Note: Descriptions are shown in the official language in which they were submitted.
1165685
This invention relates to a diagnostic reagent and method for the
immunochemical detection of a human prostate antigen which is distinct from
prostatic acid phosphatase. More particularly, this invention relates to a
novel purified human prostate antigen and antibodies specific thereto which
are suitable for use in prostatic cancer detection by labord~ory methods.
.. - I
Prostate cancer is very prevalent in old age, with approximately one
half of all males over age 70 having been shown to develop prostatic
cancer. This high incidence of prostate malignancy has led to the sèarch
for markers which may be used for its detec~ion. The elevation of serum
acid phospha~ase activity in patients having metastasized prostate
carcinoma was first reported by Gutman et al. in J. Cl;n. Invest. 17: 473
(1938). In cancer of the prostate, prostatic acid phosphatase is released
from the cancer tissue into the blood stream with the result ~hat ~he total
serum acid phosphatase leve1 greatly increases above normal values.
Numerous studies of this enzyme and its relation to prostatic cancer have
been made since that time, e.g., see the review by Yam in Amer. J~ Med. 56:
604 (1974~. However, the measurement of serum acid phosphatase by
conventional spectrophotometric methods often fails to de~ec~ prostatic
cancer in its early stages. In general, the activity of serum acid
phosphatase is elevated in about 65-90 percent of patients having~carcinoma
33 of the prostate with bone metastasis~ în about 30 percent of patiPnts
without roen~genological evidence of bone ~etas~asis; and in a~ou~ only
5-10 percent of patients lacking clinically demonstrable metas~asis.
~k
Prior art attempts to develop a specific test for pros-tatic acid
phosphatase have met with only limited success because techniques which
rely on enzyme activity on a so-called ~specific~ substrate cannot take
into account other biochemical and immunochemical differences among the
many acid phosphatases which are unrelated to enzyme activity of prostate
origin. In the case of isoenzymes, i.e. genetically defined enzymes having
the same characteristic enzyme activity and a similar molecular structure
but differing in amino acid sequences and/or content and therefore
immunochemically distinguishable, it would appear inherently impossible to
distinguish different isoenzyme forms merely by the choice of a particular
substrate. It is therefore not surprising that none of these prior art
methods is highly specific for the direct determination of prostatic acid
phosphatase activity; e.g. see Cancer 5: 236 (1952); J. Lab. Clin. Med. 82:
486 (1973); Clin. Chem. Acta. 44: 21 (1973~, and J. Physiol. Chem. 356:
1775 (1975).
In addition to the aforementioned problems o~ non-specificity which
appear to be inherent in many of the prior art reagents employed for the
detection of prostate acid phosphatase, there have been reports of elevated
serum acid phosphatase associated with other diseases, which further
complicates the problem of obtaining an accurate clinical diagnosis of
prostatic cancer. For example, Tuchman et al. in Am. J. Med. 27~ 959
(1959) have noted that serum acid phosphatase levels appear to be elevated
in patients with Gaucher's disease.
Due to the inherent difficulties in developing a "specific" substrate
for prostate acid phosphatase, several researchers have deve10ped
immunochemical methods for the detection of prostate asid phosphatase.
However, the previously reported immunochemical methods have drawbacks of
their own which have precluded their widespread acceptance. For example,
Shulman et al., in Immunology 93: 474 (1964) described an immunodiffusion
Page 2
test for the detection of h~lman prostate acid phosphatase. Usin~ antisera
prepared from a rpostatic fluid antigen obtained by rectal massage from
patients with prostatic disease, no cross-reactiv~ty precipitin line was
observed in the double diffuslon technique against extracts o~ normal
kidney, testicle, liver and lung. However,`this met hod has the disad-
vantages of limited sensitivity, even with the large amounts of antigen
employed, and of employing antisera which may cross-react with other~
antigenically unrelated serum protein components present in prostatic fluid.
Chu et al. in International Patent ~pplication Publication No.
W0 79/00475, which has matured as European Patent No. 18, 967, described a
new method for the detectlon of prostatic acid phosphatase isoenzyme :: .
patterns associated with prostatic cancer which obviates many of the above
drawbacks. However, practical problems are posed by the need for a source
of cancerous prostate tissue from which the diagnostically relevant prostatic
acid phosphatase isoenzyme patterns associated with prostatic cancer are
extracted for the preparation oE antibodies thereto.
In recent years considerable effort has been spent to identify
enzyme or antigen markers for various types of malignancies with the view
towards developing specific diagnostic reagents. The ideal tumor marker
would exhibit, a~ong other characteristics, tissue or cell-type specificity,
and would be released into the circulation or other biological milieu which
~0 is easily obtained from individuals. Previous investigators have demon-
strated the occurrence of human prostate tissue-specific antigens.
For example, R.H. Flocks et al., I. J. Urol. 84: 134 (1960)
immunized rabbits with an extract of BPH (benign prostatic hypertrophy
~ 3 ~
'
.
prostate tissue and showed the presence of tissue specific antiprostatic
antibodies by a gel diffusion technique~ Howeverr they presented on data
at all to lndicate the nature of the raactive antigen. The prescriptive
lines
'
~ ~ 3 a ~
formed by antiserum and prostate extract appear due to the reaction of
prostatic acid phosphatase and its antibodies.
R. J. Ablin et al., J. Immunol. 104: 1329 (1970) and R. J. Ablin,
Cancer 29: 1570 (1972) have also demonstrated the occurrence of human
prostate tissue-specific antigens. By using antiserum obtained from
immunizing rabbits with extracts of normal prostate, Ablin et al. showed
two antigenic components in human prostate. One of these was identified as
prostatic acid phosphatase, while the specificity of other was shown to be
a non-prostatic tissue antigen. The xenoantibodies reactive to the second
antigen could not be abolished by treating the antiserum with human
prostatic fluid. Furthermore, this antigen was shown to be deficient in
benign and malignant prostatic tissues. In contradistinction, the prostate
antigen of the present invention is present in all prostate tissue,
(normal, benign or malignant) in almost equal amounts. Further, it is
detectable in prostatic fluid and cultured human prostatic malignant cells
and its medium as well. The Ablin articles describe absorption of antisera
to the antigens described therein with prostatic fluid, after which a
precipitin line was still detected. Absorption of antibodies against the
present antigen with prostatic f~uid gives no precipitin line, indicating
that the ~resent antigen is present in prostatic fluid while that of Ablin
et al. is not.
C. W. Moncure et al., Cancer Chemother. Rep. 59: 105, (1975) also
demonstrated the occurrence of a human prostate tissue-specific antigen
preparation which does not bind to DEAE sepharose at pH 8.0, as does the
antigen of the present invention. This characteris~ic most probably
indicates a significant structural difference between the present protein
and that of Moncure et al.
Thus, there is still a need for simple, reliable, sensitive and
specific reagents and techniques to detect prostatic cancer with aoceptable
Page 4
diagnostic accuracy and without the aforementioned difficulties o the
prior art. The present invention fills such needs.
It is an object of one broad aspect of the present invention
to provide a purified human prostate antigen useful in preparing an
improved diagnostic reagent suitable for the immunochemically specific
detection of circulating human prostate antigen in blood, urine or other
body fluids.
An object of another aspect of the present invention is to pro-
vide rapid and simpLe, yet highly specific and sensitive, immunochemical
techniques and reagents useful in the early detection of prostatic cancer.
An object of a further aspect of this invention is to provide
a new marker for monitoring prostatic cancer and the effectiveness of
curative therapy therefor.
An object of an additional aspect of this invention is to pro-
vide useful monoclonal antibodies to human prostate antigen.
Briefly, the present invention provides, in one aspect thereof,
a purified human prostate antigen which is distinct from prostatic acid
phosphatase.
In a second aspect, the present invention provides antisera
which are highly specific to a purified human prostate antigen and which
do not immunochemically cross-react with prostatic acid phosphatases or
with acid phosphatases originating from other tissues.
In another aspect, the present invention provides specific
marker antibodies against human prostate antigen which are useful, e.g.,
as carriers for in vivo radioimmunodetection of prostate cancer and
immunospecific chemotherapy of prostatic cancer~
Thus, accord~ng to one broad aspect of the present invention,
a purified human prostate specific antigen is provided which occurs in
-- 5 --
. ~ q~ .,
~L6~ 5
normal and cancerous prostatic tissue and purified to show a single
protein band on analytical polyacrylamide gel electrophoresis and iso-
electric focusing, the antigen having a molecular weight [as determined
by SEPHADEX G-75 gel filtration of 33,000] and having a molecular weight
[as determined by sodium dodecyl sulfate polyacrylamide gel electro-
phoresis of 34,000~ with no subunit and having an isoelectric point pI
of 6.9, the antigen further being insoluble in perchloric acid and sub-
stantially free of normal serum protein components, water-insoluble
cellular material and of prostatic acid phosphatases. [SEPHADEX is the
trade mark of Pharmacia Fine Chemicals Inc., for a dry, insoluble powder
composed of macroscopic beads which are synthetic, organic compounds
derived from the polysaccharide dextran. The dextran chains are cross-
linked to give a three dimensional network and the functional ionic
groups are attached to the glucose units of the polysaccharide chains by
ether linkages. Available in various forms for use in many different
phases of chromatography.]
Furthermore, the present invention provides, in another aspect,
antibodies against the purified human prostate specific antigen as
described above, the antibodies being free of antibodies against human
prostatic acid phosphatases.
By a variant thereof, the antibodies are a combination of at
least two monoclonal antibodies.
By another variant, the antibodies are immunoprecipitating anti-
bodies.
By another variant, the antibodies are labeled for radioimmuno-
assay or enzyme-linked immunoassay.
By another variant, the antibodles are covalently bonded to a
water-insoluble support.
According to an alternate aspect of the present invention, a
B - 6 -
6~
process is provided for preparing immunoprecipitating antibodies to antigens
associated with prostatic tissue and fluid, which process comprises:
(a~ extracting an antigen as defined hereinabove from prostatic tissue or
separating the antigen as defined above from prostatic fluid; (b) separa-
ting the antigen from extraneous antigen proteins to obtain a composition
consisting essentially of the antigen; (c) immunizing animals with the
resultant purified antigen to form antibodies specific thereto; and (d)
recovering immunoprecipitating antibodies against the antigen from the
animals.
By a variant of the above process, the antigen is prepared
from extraneous antigenic protein material by salting out.
By another variant, the anti.gen is obtained from seminal fluid.
According to a further alternate aspect of the present inven-
tion, another process is provided for preparing antibodies to antigens
associated with prostatic tissue and fluid, which comprises: (a) cloning
hybridoma cells capable of secreting antibodies; (b) extracting antibodies
or mixture of antibodies defined hereinabove from the secretions; (c)
separating the antibodies or mixture. of antibodies from extraneous anti~
genic proteins to obtain purified such antibodies or mixture of antlbo-
dies; and (d) recovering an immunologically effective concentration andamount of such antibodies or mixture of antibodies from the cells.
By a variant of that process, the hybridoma cells are prepared
by fusing a nonsecretory myeloma cell line with primary mouse spleen
cells.
The antigenic preparations from either normal or ancerous
human cells or their media are purified to obtain a purified antigen con-
sisting essentially of a human prostate antigen free of prostatic acid
phosphatase. These antigenic preparations are employed for immunological
vaccination and diagnostic procedures, particularly for immunoprecipitin
- oa -
,
8~
testing.
Since immunological studies indicate that this antigen is
present in normal benign hypertrophic and neoplastic prostate, a pool of
various prostate tissues can be used as the starting material for subse-
quent purification. Briefly, the prostate tissue is first extracted in
aqueous media at 4 C. Although EDTA-PBSD solution is conveniently used,
saline, 3M KCl or 0.01 percent (v/v) TWEEN 80 (but not lM perchloric acid)
also can be used for extraction of the prostate antigen. The antigen is
thus also distinguished from carcinoembryonic antigens by its sensitivity
to perchloric acid. [TWEEN is the trade mark oE Atlas Chemical Industries,
Inc. for each member of a series of general purpose emulsifiers and sur-
face active agents. They are polyoxyethylene derivatives of fatty acid
partial esters of sorbitol anhydrides. GeneralLy soluble or dispersible
in water, and differ widely in organic solubilities.]
After clarification of the homogenate by centrifugation and
filtration, the supernatant from the crude tissue extract is subjected
to ammonium sulfate fractionation. Ammonium sulfate concentration at
20 - 80 percent saturation almost quantitatively recovers the prostatic
antigen from EDTA-PBS extracts, with the highest yield at 45 - 50 percent
saturation. In order to avoid contamination by other proteins as much
as possible, the precipitate is preferably collected between 35 - 55 percent
- 6b -
B
saturation of amrnonium sulfate, which contains approximately 70 percent of
the total prostate antigen in the crude extract.
Human seminal plasma has been found to contain an antigen, reacting
with rabbit anti-PA serum in double immunodiffusion, which forms a fused
immunoprecipitin line with that of crude extract of prostatic tissue. This
result revealed the presence of a protein, immunologically identical to the
human prostate antigen (PA), in seminal plasma. Further study showed that
all seminal plasma specimens examined contained a substantial amount of PA,
ranged from 0.4 to 1.8 mg/ml (n=9, mean + standard deviation = Or71 +
0.42). It is estimated that PA content in 20 to 30 ml of seminal plasma
was equivalent to that in 100 9 of prostatic tissue. The elution profile
of PA in chromatographies is similar to that using prostatic tissue as
source of the antigen. PA purified from seminal plasma and from prostatic
tissue possesses an identical mobility and isoelectric point (pI 6.87
0.09) as shown by polyacrylamide gel electrophoresis and isoelectric
focusing, respectively. Both purified PA preparations exhibit a molecular
weight of 33-34,000 as shown by Sephadex G-75 gel filtration. In ad~ition,
a line of identity was obtained in immunodiffusion when purified PA
preparations reacted with anti-PA serum.
Since seminal plasma is more readily available than prostatic tissues
and contains abundance af PA, it appears an ideal source for isolating PA.
Using seminal plasma as the source of PA isolation also appears to have an
advantage over the use of prostatic tissueO Firstly, the initial
extraction step, which requires at least 4 hours~ is eliminated. Secondly,
at the initial stage of purification~ handling a large volume of the tissue
extract is avoided since 20 to 30 ml of seminal plasma is equivalent to 100
g of tissue in terms of PA content. Thirdly, seminal plasma contains less
contaminating proteins and makes purification easier. For ins~ance~
hemoglobin in the tissue extract is precipitated concurrently with PA by
Page 7
~ 5
ammonium sulfate at concentration greater than 50 percent saturation, and
the removal of this hemoglobin in later steps of purification results in a
reduction oE the yield of final purified PA. Since sminal plasma contains
less contaminating proteins, it is possible during fractional precipitation
steps to recover a greater amount of PA by increasing the upper cut-off
point of ammonium sulfate concentration to 75 percent saturation. A better
recovery of PA in purified form is also achieved from seminal plasma.
DEAE-BIOGEL A anion exchange col~,n effectively retains the pro
state antigen and washing the column with tris-HCl buffer at pH 8.0 does not
dissociate the antigen from the column. (BIOGEL is the trade mark for an
anion exchange resin.) Elution of the antigen can be achieved with 10-78
mM of NaCl in the same buffer, followed ~y further purification by gel
chromatography. The bulk of contaminating proteins in the PA preparation
eluted from the DEAE column have a molecular weight greater than 45,000
and can accordingly be separated from PA ~molecular weight: 33,000 to
34,000) by gel filtration on a SEPHADEX G-100 column.
An additional step can be carried out with anion exchange chroma-
tography on a DEAE column using a pH gradient solution as the eluant. Two
protein peaks were detected between fractions in the pH range
Of 7.6 to 6.7, which were shown to contain PA. Upon polyac~ylamide gel
electrophoresis, protein heterogeneity was seen in the firs~t peak fractions,
while the second peak fractions gave a single protein band. Therefore,
for obtaining homogeneous PA, only second peak fractions were collected.
At the end of this elution, approximately 20 percent of total PA subjected
to chromatography was still bound to the DEAE column. This can be
recovered quantitatively by elution with O.OaM NaCl, but protein hetero-
geneity in the eluted PA preparation was observed.
An interesting observation has been made in the course of the above
PA purification; the first peak fractions contained PA which possessed a pI
different from that of the purified PA from the second peak fractions.
.. ~ - 8 -
Also, the PA retained by -the DEAE column at the end of pH gradient elution
exhibited a different pI. Similar observations have also been made in the
purification of PA from seminal plasma, and treatment with neuraminidase
has increased the pI of thse PA "isomers" to a higher pH range. It thus
appears that the human prostate antigen of the present invention may exist
in a number of different isomeric forms.
Preparative polyacrylamide gel electrophoresis of the partially
purified antigen is effective in achieving a pure antigen preparation.
This purified antigen is shown to be homogeneous by polyacrylamide gel
electrophoresis with and without sodium dodecyl sulfate. It has a
molecular weight of 33,000-34,000 with no subunit, and exhibits a single
pI of 6.9.
Using immunoprecipitation and immunocytochemical techniques~ PA has
been shown to be a prostate gland epithelial marker protein. PA is
localized in the epithelial lining of prostatic glands and ducts as well as
in prostatic secretions and concretions, but not in epithelia of
periurethral glands, seminal vesicles, vas deferens~ urinary bladder
transitional epithelium, prostatic urethra, glandular lining of vonBrunn's
nests, or in testes. These observations suggest that the PA in the seminal
plasma is of prostatic origin.
Data are now available to indicate the potential clinical spplication
of PA in prostatic cancer. Quantitation of circulating PA, with a
sensitivity of 0.1 ng/ml, has been achieved by an enzyme-immunoassay. PA
is not detected in sera from normal females or female cancer patien~s,
while sera from male patients with non-prostate cancer contain a similar
range of PA as that of normal males. Patients with prostatic disedses have
been shown to have elevated levels of circula~ing PA. Although no
quantitative difference in PA levels is found between patients with benign
prostatic hypertrophy and stage A of prostatic cancer, patients with other
Page 9
stages of prostatic cancer demonstrate significantly elevated PA levels,
both quantitatively and qualitatively. With immunocyto- chemical
procedures, all primary and secondary prostatic tumors examined reacted
positively with anti-PA serum, whereas the tumors of non-prostatic origin
did not react. These results, therefore, suggest an additional means for
diagnosis of prostate cancer and for monitoring the efficacy of its
treatment, Another possible application is the use of specifiç anti-PA
antibodies in in vivo radioimmunodetection of prosta~e cancer9 e.g.
according to the method described by D, Pressman in Cancer Research 40:
2960 (1980), particularly the micro-metastasis which is so cri~ical in
staging and treatment. Immune-specific chemotherapy also is a potential
area where much work can now be initiated with the availability of PA
immunologic reagents, e.g. see T. Ghose et al. in J. NatO Cancer Inst. 61:
657 (1378). Furthermore, the physiology of the prosta~e can be studied
with the aid of this new marker for prostate gland epithelium.
In order to produce antisera which can be used to detect antigens in
prostate tissues and fluids other than acid phosphatase, female rabbits
have been injected with a purified prostate antigen isolated from prostate
tissue. Sera is collected, heat inactivated and stored at -20 C until
use. After treatment ~ith insolubilized normal human plasma proteins
(antibodies to normal plasma constituents are removed by treatment of the
antiserum with glutaraldehyde-insolubilized normal plasma obtained from
normal male and female adults), the antiserum reacts specifically with
prostate tissue extracts (43/43) using the techniques of double
immunodifussion and rocket immunoelectro- phoresis. No immunological
reactivity was observed against a battery of extracts prepared from tissues
other than prostate. The prostate antigen from prostate tissue extracts
was characterized by gel filtration chromatography ~m.w. 30-40,000),
isoelectric focusing (pI 6.9) and agarose electrophoresis (MR 0.2
Page 10
relative to bovine serum albumin). The concentration of the prostate
antigen was not significantly different among extracts prepared from
normal, benign hypertrophic and malignant prostatic tissues and the antigen
exhibi~ed no acid phosphatase enzyme activity as determined by
histochemical staining procedures. Furthermore, its approximate molecular
weight (30-40,000) differs significantly from that of prostatic acid
phosphatase ~100,0003.
Although the antigen detected in serum had a higher apparent molecular
weight (80-100,000), antigen mixing and peak enhancement experiments
indicate that prostate antigen in circulation is immunolo~ically identical
to the human prostate antigen in prostate tissue and prostatic fluid. The
serum-borne antigen may be bound to a plasma protein. Such plasma protein
binding of a variety of antigens and hormonal substances is well known and
has been reported by T. Peters, Jr. "Serum Albumin" In: F.W. Putman
(ed.). The Plasma Proteins. Vol. 1: pp. 133-153, New York, N.Y. Academic
Press, 1975. However, SPD-PAGE revealed a molecular weight of 36,000,
similar to that in prostate tissue and seminal plasma.
Alternatively to the conventional techniques for preparing antibodies
in laboratory and farm animals, monoclonal antibodies against PA can be
prepared using known hybridoma cell culture techniques. In general, this
method involYes preparing an antibody- producing fused cell line, e.g~ of
primary spleen cells fused with a compatible continuous line of myeloma
cells, and growing the fused cells either in mass culture or in an animal
species from which the myeloma cell line used was derived or is
compatible. Such antibodies offer many advantages in comparison to those
produced by innoculation into animals, as they are highly specific and
sensitive and relatively "pure" immunochemically.
The full potential of this prostatic antigen in clinical applications
is not known at present, as it does not appear to exhibit any tumor
Page 11
specificity or patholog;cal association. However, as in the case of acid
phosphatase which is present in all prostate tissues, this prostate antigen
can provide useful clinical information upon serum monitoring. Recent
experiments in our laboratory have shown that the prostate antigen is
detected, by rocket immunoelectrophoresis and enzyme-linked immunoassay
techniques, in the sera of some prostate cancer patients but not in the
sera of normal healthy persons or of patients with other cancers. Further
experiments have shown that PA is histologically detectible in both
prostate tissue and established continuous tissue culture cell lines of
prostate origin, and that it is released by prostate tumor cells, both in
vivo and in vitro. In addition, it has been found that the PA level in
serum of patients suffering from prostatic cancer is unrelated to the serum
levels of prostatic acid phosphatase, so that simultaneous determination of
circulating PA and circulating prostatic acid phosphatase may well provide
an improved means of diagnosing prostatic cancer. The human prostate
antigen, although an eutopic component of the prostate, can thus play a
major role in the detection of prostate cancer.
For the preparation of immunogens suitable for preparing diagnostie
antibodies against the human prostate antigen, conventional vaccine
preparation techniques can be usedO Preferably a non- antigenic adjuvant,
e.g. alum, Freund's complete adjuvant, saponin, a quaternary ammonium
surfactant, an alkyl amine, etc. is admixed with the purified prostate
antigen in a suitable immunologically acceptable, non-antigenic carrier and
the resultant mixture can be sterilized, e.g. by filtration.
The vaccine can be administered parenterally following regimens
already known for immunization with other proteins to stimulate the
formation of immunoprecipitating antibodies, with the primary inoculation
being preferably followed up by at least one additional injection one to
ten weeks later. Good results have been obtained in rabbits using four
Page 12
booster injections at tWQ week intervals one month after the primary
immunization. The protein content per injection in rabbits, goats and
other mammals can be varied~ but is generally about 50 micrograms of
protein per kg. of body weight. The antibodies can be collected and worked
up using methods well known to those skilled in the art of immunochemistry7
and provide a useful reagent for the immunological detection of prostate
specific antigen in a variety of immunochemical procedures~ e.g.~
immunoprecipitin, fluorescent antibody, serum neutralization, etc. Such
antibodies are useful as a control reagent in the diagnostic test for
prostatic cancer described more particularly below.
The simplest immunoprecipitin test involves capillary tube precipitin
testing, wherein separate antibody and antigen solutions are allowed to
react at a common interface in a capillary tube and a positiYe reaction is
indicated by the formation of a precipitate at the interface. This method
is relatively insensitive and inaccurate due, inter alia, to unavoidable
diffusion of the two solutions across the interface, and furthermore the
final test results cannot be preserved.
Agar gel dif~usion is the simplest method which avoids these
drawbacks. A solution of the antigen (or serum sample) is placed in a
central well punched in a continuous agar gel and appropriate dilutions of
the serum containing antibodies (or, correspondingly, the antigen) thereto
are placed in wells concentrically surrounding the center well. A positive
reaction is noted by the formation of the preeipitin line between one or
more o~ the concentric wells and the eentral well. This method is
relatively insensitive and fairly slow, requiring 1 to 4 days to read the
test results.
Radioimmunoassay (RIA), e.g. radioimmunoprecipi-tin tests1 are
extremely sensitive (by several orders of magnitude over older methods) but
take several days to per~orm and require sophistieated equipment and highly
trained personnel not always widely available.
Page 13
,B~j
Countercurrent immunoelectrophoresis (CIEP) is a widely used
immunoprecipitirl method which takes only about an hour to perform and which
is considerably more sensitive than agar gel difFusion. Reactive
components are placed in opposing wells cut into an agar gel and a small
electrical current applied thereto, causing both the antigen and the
antibody to migrate towards each other. A positive reaction is indicated
by the formation of a precipitate at the antigen-antibody interface. Since
this test method is reasonably reliable, readily available and inexpensive~
it represents a preferred embodim~nt of this aspect of the present
invention.
For purposes of immunoelectrophoresis testing, the diagnostic antibody
preparation of the present invention when used without purification is
generally diluted with phosphate buffered saline in a volume ratio of 1:10
to 1:500, depending on the antibody titer thereof. The limiting factor at
the lower end of the range is the degree of distinction achieved in the
precipitin lines, which is a function of the antibody content in the total
protein present. Purified antibody preparations can of course have lower
total protein concentrations, and the protein content of even the
unpurified preparations can be varied to suit the particular immunochemical
test to be employed, the optimal amounts being determined7 e.g. by testing
simple serial dilutions.
In a preferred, further aspect of the present invention, circulating
human prostate antigen can now be detected by immunochemical techniques,
preferably by protein staining of the antibody-antigen precipitin complex.
Using the aforementioned specific antisera and coupling this with a
conYentional means for detecting the antigen-antibody complex, it is now
possible to immunochemically separate the specific human prostate antigen
from a serum sample.
The antigen-antibody complex can be stained by a number of known
3~ histochemical staining techniques, e.g. fluorescent antibody~ etc., to
Page 14
1.'~'6~
increase the sensitivity of this method. Alternatively, one can use radio-
active antibody for the assay, which not only provides a better quantita-
tive value but may also further increase the sensitivity of the assay If
desired, an enzyme, e.g., ~ -galactosidase or peroxidase, can be coupled
with purified antibodies for use in an enzyme-linked immunoassay, e.g.,
using techniques analogous to those described by ~ato et al. in J. Immunol.
116: 1554 (1976). Especially preferred is the method described by M.
Kuriyama et al. in Cancer Res. 40: 4658 (1980).
It is preferable to bind these antibodies onto a water-insoluble
support for use in the enzyme assay. Many suitable such supports and
techniques for binding proteins thereto are well known in the art and
include inorganic as well as organic supports. Presently preferred are
those water-insoluble supports which can be activated with a cyanogen
halide, preferably cyanogen bromide, prior to the covalent bonding of the
antibodies thereto, e.g., as taught by Axen et al. in U.S. Patent No.
3,645,852. Such supports are commercially available, e.g., that known by
the trade mark of ENZYMOBEADS available from Bio-Rad Laboratories.
Without further elaboration, it is believed that one skilled in
the art can, using thè preceding description, utilize the present inven-
tion to its fulLest extent. The following preferred specific embodimentsare, therefore, to be construed as merely illustrative and not limitative
of the remainder of the disclosure in any way whatsoever. In the following
Examples, the temperatures are set forth uncorrected in degrees Celsius;
unless otherwise indicated, all parts and percentages are by weight.
Protein concentration was determin~d by the method of Lowry et
al. described in J. Biol. Chem. 193: 265 (1951), using bo-vine serum albomin
as
- 15 -
,
the standard. Acid phosphatase activity was measured by the method of
Babson and Phillips using ~-naphthyl phosphate as the substrate.
Immunoprecipitation techniques were modified from B. Weeke, Scand. J.
Immunol. (Supplement 1~ 2: 37-46 (1973). Rocket-IEP was performed on
cellulose acetate membranes using 0.83 percent agaroC.e (Sigma low
electro-endo-osmosis) in 0.08 M tris-0.024 M tricine-0.024M sodium barbital
containing l.3 mM calcium lactate and 0.02 percent sodium azide (TTB
buffer). Antiserum at various final concentrations (0.5-2.0 percent) was
incorporated into the agarose at 55 C prior to plating. Rocket-IEP samples
were applied to circular wells (5.0 mm) and electrophoresed at 5 volts/cm
overnight at 4 C using TTB running buffer.
Crossed-immunoelectrofocusing was performed in the second dimension as
for rocket-IEP. Isofocused acrylamide gels (5 x 100 mm) were molded into
the antibody-agarose prior to immunoelectrophoresis modified from J.
Soderholm et al., Scand. J. Immunol. (Supplement 2) 4: 107-113 (1975).
The first dimension isofocusing was done in 7.5 percent polyacrylamide
containing 2 percent ampholytes9 pH range 4-8 at 200 volts overnight at
4 C. For some experiments, isofooused gels were laid on cellulose acetate
membrances and covered with 1 percent agrose. Troughs were cut adjacent to
the embedded acrylamide gel, the antiserum placed in the parallel troughs
was allowed to diffuse for 24-48 hours at 25 C and the resulting
immunoprecipitin lines were recorded.
Crossed-immunoelectrophoresis (cross-IEP) was performed in the second
dimension as for rocket-IEPO The first dimension electrophoresis was
performed with 1 percent agarose, and samples ~75 ~l in rectanguldr wells)
were electrophoresed at 15 volts/cm for 2 hours at 4 C. Migration was
monitored with Bromphenol Blue dye.
The abbreviations used are: PB-NaCl, 0.05 M phosphate buffer
containing 0.15 M NaCl, pH 7.2; IEP, immunoelectrophoresis; TTB,
Page 16
Tris-Tricine-Barbital (0.08M, O.Oq2M1 0.024M) buffer containing 0.3 mM
calcium lactate and 0.02 percent sodium azide, pH 8.8~ MR, relative
immunoelectrophoretic mobility; pI, isoelectric point; m.w., molecular
weight.
Example 1
Extraction of Tissues
Human prostatic tissues (normal~ benign hypertrophic, malignant) and
other human tissues were obtained during autopsy or surgery~ The histology
of each issue was confirmed by pathologists. Approximately 10 g of tissues
were minced and washed three times with 30 ml of physiologioal saline, then
mixed with 30 ml of 0~02 percent (wlv) disodium ethylenediamine
tetraacetate (EDTA)-O~lM phosphate buffered saline (PBS~, pH 6.8. The
mixture was placed in an ice-water-chilled blending chamber of a Sorvall
Omnimixer and subjected to three 5-minute blendings at a blade speed of
25,000 rpm with intermittent cooling time of 3 minutes. The homogenate was
stirred overnight at 4 C~ then centrifuged at 25~000 9 for 30 minutes. The
resultant supernatant constituted the crude tissue extract.
Example ?
Ammonium_S fate Fractionation
Pooled prostate tissues, lno 99 were extracted with 30Q ml of EDTA-PBS
as described above~ Ammonium sulfate (60 9) was added to 285 ml of crude
extract (35 percent saturation), mixed for 30 minutes and centrifu~ed
~26,000 9, 30 minutes). 38 9 of ammonium sulfate were added to 295 ml of
the resulting supernatant, (55 percent saturation)~ mixPd for 30 minutes
and centrifuged. The precipitate was dispersed in 200 ml of 55 percent
saturated ammonium sulfate solution and centrifuged. Washing of the
precipitate was repeated twice, then the precipitate was dissolved in 20 ml
of 0.01M tris-HCl buffer, pH 8.0O The residual ammonium sulfate was
removed by dialysis against 4 liters of the tris~HCl buffer for 48 hours
Page 17
with one change of buffer during dialysis~ The dialyzed solution was
centrifuqed at 46,000 g ~or 30 minutes to remove any precipitate formed
during dialysis.
Examplè 3
Ion Exchange Chromogràphy-of prostate Antigen
he supernatant from Example 2, 28 ~1, was applied onto a DEAE-
BIOGEL A column (2.5 x 93 cm) pre~equilibrated with 0.01M tris~HCL buffer,
pH 8Ø The column was first washed with 420 ml of buffer, then eluted with
0-0.2M NaCl gradient (1 liter 0.01M tris~HCL buffer, pH 8.0 in mixer ana 1
liter of 0.2M NaCl 0.01M tris-HCl, pH 8.0 in the reser~oir) at a flow rate
of 6.5 ml/hr/cm . The effluent-was collected (10 ml/fraction) and moni-
tored for the prostate antigen. -Gel diffusion according to the method of
T. M. Chu et al, in Cancer Treat~ Reports 61: (1977~ was used to measure
the presence of prostate antigen in various chromatographic fractions. The
entire procedure was carried out at 4~C. The prostate antlgen~containiDg
fractions were pooled and concentratea with an Amicon concentrator fitted
with a UM2 ultrafiltration membrane to 4.5 ml.
Example 4
Gel Filtration of Prostate Antigen
-
A portion (4.3 ml) of the concentrated solution was applied onto
a SEPHADEX G-100 column (2.5 x 110 cm) pre-equilibrated with 0.01M tris-HCl
buffer, pH 8.0, which was eluted with same buffer at a flow rate of 3.5 ml/
hr/cm , The pooled DEAE-BIOGEL A fractions 100~150, containing the pros-
tate antigen upon SEPHADEX G~100 chromatography~ were resolved into several
protein peaks. The prostate antigen was found to be in fractions 46-56.
Again, the fractions exhibiting prostate antigen reactivity were pooled and
concentrated to 3.8 ml.
-Example 5
Further Purification of Prostate Ant;~gen
~ .
The above fractions containing prostate antigen were applied onto
a SEPXA~EX G 75 column (2.5 x 113 cm) pre~equilibrated with tris-HCl buffer;
the column was eluted at a'flow rate of 5,5 ml/hr/cm . This resulted in
one ma~or symmetrical protein peak associated with the prostate antigen
reactivity and two minor protein peaks. When the fractions containing the
prostate antigen were re-chromatographed on the same column, an identical
symmetrical protein peak was obtained and the ~minor protein peàks were
eliminated. Acid phosphatase activity was measured colorimetrically and
histochemically as described by sabson et al. in Clin. Chim. Acta 13; 264-
265 (1966) using ~ -naphthyl phosphate as the substrate. No prostatic
acid phosphatase was detected in the chromatographic fractions of this
protein peak.
Example 6
Analytical Polyacrylamide Electrophoresis
Acrylamide gel (7.5 percent) columns (50 x 60mm) were made
according to the company's instruction manual (Shandon southern Instru-
ments, Ltd., Camberley, Surrey, England), and 50~ul of sample (10-40~ug
protein in 25 percent sucrose solution) were applied onto each gel column.
Ten percent sucrose solution was then layered carefully above~the sample
solution, followed by layering 0.05 M tris-glycine buffer~ pH 8.5 to the
top of the gel containing tube. Tris-glycine buffer was used in preparing
the sucrose solutions and used also as the electrolyte~ After electro~
phoresis with a constant current of 5mA per tube for 40 minutes~ the gels
were stained for protein by Coomassie brilliant blue G-25~ perchloric acid
-' 19 ~
solution.
~ ~Example 7
Sodium`Dodecyl`Sulfate (SDS) Polyacrylamide Gel Eléctrophoresis
The method was essentially the procedure of Weber and Osborn
described in J. Biol. Chem. 244: 4406 (1969). A sample (10-20 ~g protein)
in 50 ul
.
19 a =
of 0~05M tris-glycine buffer, pH 8.5~ containing 250~ul SDS and 2-
mercaptoethanol each, was incubated at 37C for 2 hours~ After incubation,
the sample was mixed with an equal volume of 50 percent sucrose, and 50~ul
were subjected to ~he polyacrylamide gel (containing 0.1 percent ~DS)
electrophoresis as described before~ After electrophoresis, the gels were
stained for protein with 0.5 percent (w/v) Coomassie brilliant blue G-
250 in ethanol-acetic acid-water (45:10;45, v/v).
Example 8
.
Preparation Polyacrylamide Gel Electrophoresis
Although SEPHADEX G-75 chromatography produced a symmetrical
protein peak with exhibited immunological reactivity of the prostate angi-
gen, the analytical polyacrylamide gel electrophoresis of Éxamples 6 and 7
revealed several protein components in this preparation. Therefore, a
preparative polyacrylamide gel electrophoresis (PAGE) was further used in
the purification of prostate antigen.
One ml of specimen (4.5 mg protein) was mixed with 1 ml of 50
percent (w/v) sucrose solution and applied onto an anmllar 7.5 percent gel
column (cross-sectional gel area, 4~4 cm ; height, 9 2 cm), followed by
successive layerings of 2 ml of 10 percent sucrose and 0.05M tris-glycine
buffer, pH 8.5. -A constant current of 30mA was flrst applied for 1 hour,
and 80 m~ thereafter. The tris~glycine buffer was continuously pumped at
a flow rate of 14-15 ml/hour into the elution plate located at the bottom
of the column to carry materials emerged from the column into a fractional
collector ~ractions containing the prostate antigen, representing the
final purified preparation, were pooled and concentrated to 0.5 ml. m e
prostate antigen was found in fractions 28-62 (eluates obtained between 4-
i68~
9 hours after electrophoresis). When the~e fractions were pooled and sub-
jected to analytical polyacrylamide gel electrophoresis, ~ultiple protein
bands still existed. However, by poolin~ fractions 56-62 (~etween
'~;
~`
-: :
' ' ~ :
:
:
: ~`
':
:
,
:
~: :
20 a -
.
.
~$6l!3~
8.1-9 2 hours of electrophoresis) only a single protein band was obtained
upon analytical polyacrylamide gel'electrophoresis and sodium dodecyl
, :
sulfate polyacrylamide gel electrophoresis.
''That the final prostate antigen preparation was o high purlty
was indicated by a single protein band without any contaminating components
in polyacrylamide gel electrophoresis in the absence and the presence of
sodium dodecyl sulfate. The purified prostate antigen was shown to'have a -
molecular weight o 33,000 by gel filtration on SEPHADEX~G-75, and 39,000
by sodium dodecyl sulfate polyacrylamide gel electrophoresis with no
subunit component. ~Isoelectric focusing revealea a single pI of 6~9.
Example 9
'"' Simplified PuriflOEation~of Antlgen'from'Tissue
The procedure for the purification of PA reported in the preced-
ing Examples takes at least two weeks and yields less than 0.5 mg of
purified PA per 100 g of prostatic tissue. Furthermore~ for~the final
-
step of purification, a painstaking construction~of the polyacrylamide gel
column as well as a special electrophoretic apparatus are~necessary, and
.
problems such as clogging of-the elution ohannel during operation often
developed. In this simplified procedure, in addition to the elimination
: ' . ~ :
of SEPHADEX G-75 chromatography and the~application of short D~AE columns~
the tedious preparative gel electrophoresis has been replaced by a simple
pH gFadlent elution o the DE~E column. The yleld of puriied PA has~in-
creased to 1 to 2 mg per 100 g of prostatic tissue and the entire procedure
has been shortened to 8 to 9 days. ~ ~
Benign hypertrophic prostatic tissues (approximately 100 g) were
extracted with disodium tetraacetate~phosphate buffered slalne as descrlbed
~ ~ : : :.
:
:
21
.~,~,~, :
.
'
,
in Exa~ple 1 except that the ho genate was stirred for o~ly 2 hours
rather than overnight.. The crude extract was then.subjected to fractional
precipitation by a _ onium.sulfate ~30 to.SO percent sa=uratic~). Ihe
~.
21 a -
:
precipitate was dissolved in 10 to 15 ml of 0.01M tris HCl buffer, pH 7~8,
and sialyzed against 4 ml of the same buffer for 48 hours with one change
of buffer. The dialyzed materials were centrifuged (46,000g, 30 min) and
the supernatant-(12 to 18 ml) was applied onto a 2.5 x 15 cm DEAE-BIOGEL A
column pre-equilibrated with 0.01M tris-HCl bufferr pH 7.8. The colurnn
was washed with ca. 300 ml of the same buffer, followed by elution with
0 08 M NaCl-0.OlM tris-HCl buffer, pH 7.8. Fractions containing PA were
pooled, concentrated (5 to 5.5 ml~, and applied to a 2.5 x 110 cm SEPHADEX
G-100 column pre-equilibrated with 0.01M tris HCl buffer, pH 7.8, which
was then eluted with the same buffer at a flow rate of 5 ml per hour per
cm . Fractions covering a molecular weight region of 26,000 to 37,000
which contained PA were pooled, concentrated (S ml) and applied to a 2.5
x 20 cm DEAE column pre-equilibrated with 0.01M tris-HCl buffer, pH 7.8.
The column was washed with 10 ml of the same buffer, followed by elution
with pH gradient solution (mixer: 0.01M tris-HC1, pH 7.0, 3Q0 ml; reser-
voir: 0.01M tris~HCl, pH 6,0, 500 ml) at a flow rate of 5 ml per hour per
cm . Second peak fractions which contained pA were pooled and concentrated.
Table 1 summarizes the results of a typical purification of PA
from prostatic tissue by this simplified procedure. Although modifica-tion
at the salt precipitation step led to lesser recovery of PA, a large
, quantity of contaminating proteins such as hemoglobin were removed, thereby
facilitating further purification of PA. Raaial immunodiffusion used in
this study was the technique of Mancini et al described in Immunochem. 2:
235~254 (1965), with modifications. Anti-PA serum (100 ~1) was mixed
with 10 ml of 1 percent agarose solution (in 0.154M NaC1~0.017M sodium
phosphate, pH 7.0) at 55bC and poured into a Petri dish (diameter; 8.3 cm).
~ 22 ;
~i
3'6'~
Wells (diameter; 2 mm) were then made on the agarose gel and to each wel:L,
10 ~1 of sample was added. Por monitoring PA in the chromatographic
fractions, overnight diffusion was sufficient to reveal precipitin; for
.
- 22 a ~
.~. . ;
1~6~i6~
quantitation of PA, the diffusion was allowed to continue for 48 hoursg
followed by washing of the gel for 2 days with 0.154M saline and subsequent
staining with Coomassie Brilliant Blue G-250.
Page 23
Table 1 Purification of Prostate Antigen from
_
- ~uman Prostatie Tis5ue
Total Total Total
volume protein PA Folds Recovery
Steps (ml) ~mg) (mg) purification t percent)
-
Crude extracta 258.0 1754.4 20.6 1 . 100
Ammonium sulfate
10 (30-50 percent) 13.8 278.8 10.0 3.1 48.5
First DEAE 5.5 80.9 7.9 8.3 38.3 }
SEPHADEX G - 1 00 5.0 9 3 4 3 39 4 20.9 t
Second DEAE 5.0 1.5 1.5 85.2 7.3
a. From 118 9 of benign hypertrophic prostate tissues.
Page 24
:,,
.
'
6~
Example 10
Purification of Antigen from Fluid
PA w~s also purified from 20 ml of seminal plasma according to the
procedure of Example 9 except that the extraction step was omitted and lt
that, in the second step, PA was precipitated by am~onium sulfate at a
concentration of 30 to 75 percent saturation. The results are shown in
Table 2.
Table 2. Purification of Prostate Antigen from
Human Seminal Plasma ¦,-
Total Total Total
volume protein PA Folds Recovery
Steps ~ml) (mg) ~mg) purification (percent)
Seminal plasma 16.2 595.2 8.8 1 100
Ammonium sulfate
(30-75 percent) Z0.0 266.2 7.4 1.9 84.1
First DEA~ 5.3 61.0 5.7 6.3 64.8
SEPHADEX 6-100 6.4 12.8 q.5 23.8 Sl.l
Second DEAE 4.5 1.5 l.S 67.6 17.0
~ .
Page 25
~5'6E3
Example 11
-
Molecular Weight Determinatio_
In order to obtain an approximate molecular weight of the prostat~
antigen present in crude prostate tissue extracts and in antigen-positive
patients' sera, these samples were subjected to a SEPHADEX G-200 gel
filtration chromatography. Serum samples (0.5 ml) previously shown to be
prostate antigen-positive by rocket-IEP, or prostate tissue extracts (0.5
ml containing ~ mg prDtein) were applied to a column (0.9 ~ 60 cm) packed
with SEPHADEX G-200 gel in P8-NaCl. Eluted sam~les were analyzed for
absorbance at 280 nm and prostate antigen level was determined by
rocket-lEP using a sample size: 0.5 ml; equilibration buffer: PB-NaCl (pH
7.2); fraction si7e: 0.8 ml; elution rate: 10 ml per hour. Peak antigen
activity as exhibited in prostate tissue extracts eluted between 30-40,000
m.w. Moiecular weight reference markers included human immunoglobulin G
(160,000). bovine serum albumin (68,000), ovalbumin (43,000),
chymotrpysinogen A (25,000) and ribonuclease A (13,700). In
antigen-positive patient sera examined by gel filtration, prostate antigen
eluted as a single symmetrical peak between 80-X00,000 m.w.
Example 12
Preparation of Antisera
-
Female rabbits were immunized as described previously by T.M~ Chu et
al. in Investigative Urology 15: 319-323 (1978) with the crude extract of
normal human prostatic tissue (for antiserum P8), or with a purified
prostate antigen (for antiserum P17) obtained at the SEPHADEX G-75 step
described above. Sera were collected, heat inactivated and stored at -20 C
until use. Absorption of the antiserum with normal female serum (NFS) or
tissue extracts (10 mg protein/ml) was carried out as described by T. M.
Chu et al. in Cancer Treatment Reports 61: 1~3-200 (1977).
Page 26
'
'~
Example 13
Specificity of Antiserum
, .. .. .. .
Immunoelectrophoresis was performed on a 9.5 x 10.2 cm agarose (0.65
percent, w/v) plate. Barbital buffer, ~pH 8.2, ionic strength 0.04~ was
used as the electrolyte and a constant voltage of 90 V. was applied for one
hour. After electrophoresis and gel diffusion ~20 hours), the plate was
washed with 0.154 M saline for 2 days and stained first for acid
phosphatase with a solution of ~-naphthyl phosphate~fas-t garnet GBC salt in
O.lM ammonium acetate9 pH 500, and then for protein with Coomassie
brilliant blue G-250-perchloric acid solution.
Immunoelectrophoresis of the crude extract prepared from normal
prostatic tissues and antiserum P8t an antiserum raised against the crude
extract of normal prostate, resulted in three precipitin arcs. One of
these arcs was formed by a normal human serum component7 as it disappeared
after the antiserum was absorbed with normal ~emale serum (NFS-P8).
Absorption of the antiserum NFS P8 with various normal human tissue
extracts (urethra, bladder, heart, lung, pancrease, bone, kidney,
intestine, liver and spleen, 10-20 mg each per ml) failed to eliminate the
two remaining precipitin arcs, one of which was identified as prostatic
~o acid phosphatase since it was stained with the -naphthyl phosphate-fastgarnet GBC salt solution. The other precipitin arc was shown to be a
prostate tissue-speclfic antigen, identified as a protein, not stainable
for acid phosphatase activity, which migrated with ~-mobility ~pon IEP
analysis. Absorption with an extract of normal human prostate removed the
reactive antibodies from the antiserum NFS-P8 and abolished both
precipitin arcs. Furthermore~ this antigen was demonstrated in all 20 of
20 normal prostates and identical results were obtained with the extracts
of benign hypertrophic (15/15~ a~d cancerous prostatic (8/8) tissues.
These data demonstrate that normal~ benign hypertrophic and malignant
Page 27
6~i
prostate contain a prostatic tissue-specific antigen in addition to
prostatic acid phosphatase.
Example 14
Specific;ty of Antiserum Raised_Against Purified Prostate Antigen
Following the procedure of Example 11, additional confirmation was
provided with the use of antiserum P17 ra;sed ayainst a purified
preparation of the prostate antigen, which was devoid of prostatic acid
phosphatase. Boih gel diffusion and immunoelectrophoresis employing the
normal adult male and female serum-absorbed antiserum P17 resulted in a
single immunoprecipitation of the prostate antigen. The extracts prepared
from tissues other than the prostate did not react with the normal female
serum-absorbed antiserum P17.
Example 15
Immunological Identity of
Tissue and Serum Prostate Antigen
In order to determine if the serum~borne antigen shared immunological
or biochemical characteristics with the antigen detected in prostate tissue
extracts, the following experiments were performed. Samples of sera and
prostate tissue extracts were subjected to rocket-lEP both individually and
immediately after combination of both antigen sources. In each experiment,
the samples were adjusted to the same final protein concentration by the
use of buffer dilutions. A representative experiment employed two sample
wells containing patient sera, sample wells containing prostate tissue
extract and two sample wells containing a mixture of patient's serum and
tissue extract to demonstrate peak enhancement. Sera and tissue extracts
were at the same final protein concentration in each well by the use of
appropriate volumes of dilu$ing buffer (PB-NaCl)~ All samples were 25 ~lj
one percent antibody in 0.83 percent agarose; 5 V per cm, 20 hours at 4 C.
All samples produced a single immuno- precipitate when assayed
Page 28
~ ! ~ ~
individually. Mixing samples, in peak enhancement experlments, produced a
single reaction whose height was greater than that of individua1 samples.
When antigen-positive serum samples and prostate tissue extracts were mixed
and immediately subjected to crossed-IEP, a fused immunoprecipitation peak
resulted. Both methods of quantitative immunoelectrophoresis, showing peak
enhancement and immunoprecipitate fu~ion, confirmed the immunological
identity of the prostate antigen as it occurrs in tissue and serum
according to the me~hod of N.H. Axelson et al. in Scand. J. Immunol.
(Supplement l) 2: 9I-g4 (1973).
It should be noted that results regarding prostate antigen, as
detected in tissue, apply to extracts prepared from normal, benign
hypertrophic and malignant prostate specimens; no physical differences in
prostate antigen ti.e.~ MR, pI or m.w.) were observed among these antigen
sources, and extracts prepared from benign and malignant prostate specimens
exhibited immuno-precipitation lines of identity with normal prostate when
tested against anti-prostate antigen antiserum by double immunodiffusion.
In addition, the relative level of prostate antigen was compared among
extracts prepared from normal, benign hypertrophic and mal19nant prostate
tissues. As determined by rocket-I~P (Table 3) no statistically
significant difference of antigen level was observed between these antigen
sources and a wide variation in antigen level tindicated by the large
standard deviations calculated) was also noted to occur for each category
of tissue extract examined.
Page 29
TABLE 3
PROSTATE ANTIGEN LEVELS IN PROSTATE TISSUE EXTRACTSa
Tissue Pathologyb(Number) Prostate Antigen Level ~ S.D.
Normal (4) 14.3 unitsC (~ 8~7)
Benign hypertrophic (8) 18.4 unitsC (~ 21.3)
Primary carcinoma ~8) 13.0 unitsC (~ 13.8)d
a) Each tissue extract was adjusted to 1 mg protein per ml prior to
analysis for prostate antigen by rocket immunoelectrophoresis.
b) All tissue specimens were histopathologically confirmed.
c) For this expPriment, one unit was arbitrarily chosen to represent one
percent of the antigen level of a reference prostate extract. The
reference extract was assayed in a dilu~ion series and run on the same
plates as experimental extracts.
d) As analyzed using Student's t-test, no statistically significant
difference was found among these groups.
Page 30
Immunological Identity of
Fluid and Serum Prostate Antigen
Since prostatic acid phosphatase has been reported in both prostatic
tissue and seminal fluid, we wished to determine whether PA also exists in
the seminal fluld. Anti-PA serum9 raised against PA isolated from
prostatic tissue, reacted in immunodiffusion with crude extract of
prostatic tissue, as well as with seminal plasma, and formed a fused line,
indicating immunologic identity of the PA in prostatic tissue with that in
seminal plasma. By the previously described radial immunodiffusion
procedure, the PA concentration in seminal plasma was found to be 0.4 to
1.~ mg/ml, while 1 g. of prostatic tissue contained d.l5 to 0.45 mg of PA
extractable with disodium ~thylenediamine tetraacetate-phosphate buffered
saline (EDTA-PBS).
Example 18
Histolo~ical Localization of Prostate Anti~en
_. , _ ~ . .
Localization of PA in the prostate gland as well as specificity of PA
have been probed with the i~munoperoxidase staining technique of Heyderman
and Neville reported in ~. Clin. Path. 30: 138-143 (1977~. Sections of
freshly fixed prostatic tissue were deparrafinized in two changes of xylol9
rehydrated thru a graded series of ethanol9 and washed with distilled
water. Following the inhibition of endogeneous peroxidase activity by 7.5
percent H202~ the reactivity of anti-PA was assessed with rabbit
anti-PA serum. After incubation and subsequent washing~ the tissue
sections were further incubated with peroxidas~- labelled goat anti-ra~bit
~-globulin. Excessive enzymerlabelled secand antibody was then removed by
washing, and ~he tissue sections were stained for perox1dase activity. As
controls9 the substrate aloneg substrate plus conjugate, pre-immune rabbit
serum, and anti-PA preabsorbed with specific antigen were used.
Page 31
S
..
lt was observed that the staining was restricte~d to epithelia1 cells
cwnprising the prostatic ductal elements. An intense staining was shown in
the dpical cytoplasma of these cel ls, but no 5taining was Seen in the
nuclei. Within several ductal elements, positiYely staining secretory
material was ob5erved in the Section examined. Specific staining was not
observed for other cellular elements, includ;ng stromal and vascular
elements. Using the same tec~nique, PA could not be detected in sections
derived fro~ other organ tissues, including the pancreas, colon, stomach~ i
liver, seminal vesicles, and testes. Employing similar immunohistochemical
techniques, PA has been demonstrated in all primary and metastatic
prostatic tumorS tested, but not in non-prostatic cancer tissues.
Example 19
Detection of Prostate Antigen in
Established Tissue Cu1ture Cell Lines
. .
To determine if malignant cells in long-term culture retain the
expression of PA, three e5tablished strains of prostate cells (LNCaP, PC-3,
and Du-145) were examined. Extracts prepared from these cells and other
cultured cells of nonprostate origins were assayed for the presence of PA
by the previously mentioned Enzyme Immunoassay (EIA) procedure capable of
detecting 1 ng PA/ml.
Immobi1ized anti-PA was prepared using cNsr-activated SEP}IAROSE lB.
[SEPHAROSE is the trade mark of Pharmacia Fine Chemicals for the neutral gelling
fraction (agarose) of the polysaccharide complex known as agar]. The reaction
mixture consisted of 5 g of CNBr-activated SEPIIAROSE and 130 rng of IgG (anti-PA)
in O.I N borate buffer (pH 8.5) containing 9.5 M NaCI. After incubation at 4 C.
for 18 hours, the beads were washed with borate buffer and post-treated with a l-N
ethanolamine solution (pH 9.0) to block unreacted groups. Covalently coupled
beads werefurther washed and stored in PBS at 40C.1
For the EIA, 100~1 of an antigen samp1e was mixed with 300 ~1 of a fifty
fo1d-diluted immobilized antibody and incubated for 3 hours at room
- 32 -
temperature. After the addition of l ml of assay buffer (PBS containing 1
percent BSA), the mixture was centrifuged at l,OOOg to wash the beads.
This procedure was repeated twice. To the washed beads was added 100 ~l of
peroxidase-IgG (anti-PA~ conjugate in assay buffer. A~ter a further
incubation for 18 hours at room temperature, the beads were again washed as
described above and assayed for the amount of bound peroxidase activity
present. The reaction mixture contained 0.08 percent dianisidine and 0.003
percent H202 in 0.01 M sodium phosphate buffer ~pH 6.03 and was allowed
to react with the beads for 90 minutes. The enzyme reaction was then
stopped with 100 ~l of 1 N HCl, and the absorbance at 403 nm was
determined. Each run included standards of known prostate antigen
concentration. Using this assay procedure, linearity of of the
dose-response curve was achieved between 1 and 20 ng PA/ml. Before
reexamination, samples containing higher levels o~ antigen were serially
diluted with assay buffer. The results indicated that two lines of
prostate cancer cells, LNCaP and PC-3, contained significant levels of PA
t50-700 ng/ml) as compared with those found in Du-145 and other cell lines
examined (less than 4 ng/m7)~ PA was also present in cond1tioned "spent"
media derived from prostate cultures producing the antigen.
Example 20
Immunoloaical Reactivity of Cultured Prostate Cells
with Immunoglobulin Antiserum Fra~ments
To further assess antibody specificity~ we examlned cultured cells for
their ability to specifically acorete radiolabeled antibody fragments.
Included in these experiments were cells derived from the prostate gland
(LNCaP)~ colon (HT-29), and breast (MCF-7). For the preparation of
immunoglobulin fragments, samples of IgG, anti-PA and rabbit pre-immune
serum were dissolved in 0.1 M sodium acetate buffen (pH 4.0). For every 50
mg of IgG, 1 mg of crystallized pepsin was added and the reaction mixture
Page 33
was incubated overnight at 37 C. F(~b')2 fragments we~e separated from unreactedIgG and from smaller peptides by gel flltration ov~r SEPHACRYL S~300 ~SEPHACRYL
is the trade marh of Phar~acia Fine Chemicals for an acrylic base eiltration
medium] equilibrated in PBS buffer~ The column (2.6X70 cm) was calibrated by the
chromatography of molecular weight standards, including IgG, BSA, and egg
albumin. Isolated F(ab')2 fragments were concentrated by ultrafiltration with the
use of an Amicon PM-lO membrane and positive pressure.
Trace labeling of F(ab )2 fragments was accomplished with the use of
solid-phas2 lactoperoxidase-glucose oxidase ENZYMODBEADS; Bio-Rad
Laboratories). To 1 mg of F(ab )2 were added S0 ~1 of ENZYMOBEADS- ~5 ~1
of 1 percent B-D-glucose~ 200 ~1 of 0.2 M sodium phosphate (pH 7.0) and 1
mCi of carrier-free 1311 or 1251. The reaction was allowed to proceed
for 30 minutes at roo~ temperature. Unreacted iodide was separated from
labeled protein by gel filtration over SEPHADEX G-25~ Specific activities
of trace-labeled F(ab )2 preparations ranged between 0.6 and 0.8 ~Ci/~g.
Cells grown in 24-well culture dishes with each well containing
l.SxlO cells were incubated with a paired radiolabeled mixture of
51-labeled F(ab )2 (anti-PA) and 131I-labeled F(ab )2 (preimmune)
in fresh media. This mixture contained l.sx105 cpm for each radioactive
nuclide and was allowed to react with cultured adherent ce115 overnight
under standard conditions. To determine radioactive uptake7 cells were
scraped from the wells and washed three times in PBS before countiny for
each radioactive nuclide in a Packard automatic y-scintillation counter.
Preferential uptake of a specific radioactive nuclide ~as calculated as a
localization ratio: (1251 cell bound 1311 cell bound) : (1251 added
1311 added). A specific localization ratio was calculated for each cell
type and the results indicated a significantly higher antibody uptake by
the LNCaP cells of prostate origin than for the HT-29 or MCF-7 cells.
- 34 _
Example 21
Release of Prostate Anti~en by _n _ o Tumor Cells
When established as tumor cell xenografts in nude mice, LHCaP cells
released detectable levels of PA into the circulation o~ these animals.
Congenitally athymic nude mice homozygous for the nu/nu allele were bred at
Roswell Park Memorial Institute from matings of BABL/c nu/nu homozygous
males and BALBtC ~/nu heterozygous females. Human tumors were established
subcutaneously in the nude mice before sera were obtained for PA analysis
by the injection of cu)tured cell suspensions. For this, the cells were
washed in PBS, counted for viability, and adjusted to the desired
concentration in sterile 0.9 percent NaCl solutions. Subsequent to nodule
formation, nude mouse serum was collected by severing the retro-orbital
plexus and stored at -20 C. Cell cultures used for the establishment of
human tumor grafts included LNCaP (adenocarcinoma of the pr~state gland),
RT-4 (transitional cell carcinoma of bladder), Palarmo ~malignant
melanoma), and AsPC-1 (pancreatic carsinoma).
Of the human tumors examined, only the LNCaP prostate line released PA
into the circulation, which correlates with results obtained from the assay
of cultured cells. The control mice given preimplants of RT-4 cellsl
Palarmo cells, or AsPC-l cells showed no detectable PA in their
circulation. It is not presently appreciated if serum antigen levels are
commensurate with the tumor load, a phenomenon previously reported by E.J.
Pesce et al. in Cancer Res. 37: 1998-2003 (1977) for the lactate
dehydrogenasde en~yme released by human xenografts. If this is so, then
the human prostate tumor-nude mouse system, coupled with detection of PA,
can provide a valuable clinical model to monitor the effects of antitumor
modalities and the effects of biologic response modifiers for human
prostate cancer.
Page 35
Example 22
Antiserum Specificity
The optimal concentration of adsorbed antiserum required in the
rocket-IEP procedure was determined by examining the migration of prostate
antigen in gels containing varying concentrations of anti-prostate antigen
antiserum. The reactivity of various tissues was examined by rocket-IEP.
As shown in Table 4, all extracts prepared from normal prostate, benign
hypertrophic and malignant prostate tissues showed reactivity9 producing a
single immunoprecipitin reaction. Extracts prepared from the non-prostate
tissues, whether normal or neoplastic in nature, gave no immunologic
reactivity.
Page 36
B~
TABLE 4
REACTIVITY OF ANTI-PRVSTATE ANTIGEN ANTISERUM WITH HUMAN TISSUE
EXTRACTSa
Percent Positive Reaction
Tissue Pathology Rocket-IEP Immunodi~ussion
Liver Normal O (0/2) 0 50/1)
Spleen Normal O (0/1) 0 (0/1)
Lung Normal O (Q11) 0 (0/1)
Adenocarcinoma0 (0/1) 0 (0/1)
Bone Marrow Normal O (0/1)
Bladder Normal o (O/l) O (O/l)
Breast Normal O (0/1)
AdenocarcinomaO (0/5)
Intestine Normal O (Oll) O (0/1)
AdenocarcinomaO (0l1) 0 (0/1)
Heart Nor~al o (Oj1) o (Otl)
Pancreas AdenocarcinomaO (0/~) 0 ~0/1
Kidney Normal O (0/1) 0 (0/1)
Cerebral Cortex Normal O (0/1) 0 ~0/1)
Prostate Normal 100 (~/4) 100 (20/20)
Benign Hypertrophic 100 (8/8) 100 (15115)
Adenocarcinoma100 (8/8) 100 (8/8)
.. . ,. " _
a) All tissue extracts were adjusted to 10 mg protein/ml prior to analysis
Page 37
Example ?3
~ ic Specificity
To examine the potentia7 diagnostic value of the prostate antigen,
serum samples were examined for its presence by the method of rocket-IEP,
using anti-prostate antigen antiserum treated with normal human plasma to
remove antibodies against normal plasma proteins ~Table 5) Serum samples
obtained from 20 normal healthy adults and 20 male volunteers over the age
of 55 years showed no reactivity against the antlserum with this assay.
Also, serum was drawn from a total of 175 patients with various advanced
malignancies, including patients with malignancies of lung, colon, rectum,
stomach, pancreas, thyroid, breast and with myeloma. All sera obtained
from patients with non-prostatic malignancies were prostate
antigen-negative when assayed by the rocket-IEP procedure. However, out of
a total of 219 sera examined from advanced prostatic cancer patients, 17 or
approximately 8 percent showed the presence of prostate antigen in
circulation. All sera showing a positive reaction for prostate antigen
were subsequently subjected to the same assay, and reproducibility of the
test was 100 percent.
Pa~e 38
TABLE 5
REACTIVITY OF ANTI-PROSTATE ANTIGEN ANTISERUM WITH HUMAN
SERA BY ROCKET-IEPa
Serum Donors Percent Positive Reaction
Normal adults (male and female~ O ~0/20)
Age-and sex-matched controls O (0/20)
Patients with advanced malignanciesb
Lung carcinoma O (0/83)
Thyroid carcinoma O (O/l)
Colon-rectal carcinoma O (0/22)
Stomach-pancreas carcinoma O (0/34)
Breast carcinoma O (0/33)
Myeloma O (0/2)
Prostate carcinoma 8 (171219)
a) All sera were aliquoted and s~ored at -20 C or -70 C until required. A
sample volume of 25 ~l was used for all studies.
b) Each case was pathologically confirmed.
Page 39
Example 24
En~yme-Linked Immunoassay Tests
Using IgG antibodies against the purified prostate-specific antigen
with horseradish peroxidase and CNBr-activated Sepharose 4B as reagents, a
sensitive sandwich-type ~sEPHARosE 4B-anti-prostate antlgen IgG: prostate
antigen: anti-prostate antigen IgG-peroxidase~ enzyme-linked immunoassay
capable of detecting 0.1 ng of prostate antigen/ml. was evaluated. Of the
various normal and tumor tissues examined, on)y human prostate tissue was
shown to contain the prostate antigen (normal prostate 10 ~ 21.9~9 prostate
antigen/mg. protein, n=6; benign hypertrophy 18.3 ~ 29.5, n=12; malignant
prostate 19.1 ~ 15.3, n=13). Circùlating le~els of prostate antigen were
also quantitated by the same assay; no prostate antigen was detectable in
sera from normal females (n=17) or female cancer patients (n=25).
Additionàl results (in ng/ml) are shown in Table 6.
)3? ~ ~s
Page 40
~l~65~
TABLE 6
ENZYME-LINKED IMMUNOASSAY RESULTS
Group Mean S.D. ~ n ? . 30 p
Normal males 0.47 0.66 0.1-2.6 51 2
Non-prostate
cancer males 0.52 0.62 0.1-3.0 92 3 N.S~
Prostate cancer
Stage A 8.00 5.64 2.8-14 3 3 ~.02
Stage B 7.15 3.05 ~.6-ll 4 4 o.ool
Stage C 10.52 17.06 0.3-100 44 31 0.001
Stage D 22.84 30.97 0.2-270 2s0 193 o.ool
(p vs. normal males by Student's t test; N.S. = not significant)
The prostate antigen from the serum of prostate cancer patients was
purified and shown to be im~unochemically identical to the prostate anti~en
of normal prostate tissue. This data, therefore, demonstrate that the
prostate antigen described herein, although a hlstotype-specific antigen of
the normal prostate, can be used in the immunological detection of
prostatic cancer.
Example ?5
Prognostic Value of Enzyme~Linked Im~unoassay Tests
Using the sensitive en7yme immunoassay reported in Cancer Res. 40:
4658(1980), the circulating PA in prostatic cancer patients has been
evaluated clinically. In 36 patients in advanced stage of disease ~D~)
and receiving chemotherapies, the pretrea~ment serum PA levels were found
to be of prognostic value in regard to patients' survival. Patients who
survived more than 12 months tn=10) had serum PA levels of 11.2 ng/ml
12.7 (mean ~ S.D.)7 and those (n=59~ who expired within 5 months exhibited
serum PA values of 28.5 ~ 31.9; while levels of 13.7 t 18.2 were ~ound in
Page 41
the patients (n=27) who survived 6-11 months. Nineteen of these patients
were monitored by 220 serial PA assays for more than 6 months, and a
clinicopatholo~ical correlation between PA levels and clinical course was
found in 14 patients (74 percent). Additionally, in another group of 32
patients who underwent curative therapies for localized prostate cancer,
161 serum samples were analy2ed during a period of 12 to 114 weeks (average
56 weeks). Of these patients, 5 developed metastasis during follow-up and
all were shown to exhibit increasingly elevated serum PA values 0-68 weeks
preceding the clinical diagnosis of disease recurrence. These results
demonstrate that PA is a ùseful new marker for monitoring prostate cancer.
Example 26
Establishment of a Hybridoma Cell Line
n~ .
A nonsecreting myeloma cell line of Balb/c origin described in J.
Immunol. 123: 1548-1550 (1979) was used. This line, P3~63A98.653, is
derived from the y-l K-type producing P3X63Ag8. P3X63Ag8.653 cells were
maintained in Dulbecco's modified Eagle's medium (DMEM) contain1ng 15
percent heat-inactivated fetal bovine serum (FBS), 2mM glutamine, 100 U/ml
penicillin, and 100 U/ml streptomycin in a 10 percent C02/air humidified
incuba~or at 37 C.
Female Balb/c mice (8-10 weeks old) were intraperitoneally injected on
days 1 and 30 with 10 ~9 Prostate Antigen (PA) purified as described in
Investigative Urology 17:159-163 (1979). Three to six weeks later, the
mice received an intravenous injection of 10 ~9 PA in sterile saline. All
mice used for fusion experiments showed the presence of serum antibodies
against PA, as detected using double immunodiffusion against purified PA.
Spleens were aseptically removed from immune mice after the last
immunization, placed in 10 ml DMEM-15 percent FBS, and cell suspensions
made by teasing with curved forceps. Clumps and membrane fragments were
allowed to settle and the resulting single cells were washed once by
Page 42
6;568S
centrifugation at 6009 for 10 min. Red cells were lysed by incuhaki~n in
0.84 percent NH4Cl, and the cells were washed and resuspended in DMEM at
a concentration of 2 X 107 cells/ml. The myeloma cells were similarly
washed and adjusted to 2 X 107 cells/ml of DMEM. Three ml of spleen
cells were added to 3 ml of myeloma cells and co-pelleted at 6009 for 10
min.~ and the medium was completely removed. For fusion, the cells were
gently resuspended in 1 ml of 30 percent (w/v) polyethylene glycol (PEG),
average molecular weight 1,000, in DMEM a$ pH 8Ø The tube was rocked for
1 min. and centrifuged for 6 min. at 6009. After a total exposure time to
the PEG of 8 minutes, the supernatant was removed and 10 ml DMEM slowly
added with continuous circular agitation of the tube. Cells were then
centrifuged for 10 mln. at 6009, and gently resuspended in SO ml of HAT
selection medium (DMEM-15 percent FBS containing 1.6 X 10-5M thymidine, 1
X 10-4M hypoxanthine, and 1 X 10-7M aminopterin~. One ml amounts were
dispensed into 48 wells of multiwell dishes and plates were incubated at
37 C in a humidified atmosphere of 10 percent C02 in air. HAT medium was
replenished every 4 days until day 21 when hypoxanthine/thymidine (HT)
medium was added. As wells turned acid (usually 8-12 days after fusion),
supernatants were tested for antibody activity. Samples of hybrid cells
from wells which showed positive antibody activity were clcned by limiting
dilution and some cells from the same well were transferred to 25cm2
tissue culture flasks until 2 x 107 cells were obtained, which were
frozen in liquid nitrogen in 10 percent dimethylsulfoxide ~ 90 percent FBS.
Example ?7
Selection of Hybridoma Cells
Fusion supernatants were screened for anti-PA antibodies by a
solid-phase enzyme immunoassay. Disposable 96-well microtiter plates were
used as the solid adsorbing surface. The wells were filled with 100 ml
poly-L-lysine succinate (0.25 mg/ml H20) and incubated at 23 C for 15
min. Following 3 washes with PB-NaCl pH 7.2 (50 mM sodium phosphate:l4~ mM
Page 43
sodium chloride), 100 ml of purified PA were added at a concentration of 50
~gl~l 10 mM carbonate buffer, pH 9.6. After incubation for 24 hr. at 37 C,
100 ml of 1 percent (w/v) bovine serum albumin solution in PB-NaCl buf~er
were added for a further 3 hrs. at 37 C.
Prior to performing the antibody screening ass~y, the microtiter wells
were washed 3 times using PB-NaCl buffer (200 ~l). Culture fluids ~100 ~l)
to be tested were applied to individual wells and allowed to incubate at
37 C for 3 hrs., followed by 3 washes with PB-NaCl buffer. Subsequently,
100 ~1 of peroxidase-conjugated antiserum to mouse lg prepared according to
the procedure described in J. Immunol. Methods 15: 305-310 (1977) were
added to each well and incubated at 37 C for 3 hrs. Aft~r 3 washes with
PB-NaCl buffer, en~yme activity was revealed using substrate solution
containing 0.08 percent o-dianisidine : 0.003 percent H202 : 0.01M
sodium phosphate, pH 6Ø The enzyme reaction was stopped after 60 min. at
23 C using 25 ~l of lN HCl.
In each assay, positive control samples were included and consisted of
serially diluted hyperimmune mouse serum raised against purified PA.
Tissue culture supernatants ~rom P3X63A98 cultures ~y 1-Kappa) and from the
parental nonsecreting myeloma line were used as negative controls. This
screening assay detects immunoglobulins o~ the IgG, IgM and IgA classes.
Example 28
:
'!9~
Desired myeloma hybrids were c10ned by limiting dilution in the
presence of peritoneal macrophages to increase cloning efficiency. Hybrid
cells were plated in 96-well culture dishes at a density of 0.5 cellstwell
in DMEM-15 percent FBS complete medium. Vigorous growth was observed after
8-12 days, at which time supernatants were tested from wells showing single
colonies. Cultures exhibiting antibody activity were re-cloned by this
procedure to ensure population homogeneity.
Page 44
~65~
- Peritoneal macrophages were prepared by flushing the peritoneal
cavity of Balb/c mice with 5 ml of ice coldr sterile 0.34M sucrose. To
attain high yields of macrophages (5~15xl0 /mouse), animals reae;ved an
intraperitoneal injection of 0.5 ml sterile thioglycolage medium 4 days
.
prior to harvesting the cells. Macrophages were washed~once by centri~
fugation at 600g for 5 min. and were resuspended~at 10 /ml in tlssue
culture medium. Each tissue culture well received 100 ul of macrophage
suspension. The hybridoma described herein has been deposited with the
American Type Culture Collection, Rockville, Maryland 20850, U,S.A. and
is designated ATCC No. HB 8051.
Example 29
Purification'of Monoclonal Antibodies
Monoclonal antibodies from spent culture fluids were purified
uslng immuno-affinity chromatography with rabbit antimouse Ig: SEPHAROSE
4B gel matrix (R MIg:SEPHAROSE). Twenty ml of R~ MIg:SEPHAROSE were
~packed into a chromatographic column (1.5 x 30 cm) and equilibrated with
PB-NaCl'buffer. Samples of culture fluids were slowly,passed through the
column (5 ml/hr), followed by removal of non-specific reactants by elu-
tion with 100 Inl of 0.lM glycine lM NaCl, pH 9Ø Reactive proteins were
then eluted with 3 bed volumes of 4M KSCN: 0.DlM sodium~phosphate,~Ph~7.2.
Dialyzed materials were concentrated using positive-pressure ultrafiltra-
tion.
Example 30
- ~
Immu~odiffuslon'Analysis of Secreted Hybridoma Products
An affinity matrix was prepared by coupling 200 mg Ig (R- MIg)
to 5 grams of cyanogen bromide-activated SEPHAROSE 4B; an IgG fraction of
~ 45 _ ' ~
. ~ '
: .
rabbit antisera to mouse ~gG (H ~ L) was prepared by a Rivanol procedure
described in J. Immunol. Methods~15 305~310 (1977). Coupling was per-
formed at pH 8.0 ln bicarbonate buffer according to directions providea
by the
.. . . .
': ,,
.
'
,
- 45 a ~ ~
-
.
,
6~5
manufacturer (Pharmacia; Piscataway, N.J.). The class and subclass of
isolated anti-PA antibodies were determined by double immunodif~usion in
0.6 percent agarose using rabbit anti mouse IgG1, IgG2a, IgG2b, IgG3, IgM,
anti-K and anti-~ chain.
Supernatants from 96 of the most vigorously growing cultures were
screened in duplicate for antibody activity against PA, using a solid phase
enzyme-immunoassay. Results indicated that approximately 4 percent (4/96)
of cultures were initially antibody-positive. Upon re-assay of these
cultures, one hybridoma (F5-A-1/22) remained positive. This culture was
cloned by limiting dilution over peritoneal macrophages and yielded 30
cultures which macroscopically exhibited single colonies of growth. One
positive culture was re-cloned and all cultures derived therefrom were
antibody-positive. These cells (clone F5-A-1122.8.13, termed Cl. 8.13)
were expanded to mass culture and examined for antibody content (Table 7).
As a positive control for this experiment, culture fluids from P3X63Ag8
cells, known to produce IgGl kappa-type immunoglobulen9 were used~ Cells
of the P3X63Ag8 strain produced approximately 12 ~g/ml of isolated
immunoglobulin which was reactive against specific antisera to IgG1 and
K-chain (Table 8j. No precipitation occurred with other immunoglobulen
subclass antiserum reagents. Culture fluids from Cl. 8.13 showing anti-PA
activity contained about 10 ~g/ml of isolated material using R-~MIg.
Sepharose purification. This isolate WdS identified as mouse
immunoglobulin of the IgM K-type subclass (Table 8). This preparation did
not precipitate against other subclass of antisera, indicating
monoclonality of the isolated immunoglobulin preparation.
Page 46
~able 7. Affinity purification of monoclonal antibodies using R-~Mlg:
SEPHAROSE 4B chromatography.
Culture Fluid
(ml) applied to Yield ~glg/ml l;
Culture affinity adsorbent Ig(mg)b culture fluid
P3X63Ag8 125 1~6 13
F5-A-1/22.8.13 300 3.1 10
. . ~
(a3 Clarified culture fluids were passed through R-~Mlg: SEPHAROSE 4B
adsorbent as described. The gel was washed with lM NaCl pH 9.0 until ~A;
absorbance at 280 nm reached baseline (less than 0.0200.D. units).
(b) R-nMIg-reactive substances were eluted under chaotropic
dissociating conditions of 4M KSCN. Yield of Ig isolated was
determined by optical density measurements where a 1 percent (w/v)
solution of mouse IgG shows an extinction coefficient of 14.
~'
Page 47
,
. ' ', ''
-:
.
Table 8. Immunodiffusion analysis of afinity puriied immunoglobulins
fro~ spent culture fluias.
'Affinity'isolates from cultures ~ '
~ ~ F5-a~l/22.~.13
R-C~ IgGl ~ +
R-C~ IgG2a
R-o~ IgG2b
R- ClIgG3
R-~ K chain ,+ - +
R-C~ ~ chain ' -
R ~ IgM ~ ~ ~ +
The preceding examples can be repeated,with similar success.~
by substituting the generically or~speciflcalIy described reactants and/or
operating conditions of this invention for those specifically used in the
examples. From the foregoing description, o~e skilled in the art to which -
this invention pertains can easily ascertain the essential characteristics
thereof and can make various changes and modifications to adapt it to
various usages and conditions. ~ ~
, Industrial Applicability
As can be seen from the present specification and examples, the
present invention lS industrially useful in several respects. Although
this prostate antigen is an eutopic product, it appears to be a useful
tumor marker such as other differentiated cell productsj e.g., prostatic
acid phosphatase and thyrocalcitonin~ The speclficity of prostate anti-
gen antiserum can allow for the identification of neoplastic cells in
isolated metastases with unknown primar~-origin. Anti~prostate antigen
- 48 -
-. .. : - ' :
antiserum can also be used as a vector to localize and~or carr,v cytotoxic
substances to neoplastic prostatic tissue~ ~urther~ the study of
variations in the
.
~ 48 a ~
~ .
expression of normal differentiation between antigens of prostatic tissue
during development of the gland and in prostatic lesions can provide
insight into the phenomenon of growth regulation and metastatic ability of
prostate cells.
Page 49
