A method for detecting an antigen in a plasma or serum sample,
which comprises conditioning the sample, wherein conditioning comprises
either diluting the sample by a factor of at least 100 or dissociating
the antigen in the sample from binding proteins with acid, base,
or a chaotropic agent; drying the conditioned sampled; and detecting
the antigen in the dried sample using an immunological assay is
disclosed. The method is particularly useful for the detection of
antigens associated with breast cancer.
What is claimed as new and desired to be secured by Letters Patent
of the United States is:
1. A method for the detection of a human mammary epithelial differentiation
antigen in a blood sample, which comprises:
(a) conditioning said sample, by dissociating said antigen in said
sample from binding proteins by adjusting the pH of said sample
to within a range of from about 2 to 5;
(b) drying said conditioned sample onto the surface of a test container
to give a dried sample; and
(c) adding a labeled antibody and detecting the label.
2. The method of claim 1, wherein said sample is conditioned by
adjusting the pH to within a range of from about 2 to 4.
3. The method of claim 1, wherein said sample is conditioned by
adjusting the pH to within a range of from about 2 to 3.
4. The method of claim 1, wherein said detecting comprises employing
an immunological assay which utilizes a monoclonal antibody.
5. The method of claim 1, wherein said sample is plasma.
6. The method of claim 1, wherein said drying comprises heating
at a temperature of no more than 55.degree. C.
7. The method of claim 1, wherein said drying comprises heating
at a temperature of no more than 42.degree. C.
8. The method of claim 1, wherein said heating continues for no
more than 36 hours at dryness.
BACKGROUND OF THE INVENTION
1. Field of the Invention:
This invention is related to diagnostic assays for the detection
of antigens in blood and particularly to diagnostic assays relating
to the detection of antigens associated with breast cancer.
2. Description of the Prior Art:
Carcinoma of the breast is the most frequent form of cancer in
women in North America and Europe. The traditional diagnostic technique
of palpation is often insuffient for the detection of early lesions.
Often, by the time that a malignant mass becomes distinguishable
from a benign nodule of breast tissue, the cancer has metastasized.
The principal alternative merhod of detecting breast malignancies
in recent years is the mammogram, a low-voltage X-ray procedure.
However, because of the oncogenic potential of this X-ray procedure,
the method is of limited usefulness in premenopausal women. Current
guidelines of the National Cancer Institute recommend that routine
mammography be restricted to women over 50 and to high-risk patients
under 50. There accordingly remains a need for a non-invasive method
of detecting early malignancies of the breast.
One method of detecting cancer which has received considerable
attention in recent years is the use of immunological techniques
for detecting antigens associated with cancers. The best known such
antigen is carcinoembryonic antigen (CEA). When first discovered,
CEA was thought to be specific to cancers of the digestive system.
However, CEA has since been detected in normal adults as well as
in patients with benign liver disease, such as alcoholic hepatitis
or biliary obstruction. Because of the overall lack of specificity
and sensitivity, there being no threshold difference in CEA levels
that serves to separate benign from malignant conditions, CEA cannot
be used as a general diagnostic test. It is principally used in
the monitoring of response to treatment.
Similar antigens are now known to exisr in breast cancer. Breast
tissue markers such as casein [Franchimont et al, Cancer, 39, 2806-2812
(1977)] and .alpha.-lactalbumin [Kleinberg et al, Science, 190,
276-278 (1975)] and proported cancer markers such as glycosyl transferases
[Ip et al, Cancer Res., 38, 723-728 (1978); Dao et al, J. Natl.
Cancer Inst., 65, 529-534 (1980)], glycolipids [Kloppel et al, Proc.
Natl. Acad. Sci. USA, 74, 3011-3013 (1977)], and phospholipids [Skipsky
et al, Proc. Soc. Exp. Biol. Med., 136, 1261-1264 (1971)] have all
been used in various diagnostic techniques for breast cancer without
gaining widespread acceptance as a breast cancer marker. More recently,
circulating human mammary epithelial antigens have been proposed
as specific markers for breast cancer [Ceriani et al, Proc. Natl.
Acad. Sci. USA, 79, 5420-5424 (1982)].
However, one recurring problem in the immunological detection of
any antigen present in blood (not just in tests for antigens associated
with cancer) is the occurrence of false positive and false negative
reactions. For example, in the area of cancer diagnosis, false positives
can result in unnecessary diagnostic surgery or mammography of a
patient who does not have cancer, while false negatives result in
cancer that goes undetected. Accordingly, a method for improving
the discrimination of immunological assays and eliminating false
positives and false negatives to the maximum extent possible is
needed both in the general area of antigen detection and particularly
in the detection of life-threatening malignancies.
SUMMARY OF THE INVENTlON
Accordingly, it is an object of the present invention to provide
a diagnostic technique for use with immunological assays for antigens
present in blood which increases the reliability of the assay technique.
It is a further object of this invention to provide a method specific
for the diagnosis of breast cancer utilizing this method of improved
These and other objects of the invention as will hereinafter become
more readily apparent have been accomplished by providing a method
for antigen detection in a blood sample, which comprises conditioning
said sample, wherein conditioning comprises either diluting said
sample by a factor of at least 100 or dissociating said antigen
in said sample from binding proteins with acid, base, or a chaotropic
agent to give a conditioned sample; drying said conditioned sample
onto the surface of a test container to give a dried sample; and
detecting said antigen in said dried sample using an immunological
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention arose as the result of studies in the laboratories
of the inventors which indicated that the specificity of a diagnostic
test for an antigen present in blood could be improved by using
a process which dissociates antigens from antibodies already present
in the blood sample. This dissociation process of the invention
is usually associated with a concurrent dilution of the sample,
and the entire process of diluting and dissociating is termed "conditioning"
for the purposes of this application. After the sample is conditioned,
it is dried onto the surface of a test container, after which the
antigen can be identified by standard methods of immunological diagnosis.
Surprisingly, the combination of conditioning and drying the sample
prior to immunological analysis greatly enhances the reliability
of the immunological test procedure. This discovery has now been
developed into the complered invention which is set forth in this
application for patent.
The present assay technique is designed for utilization with a
plasma or serum blood sample. Plasma samples are preferred since
the clotting process often removes antigens from the aqueous phase
of blood. Although the principal objective of this invention is
the detection of antigens in human blood, blood from animals, particularly
mammals, can also be tested in the same way.
When the conditioning process of this invention involves dilution,
dilution by at least a factor of 10 and more preferably by a factor
of at least 100 is preferred. Particularly preferred are dilutions
in the range of 100- to 1000-fold. The conditioning process may
optionally comprise the use of a chaotropic agent or a disruptive
condition (whether or not a dilution is used) which results in the
dissociation of immunological complexes in the blood sample. A chaotropic
agent or disruptive condition is required if dilutions of less than
100-fold are used. A chaotropic agent or disruptive condition is
a chemical agent or a condition that disrupts the forces involved
in the bonding of antigen/antibody complexes. They include vander
Waals forces, coulombic forces, ion attractive forces, and hydrogen
bond forces. Chaotropic agents interact with and dissociate immune
complexes by ionic substitution. Typical chaotropic agents include
solutions of KI, KCN, and KSCN and will be described in more detail
at a later time. Typical disruptive conditions are the use of acid
or base which result in pH values outside the normal range of physiological
pHs. In this application, adjustment of pH by means of an appropriate
buffer is referred to as a "condition" rather than as
the use of an "agent" since the condition is described
in terms of the pH value obtained and does not depend on the particular
agent used to obtain the stated pH. Chemical compounds whose structure
and individual properties are important in obtaining the resulting
dissociation of immunological complexes are referred to as "agents",
more specifically as "chaotropic agents", rather than
When the sample is diluted by a factor of 100 or more, chaotropic
agents or disruptive conditions are not essential but may be used
if desired. However, when dilutions are not carried out, it is essential
that a chaotropic agent or disruptive condition suitable for dissociating
immunological complexes be present. A combination of dilution in
the range of 10-to 1000-fold and use of a chaotropic agent or disruptive
condition is especially preferred.
The preferred disruptive condition for use with this aspect of
the invention is low pH. By low pH is meant from 2 to 5, preferably
from 2 to 4, and more preferably from 2 to 3. Suitable buffers for
establishing the necessary pH ranges include citrate, citric acid,
or a mixture of dilute hydrochloric acid and glycine. Buffers are
made up according to standard methods known to those skilled in
the art thereof and can be prepared for any desired value in the
stated ranges. Suitable techniques are well known and are described
in, for example, Handbook of Chemistry and Physics, 48th Ed., Weast
and Selby, eds., The Chemical Rubber Co., Cleveland, Ohio, 1967,
pages D-78 and D-79, which are herein incorporated by reference.
Buffers having a concentration of 20 to 100 mM are preferred. Examples
of chaotropic agents suitable for this conditioning step include
SCN.sup.-, CN.sup.-, I.sup.-, Br.sup.-, and Cl.sup.-, which decrease
in disruptive activity in the order given. Typical concentrations
of such agents are from 0.5 to 2.5M. Particularly preferred agents
and concentrations include 0.5M KI, 2.5M NaSCN, and 2.5M PVP-Iodide
(a commercially available polyvinylpyrrolidine composition containing
10.3% iodine available from Sigma Chemical Co.). When high pH is
used as a disruptive condition, values of 9 to 12 are preferred
with values of 9 to 11 and 9 to 10 being more preferred and most
It will be apparent to those skilled in the immunological art that
the exact chaotropic agent or condition and the paramerers thereof
which work best for any particular antigen will vary with the antigen.
However, the best conditions for a particular antigen can easily
be determined if the following procedure is carried out. Precipitate
the immune complexes in a sample with any suitable agent, such as
polyethylene glycol. Divide the precipitated complexes into aliquots
and treat the individual aliquots with the chaotropic agent or condition
which is being tested, varying the concentration, temperature, etc.,
as is appropriate for determining optimum conditions. Determine
the degree of dissociation of the complex by any suitable technique,
such as electrophoresis on an agarose gel. Then choose the conditions
and agents which result in maximum dissociation of the immune complex
without undue loss of the ability of the antigen to complex with
the antibody used in the analytical step (described in more detail
in a later section).
After the sample has been conditioned, it is dried onto the walls
of the container in which subsequent testing will take place. The
material from which the container is made is not important for many
antigens, although glass and plastic are preferred support materials
in general. Because of the ease with which they may be manipulated,
plastic microtiter plates are particularly preferred containers.
For some antigens and analytical processes, however, the choice
of support material will be important. For example, a particularly
preferred support material for use in the determination of human
epithelial differentiation antigens in blood (as an indication of
breast cancer) is polystyrene, especially when subjected to coronal
discharge treatment prior to use in the assay. Microtiter plates
are commercially available in this form, for example, from Costar,
205 Broadway, Cambridge, Mass. 02139 (who identifies such plates
as Tissue Culture Clusters).
After the sample is added to the container, the aqueous phase is
gently evaporated. Some heat may be applied but it is preferred
that the temperature of the samples not be raised to more than 55.degree.
C., preferably not more than 42.degree. C. One preferred method
of drying the samples which is parricularly useful when microtiter
plates are used as the sample containers is overnight drying in
an oven at 37.degree. C. Other suitable drying techniques include
use of a blow dryer ("hair dryer") on a low setting. Dried
samples can be processed through the remaining steps of the analytical
procedure immediately after drying if desired. However, satisfactory
results will also be obtained if a delay occurs between drying and
the next processing step. Delays of up to 36 hours are generally
possible, but delays of less than 24 hours are preferred. Overnight
drying and the resulting delays at dryness (e.g., 10-15 hours dryness)
are most preferred at 37.degree. C.
If a chaotropic agent or disruptive condition has been used which
will interfer with the immunological binding that rakes place during
the later detection step, it is preferred to wash the dried sample
with a suitable wash solution to remove dried salts, acids, bases,
or other inorganic or low molecular weight organic substances. Typically
this is done using a buffered solution and most typically using
the same buffered solution that is used to dilute the antibody in
the detection step. A typical wasn solution is phosphate buffered
saline (PBS); 10 mM phosphate, 0.9% NaCI, pH 7.4. The dried sample
is washed several times (typically 3-4 times) with a volume of wash
solution appropriately sized for the container and sample size being
used. If desired the discarded wash sample can be tested and washing
can be continued until the chaotropic agent or condition has been
removed completely and can no longer be detected in the discarded
wash solution, although this is generally not necessary.
After the aqueous phase has evaporated and the serum or plasma
sample has been dried onto the walls of the container (and optionally
washed), the antigen present in a sample is detected using standard
immunological techniques. Although such techniques are easily within
the skill of those knowledgeable in the immunological art, a suitable
general technique will be summarized here for convenience. For further
information, if necessary, see Chard, An Introduction to Radioimmunoassay
and Related Techniques, North-Holland Publishing Company, 1978,
which is herein incorporated by reference.
Typically, a buffered solution containing an antibody specific
for the antigen being detected would be added to the container having
the sample dried on its walls. Since immunological binding reactions
typically work best at or near physiological pH, it is preferred
to maintain a pH in the range of from 5 to 9, more preferably from
7 to 8, by use of a suitable buffer. Suitable buffers include PBS
and Tris. Buffers and other materials in the test solution should
be present in amounts such that the ionic strength of the solution
is maintained ar or near normal physiological levels.
The amount of antibody present in the diagnostic solution can be
varied in accordance with the amount of antigen expected to be present
as is well known to tnose skilled in the art. Typically, the amount
used is determined by simple experimentation for each new batch
of antibody using serial dilutions to determine the optimum amount,
as is well known to those skilled in the art.
Numerous antibodies are commercially available for diagnostic testing,
and the production of such antibodies need not be set forth here
in detail. Either polyclonal antibodies or monoclonal antibodies
may be used, depending on the antigen being detected. It is particularly
preferred to use monoclonal antibodies, especially for the antigens
associated with breast cancer, in order to maximize specificity
of the reaction and reduce the number of false positives. Particularly
preferred monoclonal antibodies for use in detecting breast cancer
are those identified as 115D8 (Mam-6a) and 67D11 (Mam-3a) by Antoni
van Leeuwenhoekhuis, Het Nederlands Kanker Instituut, Amsterdam,
Netherlands. These are monoclonal antibodies against human mammary
epithelial differentiation antigens produced by mouse/mouse hybridomas
and are readily available from the indicated source. See, for example,
Rasmussen et al, Breast Cancer Research and Treatment, 2, 401-405
(1982); Hilkens et al, Protides of the Biological Fluids, 29, 813-816
(1982); Hilkens et al, "Monoclonal Antibodies Against Human
Milkfat Globule Membranes Useful in Carcinoma Research," Proceedings
of the Biological Fluids, 31 (1983) and Hageman et al, "Sweat
Glands and Salivary Glands as Model System for the Characterization
of Monoclonal Antibodies Against Differentiation Antigens of the
Human Mammary Gland," Proceedings of the Biological Fluids,
31 (1983), all of which are herein incorporated by reference, for
a discussion of these antibodies.
Antigen is detected by utilizing a signalling reagent attached
to the antibody which, after suitable washing steps, remains in
the test conrainer only if it has become bound to antigen coated
on the test container walls. Many signalling techniques are known
to those skilled in the art and need not be innumerated here in
detail. However, examples of suitable techniques include radioactive
labeling of the antibody, attachment of an enzyme ro the antibody
(ELlSA assays), and fluorescent labeling of the antibody.
As was discussed previously, the present invention can be applied
to the detection of any antigen in blood. However, it is particularly
suited for use in the detection of human mammary gland epithelial
differentiation antigens in plasma, and the preferred conditions
set forth in this application are particularly adapted to that purpose.
By adhering to the critical condirions set forth in this application
when used for this purpose, it is possible to achieve markedly superior
reliability in a test for these antigens, which are associated with
the presence of breast cancer.
The invention now being generally described, the same will be better
understood by reference to certain specific examples which are included
herein for purposes of illustration only and are not intended to
be limiting of the invention or any embodiment thereof, unless specified.
Studies on Plasma Samples of Breast Cancer Patients Using an ELISA
Studies were carried out using an ELISA type assay and two Dutch
monoclonal antibodies. These studies demonstrated the presence of
significant levels of differentiation antigens in the plasma of
breast cancer patients. The plasma of healthy control subjects had
significantly lower antigen levels.
The procedure utilized highly diluted (1:100) human plasma samples
which were dried at a pH of 2.1 on plastic microtiter plates. Monoclonal
antibody (ascitic fluid diluted 1:1000) was then added to the microtiter
plate. The amount of monoclonal reactive antigen was measured by
a standard peroxidase enzyme system. Quantitation was achieved by
constructing a standard dosage curve using a partially purified
milk membrane preparation. Probably 1% of the membrane preparation
was monoclonal-reactive differentiation antigen estimated by an
eye scan of a Coomasse blue stained gel preparation. The position
of the reactive antigen in the gel was identified by a Western blot.
Plasma Samples: Blood samples collected in an EDTA or heparinized
tube were utilized. Cell-free plasma was obtained by a two minute
centrifugation in a Brinkman Model 5414 Table Top Cenrrifuge (15,000.times.G).
Aliquots of plasma were stored at -70.degree. C. Several studies
were also done on blood samples received from the Corning Blood
Collection Pool at Medford, Mass. These blood samples were collected
in heparinized tubes and stored at -70.degree. C.
Monoclonal Antibodies: Two monoclonal antibodies obtained from
the Netherland Cancer lnstitute in Amsterdam were utilized. They
are designated 115D8 and 67D11 and were obtained by standard hybridoma
technology utilizing human antigens from purified human milkfat
globule membranes. Spleen cells from immunized mice were fused to
myeloma cells. Antibody screening assays were carried out with solid
phase radioimmunoassays inVolving iodinated Protein A and standard
ELISA Assay for the Detection of Epithelial Antigens in Human Plasma:
Human plasma samples were diluted 1:100 in 20 mM citrate buffer
at pH 2.1. Five (5) lambda of this diluted plasma was added to 45
lambda of citrate buffer in the well of a Costar microtiter plate.
At this dilution of plasma, approximately 1700 nanograms of plasma
protein were deposited in each microtiter well. The sample was dried
overnight at 37.degree. C. Plates were washed 3 times with PBS,
pH 7.6, and 0.1% gelatin in PBS was added to fill the microtiter
well (approx. 350 lambda). After 18 hours at 4.degree. or 5 hours
at 37.degree. the plates were washed 10 times with PBS, pH 7.6,
and 50 lambda of diluted monoclonal antibody (ascitic fluid diluted
1:1000 in 0.1% BSA, PBS, pH 7.6) was added. The plates were allowed
to stand overnight at 4.degree. C. and then washed 10.times. with
PBS, and 50 lambda or sheep antimouse IgG conjugated with horseradish
peroxidase was added for 60 minutes at room temperature. Cappel
affinity-purified sheep antimouse IgG was used since it did not
cross react with human IgG. The plates were washed 3 times with
PBS, and 100 lambda of peroxidase substrate solution continuing
equal parts of 0.03% Hydrogen peroxide and 0.2 mg/ml of ABTS [2',
2'-azino-di(3-ethylbenzthiazoline-6-sulfonic acid)] were added.
After 60 minutes the reaction was sropped with 50 lambda of 3 N
sulfuric acid. The optical densities were read on a Dynatech Micro
ELISA reader at a wavelength of 514 .ANG.. From the standard curve,
the nanogram/ml antigen level for each patient was calculated.
Standard curve: A standard dosage curve was established using partially
purified antigen obtained from a human milk membrane preparation.
The antigen level in the standard preparation was estimated to be
1% using an eye scan of an SDS gel. SDS-Western blot studies had
shown the position of the reactive antigen in the gel and that it
had a molecular weight greater than 200,000 daltons. The standard
curve was established by adding known amounts of the antigen preparation
to different microtiter wells. To this were added 1, 5, 10, 50,
100, 500, or 1000 nanograms of membrane preparation to different
wells. The modified ELISA assay was performed. The OD value for
each antigen level was recorded and a standard curve was plotted.
Studies were carried out utilizing five groups of human plasma
samples: (1) 28 plasma samples from active breast cancer patients,
(2) 32 normal subjects, (3) 13 plasma samples from patients with
other types of cancer, (4) 10 plasma samples from women with benign
breast disease (fibrocystic disease), and (5) 8 plasma samples from
patients with altered physiology including 6 heparinized patients
and 2 pregnant women.
Breast Cancer Patients: Plasma samples from 28 patients with acrive
breast cancer were studied. These represented different stages of
active disease, very early through wide metastases. By using a standard
curve, the nanogram level of antigen/ml of human plasma was calculated.
For women with breast cancer, the median plasma 115 antigen level
was 500 ng/ml compared to a control median 115 antigen of 70 ng/ml.
The median combined antigen level of breast cancer patients was
1100 ng compared to a median combined level of 90 ng/ml for control
individuals. This difference between cancer patients and healthy
controls, i.e., 1100 vs. 90, is highly significant.
The diagnostic discriminatory value of the ELISA assay is shown
in Table 1. The best discrimination between cancer patients and
healthy controls is achieved by using a combined antigen levels.
It can be seen in Table 1 that using a combined antigen cutoff of
500 ng/ml, 26/28 (93%) of breast cancer patients are positive (antigens
above this level), and no healthy controls (0/32) are positive at
the 500 ng level. Using a single antigen level achieves less diagnostic
discrimination. At the 300 ng antigen level it can be seen that
22/28 breast cancer patients are positive with 1/32 healthy controls
positive, i.e., 92% breast cancer positive vs. 3% controls. Using
a single antigen at higher cutoffs, i.e. 400 and 500 ng, the number
and percent of positive cancer patients falls. With combined antigens
at 500 ng/ml cutoff, 93% of breast cancer patients are positive
and no healtny controls (0/32) are positive.
TABLE 1 ______________________________________ ELISA ASSAY DISCRlMINATION
Patients/Controls: Number Positive at Different Antigen Levels Antigen
Level Breast Cancer Controls cutoff MC115 MC67 combined combined
______________________________________ > 300 ng/ml 23/28* 22/28
24/28 1/32 > 400 ng/ml 17/24 18/24 24/28 1/32 > 500 ng/ml
12/24 12/24 26/28 0/32 ______________________________________ *No.
If the data on the ELlSA assays is reviewed according to stage
of disease, it can be seen that the combined antigen test is also
highly discriminatory in patients with early breast cancer as well
as in patients with more advanced disease. All (14/14 or 100%) early
breast cancer patients were positive; 5/5 (100%) women with local
recurrence and 8/9 (89%) of women with metastatic disease were also
positive. No healthy controls, 0/32, were positive at the 500 ng
level. The one negative patient in the metastatic category had been
recently treated with X-ray to a localized bone deposit and also
with heavy chemotherapy. She was listed clinically as "partially
regressing". The results are shown in Table 2.
TABLE 2 ______________________________________ ELISA ASSAY RESULTS
Different Stages of Breast Cancer Combined Antigen level above 500
ng/ml No. Positive % Positive ______________________________________
Early breast cancer 14/14 100 Local recurrence 5/5 100 Metastatic
8/9* 89 Controls 0/32 0 ______________________________________ *The
one negative patient in this metastatic category had been treated
with xray and heavy chemotherapy and was listed as "partially
Other types of cancers: Plasma samples from 13 patients with other
types of cancer were studied. Table 3 shows the median plasma antigen
levels of 1000 ng/ml (combined value) for colon patients; 400 ng/ml
for ovarian, esophageal and lung cancer patients; and 500 ng/ml
for leukemia patients. Melanoma patients were negative on the test
(combined median levels less than 100). Of the total of 13 other
cancer patients, 9 or 69% were positive on the combined antigen
TABLE 3 ______________________________________ OTHER CANCER PATIENT
STUDIES Median Plasma Combined Antigen Levels Cancer Type # Studied
# Positive* Median Antigen Levels+ ______________________________________
Colon 4 4 1000 Melanoma 2 0 100 Ovary 2 1 400 Lung 2 1 400 Leukemia
2 2 500 Esophagus 1 1 430 Control 33 0* 100 ______________________________________
*For cancer patients, based on a combined cutoff of 500 ng/ml. For
control combined cutoff of 500, no controls had combined levels
above 500. +Median antigen levels were based on combined 115D8 and
67D11 antigen levels.
Healthy control subjects: Plasma samples from 31 healthy control
subjects were studied. The median plasma antigen levels for controls
with the monoclonal 115D8 antigen was 70 ng/ml, the median 67D11
antigen level was 35 ng/ml, and the median combined antigen level
was 90 ng. Table 1 (above) shows the number of control subjects
positive at different antigen level cutoff values. It can be seen
that in the combined antigen test at a cutoff value of 500 ng/ml,
no control subjects (0/32) were positive.
Benign breast disease: Of the 10 women with benign breast disease
studied, 4 had combined antigen levels above 500 ng. Thus, 4/10
or 40% were positive on the combined antigen test. The discrimination
between benign and malignant breast disease by the monoclonal ELISA
assay presents some problems. One helpful note is that most women
with benign disease had elevated levels of 67D11 antigen, i.e.,
300-350 ng, but had low 115D8 antigen levels, i.e., less than 100
Altered physiology: Two non-cancer categories of patients gave
positive ELISA monoclonal assays: (1) Hospitalized patients receiving
heparin and (2) pregnant women.
Heparin: Plasma samples from 6 hospitalized patients receiving
heparin were studied. Table 4 shows the results of these studies.
It can be seen that 3/6 (50%) of heparinized patients showed combined
antigen levels above 500 ng/ml. Clinically this false positive category
of patients would present few problems for the clinician in diagnostic
TABLE 4 ______________________________________ ELISA RESULTS Other
Conditions/Combined Antigen Levels > 500 ngm. No. Positive/No.
Studied % pos. ______________________________________ Benign breast
disease 4/10 40 Heparinized patients 3/6 50 Pregnant women 2/2 100
Pregnancy: Plasma samples from 2 pregnant women were studied on
the ELISA assay, and both had combined antigen levels above 500
ng/ml. These results, are also shown in Table 4. Presumably these
women are positive because of very high metabolic activity in the
mammary gland tissue. Clinically false positive tests of pregnant
women should not present diagnostic problems. very few breast cancers
occur in women in the child bearing age, i.e., <30 years of age.
The invention now being fully described, it will apparent to one
of ordinary skill in the art that many changes and modifications
can be made thereto without departing from the spirit or scope of
the invention as set forth herein.