U.S. patent number 3,697,638 [Application Number 05/133,404] was granted by the patent office on 1972-10-10 for antigens.
This patent grant is currently assigned to Hoffmann-La Roche Inc.. Invention is credited to Hans John Hansen.
United States Patent |
3,697,638 |
Hansen |
October 10, 1972 |
**Please see images for:
( Certificate of Correction ) ** |
ANTIGENS
Abstract
Antigens associated with carcinomas and adenocarcinomas as well
as methods for isolating, identifying and detecting them are
disclosed.
Inventors: |
Hansen; Hans John (Allendale,
NJ) |
Assignee: |
Hoffmann-La Roche Inc. (Nutley,
NJ)
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Family
ID: |
27366145 |
Appl.
No.: |
05/133,404 |
Filed: |
April 12, 1971 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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110288 |
Jan 27, 1971 |
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42526 |
Jun 1, 1970 |
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Current U.S.
Class: |
435/7.23;
436/542; 436/547; 436/813; 530/380; 530/416; 530/389.7; 436/539;
436/545; 436/804; 436/825; 530/403; 530/806; 530/828 |
Current CPC
Class: |
A61K
51/1048 (20130101); C07K 16/3007 (20130101); B01J
47/014 (20170101); G01N 33/57473 (20130101); B01J
39/26 (20130101); B01J 47/04 (20130101); Y10S
436/813 (20130101); Y10S 436/825 (20130101); Y10S
530/806 (20130101); Y10S 530/828 (20130101); A61K
2123/00 (20130101); A61K 39/00 (20130101); Y10S
436/804 (20130101) |
Current International
Class: |
A61K
51/02 (20060101); A61K 51/10 (20060101); B01J
39/26 (20060101); B01J 47/04 (20060101); B01J
47/00 (20060101); C07K 16/18 (20060101); C07K
16/30 (20060101); G01N 33/574 (20060101); A61K
39/00 (20060101); A61k 027/04 () |
Field of
Search: |
;424/1,8,12,88
;252/31.1R,408 ;23/23B,253TP |
Other References
thomson et al., Proc. Natl. Acad. Sci. U.S., 64:161-167 (1969) "The
Radioimmunoassay of Circulating CEA of the Human Digestive
System.".
|
Primary Examiner: Sebastian; Leland A.
Assistant Examiner: Hellman; S. R.
Parent Case Text
RELATED APPLICATIONS
This is a continuation-in-part of U.S. Pat. Application Ser. No.
110,288, filed Jan. 27, 1971 which in turn is a
continuation-in-part of U.S. Pat. Application Ser. No. 42,526,
filed June 1, 1970.
Claims
What is claimed is:
1. A method for detecting the presence of carcinoembryonic antigen
material, component A and/or component B in blood which
comprises
a. adding a measured amount of antibody to a solution of a
perchloric acid extract of blood serum or blood plasma in a
buffered solvent at pH 6.25 of a maximum normality of 0.01:
b. incubating the mixture;
c. adding a measured amount of radioactive tagged carcinoembryonic
antigen material, component A or component B to the incubated
mixture;
d. incubating the resulting mixture;
e. adding a precipitating agent to the incubated mixture thereby
precipitating all the CEA-anti CEA complexes; and
f. measuring the radioactive content of the precipitate.
2. The method of claim 1 wherein in step (a) the buffer is a borate
buffer.
3. The method of claim 1 wherein in step (a) the buffer is ammonium
acetate.
4. The method of claim 1 wherein in step (a) a perchloric acid
extract of blood plasma is used.
5. The method of claim 1 wherein in step (c) the amount of CEA
material, component A or component B added is about 50
nanograms.
6. The method of claim 1 wherein the in step (e) the precipitating
agent is zirconyl phosphate gel.
7. The method of claim 1 wherein in step (c) radioactive tagged CEA
component A is added.
8. The method of claim 1 wherein in step (c) radioactive tagged CEA
component B is added.
9. The method of claim 1 wherein in step (c) radioactive tagged CEA
material is added.
10. A method for detecting the presence of carcinoembryonic antigen
material, CEA component A and/or CEA component B in blood which
comprises
a. adding a measured amount of tagged CEA material, component A or
component B to a perchloric acid extract of blood serum or blood
plasma prior to dialysis;
b. dialyzing the mixture against a polyethylene glycol with an
average molecular weight of about 15,000 to 20,000 and a softening
point of about 60.degree. C.;
c. adjusting the pH of the retentate to about pH 6-7;
d. adding a measured amount of antibody to the adjusted
mixture;
e. dialyzing against polyethylene glycol of molecular weight of
about 15,000 to 20,000 and having a softening point at 60.degree.
C.;
f. dissolving the resulting retentate in borate buffer of pH
6.25;
g. adding a precipitating agent to the solution thereby
precipitating all the CEA-anti CEA complexes; and
h. measuring the radioactive content of the precipitate.
11. The method of claim 10 wherein in step (c) the pH of the
mixture is adjusted by a borate buffer of pH 6.25.
12. The method of claim 10 wherein in step (f) the precipitating
agent is zirconyl phosphate gel.
13. The method of claim 10 wherein in step (a) I.sup.125 tagged CEA
material is used.
14. The method of claim 10 wherein in step (a)I.sup.125 tagged CEA
component A is used.
15. The method of claim 10 wherein in step (a) I.sup.125 tagged CEA
component B is used.
16. The method of claim 10 wherein in step (a) blood plasma is
used.
17. A method for detecting the presence of carcinoembryonic antigen
material, component A and/or component B in blood which
comprises
a. diluting blood serum or blood plasma with at least 100 volumes
of water;
b. adding a measured amount of CEA antiserum to the dilution;
c. incubating the mixture;
d. adding a measured amount of radioactive tagged carcinoembryonic
antigen material, component A or component B to the incubated
mixture;
e. incubating the mixture;
f. adding a precipitating agent to the incubating mixture thereby
precipitating all the CEA-anti CEA complexes; and
g. measuring the radioactive content of the precipitate.
18. The method of claim 17 wherein in step (f) the precipitating
agent is zirconyl phosphate gel.
19. Radioiodinated carcinoemryonic antigen component A
characterized by having a molecular weight of between 120,000 and
240,000, being elutable from a mixed bed ion exchange column
comprising an equal mixture by weight of either preswollen or dry
microgranular carboxymethyl cellulose having rod shaped particles
with a particle size distribution with a range of about 20 .mu. to
about 60 .mu., a capacity of 1.0 .+-. 0.1 meq./gm. and a water
regain of 2.3-2.7 gm./gm. dry exchanger, and microgranular
diethylaminoethyl cellulose, in free base form, having rod shaped
particles, a particle size distribution with a range of about 20
.mu. to about 60 .mu., a capacity of 1.0 .+-. 0.1 meq./gm. and a
water regain of 2.3-2.8 gm./gm. dry exchanger with ammonium acetate
at pH 4 in 0.05 M sodium chloride, forming a single line
precipitate with its specific antibody in unabsorbed antiserum in
gel diffusion tests, being soluble in perchloric acid, having a
spectrophotometer absorption peak wave length of 280 m.mu. and
having a specific activity of 5-10 m.mu. Ci/ng. of carcinoembryonic
antigen component A.
20. Radioiodinated carcinoembryonic antigen component B
characterized by having a molecular weight of between 120,000 and
240,000 being elutable from a mixed bed ion exchange column
comprising an equal mixture by weight of either preswollen or dry
microgranular carboxymethyl cellulose having rod shaped particles
with a particle size distribution with a range of about 20 .mu. to
about 60 .mu., a capacity of 1.0 .+-. 0.1 meq./gm. and a water
regain of 2.3-2.7 gm./gm. dry exchanger, and microgranular
diethylaminoethyl cellulose, in free base form, having rod shaped
particles, a particle size distribution with a range of about 20
.mu. to about 60 .mu., a capacity of 1.0 .+-. 0.1 meq./gm. and a
water regain of 2.3-2.8 gm./gm. dry exchanger with ammonium acetate
at pH 4 in 0.1 M NaCl, forming a single line precipitate with its
specific antibody in unabsorbed antiserum in gel diffusion tests,
being soluble in perchloric acid, having a spectrophotometer
absorption peak wave length of 280 m.mu. and having a specific
activity of 5-10 m.mu. Ci/ng. of carcinoembryonic antigen component
B.
Description
BACKGROUND OF THE INVENTION
The neoplastic process in human beings has been and still is the
subject of intensive study. In order to obtain a better
understanding of the disease, human cancer tissue has been studied
in an effort to discover the cause, treatment, prevention and/or
diagnosis of cancer. Early diagnosis of cancer is very important
since it increases the chances of effecting a complete remission of
the disease.
In an effort to utilize known diagnostic tools to detect the
presence of cancer tumors, attempts have been made to demonstrate
tumor specific antigens to human carcinomas. These attempts have
previously been unsuccessful with many types of carcinomas since it
has not been possible to segregate normal tissue antigens from
abnormal cancer antigens and demonstrate the specificity of the
cancer antigens.
In the efforts to isolate abnormal cancer antigens and demonstrate
their specificity, attempts have been made to cause the formation
of tumor specific antibodies and demonstrate their presence in sera
obtained from animals immunized with preparations of human cancer.
If consistently reproducible, the demonstration of the presence of
tumor-specific antibodies in animal antisera would lead to the use
of a valuable diagnostic tool.
In order to fully utilize the existence of tumor-specific
antibodies in animal antisera as a diagnostic tool, a test must be
developed which will demonstrate the presence of the tumor antigen
in the blood of the patient. Procedures which have been devised
have not proven efficient or sensitive in the detection of and
differentiation between carcinomas originating at different
locations within the body, or metastatic conditions.
Among the possible sources of antigens associated with human
carcinomas which have been most extensively studied by
investigators are adenocarcinoma of the colon and digestive tract,
meconium, carcinoma of the liver, ovarian cysts and carcinoma of
the breast. Since adenocarcinoma of the colon is one of the most
widespread cancers and usually requires a surgical procedure for
definitive diagnosis, after some gross symptomatology has
developed, it has been among the most extensively studied.
Efforts to extract a relatively pure antigen associated with
carcinomas or adenocarcinomas have met with either no success or
are impractical from a commercial point of view since a process has
not been found to make it possible to completely segregate such an
antigen from normal tissue antigens and non-antigenic
materials.
The presence of antigens which are stated to be specific to
adenocarcinomas of the colon and digestive system by means of
immunological tolerance and absorption techniques have been
demonstrated, Gold et al., J. Expt. Med. 121 439-462 (1965).
However, the practical and commercially feasible isolation of the
antigen itself as well as its association with carcinomas and
adenocarcinomas had, until the present invention, not been
achieved.
The tumor-specific antigens have been previously shown to be
present only in patients who have adenocarcinoma which originate in
digestive system epithelium derived from embryonic entodermal
tissue, i.e., esophagus, stomach, duodenom, pancreas and
rectum.
It has also previously been demonstrated that the tumor-specific
antigen is also present in the digestive organs of fetuses between
2 and 6 months of gestation, Gold et al., J. Exptl. Med. 122
467-487 (1965). Thus, for convenience, the antigen has been
designed as carcinoembryonic antigen (CEA).
Not only has it heretofore not been possible to isolate and
characterize the CEA by practical rapid methods, but it has not
been possible to demonstrate its presence in the blood of persons
having adenocarcinoma with a diagnostic test suitable for large
screening programs.
DETAILED DESCRIPTION OF THE INVENTION
I have discovered that the material heretofore known as
carcinoembryonic antigen (CEA) is a mixture of several components,
at least two of which have antigenic activity which is associated
with human carcinoma generally. These two active components are
called carcinoembryonic antigen component A and carcinoembryonic
antigen component B.
This invention in one significant aspect relates to a method of
fractionating material having carcinoembryonic antigen activity
into its component parts, e.g., carcinoembryonic antigen component
A and carcinoembryonic antigen component B.
This invention in further aspects relates to methods of isolating
and characterizing the carcinoembryonic antigen components
associated with carcinomas, for diagnostic test procedures and for
utilizing either radioactive tagged carcinoembryonic antigen
material, component A or component B to detect circulating
carcinoembryonic antigen material, component A and/or component
B.
This invention also relates to a diagnostic test method useful in
the detection of carcinoma and suitable for post-operative
monitoring of carcinoma patients. "Carcinoma," as used herein,
includes all carcinomas and adenocarcinomas present in humans. As
used herein, "carcinoembryonic antigen material," means the
material with carcinoembryonic antigen activity which contains
component A and/or component B.
In order to produce radioactive tagged carcinoembryonic antigen
material, component A or component B individually and utilize each
separately in the improved diagnostic tests of this invention, it
is first necessary to isolate and purify each entity and confirm
its identity by means of specific anti-bodies.
According to this invention, practical processes have been
discovered for:
a. Isolating, purifying, characterizing and confirming the identity
and specificity of carcinoembryonic antigen material, component A
and component B;
b. Utilizing radioactive tagged carcinoembryonic antigen material,
component A and/or component B to detect the presence of carcinoma
by the detection of circulating antigens; and
c. Differentiating between circulating free and total
carcinoembryonic antigen material, component A and/or component
B.
While this invention is concerned with antigens associated with
carcinomas generally the isolation and purification procedures
described herein will refer to colon carcinoma tissue and meconium
which are representative of the materials containing CEA material,
component A and/or component B.
Material having carcinoembryonic antigen activity is isolated and
purified according to the process of this invention by homogenizing
adenocarcinoma tissue from primary or metastatic tumors, preferably
those originating within the digestive system, with tumors from the
colon, for example, being suitable, or by homogenizing
meconium.
In order to isolate the carcinoembryonic antigen material,
component A and/or component B associated with the homogenized
material, it is necessary to first separate all other material from
the homogenate, isolate the carcinoembryonic antigen material, then
isolate the individual components of the carcinoembryonic antigen
material thereof. This is accomplished by chemical and physical
extraction and purification procedures. If, for example, only
component A or component B are present in the homogenate, then the
isolation and purification procedures will produce the component
without fractionation.
Once the extraction and purification procedures are completed, the
identity of the finally isolated fractions as carcinoembryonic
antigen material or a component must be confirmed. This can be
accomplished by various known techniques, e.g., double diffusion in
agar gel, immuno-electrophoresis, hemagglutination, passive
cutaneous anaphylaxis and the like.
In order to utilize these techniques, the antibodies used must be
confirmed to be specific for the CEA material, component A and/or
component B. Antibodies which meet this criteria can be produced by
immunological tolerance or absorption techniques.
In the absorption technique, tumor antiserum is absorbed with
normal tissue and normal fluids (saliva, serum, plasma) in order to
remove anti-normal components of the antiserum. Any residual
antibody activity in the absorbed antiserum which is directed
against tumor material is then considered to be tumor specific.
This method is not without its faults since there is the
possibility that tumor specific antibodies may have been removed or
inactivated by normal tissue components similar to, but not
identical with, the tumor antigens which initially stimulated the
antibody production.
In the immunological tolerance technique, animals are rendered
immunologically tolerant to normal tissues during neonatal life.
The tolerant animals are then immunized with tumor preparations of
the same donor species. Where adequate suppression of the immune
response to normal tissue components has been achieved, the
development of antibodies apparently specific for the
carcinoembryonic antigen activity has been achieved.
Colon adenocarcinoma tumor tissue and normal colon tissue from the
same individual can be utilized to illustrate this technique
because adenocarcinoma of the colon almost never extends
submucosally more than 6 to 7 cm. on either side of a tumor visible
in the gross.
The colon adenocarcinoma tumor tissue and normal colon tissue from
the same individual are treated separately but in parallel fashion.
The tissue is ground up, suspended in a buffer, then homogenized.
The homogenate is then treated to remove solid particles.
Centrifugation or filtration through successively smaller filter
openings are preferred. The purpose is to remove all particles
about 0.22 .mu. or larger, thus removing all the bacteria present.
The supernatant or filtrate is thereby sterilized to insure against
bacterial contamination.
Test animals divided into appropriate groups are then immunized
with the extracts and, after a suitable time interval, serum is
obtained from the animals. The presence of antibodies in the test
sera is demonstrated by either the Ouchterlony technique of double
diffusion in agar gel, immunoelectrophoresis, hemagglutination
reactions or passive cutaneous anaphylaxis. The preferred practical
method, because of its simplicity and reproducible results is the
Ouchterlony technique.
Once the antibodies are demonstrated to be present, it is possible
to determine if a particular extraction technique does, in fact,
isolate a fraction which contains carcinoembryonic antigen
material, component A or component B.
I have discovered an extraction and purification technique which
finally results in two separate fractions each of which invariably
produces one precipitant line in the Ouchterlony technique when
tested against non-absorbed antisera. The technique, according to
this invention, also provides a means wherein CEA components A and
B are separated from each other and from materials of the same
molecular weight and thus are isolated in substantially pure
form.
CEA materials as well as components A and B are isolated and
purified, according to the preferred process of this invention,
from primary or metastatic carcinoma tissue. Also, CEA material as
well as components A and B can be isolated and purified, according
to the process of this invention, from embryonic digestive organs
of fetuses in the second to seventh month of gestation and from
meconiums. The following description will in most respects be
directed to extraction from cancer tissue; however, the process may
also apply to embryonic tissue or meconium.
CEA material, CEA component A or CEA component B in either
embryonic digestive organ tissue from the first and second
trimester, meconium or adenocarcinoma tissue are extracted with a
glycoprotein solvent in which CEA material, component A and
component B are soluble. This is required so that precipitable
normal proteins and interfering antigenic materials can be
separated from the CEA material or components A and B. Glycoprotein
solvents which are suitable are, e.g., perchloric acid,
trichloroacetic acid, phosphotungstic acid and the like. However,
perchloric acid, because of its availability and ease of use is
preferred.
Prior to the addition of the glycoprotein solvent, the material
which is being treated is homogenized with water in order to
solubilize the CEA material or component A or component B,
whichever is present. The amount of water should be sufficient to
solubilize all of the carcinoembryonic antigen material or
component A or component B. Generally, about two liters of water
per about every kilogram of treated material is sufficient. More
water can be used, however, it is usually not necessary. It is
preferred to use distilled water since the chances of contamination
are thereby reduced. The homogenization can be carried out at from
about 4.degree. C. to about 60.degree. C., however, from about
4.degree. C. to about room temperature (about 20.degree. C. to
about 25.degree. C.) is preferred.
The solid particles are then removed from the homogenate. Since the
CEA material, component A and component B are water soluble, this
can be accomplished by any convenient method of separation, e.g.,
filtration or centrifugation and the like. Centrifugation is
preferred because it is faster and sufficient force can be
developed to remove substantially all the solid particles.
Generally, about 3,000 to about 8,000 revolutions per minute are
sufficient to accomplish this. The separation is preferably carried
out at cold temperatures, e.g., about 4.degree. C. to about
10.degree. C., to prevent loss of activity and at these temperatues
the time for sedimentation may be decreased.
The supernatant from the centrifugation is then treated with a
glycoprotein solvent to remove protein materials and interfering
antigenic materials.
Any temperature below room temperature is suitable for the addition
of the glycoprotein solvent to the supernatant of the homogenate.
Preferably, however, from about 4.degree. C. to about room
temperature is used. The temperature of the glycoprotein solvent
which is added to the supernatant can also be variable, preferably,
however, the same temperature as the extracting temperature is
utilized. Generally, a concentrated acid is used as the
glycoprotein solvent, e.g., about 0.5N to about 2N with 2N being
preferred. The solvent is added in about equal volume to the
supernatant. The time in which the reaction takes place is usually
about 5 to about 30 minutes. Longer times are undesirable since
they can result in loss of antigencity.
A precipitate results. This precipitate is separated from the
supernatant containing the dissolved CEA material, component A or
component B. Any convenient method of separation is suitable, e.g.,
filtration, centrifugation and the like, however, centrifugation
under the same conditions as used with the homogenate is
preferred.
Perchloric acid, salts such as sodium chloride and other low
molecular weight materials are then removed in order to further
purify the system. While it may be possible to accomplish this by
precipitating the remaining proteins, I have discovered a fast,
efficient method comprising dialysis through a semipermeable
membrane against a polyethylene glycol with an average molecular
weight of about 15,000 to 20,000 and a softening point at
60.degree. C. A typical suitable commercial product useful for this
dialysis is "20 M Carbowax" marketed by Mann Research Laboratories.
The dialysis is a critical part of the process since it is fast and
efficient and eliminates substantially all diffusible soluble
materials except the higher molecular weight materials which
include the materials containing CEA activity. The dialysis is
carried out at 4.degree. C. to 10.degree. C., preferably 4.degree.
C. and is completed in about 18 hours. The process to this point
takes about 24 hours to complete.
The use of the 15,000 to 20,000 molecular weight polyethylene
glycol in the dialysis step is critical to this invention since it
aids in speeding up the isolation of the CEA material, component A
or component B, by the use of only one dialysis step rather than
time consuming multiple dialysis steps against water and eliminates
the need for lyophylizing the retentate.
The resulting retentate is substantially solid in character and
contains several materials having both higher and lower molecular
weights than the CEA material, component A or component B.
The separation of the portion of the resulting retentate which
contains the CEA material, or component A or component B to the
substantial exclusion of other materials is accomplished according
to this invention by sequential chromatography with two different
gel columns followed by chromatography with an appropriate ion
exchange column. The eluted fractions from the column
chromatography which have a molecular weight of about
200,000-500,000 and a definite peak at the spectrophotometric
absorption wave length of 280 m.mu. are those containing the CEA
material, component A or component B.
The column chromatography can be accomplished by subjecting the
retentate, in solution, to sequential chromatography on two
different gel columns in any order. Practically, however, when
using carcinoma tissue, a gel column which is used first in
accordance with this invention is an agarose gel. Agarose is the
neutral portion of agar. The gel material is commercially available
from AB Pharmacia, Uppsala, Sweden, under the trade name
"Sepharose." The gels are available as aqueous suspensions in 0.02
percent sodium azide as a preservative. The gel structure is due to
hydrogen bonding. The gel is prepared in beaded form having a
selected particle size and percent agarose. The concentration of
the agarose in the gel determines its fractionation range.
The gels most suitable for use in this invention are those which
have a particle size of from 40 to 210 microns and contain 6
percent by weight agarose. These materials named "Sepharose 6B"
have a fractionation range which separates materials of molecular
weight 4 .times. 10.sup.6 or less. In the process of this
invention, Sepharose 6B is used since, when carcinoma tissue is
used, it permits the separation of the fraction containing the CEA
material or its components from extraneous materials of
substantially higher or lower molecular weight as well as from
colloidal particles.
The second column contains a gel filter material which is a
hydrophilic water-insoluble cross-linked dextran polymer gel. This
material and the method of its manufacture are described in British
Pat. No. 854,715. The gel material, which is commercially available
from AB Pharmacia, Uppsala, Sweden, under the name "Sephadex,"
comprises a three dimensional macroscopic network of dextran
substances bonded or cross-linked together, being capable of
absorbing water with swelling. The ability of the gel material to
take up water is inversely proportional to the degree of
cross-linkage of dextran substances in the gel material. The gel
material is available in a variety of grades differing with respect
to degree of porosity. The gel preferred for use in this invention
has an approximate molecular weight exclusion limit of 100,000, a
water regain (g. H.sub.2 O/g. dry gel) of 10 .+-. 1.0, a particle
size of 40-120 microns and a bed volume/ml./g. dry gel of 15-20.
The gel is named "Sephadex G-100."
Sephadex G-100 is employed to further purify the fraction
containing the CEA material or component A or component B. Since
the column has greater resolving power than the first column for
the molecular weight range of 100,000 to 200,000, further
separation of the CEA material or component A or component B from
lower molecular weight materials is achieved. The second column,
for all practical purposes, should be used only after the colloidal
particles are removed by the first column since these particles
will clog the column and make it ineffective. The problem of
colloidal particles is applicable to the treatment of tumor tissue.
However, when, for example, meconium is used, it is preferred to
use the Sephadex G-100 column first since it removes bile salts.
After the removal of the bile salts, then the Sepharose 6B column
is advantageously used.
The chromatography is accomplished by dissolving the retentate in
aqueous buffer at a pH of from about 5 to 9, preferably pH 7. A
typical suitable buffer composition useful in the process of this
invention is composed of 0.1 M Tris-OH adjusted to pH 7 with HCl
made in 0.135 M NaCl with 0.02 percent of sodium azide as a
preservative. The thus formed buffer solution is then run through
the first column, eluted with the same buffer solvent and the
eluates collected. The eluates are then dialyzed against the
polyethylene glycol as described above. The collected active
fraction is then redissolved in an aqueous Tris-OH buffer of pH 5
to 9 of the same composition as described above, the solution is
run through the second column, eluted with a buffer of pH 5 to 9 of
the same composition as described above and the active fractions
are collected and dialyzed as before.
The advantage of utilizing low temperatures, i.e., from about
4.degree. C. to about 10.degree. C. is that it maintains stability
and can result in increased resolution. The fractions collected
from the second column are those which have a molecular weight of
200,000-500,000 and have a reading with a peak at 280 m.mu. on a UV
spectrophotometer. Those fractions collected from the first column
are selected based on the same criteria, however, they contain
material slightly greater and slightly less (as low as 70,000 MW)
than 200,000-500,000 MW. The collected fractions contain the CEA
material or component A or component B depending on the origin of
the treated material. This is shown by either the precipitin
inhibition or direct Ouchterlony testing against unabsorbed tumor
antiserum. A single line precipitate indicates pure CEA
activity.
The active fraction from the second gel column is then subjected to
chromatography on an ion exchange column in order to further purify
and fractionate the CEA active fraction and separate it from other
materials which are present. I have found that in most cases the
fraction containing the CEA activity which is derived from colon
adenocarcinoma tissue from the second gel column contains three
different materials (unless, as in some cases of colon
adenocarcinoma, only component A or component B are present
singly), all having molecular weights between about 200,000 and
500,000. Of these materials, one comprising about 5 percent by
weight of the fraction is non-reactive. A second material,
comprising about 10 percent by weight of the fraction has antigenic
sites which react with the CEA specific antibody and is identified
here as CEA component B. A third material, comprising about 85
percent by weight of the fraction also has antigenic sites which
react with the CEA specific antibody and is identified here as CEA
component A.
It has also been found that other materials, e.g., meconium, lung
tumors, breast tumors, have different proportions of the components
and as a general rule these amounts vary from patient to patient
and from tumor to tumor. Meconium, for example, has only component
B.
In order to obtain the pure CEA components it has been found
necessary to utilize an ion exchange column. If only one component
is present, then the ion exchange column is used to purify it and
confirm its presence as the sole CEA active material present.
The ion exchange column found suitable for use in accomplishing the
desired separation is a mixed bed column composed of a cation
exchanger, carboxymethyl cellulose, and an anion exchanger,
diethylaminoethyl cellulose.
The carboxymethyl celluloses most suitable for use in this
invention are those which are microgranular in form, have rod
shaped particles with a particle size distribution expressed as
diameter of equivalent spheres within a range of about 20 .mu. to
about 60 .mu., have a capacity of 1.0 .+-. 0.1 meq./gm. and a water
regain of 2.3-2.7 gm./gm. dry exchanger. The preferred ionic form
is the Na.sup.+ form. A suitable ion exchanger is commercially
available in a preswollen form from H. Reeve Angel Inc., Clifton,
N.J., under the trade name "CM 52."
Another suitable carboxymethylcellulose is "CM 32." This does not
have the capacity per volume of CM 52 but is otherwise similar,
i.e., it is microgranular in form, has rod shaped particles with a
particle size distribution expressed as diameter of equivalent
spheres within a range of 20 .mu. to 60 .mu., has a capacity of 1.0
.+-. 0.1 meq./gm. and a water regain of 2.3-2.7 gm./gm. dry
exchanger. "CM 32" is available in dry from from H. Reeve Angel
Inc., Clifton, N.J.
The diethylaminoethylcelluloses most suitable for use in this
invention are those which are microgranular in form, have rod
shaped particles with a particle size distribution expressed as
diameter of equivalent spheres within a range of about 20 .mu. to
about 60 .mu., have a capacity of 1.0 .+-. 0.1 meq./gm., a water
regain of 2.3-2.8 gm./gm. dry exchanger and are in the free base
form. A suitable ion exchanger is commercially available from H.
Reeve Angel Inc., Clifton, N.J. under the trade name "DE 52."
The mixed column is produced by removing the fines from each
exchanger by, for example, aspiration of the supernatant resulting
from adding a 10-fold volume of water, stirring and allowing to
settle. Subsequently, a solution made from ammonium acetate in 1.0
M sodium chloride is added to each column and equal volumes of each
of the resulting slurries are then combined and poured in a 2.5
.times. 40 cm. column to give a 2.5 .times. 18 cm. mixed
column.
The eluate from the gel columns is dialyzed against the
polyethylene glycol as described above. The resulting material is
then dissolved in an aqueous buffered solvent which solubilizes
proteins and does not have affinity for the column.
A typical suitable solvent is ammonium acetate at pH 4. The
buffered ammonium acetate solvent can be formed by adjusting the pH
of 0.1 M acetic acid with ammonium hydroxide.
The resulting solution is then clarified. The preferred
clarification method is centrifugation which effectively removes
all the undissolved particles. High speed centrifugation is most
effective for this clarification, preferably at speeds which
produce at least 100,000 times gravity.
The resulting supernatant is then applied to the mixed bed ion
exchange column and eluted with an ammonium acetate-sodium chloride
eluant at pH 4. Other alkali metal chloride salts such as potassium
chloride are also suitable. The relative amounts of ingredients in
the eluant compositions are varied. The variations in the
composition results in a fine separation of the active fraction
into its major components and numerous minor components. This is
accomplished by utilizing compositions containing the ammonium
acetate solvent in 0.05, 0.1, 0.25 and 1.0 M sodium chloride
solutions. The specific relationship of the ammonium acetate to the
sodium chloride is interrelated to the pH of the system, thus, if a
different pH is utilized, then the relationship must be changed to
accomplish the same purpose. The identity and relative amounts of
the major components varies with the identity of their source. For
example, in a typical case wherein colon carcinoma is the source of
antigen activity, about 85 percent by weight of the material
present in the active fraction is eluted when the eluant contains
ammonium acetate in 0.05 M sodium chloride. This is CEA component
A. About 10 percent by weight of the material present in the active
fraction is eluted when the eluant contains ammonium acetate in 0.1
M sodium chloride. This is CEA component B. The remaining material
is eluted when the eluant contains ammonium acetate in 0.25 M
sodium chloride. In cases wherein only component A is present or
only component B is present, then the component present will be
eluted with the eluants as described for each component.
In addition to having the same electrophorectic characteristics as
CEA material, i.e., migrating anodally 10-14 cm. in block
electrophoresis at the same time as ferritin marker migrates 18 cm.
anodally, using 400 volts and about 20mA with a borate buffer of pH
8.6 and ionic strength 0.05, CEA component A has a molecular weight
of between 120,000 and 240,000, is eluted from a mixed bed ion
exchange column having the composition as described with an
ammonium acetate-sodium chloride eluant at pH 4 wherein the eluant
contains ammonium acetate in 0.05 M sodium chloride. Component A
also forms a single line precipitate with its specific antibody in
unabsorbed antiserum in gel diffusion tests, is soluble in
perchloric acid and has a spectrophotometer absorption peak wave
length of 280 m.mu..
Also, CEA component B in addition to having the described
electrophoretic characteristics of CEA material has a molecular
weight of between 120,000 and 240,000, is eluted from a mixed bed
ion exchange column having the composition as described with an
ammonium acetate-sodium chloride eluant at pH 4 wherein the eluant
contains ammonium acetate in 0.1 M sodium chloride. Component B
also forms a single line precipitate with its specific antibody in
unabsorbed antiserum in gel diffusion tests, is soluble in
perchloric acid and has a spectrophotometer absorption peak wave
length of 280 m.mu..
The material or components containing CEA activity are determined
by either the precipitin inhibition or direct Ouchterlony test
against unabsorbed tumor antiserum. A single line precipitate
indicates pure CEA activity. Thus, any material which forms a
single line precipitate with unabsorbed CEA antiserum by either the
precipitin inhibition or direct Ouchterlony technique of double
diffusion in agar gel is included within the scope of this
invention and is suitable for use in the diagnostic tests described
herein.
In order to utilize these techniques, the antibodies used must be
confirmed to be specific for CEA material, component A and/or
component B. Antibodies which meet this criteria can be produced by
immunological tolerance or absorption techniques as described
above.
Once the antibodies are demonstrated to be present, it is possible
to determine if a particular extraction technique does, in fact,
isolate carcinoembryonic antigen material, component A or component
B. Using these techniques, I have found that when the CEA material
is present, component A and component B respectively, obtained from
the mixed bed ion exchanger contains substantially all the CEA
activity present in the CEA active fraction. The component which is
preferred for use in the radioimmunoassay of CEA is component B.
However, either the CEA material or component A can be
satisfactorily utilized.
In another aspect of this invention, I have discovered
radioimmunoassay techniques which are simple to perform and have a
high degree of reproducibility and specificty.
In radioimmunoassays, it is important that the radioactive atom be
sufficiently reactive with the molecule to be tagged to provide an
adequate concentration of radioactivity for determination and the
radioactive atom must provide a sufficient number of
disintegrations per unit of time to provide sufficient sensitivity
for accurate determinations. Further, in the case of
radioimmunoassay of antigens, the antigenicity must not be
deleteriously affected by the conjugation of the radioactive atom
to the antigen.
By means of the present invention, it is possible to detect the
existence of human carcinoma growth by assaying a circulating tumor
associated antigen. This invention provides a test sufficiently
sensitive to detect at least 1 ng. of CEA material, component A or
component B per ml. of serum or plasma. This sensitivity has been
found sufficient to detect abnormal amounts of CEA activity. A very
minor amount, e.g., less than 0.05 ng. of CEA activity may be
present in normal situations. The sensitivity of the assay is
limited only by the specific activity of the radioactive atom.
The CEA material, component A or component B can be tagged with
radioactive atoms which will react with their chemically reactive
groups and not substantially diminish their antigenicity. I.sup.125
has been found to be a particularly suitable radioactive atom.
The CEA material, component A or component B can be radio-iodinated
by methods known in the art, with minor modifications to
concentration and volumes. The Chloramine T method of Hunter and
Greenwood, Biochem. J. 91, 46 (1964) using iodine 125 is
particularly useful.
A radioiodination efficiency of about 20 percent to 50 percent can
be obtained by the process described herein. The radioiodination
process is equally applicable to the CEA material which is purified
and isolated prior to its fractionation into components A and B, or
each of the components. Preferred for use in this invention,
however, is component B.
The reaction is effected, for example, by using a 200 .mu. l.
reaction mixture containing 100 .mu.g. of Chloramine T (sodium
p-toluenesulfo-chloramine); 0.025-0.4 mg. of CEA material or an
individual component thereof and 4 mCi of I.sup.125 in the form of
KI or NaI. The reaction takes place in about 1 minute at room
temperature and is stopped by the addition of sodium metabisulfite.
The function of the Chloramine T is to oxidize the iodide salt to
iodine. The function of the sodium metabisulfite is to reduce
unreacted I.sup.125 back to its salt. Other reducing agents can
also be used, e.g., potassium metabisulfite. The oxidizing and
reducing agents used should not be so strong that they damage the
antigenicity of CEA material or its components. The radioiodinated
product can be separated from residual unreacted I.sup.125 by
chromatography in a cross-linked dextran gel column, e.g., Sephadex
G-100, and removing the tube with the greatest radioactivity in the
first peak. The resulting product has a specific acitivity of
between about 1,000-25,000 dpm./ng., preferably between about
10,000 and 20,000 dpm./ng., i.e., about 5-10 m.mu. Ci/ng. of CEA
material, component A or component B.
It is necessary, in order to achieve success in the aforesaid
diagnostic technique, to treat the patient's blood in a manner
which will insure that all the CEA material, component A or
component B, to the exclusion of interfering materials, is in the
finally used serum or plasma. This can be accomplished by treating
blood serum or plasma from the patients with a glycoprotein solvent
which solubilizes the CEA material, component A or component B, and
then clarifying the resulting solution. It has been found that both
serum and plasma from the blood of patients are suitable for use in
this process, however, plasma is preferred.
The glycoprotein solvent which has been found suitable for this
process is perchloric acid. Perchloric acid of 1.2 M or a
sufficient amount to provide a concentration of about 0.6 M or less
of perchloric acid is the preferred solvent since it removes
interfering substances, frees antigenic sites ad lowers ionic
strength. The resulting solution containing dissolved CEA and
component A or component B, if any are present, is then clarified.
The preferred clarification method is to centrifuge, collect the
supernatant and dialyze against distilled water, then against
buffered water (pH 6-6.25, ammonium acetate with 0.01 M acetate).
This usually takes about 6 to 10 hours. The dialysis residue
(retentate) can then be dried by lyophylization, this is not
essential however. By using this method a purified extract
containing greater than about 95 percent of the CEA material,
component A or component B originally present is produced.
It is important to this process that the extract is treated as
described since the glycoprotein solvent which solubilizes the CEA
material, component A or component B in the initial step
dissociates any pre-existing CEA-anti-CEA complexes and activates
the antigenic sites in the patient's serum or plasma, enabling the
recovery of substantially all the CEA activity originally present.
This provides a method for detecting CEA activity in patients with
primary carcinomas and metastatic carcinomas of varying origin.
It is also possible in another aspect of the radioimmunoassay
techniques of this invention to add the antibody directly to the
supernatant resulting from the glycoprotein solvent extract of the
patient's blood serum or plasma. This eliminates the need for time
consuming dialysis procedures and provides a method for detecting
CEA materials, component A and/or component B in patients having
carcinoma.
It is further possible in a preferred aspect of the
radioimmunoassay techniques of this invention to treat the blood
serum or plasma by diluting in such a manner that its ionic
strength is reduced, then add the antibody directly to the
dilution.
The dilution can be accomplished by adding at least 100 volumes of
either water or a salt solution of low ionic strength to each
volume of the blood serum or plasma. It is preferred to use plasma.
Generally any convenient salt can be used as long as it does not
interfere with the subsequent treatment with zirconyl phosphate.
The salts found suitable are, for example, ammonium acetate, sodium
chloride, sodium borate (pH 8.4) and the like. Ammonium acetate of
0.01 M or less is preferred.
The dilution of the blood serum or plasma is for the purpose of
lowering the ionic strength of the solution in order to free or
activate antigenic sites of any free CEA material or component
which is present. This technique does not dissociate any
pre-existing CEA-anti CEA complex but makes possible detection of
free circulating CEA activity. It is important when using salt
solutions as the diluent, that the molarity of the salt be
sufficient to lower the ionic strength of the serum or plasma to a
level which will activate the antigenic sites.
Since no dialysis procedures are required, this procedure saves
considerable time and is suitable for initial screening procedures
to detect free circulating CEA activity.
Further, in order to effectively conduct the radioimmunoassay, a
supply of antibodies specific to the CEA material, component A
and/or component B must be assured. This is accomplished by
immunizing animals with the purified CEA material or a component in
conventional manner as follows.
An emulsifier, e.g., Freunds adjuvant (complete) is added to the
CEA material or either component in a saline solution. The emulsion
can be injected in animals intramuscularly, subcutaneously, in the
foot pad or any combination of these methods. Animals such as fowl,
rabbits, horses, goats, sheep and the like are suitable. The
regimen in rabbits, for example, is injections twice a week until
five injections are made. After the last injection, blood is
collected from the animal. The serum from this blood is unabsorbed
CEA antiserum.
In one method, 400 .mu.g. of CEA material or a component in 1 ml.
saline solution (0.9 percent) is utilized. The injection is made
intramuscularly using a volume about four times that injected in
the foot pad.
The antibody present in the antiserum, after absorption with normal
tissue components, is specific in its activity against the CEA
material, component A and/or component B to the exclusion of other
antigens.
In conducting the radioimmunoassay of CEA, procedures based on both
the techniques of isotope dilution and competitive-inhibition can
be used. However, the competitive-inhibition method is the
preferred method of this invention. In these methods, a titration
curve, then a standard inhibition curve are obtained.
The standard inhibition curve can be made by the Farr procedure. It
is a measure of the complex formation with specific antibodies. The
curve reflects the amount of CEA material, component A and/or
component B present per unit of serum. The measurement is in
nanograms per ml., which is plotted against a known percentage of
radioactive tagged CEA material, component A or component B. The
resulting curve is used to plot the amount of CEA material,
component A or component B in a patient's serum.
In a preferred method, a standard inhibition curve can also be
obtained by the competitive-inhibition method by adding standard
CEA material, component A or component B to a series of tubes
containing powdered perchloric acid extract of normal human serum
or plasma. A measured amount of CEA antiserum which had previously
been determined from a standard dilution curve is added to the
series of tubes containing a dialyzed perchloric acid extract of
normal blood serum or plasma described above, or alternatively
serum or plasma diluted with 0.01 M (0.01 N) ammonium acetate
buffer at pH 6-6.25. In this alternative method wherein the test
fluid is diluted, a maximum normality of buffer should not be
greater than 0.01. Lower normalities are suitable. Where
appropriate, molarity can be used to describe the concentration,
equivalent normalities can be calculated by conventional means.
The resulting solutions are incubated at about 45.degree. C. for a
sufficient time to complete the reaction, usually about 30-45
minutes is sufficient. Following the incubation, a measured amount
of radioiodinated CEA material, component A or component B is added
to each of the tubes. The incubation is then continued for about an
additional 30 minutes at about 45.degree. C. When the incubation is
completed, a precipitant which precipitates the antibody and
antigen-antibody complex but not the antigen, is added to the
solution to coprecipitate the antibody bound CEA material,
component A or component B. Preferably, a zirconyl phosphate gel is
used.
Under the conditions described above, free CEA material, component
A or component B remains in solution, I.sup.125 content of the
precipitate or supernatant is then determined from a reading on a
suitable instrument and the amount of CEA material, component A or
component B in the serum or plasma is then determined by reference
to a standard.
The assay performed on the powdered perchloric acid extracts of
serum or plasma processed in the same manner as the standard CEA
material, component A or component B, results in a determination of
the amount of CEA material, component A or component B in the
patient's blood. This in turn is indicative of the presence or
absence of carcinoma in the patient.
According to this invention, the radioimmunoassay can be
accomplished by either a routine isotope dilution procedure or the
competitive-inhibition assay method described above.
The isotope dilution assay method is carried out by adding a
measured amount of tagged CEA material, component A or component B
to a perchloric acid extract of blood serum or plasma which is then
dialyzed. The extract is then neutralized with, e.g., NaOH, and a
measured amount of antibody is added. The mixture is then dialyzed
against the polyethylene glycol described previously, driving the
antibody-antigen reaction to completion.
The resulting precipitate is then dissolved in boric acid buffer at
pH 6.25. The radioactivity is then determined by adding zirconyl
phosphate gel to the solution, then centrifuging and assaying the
precipitate for radioactivity.
The preferred competitive-inhibition assay method described above
is carried out by dissolving the solid perchloric acid blood serum
or plasma extract in a suitable buffered solvent at a pH of 5-8,
preferably 6.25. While any conventional buffer is suitable, e.g.,
phosphate buffer, I have found that buffered solvents containing
boric acid are preferred. This is surprising since heretofore
borate buffers have been considered unsuitable for use in
radioimmunoassay or isotope dilution assay at an acid pH. The use
of acidic conditions is dictated by the fact that the CEA material,
component A or component B are not sufficiently stable at neutral
or alkaline pH's to maintain their antigenicity.
A measured amount of antibody is then added to the solution. While
any amount is suitable, 30 units is used for convenience and ease
of measurement, however, from about 30 to about 300 units are
suitable.
A unit of CEA activity is a nanogram of CEA material, or the
equivalent amount of component A or component B. A unit of antibody
is the amount of antibody which is bound by a nanogram of CEA
material, or the equivalent amount of component A or component
B.
The resulting mixture is then incubated for about 24 hours. 50
Units of tagged CEA material or the equivalent amount of component
A or component B are then added and the mixture is again incubated
for about 24 hours. It is possible, however, to use from about 20
to about 500 units, however, 20-50 units have been found to be
preferred. If there is some CEA material, component A or component
B in the serum or plasma, then the amount of unreacted tagged CEA
material, component A or component B in the serum or plasma can be
determined either qualitatively or quantitatively. The
radioactivity is determined by adding zirconyl phosphate gel to the
solution, then centrifuging and assaying the precipitate for
radioactivity.
In another preferred aspect of this invention, the assay for
determining free circulating CEA material, component A or component
B is carried out by diluting either blood serum or plasma with at
least 100 volumes of water or a low ionic salt solution as
described previously.
The solution is then transferred into suitable test tubes, 30 units
of CEA antiserum are added and the mixture is incubated at about
45.degree. C. for 30-45 minutes. 20 to 50 Nanograms of
radioiodinated CEA material or equivalent amounts of component A or
component B, having 10,000 to 20,000 dpm./ng. are then added and
the mixture is incubated for about 30 minutes at about 45.degree.
C. If there is some free CEA material, component A or component B,
in the blood serum or plasma then the amount of unreacted tagged
CEA material, component A or component B in the blood serum or
plasma can be determined either qualitatively or quantitatively.
The radioactivity is determined by adding zirconyl phosphate gel to
the solution, then centrifuging and assaying the precipitate for
radioactivity.
The method is advantageous because it takes about 2 hours to
complete. It is suitable for determining only the free CEA
activity. When used in conjunction with the competitive-inhibition
assay methods, it is possible to have large scale screening for
carcinomas.
The following examples illustrate the invention.
EXAMPLE 1
150 Grams of frozen primary colon adenocarcinoma tumor was
homogenized in 5 volumes of distilled water at 5.degree. C. for 2
minutes in a homogenizer. The homogenate was then blended for about
5 minutes in a blender. The resulting material was then centrifuged
for 30 minutes at 5,000 rpm. The supernatant was decanted and a
stick was used to prevent the top fat pad which forms from breaking
and contaminating the solution. One volume of 10 percent perchloric
acid was added to the supernatant and stirred at 5.degree. C. for
10 minutes. The resulting mixture was centrifuged for 30 minutes at
5,000 rpm. The supernatant was decanted and filtered through glass
wool. The resulting filtrate was then dried by dialysis against a
20 M Carbowax solution which was prepared by filling a 10 liter
beaker with 20 M Carbowax Flakes and filling it to the 7 liter mark
with a borate buffer at pH 8.4, then stirring until the flakes
dissolved. The resulting solid dissolved in 8 ml. of
Tris(hydroxymethyl)-aminomethane-NaCl (Tris-NaCl) solution. The
resulting solution was centrifuged for 30 minutes at 105,000
gravity and 5 ml. of the resulting supernatant was applied to a
Sepharose 6B column and eluted with the Tris-NaCl solution using 80
drops per tube collected in 5 ml. fractions at the rate of 0.5
ml./minute. Tubes 45-57 were pooled and concentrated by dialysis
against 20 M. Carbowax. The resulting concentrate was then applied
to a Sephadex G-100 column. This was eluted with the Tris-NaCl
solution and 4 tubes containing 5 ml. each of the first peak were
pooled and dried by dialysis against 20 M Carbowax. The resulting
solid material was dissolved in 2 ml. of the Tris-NaCl solution and
1 ml. was labelled with I.sup.125 by conventional means. The
I.sup.125 labelled material was applied to a Sepharose 6B column
and eluted with the Tris-NaCl solution. The pooled fractions 45-57
were frozen in 5 ml. tubes and stored at -20.degree. C. This is
called Tumor Extract No. 1 (TE-1). When studied by gel diffusion
versus goat antiserum, a single strong band appeared. In certain
tumor extracts a second minor band appeared.
2 Ml. of labelled TE-1 were applied to a CM-52:DE-52 column in 50
ml. of ammonium acetate (pH 4 ) solution and the column was washed
with 150 ml. of ammonium acetate. Almost all the I.sup.125 was
retained by the mixed cellulose ion exchange column. The column was
then eluted with 500 ml. each of ammonium acetate-NaCl solutions
containing 0.05 M NaCl, 0.1 M NaCl, 0.25 M NaCl, 1.0 M NaCl. Two
peaks were eluted with the 0.05 M NaCl-buffer. The first peak is
that of CEA component A. The second peak appeared to be degraded
120,000 molecular weight material which is called M-120. A second
major peak was eluted with the 0.1 M NaCl-buffer and is a pure
material having 240,000 molecular weight, it is CEA-component B. A
third major peak was eluted with the 0.1 M NaCl-buffer and was not
reactive with the antisera. This indicates it is probably a normal
component. Thus, the first peak which was eluted with the 0.05 M
NaCl is CEA component A which contains CEA activity. The second
major peak, CEA component B, also contains the CEA activity. The
identity of the CEA material, component A and component B is
confirmed by its forming a single line in the Ouchterlony gel
diffusion test with unabsorbed anti-serum. When subjected to block
electrophoresis using Sephadex G-25 Fine [a cross linked dextran
gel having an approximate molecular weight exclusion limit of
5,000, a water regain (gH.sub.2 O/g. dry gel) of 2.5 .+-. 0.2,
particle size of 20-80 microns and a bed volume/ml./g. dry gel of 5
] on a non-conductive block, e.g., Lucite, the CEA material,
component A and component B behave identically as follows:
The block electrophoresis medium, Sephadex G-25 Fine is swollen
with water for 2 hours at 80.degree. C. and washed by decantation
with borate of pH 8.6 and ionic strength 0.05. then suction
filtered through a sintered glass disk.
A thick slurry of the gel is poured onto a Lucite block support of
61 cm. .times. 7.5 cm. .times. 1 cm. in dimensions and allowed to
distribute itself evenly along the plate to a depth of 1 cm. The
surface is then blotted with cotton gauze sponges until firm but
not completely dry.
The block is then fitted with 3 mm. chromatography paper contacts
(Whatman) all aligned in the same direction of flow of the paper.
The block is then placed in the electrophoresis apparatus and
allowed to equilibrate for 1 hour under the operating conditions of
400 volts, with a constant current of approximately 20 mA at
4.degree. C. A 1 cm. strip is then removed from the center of the
block and mixed well with a solution of 60 mg. of CEA material
produced as above, in 0.5 ml. of 0.05 M borate. The resulting
slurry is then poured back in the central strip. One to two drops
of ferritin (6 .times. recrystallized) at a concentration of 100
mg./ml. is then spotted at the cathodal extremity of the block. 24
Hours after the start of the run, the ferritin marker moves 18 cm.
anodally. At that time the block is removed from the
electrophoresis apparatus and 2 centimeter strips between the zone
of application and the anodal extremity are eluted with 2 M NaCl
passed through 0.20 .mu. disposable grid membrane (Nalgene). The
activity is localized 10-14 cm. anodal to the application zone with
weaker activity being found 8-10 cm. anodal to the application
zone.
When components A and B are treated separately in the same manner,
identical results are obtained.
EXAMPLE 2
6 ml. tubes of normal serum and six 5 ml. tubes of serum from
suspected colon cancer patients each were extracted with an equal
volume of 1.2 to 2 molar perchloric acid by shaking for 20 minutes
and then centrifuging at 8,000 gravity for 5 minutes at 5.degree.
C. The supernatants were collected and transferred to a dialysis
tubing and placed in a 250 ml. beaker of a Carbowax solution formed
by filling a 10 l. beaker with 20 M Carbowax Flakes and then
filling the beaker to the 7 liter mark with borate buffer of pH 8.4
and stirring until the flakes dissolve. After 5 hours of dialysis,
the resulting precipitate in the tubes was dissolved in 1 ml. of
borate buffer at pH 6.25 and transferred to 15 .times. 125 mm. test
tubes. 0.1 Ml. of normal human serum was added to each tube and
mixed. To each of the six tubes containing normal serum, perchloric
acid extracts, 0, 10, 50, 100, 250 and 500 nanograms of CEA was
added. Then 300 to 500 units of CEA antisera was added to each of
the 12 tubes and mixed. The tubes were then stored in an icebox at
5.degree. C. for 12 hours. Subsequently, 500 units of CEA-I.sup.125
was added to each tube and incubation was continued for 18 hours at
5.degree. C. Five ml. of zirconyl phosphate gel was added to the
tubes and the tubes were then filled with ammonium acetate buffer
at pH 6.25. The tubes were stopped with rubber stoppers, inverted
five times and centrifuged at 1,500 gravity for 5 minutes. The
resulting supernatant was then discarded. The solid gel which
remained was washed with an ammonium acetate buffer by filling the
tubes with the buffer and dispersing the gel with a mixer, then
centrifuging at 1500 times gravity for 5 minutes. The gel was
assayed for bound I.sup. 125 with a Packard 3003 Tri-carb
Scintillation Spectrometer. Other similar equipment can also assay
for the bound I.sup.125 . The results of the serum being tested for
CEA activity were compared to the standard and the amount of CEA
material, component A or component B in the unknown sera was
determined.
EXAMPLE 3
0, 10, 50, 100 and 500 Nanograms of CEA material standard were
added to separate tubes each of which contained 5 ml. of normal
sera and then mixed. The standards and serum from suspected cancer
patients were extracted with perchloric acid, centrifuged and
dialyzed against 20 M Carbowax in the same manner as in Example 2.
The resulting precipitate was dissolved in 1.0 ml. of borate buffer
of pH 6.25, then 500 units of radioactive tagged CEA material was
added, the mixture was mixed thoroughly and then 300 units of CEA
antisera were added. The mixture was dialyzed against a fresh 20 M
Carbowax solution and brought to dryness in about 2 to 3 hours. The
resulting precipitate was dissolved in 1 ml. of borate buffer of pH
6.25 and then 5 ml. of zirconyl phosphate gel were added. The assay
for the I.sup.125 was made according to the process set forth in
Example 2.
EXAMPLE 4
Human meconium is homogenized in 3 volumes of 10 percent perchloric
acid at 5.degree. C. and centrifuged at 4,000 rpm for 30 minutes.
The supernatant is then dialyzed against 20 M Carbowax.
The precipitate is taken up in a minimum volume of Tris-NaCl
solution of pH 7 and centrifuged at 105,000 g. for 30 minutes.
5 Ml. of the supernatant is then applied to a Sephadex G-100 column
and eluted with Tris-NaCl solution. Four tubes of 5 ml. each from
the first fraction were pooled and brought to dryness by dialysis
against 20 M Carbowax. The residue is taken up in 8 ml. of
NaCl-Tris solution and centrifuged at 105,000 g. for 30 minutes. 5
Ml. of the resulting supernatant is then applied to a Sepharose 6B
column and eluted with the Tris-NaCl solution using 80 drops per
tube collected in 5 ml. fractions at the rate of 0.5 ml./minute.
Tubes 45-57 were pooled and brought to 1 ml. by dialysis against 20
M Carbowax. When studied by gel diffusion versus goat antiserum,
one strong band developed. It was identical to CEA component B.
EXAMPLE 5
Carcinoembryonic antigen (CEA) material was isolated and
radiolabelled with I.sup.125 as described in Example 1.
A goat antiserum mono-specific for CEA material was reacted with
radiolabelled CEA material to form an antibody-antigen complex. The
excess radiolabelled CEA material was separated from the complex by
adsorbing the complex with zirconyl phosphate gel (pH 6.25) as
described in Examples 2 and 3.
The radiolabelled CEA was then incubated with the antiserum as
follows:
100 Ng. of radioiodinated CEA material was incubated with antiserum
diluted with water (1- 10,000) at 45.degree. C. for 30 minutes in 1
ml. each of normal serum (goat, human, rat, rabbit), 0.15 M NaCl,
0.075 M Na.sub.2 HPO.sub.4, 0.15 M Tris-HCl (pH 7.5) and 0.1 M
ammonium acetate. This resulted in minimal complex formation.
When the radioiodinated CEA material and the antiserum were
incubated in 1 ml. each of H.sub.2 O,0.01 M NaCl, 0.01 M ammonium
acetate, 1 percent normal serum diluted in H.sub.2 O, or 0.05 M
sodium borate (pH 8.4) antigen-antibody complex formation took
place.
The antiserum also formed a complex with the radioiodinated CEA
material when incubated in 10 ml. of 0.01 M ammonium acetate, 0.1
ml. of normal serum diluted to 10 ml. with water, or 0.005 M sodium
borate (pH 8.4).
10 Ng. of CEA material added to dialyzed supernatant from 5 ml. of
normal serum and 5 ml. of 1 M perchloric acid, neutralized 10
percent of the antiserum when incubated at 45.degree. C. for 30
minutes prior to the addition of measured amounts of radioiodinated
CEA material.
CEA material was detected in 28 of 30 perchloric acid extracts of
serum obtained from patients with colon adenocarcinoma and directly
in sera of metastatic patients after dilution of 0.1 ml. serum in
10 ml. of water. This indicates that dilution which weakens the
ionic strength of the serum provides access to an antigenic site on
the CEA material.
EXAMPLE 6
A 3 ml. aliquot of 2 M perchloric acid was added to 5 ml. aliquots
of serum or plasma while agitating in a mixer. The mixtures were
allowed to stand at room temperature for 15 minutes then mixed
again and allowed to settle. The mixtures were centrifuged at 1,000
times g. for 5 minutes at room temperature and the supernatants
were dialyzed for 36 hours against 25 liters of distilled water at
room temperature. The dialysis bath was changed five times during a
24 hour period. This retentate was then used for testing. All
specimens were run in duplicate.
Goat antisera monospecific for CEA material was diluted 1:2000 in
10 percent normal human serum and 0.05 M borate buffer pH 8.4. CEA
material was prepared and labelled with I.sup.125 as described in
Example 1.
A dilution curve of the antisera against a constant amount of
radioiodinated CEA material was carried out in the dialysates of
perchloric acid serum extracts to which 1 ml. of borate buffer
(0.05 M, pH 8.4) was added.
Six tubes of the serum were placed in a water bath at 45.degree. C.
for 1/2 hour. After incubation of the mixture, 5 ml. of ammonium
acetate solution (0.1 M, pH 6.25) prepared by adjusting the pH of
0.1 M acetic acid to 6.25 with conc NH.sub.4 OH, and 4 ml. of
zirconyl phosphate gel (pH 6.25) were added to each tube and the
tubes capped and inverted several times. The tubes were then
centrifuged at 3,000 rpm for 15 minutes and the supernatants were
discarded. The residue from each tube was resuspended in 10 ml. of
ammonium acetate solution, recovered by centrifugation and assayed
for bound I.sup.125 in a gamma scintillation counter.
A titration curve was carried out by adding known amounts of
unlabelled CEA material to the retentates of serum perchloric acid
extracts. 1 Ml. borate buffer (pH 8.4, 0.5 M) and 0.5 ml. of a
1:1000 dilution of antisera was added to each of 6 tubes and
incubated in a water bath at 45.degree. C. for 1/2 hour. Then 0.1
ml. of radioiodinated CEA material containing 24,000 DPM was added
to each specimen. The tubes were mixed well and reincubated for one
half hour. 5 Ml. of ammonium acetate solution (0.1 M pH 6.25) and
zirconyl phosphate gel (pH 6.25) were then added to each tube. The
tubes were capped, inverted several times and centrifuged at 1,200
times g. for 15 minutes at room temperature. The supernatant was
discarded and the gel precipitate was resuspended in 10 ml.
ammonium acetate solution (0.1 M, pH 6.25). The gel was separated
by centrifugation and assayed for bound I.sup.125.
Specimens from patients were run in exactly the same manner as
above except that unlabelled antigen was not added.
About 75 percent of the I.sup.125 labelled material reacted with
the antisera. A final dilution of 1:10,000 of the CEA-antisera in
the perchloric acid extracts from serum reacts with 70 percent of
the maximum amount of labelled antibody reactive material. The
1:10,000 dilution was therefore utilized.
Incubation of the antisera diluted with water (1:10,000) with
unlabelled CEA material prior to addition of labelled antigen shows
that the reaction of antibody with antigen is linear at antigen
concentrations of from 1.5 to 10 ng./ml. but that the reaction is
less sensitive at concentrations above 20 ng./ml.
Of 487 patients tested, those with carcinomas of the breast, lung
and colon had detectable concentrations of CEA material in their
serum.
Of 229 patients without malignant disease, 11 had detectable
antigen in their serum. Two of these later developed cancer, one
had adenomatious polyp of the colon and 5 had severe emphysema.
EXAMPLE 7
1 Ml. of plasma from suspected cancer patients was diluted with 4
ml. of physiological saline solution. An equal volume of 1.2 molar
perchloric acid was added and the mixture was agitated for 20
minutes then centrifuged at 8,000 times gravity for 5 minutes at
room temperature. The supernatant was collected and transferred to
a dialysis tubing and dialyzed overnight against distilled water.
The resulting retentate was dialyzed against an ammonium acetate
solution of pH 6 to 6.25 containing 0.01 molar acetate, for 3 hours
at room temperature. The retentate was transferred into 20 ml. test
tubes. 30 Units of CEA antiserum was then added and the mixture
incubated for 30 to 45 minutes at 45.degree. C. 50 Ng. of
CEA-I.sup.125 containing 10,000 to 20,000 dpm/ng was then added and
the mixture incubated for 30 minutes at 45.degree. C. 5 Ml. of pH
6.25 zirconyl phosphate gel was added to each test tube and 5 ml.
of ammonium acetate solution (pH 6.25, 0.1M) were then added. After
mixing the tubes were centrifuged at 1,500 times gravity for 5
minutes at room temperature and the resulting supernatant was
discarded. The solid gel which remained was washed with the
ammonium acetate buffer by filling the tubes with the buffer and
dispersing the gel with a mixer, then centrifuging at 1,500 times
gravity for 5 minutes. The gel was assayed for bound I.sup.125 with
a Packard 3003 Tri-carb Scintillation Spectrometer. If CEA is
present in the plasma, the amount of bound CEA-I.sup.125 will be
reduced proportionately.
EXAMPLE 8
10 Ml. of water were added to 0.1 ml. of plasma in a 20 ml. test
tube. 30 Units of CEA antiserum were then added and the mixture
incubated for 30-45 minutes at 45.degree. C. 50 Ng. of
CEA-I.sup.125 containing 10,000 to 20,000 dpm/ng. were then added
and the mixture incubated for30 minutes at 45.degree. C. 5 Ml. of
pH 6.25 zirconyl phosphate gel and 5 ml. of ammonium acetate
solution (pH 6.25, 0.01 M) were then added to each test tube. After
mixing, the tubes were centrifuged at 1,500 times gravity for 5
minutes at room temperature and the resulting supernatant was
discarded. The solid gel which remained was washed with the
ammonium acetate buffer by filling the tubes with the buffer and
dispersing the gel with a mixer, then centrifuging at 1,500 times
gravity for 5 minutes. The gel was assayed for bound I.sup.125 with
a Packard 3003 Tri-carb Scintillation Spectrometer. If CEA is
present in the plasma, the amount of bound CEA-I.sup.125 will be
reduced accordingly.
* * * * *