit immunoglobulin Ig was prepared as follows: a saturated (NH4)2SO4 solution (760 gm/liter, was prepared and an equal volume of cold saturated (NH4)2SO4 was added drop by drop to the antiserum with stirring. A white precipitate formed and the precipitate was allowed to aggregate for 1-1/2 - 2 hours on a magnetic stirrer in the cold. The precipitated antiserum was centrifuged at 3000 x g for 20 minutes. The supernatant was removed and the pellet was resuspended in PBS, pH 7.2 (approximately half the volume of the original serum). The solubilized pellet was placed in a dialysis bag and dialyzed against 100 volumes of PBS overnight in the cold (with magnetic stirring,. The dialysis bag was placed in fresh PBS (100 x volume) the following morning and dialysis was continued for 6 hours. The immunoglobulin was carefully removed from the dialysis bag and centrifuged at 2500 x g for 20 minutes. The supernatant was collected.

The procedure described earlier (RK Busch et al, 1974; Hilgers et al, 1972) for immunofluorescence was used in this study, as follows; 150 μΐ of antinucleolar antiserum diluted 1:50 was placed on acetone fixed HeLa cells or on fixed tissue specimens (from Hilgers et al, 1972; 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 slide. Dilution of antiserum (As) is dependent on antibody (Ab, titer. Other dilutions can be used up to the point where As or Ab dilutions become too dilute to yield positive response to known positive cells (e.g. HeLa). The slides were incubated in a moist chamber for 45-50 minutes at 37°C. (The moist chamber may consist of a large petri dish to which has been added a moist paper towel., After the incubation, the antiserum was washed off the slide by the gentle addition of PBS and the slides were placed in a slide holder and washed in PBS for 1 hour. The PBS was changed three times, at 15 minutes, 30 minutes and 45 minutes. The slides were removed from PBS and dipped in distilled or deionized water ten times with rapid up and down movements. The slides were dried with cold air from a blow or hair dryer (2-3 minutes), being careful not to overdry. 150 μΐ of fluorescein labeled goat antirabbit antiserum (Hyland or Cappel) diluted 1:10 was placed on the slides and incubated in the moist chamber for 30-35 minutes at room temperature. The second antibody was removed from the slide by a gentle PBS wash. The slides were then washed in PBS for 1 hour with three changes, at 15 minutes, 30 minutes and 45 minutes; or after the first 15 minutes wash, they can be placed in fresh PBS and left in the refrigerator overnight. After the final wash with PBS, the slides were dipped in deionized or distilled water ten times with rapid up and down movements and dried with cold air from a blow or hair dryer (2-3 minutes). A solution of glycerol and PBS in a 1:1 ratio was added to the cells or tissue specimen and covered with a cover slip. The specimen can be preserved for several months if the cover slip is sealed with a sealant, such as clear nail polish and kept cold. The slide was then examined with a fluorescence microscope.

Nucleolar fluorescence was not observed with preimmune immunoglobulin or preimmune IgG fractions. The other immunological techniques used were the same as those used in earlier studies (Kendall, 1938; Lowry et al, 1951; Dale and Latner, 1969; Laurell, 1972; Wallace et al, 1974; Marashi et al, 1979). For analysis of nucleolar localization of immunofluorescence, samples were switched in and out of phase contract illumination during fluorescence observation.

Instead of fluorescein-labeled goat antirabbit, immunoperoxidase method can be used. For example, 150 pi of peroxidase labeled goat antirabbit 1:10 or 1:20 dilution was added. Localized peroxidase activity can be demonstrated by a number of redox dye systems for light or electron microscopic examination. Other enzymes can serve as labels for the indirect method and peroxidase and other enzymes can be used directly by labeling the primary antibody.

Prepare Karnofsky's incubation medium 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% by volume (in the 0.05 M Tris-HCl buffer). Mix the O.5 mg/ml DAB and the 0.02% by volune H2O2 in equal proportions a 1:1 ratio (this solution was freshly prepared each time it was used and was kept cold during the preparation). Add 200-300 ul of the DAB and H2O2n>ixture to the slide and incubate for 30 minutes in a moist chamber at room temperature.

After this incubation, remove the DAB and H20 mixture by washing the slide with the 0.05 M Tris HC1 pH 7.6 buffer to which has been added 0.1 M NaCl. The slides are given two 10 minutes washes in 0.05 M Tris HC1 pH 7.6 0.1 M NaCl. Finish processing of slides as indicated in Steps 1114 (except the PBS has been changed to Tris HC1). The completed slide is examined by light microscopy.

HeLa cell slides for immunofluorescence were prepared as follows: a stock supply of fixed HeLa cells was prepared by removing and washing with PBS, pH 7.2 actively growing cells from the HeLa culture bottle. The cells were suspended so that there were at least 1.5 x 106 cells/ml. One drop of the HeLa cell suspension was placed cn each washed slide (cleaned with detergent, rinsed with distilled or deionized water, cleaned with alcohol, rinsed and dried with heated air from hair dryer) and spread slightly and allowed to dry at room temperature (or in the cold overnight). The dried cells were fixed by placing the slides at 4°C in acetone for 12 minutes. The slides were numbered with a diamond point glass marking pencil. The slides were used as positive controls for immunofluorescence.

The present studies confirm that nucleolar antigens are present in tumor cells but are not found in nontumor tissues. Initial studies demonstrated that, both in cell cultures in human tumors and in specimens from either autopsy or biopsy samples, bright nucleolar fluorescence was produced by the double antibody technique (indirect immunofluorescence), and a corresponding result was not obtained with a series of nontumor tissues (Davis et al, 1979). In later studies, more than 60 malignant tumors were studied and a variety of tissue controls were also evaluated. It is of much interest that this broad array of malignant tumors of ectodermal, endodermal, and mesodermal origin exhibited the presence of one or more common nucleolar antigens (Table I).

Example 1 2o Normal Tissues - In 17 nontumor tissues there was no nucleolar fluorescence following incubation of the antisera or antibodies with the various fixed cell preparations. It was of particular interest that neither the Malpighian layer of the skin, nor the cells of the bone marrow, nor the crypts of Lieberkuhn demonstrated positive immunofluorescence with this procedure. Moreover, the variety of nontumor tissues adjacent to the neoplasms were also negative; these include many tissues of varying types. A group of benign tumors evaluated, including several types of thyroid adenomas, were also negative (Table II).

Example 2 Inflammatory Lesions - To ascertain whether an inflammatory response was related to the appearance of these antigens, studies were made on 8 types of inflammatory tissues. In most of these, there was no notable fluorescence in the nucleoli of the cells studied. However, sections were found in the ulcerative colitis and gastric ulcer specimens which did exhibit positive nucleolar fluorescence. Notably, 2 of 3 sections of the ulcerative colitis were negative and one showed definite positive nucleolar fluorescence. In the gastric ulcer, one of the 2 sections analyzed exhibited positive nucleolar fluorescence. These results are particularly interesting in view of the known propensity of these lesions to undergo malignant change. It was of special interest to review both the focal positive and negative regions of these slides in the hematoxylin eosin-stained sections; these showed that there were indeed regions in these lesions which exhibited not only mitotic figures but also a heaping up of the epithelial lining. This finding suggested that these cells might constitute preneoplastic lesions or carcinoma in situ. It is possible that the finding of these fluorescent regions might aid in decisions to proceed surgically with either local or more general resections of the affected lesions.

Example 3 Artifacts - In the gastric epithelium, there was a region of fluorescence in each cell which was non-nucleolar that appeared to represent a nonspecific localization of the fluorescent antibody. In a crypt of the small intestine, a nonspecific localization of the antibody appeared to occur in the form of aggregates; in most instances, prefiltration of the antibodies through a 0.45 pm Millipore (Millipore is a Trade Mark) filter eliminated these aggregates. In a sample of breast tissue, which was negative for nucleolar fluorescence, small nonspecific immunoflucrescent specks were generally distributed with no special localizing features with regard to cell morphology. The diameters of these very small nonspecific precipitates were 0.5 to 0.1 pm as compared to the nucleolar diameters in the nuclei and nucleoli which were 4 to 6 pm.

Example 4 Fluorescence during Phases of the Cell Cycle - The nucleolar fluorescence was readily visualized in the interphase nucleoli. In metaphase, the nucleolar fluorescence was not seen as a distinct entity but was visible between the chromosomes and in the junctional area between the nucleus and the cytoplasm. Inasmuch as the nucleolus largely disappears during metaphase and rRNA synthesis ceases in late prophase, it was not surprising that the nucleolar fluorescence was not visible as a distinct entity in such cells (Tan and Lerner, 1972). However, the finding that remnants of the immunofluorescent products persist throughout mitosis suggests that the nucleolar substructures (rather than the nucleolar products) contain the antigens which are persistent epigenetically.

Example 5 Malignant Tumor Negatives - In the series of malignant tumors, negative regions were found in varying extents throughout the slides. In general, these correlated with either necrotic or abscessed portions of the neoplasms. In one sample of a tumor of the brain, the mass which exhibited no positive fluorescence was necrotic; many leukocytes were present but there was no defined structure. One adenocarcinoma, which metastasized to the brain, did not exhibit positive fluorescence; the reasons were not clear. Inasmuch as 61 of 63 tumors studied had positive nucleolar fluorescence, 97% of the series studied was positive. These studies now have been broadly extended to over 300 human cancer specimens including a series of cancers of the breast, prostate, lung and hematological tumors with very similar results.

Example 6 Labelling - Direct immunochemical methods for the demonstration of the antibodies include labelling the primary antibody with one or more of the following labels: a radioisotope for autoradiography such as or 3H; a fluorescent dye such as fluorescein or tetramethyl rhodamine,for fluorescence microscopy, an enzyme which produces a fluorescent or colored product for detection by fluorescence or light microscopy, for example peroxidase, betagalactosidase, alkaline phosphatase or cytochrome C, or which produces an electron dense product for demonstration by electron microscopy; or an electron dense molecule such as ferritin, hemocyanin, virus particles or latex spheres, for direct electron microscopic visualization.

Indirect imnunochemical methods include labelling tile second antibod; or other binding protein specific for the first antibody with a fluorescent dye, an electron dense canpound, an enzyme which produces a product detectable by light, fluorescence or electron microsoopic examination or a radioisotope detectable by autoradiography.

The indirect immunochemical methods for the visualization of the antibodies include application of hybrid primary or secondary antibodies or antibody fragments (F(ab')2) wherein part of the hybrid antibody preparation is specific for the nucleolar antigens, (hybrid primary antibody) or for the primary antibody (hybrid second antibody), and part is specific for a label, such as those mentioned in the preceeding paragraph.

Labelled conjugated and nonconjugated antibodies maybe packaged separately in phosphate buffered saline (PBS) or other buffered suspending agents for distribution as diagnostic kits. Suitable suspending agents include glycerin, heparin, or sucrose. Suitable buffers include barbital buffers, morpholine buffers, MOPS-3-(N-morpholino) propane sulfonic acid, hepes-N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid and Tris carbonate.

Claims (31)

1. Human cancer cell associated antigens in substantially purified form having major and minor species, the major species having the following characteristics:a Pl on isoelectric focussing of from 6.0 to 6.7, and a molecular weight of from 50,000 to 60,000 daltons, is soluble in 0.01M Tris HC1 pH8, and is primarily localized in nucleoli of human cancer cells.

2. The major antigen species according to claim 1, in substantially purified form.

3. A method of isolating antigens claimed in claim 1 from nuclei or nucleoli of human cancer cells which method comprises extracting the antigens from nuclei or nucleoli of human cancer cells with 0.01M Tris HC1 at a pH of from 7 to 9.

4. A method as claimed in claim 3, which additionally comprises purifying the antigens by the sequential steps of precipitation with ammonium sulphate, DEAE - cellulose column chromatography, a gel exclusion technique using gel particles of cross-linked dextran, cation exchange and calcium phosphate gel chromatography and preparative isoelectric focussing.

5. A process of preparing antibodies characterized by specificity against antigens found in the nucleoli of human cancer cells which process comprises immunizing a non-human host animal with the antigens claimed in either claim 1 or claim 2, and harvesting the antibodies produced by the immunized animal.

6. A process as claimed in claim 5, wherein the host animal is immunized with a human cancer cell nucleoli preparation.

7. A process as claimed in claim 5, wherein the host animal is immunized with an extract of human cancer cell nuclei or nucleoli.

8. A process as claimed in claim 7, wherein the extract used is obtained by extracting the nuclei or nucleoli of human cancer cells with 0.01 M Tris HC1 at a pH of from 7 to 9.

9. A process as claimed in any one of claims 5 to 8, which comprises an additional step of purifying the antibodies harvested using DEAE-cellulose column chromatography.

10. Antibodies prepared by a process claimed in any one of claims 5 to 9.

11. A process for the immunological detection of cancer in a specimen of a human tissue section, smear or exfoliative cytological preparation, which process comprises contacting the specimen with the antibodies of claim 10 and demonstrating the localization of antibodies in the nucleoli of malignant cells, but not of SO 7 β Ο normal cells, in the specimen.

12. A process as claimed in claim 11, wherein the antibodies are characterized by specificity against antigens found in the nucleoli of malignant human 5 cells selected from HeLa cells, carcinoma cells, sarcoma cells and hematological neoplasm cells.

13. A process as claimed in claim 12, wherein the antibodies are characterized by specificity against antigens found in the nucleoli of HeLa cells or human 10 prostatic carcinoma cells.

14. The process claimed in any one of claims 11 to 13, wherein the antibodies are labelled and localization of antibodies in the nucleoli of malignant cells is demonstrated by a technique selected from 15. Light microscopy, fluorescence microscopy, electron microscopy and autoradiography.

15. The process claimed in any one of claims 11 to 13, wherein localization of antibodies in the nucleoli of malignant cells is demonstrated by reaction with 20 a reagent, a product of the reaction being detected by a technique selected from light microscopy, fluorescence microscopy, electron microscopy and autoradiography.

16. A diagnostic kit comprising antibodies as 25 claimed in claim 10 in a buffered suspending agent or solution, which antibodies are provided with a label which is detectable by a technique selected from light microscopy, fluorescence microscopy, electron microscopy and autoradiography.

17. A diagnostic kit as claimed in claim 16, wherein the antibodies are labelled with one or more of the following:(1) a radioisotope detectable by autoradiography, (2) a fluorescent dye detectable by fluorescence microscopy, (3) an enzyme which produces a fluorescent or coloured product detectable by fluorescence or light microscopy, (4) an enzyme which produces an electron dense product detectable by electron microscopy, and (5) an electron dense substance detectable by direct electron microscopic visualization.

18. A diagnostic kit as claimed in claim 17, wherein the antibodies are labelled with a radioisotope selected - 125 T 135 T 14 , . 3„ from I, I, C and H.

19. A diagnostic kit as claimed in claim 17, wherein the antibodies are labelled with a fluorescent dye selected from fluorescein and tetramethyl rhodamine.

20. A diagnostic kit as claimed in claim 17, wherein the antibodies are labelled with an enzyme which produces a fluorescent or coloured product detectable by fluorescence or light microscopy selected from peroxidase, Beta-galatosidase, alkaline phosphatase and cytochrome C.

21. A diagnostic kit as claimed in claim 17, wherein the antibodies are labelled with an electron dense substance selected from ferritin, hemocyanin, virus particles and latex spheres. 5

22. A diagnostic kit comprising antibodies as claimed in claim 10 in a buffered suspending agent or solution and a reagent for the detection of the antibodies localized in the nucleoli of malignant cells which reagent reacts to give a product which is detectable by 10 a technique selected from light microscopy, fluorescence microscopy, electron microscopy and autoradiography.

23. A diagnostic kit as claimed in claim 22 wherein the reagent comprises at least one of a labelled second antibody and other binding protein specific for the 15 antibodies of claim 10 or modifications thereof.

24. A diagnostic kit as claimed in claim 23, wherein the label is selected from (1) a radioactive isotope detectable by autoradiography, 20 (2) a fluorescent dye detectably by fluorescence microscopy, (3) an enzyme which produces a fluorescent or coloured product detectable by fluorescence microscopy or light microscopy, 25. (4) an enzyme which produces an electron dense product detectable by electron microscopy, and {5) an electron dense substance detectable by direct electron microscopic visualization. S0760

25. Human cancer cell associated antigens according to claim 1, substantially as hereinbefore described.

26. A method according to claim 3 of isolating antigens, substantially as hereinbefore described. 5

27. Antigens whenever isolated by a method claim in any one of claims 3, 4 and 26.

28. A process according to any one of claims 5 to 9 for preparing antibodies with specificity against antigens found in the nucleoli of human cancer cells, sub10 stantially as hereinbefore described.

29. Antibodies with specificity against antigens found in the nucleoli of human cancer cells, whenever prepared by a process claimed in claim 28.

30. A diagnostic kit according to any one of claims 15 16 to 21 substantially as hereinbefore described.

31. A diagnostic kit according to any one of claims 22 to 24 substantially as hereinbefore described.