DETECTION OF HUMAN CANCER CELLS WITH ANTIBODIES TO HUMAN CANCER NUCLEOLAR ANTIGEN(S)

DETECTION DE CELLULES CANCEREUSES HUMAINES A L'AIDE D'ANTICORPS CONTRE LES ANTIGENES NUCLEOLAIRES DE CANCERS HUMAINS

English Abstract

DETECTION OF HUMAN CANCER CELLS WITH ANTIBODIES
TO HUMAN CANCER NUCLEOLAR ANTIGEN(S)
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.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for the immunological detection of cancer
in human tissue sections, smears and exfoliative cytological
preparations employing antibodies characterized by specificity
against human cell associated antigens in substantially
purified forms which comprises,
contacting specimens with said antibodies,
said 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 pI 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 pI on isoelectric focussing of about 6.0, and
a molecular weight of about 50,000 to about 60,000
daltons, and
demonstration of the localization of said anti-
bodies in the nucleoli of malignant cells but not normal
cells.
2. The process of claim 1 wherein,
the antibodies are elicited to nucleoli isolated
from one of the family of malignant human cells
consisting of HeLa cells,
carcinomas, sarcomas, and hematological neoplasms.
3. The process of claim 1 wherein,
the antibodies are elicited to nucleoli isolated
from one of the group consisting of HeLa cells or human
26

prostatic carcinoma cells.
4. The process of claim 1 wherein,
the antibodies are elicited to extracts
of nuclei or nucleoli of malignant human cells from the
family consisting of HeLa cells, carcinomas, sarcomas,
and hematological neoplasms.
5. The process of claim 1 wherein,
the antibodies are elicited to extracts
of nuclei or nucleoli of one of the group consisting of
HeLa cells or human prostatic carcinoma cells.
6. The process of claim 1 wherein,
the antibodies are elicited to extracts of nuclei or
nucleoli of malignant human cells obtained by extraction
with 0.01 M Tris HCl, pH 7-9.
7. The process of claim 1 wherein,
the antibodies are elicited to extracts of nuclei or
nucleoli from the group consisting of HeLa cells or human
prostatic carcinoma obtained by extraction with 0.01 M
Tris HCl, pH 7-9.
8. The process of claim 1 wherein,
the localization of the antibody is demonstrated by
one of direct and indirect immunochemical methods.
9. The process of claim 1 wherein,
the immunological detection of cancer is by
antibodies of claim 1, including
labeling of the primary antibodies with one or more
of the following labels,
a radioisotope detectable by autoradiography,
a fluorescent dye detectable by fluorescence
microscopy,
an enzyme which produces a fluorescent or colored
product detectable by fluorescence or light microscopy,
27

an enzyme which produces an electron dense product
detectable by electron microscopy, and
an electron dense molecule detectable by direct
electron microscopic visualization.
10. The process of claim 9 wherein,
the radioisotope is selected from the group
consisting of 125I, 131I, 14C, and 3H.
11. The process of claim 9 wherein,
the fluorescent dye is selected from the group
consisting of fluorescein and tetra-methyl rhodamine.
12. The process of claim 9 wherein,
the enzyme which produces a fluorescent or colored
product detectable by fluorescence or light microscopy is
peroxidase, Beta-galactosidase, alkaline phosphatase or
cytochrome C.
13. The process of claim 9 wherein,
the enzyme dense molecule detectable by direct
electron microscopic visualization is ferritin,
hemocyanin, virus particles or latex spheres.
14. The process of claim 1 wherein,
the immunological detection of cancer is by indirect
immunochemical demonstration of the antibodies including,
application of at least one of a labeled second
antibody and other binding protein specific for the
primary antibodies or modifications thereof.
15. The process of claim 14 where,
the label is selected from the group
consisting of fluorescent dye, an electron dense
compound, and an enzyme which produces a product
detectable by light, fluorescence or electron microscopic
examination, and a radioisotope detectable by
autoradiography.
28

16. A process for the immunological detection of
cancer in human tissue sections, smears, and exfoliative
cytological preparations employing modified or fragmented
antibodies characterized by specificity against human cell
associated antigens in substantially purified form, which
comprises,
contacting specimens with said modified
or fragmented antibodies,
said antigen(s) having major and minor species, the
major species having the following characteristics,
a pI 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 pI on isoelectric focussing of about 6.0, and
a molecular weight of about 50,000 to about 60,000
daltons, and
demonstration of the localization of the
modified antibodies or antibody fragments by one of
light microscopy, fluorescence microscopy, electron
microscopy and autoradiography by at least one of
direct or indirect immunochemical means, the
localization occurring in the nucleoli of the
malignant human cells but not in the nucleoli of
normal cells.
17. A process of preparing antibodies with specifi-
city against antigen(s) found in nucleoli and nuclei of human
cancer cells,
said antigen(s) having major and minor species, said
minor species being in a relatively smaller concentration
29

than said major species and the major species having
the following characteristics,
a pI 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 pI on isoelectric focussing of about 6.0, and
a molecular weight of about 50,000 to about 60,000
daltons, said process comprising
immunizing non-human host animals with said
antigen(s), and
harvesting the antibodies from the immunized
animal.
18. A process of preparing antibodies with specificity
against antigen(s) found in nucleoli of human cancer cells,
said antigen(s) having major and minor species,
said minor species being in a relatively smaller concen-
tration than said major species and the major species
having the following characteristics,
a pI 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 pI on isoelectric focussing of about 6.0, and
a molecular weight of about 50,000 to about 60,000
daltons, and

said antigens having a label detectable by one of
light, fluorescence, electron microscopy, indirect assay
methods and autoradiography, said process comprising
immunizing non-human host animals with said
antigen(s), and
harvesting the antibodies of the immunized animal.
19. A diagnostic kit comprising,
antibodies characterized by specificity against
human cell associated antigens in substantially purified
form having major and minor species, the major species
having the following characteristics,
a pI on isoelectric focusing 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 nucleoli of human cancer
cells,
the antibodies having a label detectable by one of
light, fluorescence, electron microscopy, indirect
assay methods and autoradiography, and
disposed in a buffered suspending agent or solution.
20. A diagnostic kit containing,
antibodies characterized by specificity against
human cell associated antigens in substantially purified
form having major and minor species, the major species
having the following characteristics,
a pI on isoelectric focusing of about 6.0 to 6.7,
and
a molecular weight of about 50,000 to about 60,000
daltons,
31

is soluble in 0.01 M Tris HCl pH 8, and
is primarily localized in nucleoli of human cancer
cells,
the primary antibodies being unlabeled and in a
buffered suspended agent or solution, and
appropriate labeled or unlabeled reagents for
detection of the primary antibodies in fixed human
malignant cells by a method selected from the group
consisting of live microscopy, fluorescent microscopy,
electron microscopy and autoradiography.
32

Description

115'7373
FIELD OF THE INVENTION
This invention 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 these nucleolar antigen(s) for diagnostic
purposes.
BACXGROUND OF THE INVENTION
Earlier findings in experimental animals have
indicated the presence of nuclear and nucleolar antigen(s)
in tumors which were not found in non-tumor tissues (R. K.
Busch et al, Cancer Res. 34, 2362, 1974; Yeoman et al, Proc.

115`7373
1 Natl. Acad. Sci. VSA 73, 3258, 1976; ~usch and Busch, Tumori
_, 347, 1977; Davis et al, Cancer Res. 38, 1906, 1978;_ _
Marashi et al, Cancer Res. 39, 59, 197~). In 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 gels formed with anti-
sera to Novikoff hepatoma nucleolar antigents) extracted from
rat Novikoff hepatoma nucleoli differed from the corresponding
immunoprecipitin bands produced with liver nucleolar antigen(s)
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 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
--2--

115'7~73
1 human nucleolar antigen(s). Positive immunofluorescence was
then found in human tumor tissues with antisera and anti-
bodies 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 antinen(s) 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 antigen(s)
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
5. Esophagus, squamous cell carcinoma
6. Liver, primary carcinoma
7. Lung:
adenocarcinoma (2)
oat cell (2)
squamous cell (5)

l lS~373
1 ~. Melanoma, malignant, cerehral
metastases
9. Prostate, adenocarcinoma (4)
10. 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. Myoblastoma, malignant of lip
metastasis to cervical lymph node
2. Osteogenic sarcoma (3), biopsy,
tissue cluture
3. Synovial sarcoma
4- Lymphoma (4), non Hodgkins
III. Hematolo~ical Neoplasms
1~ Hodgkins disease (Reed Sternberg, 5)
2. Leukemia: CLL (5), Hairy cell (spleen)
3. Lymphoma, lymphocytic, spleen
4. Multiple myeloma (5)
5. Mycosis fungoides
6. Acute myelocytic leukemia (5)
7. Chronic myelocytic leukemia (5)
8. Acute monocytic leukemia (2)
IV. Cultures
1. Breast carcinoma
2. Colon adenocarcinoma
3. HeLa
4. HEp-2
5. Prostate, carcinoma (3)
6. Squamous cell carcinoma (3)
*Numbers in parenthesis represent number of cases
In the non-tumor tissues, benign tumors, and in-
flammatory states, negative results were generally obtai.ned
as indicated in the following Table II (Busch et al, supra).
TABLE II
-
NEGATIVE IMMUNOFLUORESCENCE IN YUMAN TISSUES
(From: Busch et al, 1979)
I. Normal Tissue
1. Bladder
2. Bone marrow (hemoblastic lines, 5)*
3. Breast
4. Buffy coat-blood (3)

I ~'7373
l 5. Gallbladder
6. Intestine, small, crypts of
Lieberkuhn
7~ Intestine, large
8. Kidney
9. Liver (2)
10. Lung (adjacent to tu~or)
ll. Lymph node
12. Lymphocytes, normal (2)
13. Pancreas
14. Pineal gland
15. Pituitary
16. Placenta
17. Prostate ~land
18. Skin
19. Stomach
20. Thyroid gland
II. Beni~n Growing Tissues
l. Breast, adenoma
2. Parathyroid, adenomas (2)
3. Prostate gland, hyperlasia (3)
4. Thyroid, adenomas (3)
nodular goiters (2)
III. Inflammatory Diseases
l. Chronic Ulcerative colitis
2. Glomerulonephitis
3. Granuloma and fibrosis of lung
4. Liver - cirrhosis, hepatitis
5. Lupus profundus (mammary gland
and skin)
6. Pemphigus - bullous
7. Ulcer, gastric
8. Inflammatory hyperplasia~lymph nodes (4)
9. Infectious mononucleosis ~5)
IV. Cultures
l. Breast fibroblasts
2. Lymphocytes, PHA stimulated
*Numbers in parenthesis represent number of cases
These results, originally obtained with immuno-
fluorescence, have been verified and extended with im~uno-
peroxidase methods.
BACKGROUND REFERENCES
Busch, H., Gyorkey, F., Busch, R. K., Davis, F. M.,
Gyorkey, P. and Smetana, K. A nucleolar antigen(s) found in
a broad range of human malignant tumor specimens. Cancer
Res. 39: 3024-3030, 197q.

1 1~'73rt3
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., Kellermayer,
M. and Busch, H. Rabbit antibodies to nucleoli of Novikoff
hepatoma and normal liver of the rat. Cancer Res. 34:
2362-2367, 1974.
Dale, G. and Latner, S. L. Isoelectric focusing of
serum proteins in acrylamide gels followed by electrophoresis.
Clin. Chim. Acta 24: 61-68, 1969.
Davis, F. M., Busch R. K., Yeoman~ L. C. and Busch,
H. Differences in nucleolar antigen(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,
H. A nucleolar antigents) found in several human tumors but
not in nontumor tissues. Proc. Natl. Acad~ Sci. USA 76:
892-896, 1979.
Garvey, J.S., Gremer, N.E. and Sussdorf, D. H.
Methods in Immunology, 1977. W. a. Benjamin, Inc. Reaping,
Mass.
Hilgers, J., Nowinski, R. C., Geering, G. and
Hardy, W. Detection of avian and mammalian oncogenic RNA
viruses (oncornaviruses) by immunofluorescence. Cancer Res.
32: 98-106, 1972.
Kendall, F. E. The use of immunochemical methods
for the identification and determination of human serum
proteins. Cold Spring Harbor Symp. Quant. Biol. 6: 376-384,
1938.
Laurell, C. B. Electroimmunoassay. Scand. J. Clin.
Lah. Invest. 29 (Suppl. 124): 21-37, 1972.

1 15'7373
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. ~ usch, R. K., Savage, H.
E. and Busch, H. Purification and partial characteri~ation
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 human hematological
malignancies. Life Sci. 25: 227-234, 1979.
Tan, ~. M. 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. ~., Dicekcert, J. P. and
Dieckert, J. W. Visualization of protein-SDS complexes in
polyacrylamide gels by chilling. Anal. Biochem. 61: 86-92,
1974.
Yeoman, L. C., Jordan, J. J., ~usch, R. K., Taylor,
C. W., Savage, H. and Busch, H. A fetal protein in the
chromatin of Novikoff hepatoma and Walker 256 carcinosarcoma
tumors that is absent from normal and regenerating rat
liver. Proc. Natl. Acad. Sci. USA 73: 3258-3262, 1976.
SUMMARY OF THE INVENTION
The present invention resides in the surprising and
unexpected discovery that common nucleolar antigen!s) are
found in a broad range of humàn cancer cells but are not
found in normal human cells. The antigen(s) 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 antigen(s) found in human cancer cells,
isolation and purification of the nucleolar antigen(s),

1 15~7373
1 production of antibodies specific to these antigen(s),
diagnostic test methods using antibodies specific to these
antigen(s) to detect human cancer cells, a diagnostic kit
containing either antibodies or antisera specific to these
nucleolar antigen(s).
Accordingly, an object of the present invention is
the provision of common nucleolar antigen(s) found in a wide
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 further object of the present invention is the
provision of these nucleolar antigen(s) in substantially
purified form.
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
provision of processes for purifying 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
to purified antigen(s) found 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 to nucleolar antigen(s).
A further object of the present invention is

1 1S 7~73
1 the provision of antisera and antibodies specific to the same
antigen(s) found in the nucleoli of a broad range of human
cancer cells.
A further object of the present invention is the
provision of antibodies and antisera specific to the common
or same antigen(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.
D~SCRIPTION OF THE P~EFERRED EMBODIMENTS
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 vitro and ln vivo.
The Antigen in the Cancer Cells
The antigen(s) have keen 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 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
invention to detect malignant cells, approximately one
percent false negatives and three percent false positives

115'~373
1 were detected. The false negatives represent necrotic tumor
tissues or non-reactive 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 antigen(s) 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 of 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 electro-
phoresis with an SDS(sodium dodecylsulfate) second dimension;
(c) is in part tightly bound to nuclear and nucleolar RNP 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.
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.
Antigen(s) are also present in nucleolar ribo-
nucleoprotein (RNP) particles obtained by ultracentrifugation
of the Tris extracts, subsequently described. The antigen
present in these particles is more tightly bound to proteins
ana ribonucleic acid (RNA) than the antigen in the Tris
--10--

l 1~7373
1 soluble fraction. It is not yet clear whether the antigen(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 antigen(s). Nucleolar antigen(s)
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 antigen(s)
represent a substance that is present in high concentrations
in cancer cells and very low concentrations in noncancerous
cells or are fetal antigen(s) as was found earlier in the
comparative studies on nucleolar antigens of the rat Novikoff
hepatoma and normal rat liver cells (Yeoman et 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 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 antigen(s) by indirect immunofluorescence
and immunoperoxidase techniques.
Purification of the Nucleolar Antigen(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 one volume

1157373
1 of nuclei or nucleoli. The extract was centrifuged first at
27,OGO g for 10 minutes and then at 100,000 g for 16 hours.
Ammonium sulfate at 40% saturation was used to remove conta-
minants. The 40 - 100~ ammonium sulfate fraction was collected
by centrifugation and dialyzed against 20 mM Tris HCl/pH 7.6.
The antigen(s) were chromatographed on DE-52 cellulose columns
(1 x 10 cm). The antigen(s) were eluted in the 0.15M NaCl/O.l
mM PMSF/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 dodecyl 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 tO.15 m NaCl,
0.01 M phosphate, pH 7.2) by gentle homogenization 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
peilets were weighed. The cell pellets were suspended by
gentle homogenization in 20 volumes of reticulocyte saline
buffer (RSB), pH 7.4 and allowed to swell for 30 minutes on
ice. The cells were then centrifuged at 1000 x g far 8
minutes and resuspended by gentle homogenization in RSB
buffer plus 1/20 volume of the detergent Nonidet P40 (10~ in
RSB). The final volume of Nonidet was 0.5~. The cells were
homogenized 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
~12-

373
1 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 ~or 20 minutes. The resulting pellet contained
the HeLa nuclei that were used to prepare the antiyen 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 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
3a steps. Nucleoli prepared from 5- to 10-g HeLa cell preparations,
rather than the 0.5- to l-g quantities used in earlier
studies, provided sufficient material for adequate purification.
~la~e~ r~ -13-

l 15 7373
1 The conditions for growing the ~leLa 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 HeLa 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-
sulfonylfluoride (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 ~M-10 or PM-10 Diaflo membrane. Generally, the
volume at the beginning is around 50 ml and this was concentrated
rr~e ~a~
-14-

11~'7373
1 down to 4-5 ml. The final concentration of protein i9 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 ul of Tris
extract (4-5 mg/ml) as prepared above with 250 ul PBS.
Combine 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 ~eLa 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 A5-50 ml) was
allowed to clot for 3-4 hours at room temperature. The serum
-15-

7 3
1 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 antigen(s) of the present invention. Antisera may
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 was first absorbed with 20%
normal human serum and 20% fetal calf serum (GIBCO). 20%
normal human serum was added to the rahbit antiserum (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/24 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
l5,000 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)2SO~ precipitation of serum, subsequently
described.
The Ig preparation from the nucleolar antiserum
which had been absorbed with normal human serum and fetal
-16-

1 15'7373
1 calf serum was now absorbed with a normal human tissue ~placenta
or liver). An equal volume of placental nuclear sonicate in
PB5 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 15,000 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, What~an 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.
Preparation of Rabbit Immunoglobulin Ig
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
-17-

1157373
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 rnorning 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.
Immunofluorescence
The procedure described 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
(from Hilgers et al, 1972; RK Busch et al, 1974). It Iray be
necessary to use more than 150 ul if the tissue specimen
covers a large section of the slide. Dilution of antiserum
2~ is dependent on 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
37C. (The moist chamber may consists 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 ~5 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
--18--

l ~7~73
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 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 PsS, 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, 193~; 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.
Immunoperoxidase Method:
Instead of fluorescein-labeled goat antirabbit 150
ul 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
--19--

1 15'~373
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 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% (in the 0.05 M
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
prepared each time it was used and was kept cold during the
preparation). Add 200-300 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 Tris HCl 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 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 Slides 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-

l 15 7373
1 slides at ~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.
Malignant 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 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
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 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. 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 antigen(s),
studies were made on 8 types of inflammatory tissues. In
-21-

~157373
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 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 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 sliaes 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 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 Millipore filter eliminated these aggregates.
In a sample of breast tissue, which was negative for nucleolar
fluorescence, small nonspecific immunofluorescent specks were
generally distributed with no special localizing features
with regard to cell morphology. The diameters of these very

ll~'7~73
1 small nonspecifie precipitates were 0.5 to 0.1 u as eompared
to the nueleolar diameters in the nuclei and nueleoli whieh
were 4 to 6 u.
Example ~
Fluoreseenee during Phases of the Cell Cyele - 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 sur-
prising that the nucleolar fluorescence was not visible as a
distinct entity in such cells (Tan and Lerner, 1972). How-
ever, the finding that remnants of the immunofluorescent
structures persist throughout mitosis suggests that the nu-
cleolar substructures (rather than the nucleolar products)
contain the antigen(s) which are persistent epigenetically.
Example 5
Maliqnant 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 hreast, prostate, lung and hematol-
ogical tumors with very similar results.
-23-

11$7373
1 ~xample6
Labeling - Direct immunochemical methods for the demonstration
of the antibodies include labeling the primary antibody with
one or more of the following labels: a radioisotope for
autoradiography 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 microscopy, or
which 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
compound, an enzyme which produces a product detectable by
light, flourescence or electron microscopic 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 primary antibody) or
for the primary antibody (hybrid second antibody~, and part
is specific for a label, such as those mentioned in the pre-
ceeding paragraph.
Diagnostic Kits
I,abelled conjugated and nonconjugated antibodies
may be packaged separately in phosphate buffered saline (PBS)
or other buffered suspending agents for distribution.
Suitable suspending agents include glycerin, heparin, or
sucrose. Suitable buffers include barbital buffers, morpholine
buffers, MOPS-3-(N-morpholino) propane sulfonic acid, hepes-~-2-
-24-

l 157373
1 hydroxyethylphiperazine-N-2-ethane sulfonic acid, ~rris carbonate
and the like.
The present invention, therefore, i5 well suited
and adapted to attain the objects and ends and has the
features mentioned as well as others inheren-t therein.
While presently preferred embodiments of the
invention have been given for purposes of the disclosure,
changes can be made therein within the spirit of the invention
as defined by the scope of the appended claims.