CA1172165A - Detection of human cancer cells with antibodies to human cancer nucleolar antegen(s) - Google Patents
Detection of human cancer cells with antibodies to human cancer nucleolar antegen(s)Info
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- CA1172165A CA1172165A CA000424445A CA424445A CA1172165A CA 1172165 A CA1172165 A CA 1172165A CA 000424445 A CA000424445 A CA 000424445A CA 424445 A CA424445 A CA 424445A CA 1172165 A CA1172165 A CA 1172165A
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Abstract
ABSTRACT OF THE DISCLOSURE
Specific common nucleolar antigen(s) are found in 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 subs-tantially 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.
Specific common nucleolar antigen(s) are found in 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 subs-tantially 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
~172~6S
I This application is a division of application Serial No. 343,088, filed January 4~ 19800 FIELD OF T~E INVENTION
; This inv~ntion relates to nucleolar antigen(s) found in a broad range of human cancers and not found in corresponding non-tumor tissues and to antibodies and antisera specific to those nucleolar antigen(s) for diagnostic purposes.
~ACKGROUND- OF THE INVENTION
Earlier finds in exper1mental animals have lndicated ~the presénce of nuclear and nucleolar antigén(sj in tumors which were not found in non-tumor tissues (R.K.
., , .. _ .. . , . . _ , ...... . .
Busch et al, Cancer Res, _, 23~2, 1974; Yeomar et al, Proc.
,~
11~7~S
1 Natl. Acad. Sci. USA 73, 3258, 1976; ~usch and Busch, Tumori 63, 347, 1977; Davis et al, Cancer Res. 38, 1906, 1978;
. .. . ................ ........ ... . . _ ,._ _ __ Marashi et al, Cancer Res. 39, 59, 1979). ~n these early studies by the inventors, antibodies were prepared to nu-cleoli of rat normal and neoplastic cells by immunization of rabbits (R. K. Busch et al, supra; Busch and Busch, Tumori, 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 Ouchterlony ~els formed with anti-sera to Novikoff hepatoma nucleolar antigen(s) extracted from rat Novikoff hepatoma nucleoli differed from the corresponding immunoprecipitin bands produced with li~ver nucleolar antigen(s) and antiliver nucleolar an~isera tBUSch and ~usch, supra).
Further specificity was shown when antitumor nucleolar antiserum absorbed ~ith liver nuclear extracts produced positive nucleolar fluorescence in Novikoff hepato~a ascites cells but not in liver cells. Conversely, antiliver nucleolar antiserum absorbefl 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 af~er absorp-tion of the antisera with normal liver nuclei and nucleoli), attempts were made to utilize these antisera to rat tumor nucleolar antigen(s~ in testing corresponding tissue samples derived from 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 ~ ~ ~ 7;~5 1 human nucleolar antig~nts). Positive immunofluorescence was then found in human tumor tissues with antisera and anti-bodies to these new human tumor nuclaolar preparations. In these studies, the antibodies were absorbed ~lith placental nuclear sonicates as well as fetal calf serum (Busch et al, 39, 3024, 197g; 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 stucies designed to utilize these new human nucleolar antigen(s) for the detection of a broad ranae of human neoplasms.
The followin~ Table I presents a summary of the human tumors in ~hich 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 antigen(s) which exhibits a positive immunofluorescence with antisera or immunoglohulin fractions of such antisera (Busch et al, supra.
:. .
I This application is a division of application Serial No. 343,088, filed January 4~ 19800 FIELD OF T~E INVENTION
; This inv~ntion relates to nucleolar antigen(s) found in a broad range of human cancers and not found in corresponding non-tumor tissues and to antibodies and antisera specific to those nucleolar antigen(s) for diagnostic purposes.
~ACKGROUND- OF THE INVENTION
Earlier finds in exper1mental animals have lndicated ~the presénce of nuclear and nucleolar antigén(sj in tumors which were not found in non-tumor tissues (R.K.
., , .. _ .. . , . . _ , ...... . .
Busch et al, Cancer Res, _, 23~2, 1974; Yeomar et al, Proc.
,~
11~7~S
1 Natl. Acad. Sci. USA 73, 3258, 1976; ~usch and Busch, Tumori 63, 347, 1977; Davis et al, Cancer Res. 38, 1906, 1978;
. .. . ................ ........ ... . . _ ,._ _ __ Marashi et al, Cancer Res. 39, 59, 1979). ~n these early studies by the inventors, antibodies were prepared to nu-cleoli of rat normal and neoplastic cells by immunization of rabbits (R. K. Busch et al, supra; Busch and Busch, Tumori, 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 Ouchterlony ~els formed with anti-sera to Novikoff hepatoma nucleolar antigen(s) extracted from rat Novikoff hepatoma nucleoli differed from the corresponding immunoprecipitin bands produced with li~ver nucleolar antigen(s) and antiliver nucleolar an~isera tBUSch and ~usch, supra).
Further specificity was shown when antitumor nucleolar antiserum absorbed ~ith liver nuclear extracts produced positive nucleolar fluorescence in Novikoff hepato~a ascites cells but not in liver cells. Conversely, antiliver nucleolar antiserum absorbefl 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 af~er absorp-tion of the antisera with normal liver nuclei and nucleoli), attempts were made to utilize these antisera to rat tumor nucleolar antigen(s~ in testing corresponding tissue samples derived from 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 ~ ~ ~ 7;~5 1 human nucleolar antig~nts). Positive immunofluorescence was then found in human tumor tissues with antisera and anti-bodies to these new human tumor nuclaolar preparations. In these studies, the antibodies were absorbed ~lith placental nuclear sonicates as well as fetal calf serum (Busch et al, 39, 3024, 197g; 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 stucies designed to utilize these new human nucleolar antigen(s) for the detection of a broad ranae of human neoplasms.
The followin~ Table I presents a summary of the human tumors in ~hich 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 antigen(s) which exhibits a positive immunofluorescence with antisera or immunoglohulin fractions of such antisera (Busch et al, supra.
:. .
2 0 TABLE
B~IGHT NUCLEOLAR IMMUNOFLUORESC~NCE IN HUMAN TU~!ORS
(From:~ Busch et al, 1979) I. Carcinomas 1. Bladder, Transitional cell 2. Brain astrocytoma glio~lastoma
B~IGHT NUCLEOLAR IMMUNOFLUORESC~NCE IN HUMAN TU~!ORS
(From:~ Busch et al, 1979) I. Carcinomas 1. Bladder, Transitional cell 2. Brain astrocytoma glio~lastoma
3. Colon, adenocarcinoma ~4 metasta~is: liver transplantable carcinoma (C-W-39)
4. Eccrine gland, carcinoma
5. Esopha~us, squamous cell carcinoma
6. Liver, primary carcinoma
7. Lung:
adenocarcinoma (2) oat cell (2) squamous cell (5) llt7~6S
1 8. Melanoma, maliynant, cerehral metastases 9. Prostate, adenocarcinoma (4) 10. Skin: basal cell carcinoma (2) s~uamous cell carcinoma (7) metastasis: lymph node 11. Stomach, adenocarcinoma metastasis: liver metastasis: lymph node 12. Thyroid, carcinoma (2) II. Sarcomas 1. Myoblastoma, malignant of lip metastasis to cervical lymph node 2. Osteogenic sarcoma (3), biopsy, tissue cluture 3. 5ynovial sarcoma 4. Lymphoma (4), non Hodykins III. Hematoloqical Neoplasms 1. Hodgkins disease (Reed Sternberg, 5) 2. Leukemia: CLL (5), Hairy cell (spleen) 3. Lymphoma, lymphocytic, spleen 4. Multiple myeloma (5) 5. Mycosis fungoiaes 6. Acute myelocytic leukemia (5) 7. Chronic myelocytic leukemia (5)
adenocarcinoma (2) oat cell (2) squamous cell (5) llt7~6S
1 8. Melanoma, maliynant, cerehral metastases 9. Prostate, adenocarcinoma (4) 10. Skin: basal cell carcinoma (2) s~uamous cell carcinoma (7) metastasis: lymph node 11. Stomach, adenocarcinoma metastasis: liver metastasis: lymph node 12. Thyroid, carcinoma (2) II. Sarcomas 1. Myoblastoma, malignant of lip metastasis to cervical lymph node 2. Osteogenic sarcoma (3), biopsy, tissue cluture 3. 5ynovial sarcoma 4. Lymphoma (4), non Hodykins III. Hematoloqical Neoplasms 1. Hodgkins disease (Reed Sternberg, 5) 2. Leukemia: CLL (5), Hairy cell (spleen) 3. Lymphoma, lymphocytic, spleen 4. Multiple myeloma (5) 5. Mycosis fungoiaes 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. Squamous cell carcinoma (3) *Nu~bers in parenthesis represent~number of cases In the non-tumor tissues, henign tumors, and in-flammatory statés,~negative results were generally cbtained as indicated in the followin~ Table II (susch et al, supra).
._ TABLE II
NEGATIVE IM~UNOFLUORESCENCE IN r~U~1AN TISStlES
(From: Busch et al, 1979) I. ~iormal Tissue 1. Bladder 2. Bone marrow Ihemoblastic lines, 5)*
3. Breast 4. Buffy coat-blood (3) l 5- Gallbladder 6. Intestine, small/ crypts of Lieberkuhn 7. Intestine, large 8. Kidney
._ TABLE II
NEGATIVE IM~UNOFLUORESCENCE IN r~U~1AN TISStlES
(From: Busch et al, 1979) I. ~iormal Tissue 1. Bladder 2. Bone marrow Ihemoblastic lines, 5)*
3. Breast 4. Buffy coat-blood (3) l 5- Gallbladder 6. Intestine, small/ crypts of Lieberkuhn 7. Intestine, large 8. Kidney
9. Liver (2)
10. Lung (adjacent to tu~or)
11. Lymph node
12. Lymphocytes, normal (2)
13. Pancreas
14~ Pineal glan~
15. Pituitary
16. Placenta
17. Prostate gland
18. Skin l9. Stomach 20. Thyroid gland II. eniq- _owiA~ e~
1. Breast, adenoma 2. Paxathyroid, 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 5. Lupus profundus (mammary gland and s~in) 6. Pemphigus - buIlous 7. Ulcer, gastric 8. Inflammatory hyperplasia-lymph nodes (4) 9. Infectious mononucleosis (5 IV. Cultures .... ~
1. Breast fihroblasts 2. Lymphocytes, PHA stimulated *Numbers in parenthesis represent number of cases These results, originally obtained with immuno-fluorescence, have been verifled and extended with immuno-peroxidase methods.
BACKGROUND REFERENCES
Busch, ~., Gyorkey, ~., Busch, R. K., ~avis, F. M., Gyorkey, P. and Smetana, K~ A nucleolar antigen(s) iound in a broad range o~ human malignant tumor specimens. Cancer Res. 39: 3024-3030, 1979.
1~7Z~S
1 Busch, R. K. and Busch, H. AntigeniC proteins of nucleolar chromatin of Novikoff hepatoma ascites cells.
Tumori 63: 347-357, 1977.
Busch, R. K., Daskal, I., Spohn, W. H., ~ellermayer, ~. and Busch, H. Rabbit antibodies to nucleoli of Novikoff hepato~a and normal liver of the rat. Cancer Res. 34:
2362-2367, 1974.
Dale, G. and Latner, S. L. Isoelectric focusing of se~um proteins in acryla~ide ~els followed by electrophoresis.
Clin. Chim. Acta 24: 61-68, 1969, Davis, F. M., Busch R. K., Yeo~an, L. C. and Rusch, ~. Differences in nucleolar anti~en(s) of rat liver and Novikoff hepatoma ascites cells. Cancer Res. 38: 1906-1915, 1978.
Davis, F. M., Gyorkey, F., Busch, R. K. and Busch, . A nucleolar antigen(s) found in several human tumors but not in nontumor tissues. Proc. Natl. Acad. Sci. ~S~ 76:
892-896, 1979.
Garvey, J.S~, Gremer, M.E. and Sussdorf, D. H.
~ethods in Immunology, 1~77. W. a. Benjamin, Inc. Reapin~, ~lass.
Hilgers, J., Nowinski, R. C., Geering, G. and Hardy, W. Detéction of avian and ma~alian oncoyenic RNA
viruses (oncornaviruses) by immunofluorescence. Cancer Res.
32: 98-106, 1972. ~
Kendall, F. E. The use of immunochemical methods for th~ identification and determination of human serum proteins! Cold Spring Harbor Symp. Quant. Biol. 6: 376-3~4, 1938.
Laurell, C. B. Electroimmunoassay. Scand. J. Clin.
Lab. Invest. 29 (Suppl. 124): 21-37, 1972.
~17~ i5 1 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951.
Marashi, F., Davis, F. rl.~ Busch, R. K., Savage, H.
F. and Busch, H. Purification and partial characterization of nucleolar antigen-l of the Novikoff hepatoma. Cancer Res.
39: 59-66, 1979.
Smetana, K., Busch, R. K., Hermansky, F. and Busch, H. Nucleolar immunofluorescence in hu~an hematological malignancies. Life Sci. 25: 227-234, 1979.
Tan, E. ~. and Lerner, R. A. An immunological study of the fate of nuclear and nucleolar macromolecules during the cell cycle. J. Mol. Biol. 68: 107-114, 1972.
Wallace, R. W., Yu, P. H., Dicekcert, J. P. and Diec~ert, J. W. Visualization of protein-SDS complexes in polyacrylamide gels by chilling~ Anal. Bioche~. 61: 86-92, 1974.
Yeoman, L. C., Jordan, JD J~ ~ Busch, R. X., Taylor, C. ~7., Savage, H. and Busch, H. A fetal protein in the ~ chromatin of NoviXoff hepatoma and Walker 256 carcinosarco~a tumors that is abs nt from normal and regenerating rat liver. Proc. Natl. Acad. Sci. USA 73: 325~-3262, 1976.
SUMMARY OF THE I~1VENTION
The present invention resides in the surprising and unexpected discovery that common nucleolar antigen(s) are found in a hroad ranae of human cancer cells but are not found in normal human cells. The antiaen(sj are proteins which ~ay 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 antigen(s) found in hu~an cancer cells, isolation and purification of the nucleolar antigen(s), l72~
1 production of antibodies specific to these antigen(s), diagnostic test methods usin~ antihodies specific to these antigen(s) to detect human cancer cells, a diaqnostic kit containing either antibodies or antisera specific to these nucleolar anti~en(s).
Accordin~ly, an object of the present invention is the provision o co~mon nucleolar anti~en(s) found in a ~Jide range of human cancer cells.
A further object of the present invention is the provision of antisera and antibodies specific to these common nucleolar antigen(s) which can be used for diagnostic and treatment purposes.
A urther object of the present invention is the provision of these nucleolar antigen(s) in substantially purified for~.
A further object of the present invention is the provision of processes for extracting and isolating these nucleolar antigen(s).
A further object of the present invention is the ~20 provision of processes for purlfying these nucleolar antigen(s).
A further object of the present invention is the provision of antisera and antibodies having high specificity to common nucleolar antigen(s) found in a broad range of human cancer cells.
A further object of the present invention is the provision of antisera and antibodies induced by and specific t~ purified antigen(s) ~ound in a broad range of human cancer cells.
A further object of the present invention is the provision of diagnostic kits comprised of antibodies or antisera induced by and specific ~o nucleolar antigen(s).
A further object of the present invention is 17Z~6i5 1 the provision of antisera and antibodies specific to the same antigen(s) found in the nucleoli of a broad ranae of human cancer cells.
A further object of the present invention is the provision of antibodies and antisera specific to the common or same antiqen(s) found in a wide range of human cancer cells which serve as a carrier for markers for diagnostic purposes.
Other and further objects, features and advantages of the invention appear throughout.
DESCRIPTION OF TIIE PP~EFERRED EMBODIMF.NTS
The present invention resides in the unexpected and surprising discovery that common specific antigen(s) are present in the nucleoli of a wide range of human cancer cells, their extraction, isolation, and substantial purification, the production of antisera and antibodies of high specificity and selectivity to these nucleolar antigen(s) which can be tagged directly or indirectly to allow diagnostic testing for human cancers in v ro and in vivo.
The Antiqen in thc Cancer Cells The anti~en(s) have been found in a broad range of human cancers including cancers of the central nervous system, gastrointestinal tractj genitourlnary tract, lung, skin, blood forming tissues and endocrine and exocrine glands. For example, the malignant human cells include ~IeLa cells, prostatic carcinoma, other carcinomas, sarcomas and hematological neoplasms. The antigen~s) can be extracted from nuclei or nucleoli of human malignant cells. The antigen ( s ) have not been found in corresponding nontumor tissues. In using the diagnostic methods of the present in~ention to detec~ malignant cells, approximately one percent ~alse negatives and three percent false positives -~7~65 1 were detected. The false negatives represent necrotic tu~or tissues or non-reactive tu~ors 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 antigen(s) have a ~ajor 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 of from 6.0 to 6.7 an~ approximately 6.3 as deter~ined by isoelectric focusing, pH 3-10, polyacrylamide gel; (b) has an approximate molecular t~eight of 50,000 to 60,000 daltons as determined by two-dimensional gel electro-phoresis with an SDS(sodium dodecylsulfate~ second dimension;
(c) is in part tightly bound to nuclear and nucleolar RMP and in part soluble in 0.01 M Tris-HCl, pH 8; (d) and is both nucleolar and extranucleolar but remains "intranuclear" or chromosone-associated during cell division.
-20 The second antigen species which has been detected has a pI of approximately 6.0 (detected by the sa~e procedures as the ~ajor 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 antiqen species.
Antigen(s) are also present in nucleolar ribo-nucleoprotein (RNP) particles obtained by ultracentrifugation of the Tris extracts, subsequently descrihed. The antigen present in these particles is more tightly bound to proteins and ribonucleic acid (P~NA) than the antigen in the Tris ~172~65 1 soluble fraction It is not yet clear whether the anti~en(s) 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 anti~en(s). Nucleolar antigen(s) present in fibrils, probably of the nuclear rihonucleoprotein network, are also seen in cancer cells by immune-light microscopy. These are e~tranucleolar structures which may represent elements from which the ~.~lP particles are derived.
It remains to be determined whether the an-ti~en(s) represent a substance that is present in high concentrations in cancer cells and very low conccntrations in noncancerous cells or are ~etal anti~en(s) as was found earlicr in the co~parative studies on nucleolar anti~ens of the rat Novikoff hepatoma and normal rat liver cells (Yeoman t al, 1976).
Human Tumors and Other Tissues All steps for obtaining and analyzing samples of human tissue, blood and serum of suspected cancer patients were approved by the Human Research Co~mittee at Baylor College of Medicine, Houston, Texas and affiliated hospitals.
Sections of human tumors were obtained from frozen sections of surgical specimens, biopsy, or pxeserved cryostat speci~.ens, ~ainly from the Department of Pathology from the Houston Veterans Ad~inistration Medical Center, and also from The Michigan Cancer Foundation in Detroit, Michigan, and the Department of Internal ~edicine, Charles University in Pra~ue, Czechoslovakia. mhese sections were analyzed for the presence of nucleolar anti~en~s) by indirect immunofluorescence and i~munoperoxidase tcchniques.
Purification of the ~Jucl~olar Anti~en(s) _ Purification of the antigen(s) was achieved by extraction of nuclei or nucleoli with 10 mM Tris HCl/0.1 M
PMSF/pH 8 for 6 times in a ratio of 20 volumes to onc volume ~7~6S
1 of nuclei or nucleoli. The extract was centrifuged first at 27,000 g for 10 minutes and then at 100,000 g for 16 hours.
Am~oniu~ sulfate at 40% saturation was used to re~ove conta-minants. The 40 - 100% am~.oniu~ sulfate fraction was collected by centrifugation and dialyzed against 20 ~il Tris HCl/pH 7.6.
The antigen(s) were chromatographed on DE-52 cellulose columns (1 x 10 crn). The antig~n(s) were eluted in the 0.15M NaCl/0.1 mM P~SF/pH 7.6 fraction. Isoelectric focusing gels were used to identify and purify the antigen(s). These contained 4%
acrylamide/8M urea/2% ampholines (pH 3.5-10). The antigen(s), pI 6.3 and 6.0 respectively, were cut out of the gels. On SDS (sodium doaecyl sulfate) gels, one major spot was found for each of these antigens.
Preparation of HeLa Cell Nuclei or Nucleoli HeLa cells were collected from Spinner culture bottles (7-8 liters). The cells should be and were in log phase 7-8 x 105 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 gentle homogeniæation with a loose Teflon 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 homogeni2ation in 20 volumes of reticulocyte saline buffer (RS~), 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 deterg~nt Nonidet P40 (10~ in RSE). The final volume of ~onidet was 0.5%. The cells were ho~ogenized with a Dounce homogenizer 20-60 strokes until the cells were broken and the nuclei released and f-eed of cytoplasm. The cells were then centrifuged 1000 x g for 8 ~7Z~65 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 sonifier) by 10-second bursts (and 10 seconds rest). Total time was between 60 and 110 seconds. The nucleoli released were monitored by microscopic examination.
.. . _, .. , . . . _ . .. _ . . . . , . . ................ .. ; . _ . . _ To visuali2e 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 mmunogen.
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-~ quantities used in earlier studies, provided sufficient material for adequate purification.
*
Trade Mark 21~5 The conditions for growing the HeTa cells and the isolation of placental nuclei were essentially the same as those reported previously (Davis et al, 1979).
Preparation of the HeLa Tris Extract The ~eLa nuclei were suspended 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 phenylmethyl-suIfonylfluoride (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 repeatea 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 OoOl M Tris-HC1; 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 examinatlon. 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 membrane. Generally, the volume at the beginning is around 50 ml and this was concentrated ~rade Mark - 14 -1 do~n to 4~5 ~1. The final concentration of protein is around 4-5 mg/ml. The rabhit 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 immuno~en, dilute 250 ul of Tris extract (4-5 mg/ml) as prepared above with 250 ul PBS.
Co~bine this with the Freund's adjuvant as described for the nucleolar immunogen below.
Immunization of Rabbits with HeLa Cell Nucleolar Preparations to Produce Antibodies The HeLa nucleoli were weighed (20-30 mg, wet weight) and suspended evenly in 0.5 ml Q.01 M phosphate buffered saline, pH 7.2. They were then mixed with 0.6 ml ~reund's complete adjuvant ~GIBCO) as follows: The suspended nucleoll were placed in a 5 ml syringe and the Freund's adjuvant in a second syringe. ~o each syringe an 18 ~auge needle from which the tip had been removed was attached. ~he needles were then connected by a piece of polyethylene~
tubing, I.D. 0.047 ~C1ay-Adams). The contents of the syrin~es were ~ixed until the preparation became thickened and wa~
difficul~ 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 bacX) and half intramuscularly (in the thi~h 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 vacuu~ pump were used to collect the blcod. The blood ~approximately 45-50 ~1) was allowed to clot for 3-4 hours at room temperature. The serum -15~
~- ~ 7216S
1 portion was removed fro~ 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 antihodies by the indirect immunofluorescence procedure.
Other non-human hosts (e.g. goat, sheep, horse, chic~en, etc.) may be immunized with human malignant cell nucleoli preparations to elicit the antisera or antibodies to the nucleolar antigen(s) of the present invention. Antisera ~ay also be prepared by immunization of non-human hosts animals with extracts (e,g. tris extract) of human mali~nant cell nuclei or nucleoli.
Absorption of Antinucleolar Antiserum The rabbit antiserum ~as first absorbed with 20 normal human serum and 20% fetal calf serum (GIBCO). 20 normal human serum was added to the rabbit an iserum (4 ml~
20 ml) and incubated for 1 hour in a 37C shaking water bath.
The flask was removed, 20% fetal calf serum (4.8 ml/29 ml) was added, and incubation was carried out for 1 hour in a 37 shaking water bath. The flask was removed and incubated an additional hour at room temperature with mixing by gentle swirling every 15 minutes. It was then centrifuged at 15,000 x g for 30 minutes, and the supernatant (absorbed serum~ was removed and save~. The absorbed seru~ was converted to the immunoglobulin (Ig) form by following the procedure yiven for the (NH4)2so9 precipitation of serum, subsequently described.
~he Ig preparation from the nucleolar antiserum which had becn absorbed with normal human serum and fetal 1~7216~
1 calf seru~ was now absorbed with a nor~al 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 37C 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 15,0Q0 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, i5 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, ~1hatman DE52). The IgG is eluted from the colu~n 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 sa~e procedure was followed for the control serum which consisted of prei~mune serum which ~as obtained by bleeding the rabbit (or other non-human host ani~al) before the immuniæation was started.
Preparation of Rabbit Immunoalobulin I~
A saturated (NH4)2SO4 solution (760 gm/liter) was prepared and an equal volume of cold saturated (NH4)2SO4 was added drop b~ drop to the antiserum with stirring. A white precipitate for~ed 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 ~7~65 1 minutes. The supernatant was removed and the pellet was resuspended in PBS, pH 7.2 (appro~imately half the volume of the original serum). The solubilized pellet was placed in a dialysis bag and dialyzed against 100 volu~es of PBS overnight in the cold (with magnetic stirring). The dialysis ba~ 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 centrifuc~ed at 2500 x g for 20 minutes. The supernatant was collected.
L0 Immunofluorescence The procedure descrihed earlier (RK Busch et al, 1974; Hilgers et al, 1972) for immunofluorescence was used in this study, as follows:
Indirect Immunofluorescence Method:
150 ul of antinucleolar antiserum diluted 1:50 was placed on acetone fixed HeLa cells or on fixed tissue specimens (fro~ ~ilgers et al, 1972; RK Busch et al, 1974). It may be necessary to use more than 150 ul if the tissue specimen covers a large section of the slide. Dilution of antiserum is dependent on Ab titer. Other dilutions can be used up to the point ~here 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 37C. (The moist cha~ber may consists of a large petri dish to which has been added a moist paper towel.) After the incubation, the antiserurl 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. ~he slides were removed from PBS and dipped in distilled or deionized water ten ti~es with rapid up and down movements.
The slides ~ere dried with cold air from a blow or hair dryer --1~--:~7Z~65 1 (2-3 minutes), being careful not to overdry. 150 ul 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 te~perature.
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 ~BS and left in the refrigerator overnight. After the final wash with PBS, the slides were dipp~d 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 sa~e as those used in earlier studies (Kendall, 193~; Lowry et al, 1951;
Dale and Latner, 1969; Laurell, 1972; ~allace et al, 197~;
Marashi et al, 1979). For analysis of nucleolar localization of immunofluorescence, samples were switched in and out of phase contract illumination during fluorescence observation.
Immunoperoxidase Method:
Instead of fluorescein-labeled goat antirabbit 150 ul of peroxidase labeled ~oat 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
1. Breast, adenoma 2. Paxathyroid, 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 5. Lupus profundus (mammary gland and s~in) 6. Pemphigus - buIlous 7. Ulcer, gastric 8. Inflammatory hyperplasia-lymph nodes (4) 9. Infectious mononucleosis (5 IV. Cultures .... ~
1. Breast fihroblasts 2. Lymphocytes, PHA stimulated *Numbers in parenthesis represent number of cases These results, originally obtained with immuno-fluorescence, have been verifled and extended with immuno-peroxidase methods.
BACKGROUND REFERENCES
Busch, ~., Gyorkey, ~., Busch, R. K., ~avis, F. M., Gyorkey, P. and Smetana, K~ A nucleolar antigen(s) iound in a broad range o~ human malignant tumor specimens. Cancer Res. 39: 3024-3030, 1979.
1~7Z~S
1 Busch, R. K. and Busch, H. AntigeniC proteins of nucleolar chromatin of Novikoff hepatoma ascites cells.
Tumori 63: 347-357, 1977.
Busch, R. K., Daskal, I., Spohn, W. H., ~ellermayer, ~. and Busch, H. Rabbit antibodies to nucleoli of Novikoff hepato~a and normal liver of the rat. Cancer Res. 34:
2362-2367, 1974.
Dale, G. and Latner, S. L. Isoelectric focusing of se~um proteins in acryla~ide ~els followed by electrophoresis.
Clin. Chim. Acta 24: 61-68, 1969, Davis, F. M., Busch R. K., Yeo~an, L. C. and Rusch, ~. Differences in nucleolar anti~en(s) of rat liver and Novikoff hepatoma ascites cells. Cancer Res. 38: 1906-1915, 1978.
Davis, F. M., Gyorkey, F., Busch, R. K. and Busch, . A nucleolar antigen(s) found in several human tumors but not in nontumor tissues. Proc. Natl. Acad. Sci. ~S~ 76:
892-896, 1979.
Garvey, J.S~, Gremer, M.E. and Sussdorf, D. H.
~ethods in Immunology, 1~77. W. a. Benjamin, Inc. Reapin~, ~lass.
Hilgers, J., Nowinski, R. C., Geering, G. and Hardy, W. Detéction of avian and ma~alian oncoyenic RNA
viruses (oncornaviruses) by immunofluorescence. Cancer Res.
32: 98-106, 1972. ~
Kendall, F. E. The use of immunochemical methods for th~ identification and determination of human serum proteins! Cold Spring Harbor Symp. Quant. Biol. 6: 376-3~4, 1938.
Laurell, C. B. Electroimmunoassay. Scand. J. Clin.
Lab. Invest. 29 (Suppl. 124): 21-37, 1972.
~17~ i5 1 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951.
Marashi, F., Davis, F. rl.~ Busch, R. K., Savage, H.
F. and Busch, H. Purification and partial characterization of nucleolar antigen-l of the Novikoff hepatoma. Cancer Res.
39: 59-66, 1979.
Smetana, K., Busch, R. K., Hermansky, F. and Busch, H. Nucleolar immunofluorescence in hu~an hematological malignancies. Life Sci. 25: 227-234, 1979.
Tan, E. ~. and Lerner, R. A. An immunological study of the fate of nuclear and nucleolar macromolecules during the cell cycle. J. Mol. Biol. 68: 107-114, 1972.
Wallace, R. W., Yu, P. H., Dicekcert, J. P. and Diec~ert, J. W. Visualization of protein-SDS complexes in polyacrylamide gels by chilling~ Anal. Bioche~. 61: 86-92, 1974.
Yeoman, L. C., Jordan, JD J~ ~ Busch, R. X., Taylor, C. ~7., Savage, H. and Busch, H. A fetal protein in the ~ chromatin of NoviXoff hepatoma and Walker 256 carcinosarco~a tumors that is abs nt from normal and regenerating rat liver. Proc. Natl. Acad. Sci. USA 73: 325~-3262, 1976.
SUMMARY OF THE I~1VENTION
The present invention resides in the surprising and unexpected discovery that common nucleolar antigen(s) are found in a hroad ranae of human cancer cells but are not found in normal human cells. The antiaen(sj are proteins which ~ay 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 antigen(s) found in hu~an cancer cells, isolation and purification of the nucleolar antigen(s), l72~
1 production of antibodies specific to these antigen(s), diagnostic test methods usin~ antihodies specific to these antigen(s) to detect human cancer cells, a diaqnostic kit containing either antibodies or antisera specific to these nucleolar anti~en(s).
Accordin~ly, an object of the present invention is the provision o co~mon nucleolar anti~en(s) found in a ~Jide range of human cancer cells.
A further object of the present invention is the provision of antisera and antibodies specific to these common nucleolar antigen(s) which can be used for diagnostic and treatment purposes.
A urther object of the present invention is the provision of these nucleolar antigen(s) in substantially purified for~.
A further object of the present invention is the provision of processes for extracting and isolating these nucleolar antigen(s).
A further object of the present invention is the ~20 provision of processes for purlfying these nucleolar antigen(s).
A further object of the present invention is the provision of antisera and antibodies having high specificity to common nucleolar antigen(s) found in a broad range of human cancer cells.
A further object of the present invention is the provision of antisera and antibodies induced by and specific t~ purified antigen(s) ~ound in a broad range of human cancer cells.
A further object of the present invention is the provision of diagnostic kits comprised of antibodies or antisera induced by and specific ~o nucleolar antigen(s).
A further object of the present invention is 17Z~6i5 1 the provision of antisera and antibodies specific to the same antigen(s) found in the nucleoli of a broad ranae of human cancer cells.
A further object of the present invention is the provision of antibodies and antisera specific to the common or same antiqen(s) found in a wide range of human cancer cells which serve as a carrier for markers for diagnostic purposes.
Other and further objects, features and advantages of the invention appear throughout.
DESCRIPTION OF TIIE PP~EFERRED EMBODIMF.NTS
The present invention resides in the unexpected and surprising discovery that common specific antigen(s) are present in the nucleoli of a wide range of human cancer cells, their extraction, isolation, and substantial purification, the production of antisera and antibodies of high specificity and selectivity to these nucleolar antigen(s) which can be tagged directly or indirectly to allow diagnostic testing for human cancers in v ro and in vivo.
The Antiqen in thc Cancer Cells The anti~en(s) have been found in a broad range of human cancers including cancers of the central nervous system, gastrointestinal tractj genitourlnary tract, lung, skin, blood forming tissues and endocrine and exocrine glands. For example, the malignant human cells include ~IeLa cells, prostatic carcinoma, other carcinomas, sarcomas and hematological neoplasms. The antigen~s) can be extracted from nuclei or nucleoli of human malignant cells. The antigen ( s ) have not been found in corresponding nontumor tissues. In using the diagnostic methods of the present in~ention to detec~ malignant cells, approximately one percent ~alse negatives and three percent false positives -~7~65 1 were detected. The false negatives represent necrotic tu~or tissues or non-reactive tu~ors 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 antigen(s) have a ~ajor 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 of from 6.0 to 6.7 an~ approximately 6.3 as deter~ined by isoelectric focusing, pH 3-10, polyacrylamide gel; (b) has an approximate molecular t~eight of 50,000 to 60,000 daltons as determined by two-dimensional gel electro-phoresis with an SDS(sodium dodecylsulfate~ second dimension;
(c) is in part tightly bound to nuclear and nucleolar RMP and in part soluble in 0.01 M Tris-HCl, pH 8; (d) and is both nucleolar and extranucleolar but remains "intranuclear" or chromosone-associated during cell division.
-20 The second antigen species which has been detected has a pI of approximately 6.0 (detected by the sa~e procedures as the ~ajor 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 antiqen species.
Antigen(s) are also present in nucleolar ribo-nucleoprotein (RNP) particles obtained by ultracentrifugation of the Tris extracts, subsequently descrihed. The antigen present in these particles is more tightly bound to proteins and ribonucleic acid (P~NA) than the antigen in the Tris ~172~65 1 soluble fraction It is not yet clear whether the anti~en(s) 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 anti~en(s). Nucleolar antigen(s) present in fibrils, probably of the nuclear rihonucleoprotein network, are also seen in cancer cells by immune-light microscopy. These are e~tranucleolar structures which may represent elements from which the ~.~lP particles are derived.
It remains to be determined whether the an-ti~en(s) represent a substance that is present in high concentrations in cancer cells and very low conccntrations in noncancerous cells or are ~etal anti~en(s) as was found earlicr in the co~parative studies on nucleolar anti~ens of the rat Novikoff hepatoma and normal rat liver cells (Yeoman t al, 1976).
Human Tumors and Other Tissues All steps for obtaining and analyzing samples of human tissue, blood and serum of suspected cancer patients were approved by the Human Research Co~mittee at Baylor College of Medicine, Houston, Texas and affiliated hospitals.
Sections of human tumors were obtained from frozen sections of surgical specimens, biopsy, or pxeserved cryostat speci~.ens, ~ainly from the Department of Pathology from the Houston Veterans Ad~inistration Medical Center, and also from The Michigan Cancer Foundation in Detroit, Michigan, and the Department of Internal ~edicine, Charles University in Pra~ue, Czechoslovakia. mhese sections were analyzed for the presence of nucleolar anti~en~s) by indirect immunofluorescence and i~munoperoxidase tcchniques.
Purification of the ~Jucl~olar Anti~en(s) _ Purification of the antigen(s) was achieved by extraction of nuclei or nucleoli with 10 mM Tris HCl/0.1 M
PMSF/pH 8 for 6 times in a ratio of 20 volumes to onc volume ~7~6S
1 of nuclei or nucleoli. The extract was centrifuged first at 27,000 g for 10 minutes and then at 100,000 g for 16 hours.
Am~oniu~ sulfate at 40% saturation was used to re~ove conta-minants. The 40 - 100% am~.oniu~ sulfate fraction was collected by centrifugation and dialyzed against 20 ~il Tris HCl/pH 7.6.
The antigen(s) were chromatographed on DE-52 cellulose columns (1 x 10 crn). The antig~n(s) were eluted in the 0.15M NaCl/0.1 mM P~SF/pH 7.6 fraction. Isoelectric focusing gels were used to identify and purify the antigen(s). These contained 4%
acrylamide/8M urea/2% ampholines (pH 3.5-10). The antigen(s), pI 6.3 and 6.0 respectively, were cut out of the gels. On SDS (sodium doaecyl sulfate) gels, one major spot was found for each of these antigens.
Preparation of HeLa Cell Nuclei or Nucleoli HeLa cells were collected from Spinner culture bottles (7-8 liters). The cells should be and were in log phase 7-8 x 105 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 gentle homogeniæation with a loose Teflon 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 homogeni2ation in 20 volumes of reticulocyte saline buffer (RS~), 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 deterg~nt Nonidet P40 (10~ in RSE). The final volume of ~onidet was 0.5%. The cells were ho~ogenized with a Dounce homogenizer 20-60 strokes until the cells were broken and the nuclei released and f-eed of cytoplasm. The cells were then centrifuged 1000 x g for 8 ~7Z~65 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 sonifier) by 10-second bursts (and 10 seconds rest). Total time was between 60 and 110 seconds. The nucleoli released were monitored by microscopic examination.
.. . _, .. , . . . _ . .. _ . . . . , . . ................ .. ; . _ . . _ To visuali2e 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 mmunogen.
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-~ quantities used in earlier studies, provided sufficient material for adequate purification.
*
Trade Mark 21~5 The conditions for growing the HeTa cells and the isolation of placental nuclei were essentially the same as those reported previously (Davis et al, 1979).
Preparation of the HeLa Tris Extract The ~eLa nuclei were suspended 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 phenylmethyl-suIfonylfluoride (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 repeatea 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 OoOl M Tris-HC1; 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 examinatlon. 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 membrane. Generally, the volume at the beginning is around 50 ml and this was concentrated ~rade Mark - 14 -1 do~n to 4~5 ~1. The final concentration of protein is around 4-5 mg/ml. The rabhit 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 immuno~en, dilute 250 ul of Tris extract (4-5 mg/ml) as prepared above with 250 ul PBS.
Co~bine this with the Freund's adjuvant as described for the nucleolar immunogen below.
Immunization of Rabbits with HeLa Cell Nucleolar Preparations to Produce Antibodies The HeLa nucleoli were weighed (20-30 mg, wet weight) and suspended evenly in 0.5 ml Q.01 M phosphate buffered saline, pH 7.2. They were then mixed with 0.6 ml ~reund's complete adjuvant ~GIBCO) as follows: The suspended nucleoll were placed in a 5 ml syringe and the Freund's adjuvant in a second syringe. ~o each syringe an 18 ~auge needle from which the tip had been removed was attached. ~he needles were then connected by a piece of polyethylene~
tubing, I.D. 0.047 ~C1ay-Adams). The contents of the syrin~es were ~ixed until the preparation became thickened and wa~
difficul~ 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 bacX) and half intramuscularly (in the thi~h 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 vacuu~ pump were used to collect the blcod. The blood ~approximately 45-50 ~1) was allowed to clot for 3-4 hours at room temperature. The serum -15~
~- ~ 7216S
1 portion was removed fro~ 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 antihodies by the indirect immunofluorescence procedure.
Other non-human hosts (e.g. goat, sheep, horse, chic~en, etc.) may be immunized with human malignant cell nucleoli preparations to elicit the antisera or antibodies to the nucleolar antigen(s) of the present invention. Antisera ~ay also be prepared by immunization of non-human hosts animals with extracts (e,g. tris extract) of human mali~nant cell nuclei or nucleoli.
Absorption of Antinucleolar Antiserum The rabbit antiserum ~as first absorbed with 20 normal human serum and 20% fetal calf serum (GIBCO). 20 normal human serum was added to the rabbit an iserum (4 ml~
20 ml) and incubated for 1 hour in a 37C shaking water bath.
The flask was removed, 20% fetal calf serum (4.8 ml/29 ml) was added, and incubation was carried out for 1 hour in a 37 shaking water bath. The flask was removed and incubated an additional hour at room temperature with mixing by gentle swirling every 15 minutes. It was then centrifuged at 15,000 x g for 30 minutes, and the supernatant (absorbed serum~ was removed and save~. The absorbed seru~ was converted to the immunoglobulin (Ig) form by following the procedure yiven for the (NH4)2so9 precipitation of serum, subsequently described.
~he Ig preparation from the nucleolar antiserum which had becn absorbed with normal human serum and fetal 1~7216~
1 calf seru~ was now absorbed with a nor~al 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 37C 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 15,0Q0 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, i5 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, ~1hatman DE52). The IgG is eluted from the colu~n 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 sa~e procedure was followed for the control serum which consisted of prei~mune serum which ~as obtained by bleeding the rabbit (or other non-human host ani~al) before the immuniæation was started.
Preparation of Rabbit Immunoalobulin I~
A saturated (NH4)2SO4 solution (760 gm/liter) was prepared and an equal volume of cold saturated (NH4)2SO4 was added drop b~ drop to the antiserum with stirring. A white precipitate for~ed 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 ~7~65 1 minutes. The supernatant was removed and the pellet was resuspended in PBS, pH 7.2 (appro~imately half the volume of the original serum). The solubilized pellet was placed in a dialysis bag and dialyzed against 100 volu~es of PBS overnight in the cold (with magnetic stirring). The dialysis ba~ 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 centrifuc~ed at 2500 x g for 20 minutes. The supernatant was collected.
L0 Immunofluorescence The procedure descrihed earlier (RK Busch et al, 1974; Hilgers et al, 1972) for immunofluorescence was used in this study, as follows:
Indirect Immunofluorescence Method:
150 ul of antinucleolar antiserum diluted 1:50 was placed on acetone fixed HeLa cells or on fixed tissue specimens (fro~ ~ilgers et al, 1972; RK Busch et al, 1974). It may be necessary to use more than 150 ul if the tissue specimen covers a large section of the slide. Dilution of antiserum is dependent on Ab titer. Other dilutions can be used up to the point ~here 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 37C. (The moist cha~ber may consists of a large petri dish to which has been added a moist paper towel.) After the incubation, the antiserurl 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. ~he slides were removed from PBS and dipped in distilled or deionized water ten ti~es with rapid up and down movements.
The slides ~ere dried with cold air from a blow or hair dryer --1~--:~7Z~65 1 (2-3 minutes), being careful not to overdry. 150 ul 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 te~perature.
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 ~BS and left in the refrigerator overnight. After the final wash with PBS, the slides were dipp~d 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 sa~e as those used in earlier studies (Kendall, 193~; Lowry et al, 1951;
Dale and Latner, 1969; Laurell, 1972; ~allace et al, 197~;
Marashi et al, 1979). For analysis of nucleolar localization of immunofluorescence, samples were switched in and out of phase contract illumination during fluorescence observation.
Immunoperoxidase Method:
Instead of fluorescein-labeled goat antirabbit 150 ul of peroxidase labeled ~oat 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
-19-~L~7;~165 1 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 Xarnofsky's incubation medium as follows:
weigh out enough diaminoben~idine (Sigma) and suspend i.n 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 ~1 Tris-HCl buffer). Mix the 0.5 mg/ml DAB and the 0.02~ H2O2 in equal proportions a 1:1 ratio (this solution was freshly o prepared each time it was used and was kept cold during the preparation). Add 2Q0-300 Ul of the DAB and H2O2 mixture to the slide and incubate for 30 minutes in a moist chamber at room temperature.
After this incubation, remove the DAB and H2O
mixture by washing the slide with the 0.05 M mris E~Cl pH 7.6 buffer to which has been added 0.1 M NaCl. The slides are given two 10 minutes washes in 0.05 ~ Tris HCl pH 7.6 0.1 M
NaCl. Complete slides as indicated in Steps 11-14 (except the PBS has been changed to Tris HCl). The final slide is examined by light microscopy.
Preparation of HeLa Cell Slldes for Immunofluorescence A stock supply of fixed HeLa cells was prepared as follows: Actively growing cells from the HeLa culture bottle were removed and washed one time with PBS, pH 7.2.
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 on 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
Prepare Xarnofsky's incubation medium as follows:
weigh out enough diaminoben~idine (Sigma) and suspend i.n 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 ~1 Tris-HCl buffer). Mix the 0.5 mg/ml DAB and the 0.02~ H2O2 in equal proportions a 1:1 ratio (this solution was freshly o prepared each time it was used and was kept cold during the preparation). Add 2Q0-300 Ul of the DAB and H2O2 mixture to the slide and incubate for 30 minutes in a moist chamber at room temperature.
After this incubation, remove the DAB and H2O
mixture by washing the slide with the 0.05 M mris E~Cl pH 7.6 buffer to which has been added 0.1 M NaCl. The slides are given two 10 minutes washes in 0.05 ~ Tris HCl pH 7.6 0.1 M
NaCl. Complete slides as indicated in Steps 11-14 (except the PBS has been changed to Tris HCl). The final slide is examined by light microscopy.
Preparation of HeLa Cell Slldes for Immunofluorescence A stock supply of fixed HeLa cells was prepared as follows: Actively growing cells from the HeLa culture bottle were removed and washed one time with PBS, pH 7.2.
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 on 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
-20-721f~;5 1 slides at ~C in acetone for 12 minutes. Lhe slides were num~ered with a diarnond point glass marking pencil. The slides were used as positive controls for immunofluorescence.
Maliqnant Tumors as Detected with the Test Antibody The present studies confirm that nucleolar antigen(s) are present in tumor cells but are not found in nontumcr tissues Initial studies demonstrated that, both in cell cultures in human tumors and in specimens from either autcpsy 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 Normal Tissues - In 17 nontumor tissues there ~as no nucleolar ~luorescence following incubation of the antisera or antibodies wlth the various fix~d 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 Lieherkuhn 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. ~ group of benign tumors evaluated, including several types of thyroid adeno~as, were also negative (Table II).
Example 2 Inflammatory L~sions - To ascertain whether an inflammatory response was related to the appearance of these antigen(s), studies were rnade on 8 types of inflamma~ory tissues. In ~21-7~1~iS
1 most of these, there was no notable fluorescence in the nu-cleoli of the cells studied. However, sections were found in the ulcerative colitis and gastric ulcer speci~ens which did exhibit positive nucleolar fluorescence. Notably, 2 of 3 sections of the ulcerative colitis were ne~ative and one showed definite positive nucleolar fluorescence. In the gastric ulcer, one of the 2 sections analyzed exhibited positive nucleolar fluorescence. These results are particu-larly 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 t~ese lesions which exhibited not only mitotic figures but also a heaping up of the epithelial lining. Lhis finding suggested that these cells might constitute preneoplastic lesions or carcinoma ln situ. It is possible that the finding of these fluorescent regions might aid in decisions to procee~
surgically ~iith elther local or more general resections of the affected lesions.
~xample 3 Artifacts - In the ga~tric epithelium, there was a region of fluorescence in each cell which was non-nucleolar that ap-peared 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 u ~illipore filter eliminated thesc aggregates.
In a sample of breast tissue, ~lhich was negative for nucleolar fluorescence, small nonspecific immunofluorescent specks wexe generally distributed with no special localizing features with regard to cell morphology. The diameters of these very ~ ~ 72~65 1 srnall nonspecific precipitates were 0.5 to 0.1 u as compared to the nucleolar diameters in the nuclei and nucleoli which were ~ to 6 u.
Example_~
Fluorescence durinq 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 nuclcus and the cytoplasm.
~ Inasmuch as the nucleolus largely disappears during metaphase and rR~A synthesis ceases in late prophase, it was not sur-prising that the nucleolar fluorescence was not visible as a dlstinct entity in such cells (Tan and Lerner, 1972). ~ow-ever, the finding that remnants of the immunofluorescent structures persist throu~hout mitosis suggests that the nu-cleolar substructures (rather than the nucleolar products) contain the anti~en(s) which are persistent epigenetically.
Example 5 Maliqnant Tumor Negatives - In the series of mali~nant tumors, negative re~ions were found in varying extents throu~hout the slides. In general, these correlated with either necrotic or abscessed portions of the neoplasms. In one sarnple of a tumor of the bra1n, the mass which e~hibited 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 hreast, prostate, lung and hematol-ogical tumors with very siDlilar results.
_ 1*7Z165 1 ~xam~le6 I,abelin~ - Direct immunochemical methods for the demonstration of the antibodics include labeling the pri~ary antibody tJith one or more of the following labels: a radioisotope for autoradio~raphy such as 125I, 131I, 14C, 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 ~icroscopy, or ~hich produces an electron dense product for demonstration by electron microscopy; or an electron dense molecule such as ferritin for direct electron microscopic visualization.
Indirect immunochemical methods include labelling the second antibody or other binding protein specific for the first antibody with a fluorescent dye, an electron dense co~pound, an enzy~e which produces a product detectable by light, flourescence or electron ~icroscopic examination or a radioisotope detectable by autoradiography.
The indirect immunochemical methods for the visuali-zation 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 antigen(s), (hybrid prl~ary antibody) or for the primary antibody (hybrid second antibody), and part is specific fo~ a label, such as those mentioned in the pre-ceeding paragraph.
Diaqnostic Xits Labelled conjugated and nonconjugated antibodies may be packaged separately in phosphate buffered saline (PaS) or other buffercd suspending agents for distribution.
Suitable suspending agents include glycerin, heparin, or sucrose. Suitable buffers include barbital buffers, morpholine buffers, ~OPS~3-(N-morpholino) propan~ sulEonic acid, hepes-~-2-7~16S
1 hydroxyethylphipera~ine-N-2-ethanc sulfonic acid, Tris carbona~e and the like.
The present invention, therefore, is well suited and adapted to attain the objects and ends and has the features mentioned as well as others inherent therein.
~lhile presently preferred embodiments of the invention have been ~iven for purposes of the disclosure, changes can he made therein within the spirit of the invention 0 as defined by the scope of the appended claims.
Maliqnant Tumors as Detected with the Test Antibody The present studies confirm that nucleolar antigen(s) are present in tumor cells but are not found in nontumcr tissues Initial studies demonstrated that, both in cell cultures in human tumors and in specimens from either autcpsy 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 Normal Tissues - In 17 nontumor tissues there ~as no nucleolar ~luorescence following incubation of the antisera or antibodies wlth the various fix~d 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 Lieherkuhn 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. ~ group of benign tumors evaluated, including several types of thyroid adeno~as, were also negative (Table II).
Example 2 Inflammatory L~sions - To ascertain whether an inflammatory response was related to the appearance of these antigen(s), studies were rnade on 8 types of inflamma~ory tissues. In ~21-7~1~iS
1 most of these, there was no notable fluorescence in the nu-cleoli of the cells studied. However, sections were found in the ulcerative colitis and gastric ulcer speci~ens which did exhibit positive nucleolar fluorescence. Notably, 2 of 3 sections of the ulcerative colitis were ne~ative and one showed definite positive nucleolar fluorescence. In the gastric ulcer, one of the 2 sections analyzed exhibited positive nucleolar fluorescence. These results are particu-larly 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 t~ese lesions which exhibited not only mitotic figures but also a heaping up of the epithelial lining. Lhis finding suggested that these cells might constitute preneoplastic lesions or carcinoma ln situ. It is possible that the finding of these fluorescent regions might aid in decisions to procee~
surgically ~iith elther local or more general resections of the affected lesions.
~xample 3 Artifacts - In the ga~tric epithelium, there was a region of fluorescence in each cell which was non-nucleolar that ap-peared 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 u ~illipore filter eliminated thesc aggregates.
In a sample of breast tissue, ~lhich was negative for nucleolar fluorescence, small nonspecific immunofluorescent specks wexe generally distributed with no special localizing features with regard to cell morphology. The diameters of these very ~ ~ 72~65 1 srnall nonspecific precipitates were 0.5 to 0.1 u as compared to the nucleolar diameters in the nuclei and nucleoli which were ~ to 6 u.
Example_~
Fluorescence durinq 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 nuclcus and the cytoplasm.
~ Inasmuch as the nucleolus largely disappears during metaphase and rR~A synthesis ceases in late prophase, it was not sur-prising that the nucleolar fluorescence was not visible as a dlstinct entity in such cells (Tan and Lerner, 1972). ~ow-ever, the finding that remnants of the immunofluorescent structures persist throu~hout mitosis suggests that the nu-cleolar substructures (rather than the nucleolar products) contain the anti~en(s) which are persistent epigenetically.
Example 5 Maliqnant Tumor Negatives - In the series of mali~nant tumors, negative re~ions were found in varying extents throu~hout the slides. In general, these correlated with either necrotic or abscessed portions of the neoplasms. In one sarnple of a tumor of the bra1n, the mass which e~hibited 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 hreast, prostate, lung and hematol-ogical tumors with very siDlilar results.
_ 1*7Z165 1 ~xam~le6 I,abelin~ - Direct immunochemical methods for the demonstration of the antibodics include labeling the pri~ary antibody tJith one or more of the following labels: a radioisotope for autoradio~raphy such as 125I, 131I, 14C, 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 ~icroscopy, or ~hich produces an electron dense product for demonstration by electron microscopy; or an electron dense molecule such as ferritin for direct electron microscopic visualization.
Indirect immunochemical methods include labelling the second antibody or other binding protein specific for the first antibody with a fluorescent dye, an electron dense co~pound, an enzy~e which produces a product detectable by light, flourescence or electron ~icroscopic examination or a radioisotope detectable by autoradiography.
The indirect immunochemical methods for the visuali-zation 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 antigen(s), (hybrid prl~ary antibody) or for the primary antibody (hybrid second antibody), and part is specific fo~ a label, such as those mentioned in the pre-ceeding paragraph.
Diaqnostic Xits Labelled conjugated and nonconjugated antibodies may be packaged separately in phosphate buffered saline (PaS) or other buffercd suspending agents for distribution.
Suitable suspending agents include glycerin, heparin, or sucrose. Suitable buffers include barbital buffers, morpholine buffers, ~OPS~3-(N-morpholino) propan~ sulEonic acid, hepes-~-2-7~16S
1 hydroxyethylphipera~ine-N-2-ethanc sulfonic acid, Tris carbona~e and the like.
The present invention, therefore, is well suited and adapted to attain the objects and ends and has the features mentioned as well as others inherent therein.
~lhile presently preferred embodiments of the invention have been ~iven for purposes of the disclosure, changes can he made therein within the spirit of the invention 0 as defined by the scope of the appended claims.
Claims (4)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1, A method of preparing and isolating nucleolar antigen(s) comprising extracting the antigen(s) from nuclei or nucleoli of human cancer cells with 0.01 M Tris HCl pH 7-9, said antigen(s) having major and minor species, said minor species being in a relatively smaller concen-tration than said major species the major species having the following characteristics, a pH on isoelectric focussing of about 6.0 to 6.7, and a molecular weight of about 50,000 to about 60,000 daltons, is soluble in 0.01 M Tris HCl pH 8, and is primarily localized in the nucleoli, the minor species having the following characteristics, a pH on isoelectric focussing of about 6.0, and a molecular weight of about 50,000 to about 60,000 daltons.
2. A method of purifying a nucleolar antigen(s), comprising sequential purification thereof by ammonium sulfate precipitation, DEAE-column chromatography, Sephadex (trade mark) gel exclusion; cation exchange and calcium phosphate gel chromatography, and preparative isoelectric focussing, said antigen(s) having major and minor species, said minor species being in a relatively smaller concen-tration than said major species the major species having the following characteristics, a pH on isoelectric focussing of about 6.0 to 6.7, and a molecular weight of about 50,000 to about 60,000 daltons, is soluble in 0.01 M Tris HCl pH8, and is primarily localized in the nucleoli, the minor species having the following characteristics, a pH on isoelectric focussing of about 6.0, and a molecular weight of about 50,000 to about 60,000 daltons.
3. Isolated human cancer cell associated antigen(s) having major and minor species, said minor species being in a relatively smaller concentration than said major species and the major species having the following characteristics, a pH on isoelectric focussing of about 6.0 and 6.7, and a molecular weight of about 50,000 to about 60,000 daltons, is soluble in 0.01 M Tris HCl pH 8, and is primari-ly localized in the nucleoli, the minor species having the following characteristics, a pH on isoelectric focussing of about 6.0, and a molecular weight of about 50,000 to about 60,000 daltons, when prepared by the process of claim 1 or by an obvious chemical equivalent thereof.
4. Isolated human cancer cell assooiated antigens in substantially purified form having major and minor species, said minor species being in a relatively smaller concentration than said major species and said major species having the following characteristics, a pH on isoelectric focussing of about 6.0 to 6.7, and a molecular weight of about 50,000 to about 60,000 daltons, is soluble in 0.01 M Tris HCl pH 8, and is primari-ly localized in the nucleoli, the minor species having the following characteristics, a pH on isoelectric focussing of about 6.0, and a molecular weight of about 50,000 to about 60,000 daltons, when prepared by the process of claim 2 or by an obvious chemical equivalent thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000424445A CA1172165A (en) | 1980-01-04 | 1983-03-24 | Detection of human cancer cells with antibodies to human cancer nucleolar antegen(s) |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000343088A CA1157373A (en) | 1980-01-04 | 1980-01-04 | Detection of human cancer cells with antibodies to human cancer nucleolar antigen(s) |
| CA000424445A CA1172165A (en) | 1980-01-04 | 1983-03-24 | Detection of human cancer cells with antibodies to human cancer nucleolar antegen(s) |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000343088A Division CA1157373A (en) | 1980-01-04 | 1980-01-04 | Detection of human cancer cells with antibodies to human cancer nucleolar antigen(s) |
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| Publication Number | Publication Date |
|---|---|
| CA1172165A true CA1172165A (en) | 1984-08-07 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000424445A Expired CA1172165A (en) | 1980-01-04 | 1983-03-24 | Detection of human cancer cells with antibodies to human cancer nucleolar antegen(s) |
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1983
- 1983-03-24 CA CA000424445A patent/CA1172165A/en not_active Expired
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