Methods of recovering colorectal epithelial cells or fragments thereof from stool

A method of recovering colorectal epithelial cells or fragments thereof from a stool sample is provided. The method involves contacting a stool sample with a specific binding reagent having specificity for colorectal epithelial cells or membrane fragments thereof to form a complex containing the specific binding reagent and the colorectal epithelial cells or fragments thereof, and separating the complex from the sample. A method of detecting dysplastic colorectal epithelial cells or fragments thereof, wherein a specific binding reagent employed in the method has specificity for dysplastic colorectal epithelial cells or membrane fragments thereof, is also provided. An article of manufacture containing reagents for performing the method is further provided.

FIELD OF THE INVENTION 
The invention relates to methods of recovering colorectal epithelial cells 
or fragments thereof from stool. Specifically, the invention relates to 
methods of using specific binding reagents to recover colorectal 
epithelial cells or fragments thereof from stool samples, and further to 
the use of such methods in the early detection of colorectal neoplasia by 
the presence in stool of dysplastic epithelial cells or fragments thereof. 
BACKGROUND OF THE INVENTION 
Colorectal cancer mortality ranks second among all causes of malignant 
death in industrialized nations. Recognition and curative treatment of 
precursor (premalignant) adenomas and localized cancers is possible with 
early detection. 
Currently fecal occult blood testing is a widely utilized screening tool 
for the early detection of colorectal neoplasia, but suffers from several 
disadvantages. Fecal blood testing measures an ambiguous marker for 
colorectal neoplasia--hemoglobin--and tests for hemoglobin in stool 
samples have proved to be neither specific nor sufficiently sensitive. 
Recent reports indicate that fecal blood testing fails to detect over 70 
percent of pre-malignant polyps. Colorectal neoplasms bleed 
intermittently, and thus fecal occult blood is not a reliable marker for 
colorectal neoplasia. Moreover, there are many causes of gastrointestinal 
bleeding unrelated to colorectal neoplasia, resulting in a very high level 
of false positives using the fecal occult blood screening method. False 
positives result in unnecessary and costly follow-up procedures such as 
colonoscopy. 
In terms of simplicity, however, stool testing approaches the ideal for 
colorectal cancer screening; it is non-invasive, has a low unit cost, 
generates reasonably high compliance, and reflects the entire colorectal 
surface. A specific and sensitive stool screening test for colorectal 
neoplasia would thus be extremely valuable. 
Exfoliated dysplastic epithelial cells represent a possible stool marker 
for colon cancer. Such cells obtained from lavage effluents have been 
shown to be reliable markers of colorectal cancer, but specimen collection 
required the invasive technique of colorectal purgation, limiting the 
clinical utility of this approach. Recently, a density gradient 
centrifugation technique was developed to collect sloughed colonocytes 
from routine stool samples. This technique, however, is time-consuming and 
technically complex, and therefore does not allow for an efficient and 
selective recovery of colorectal epithelial cells, is not amenable to 
large volume processing, and thus is not useful as a stool screening 
technique of the type currently needed. 
SUMMARY OF THE INVENTION 
The present invention provides an efficient and selective method of 
recovering colorectal epithelial cells or fragments thereof from stool, 
and overcomes the disadvantages associated with the density gradient 
centrifugation technique currently available in the art. The present 
invention further provides a screening technique for colorectal neoplasia 
that is markedly superior to the fecal occult blood detection technique 
currently utilized. In one aspect, the present invention relates to a 
method of recovering colorectal epithelial cells or fragments thereof from 
a stool sample. The method involves the steps of (a) contacting a stool 
sample containing colorectal epithelial cells or fragments thereof with a 
specific binding reagent having specificity for colorectal epithelial 
cells or fragments thereof to form complexes comprising said specific 
binding reagent and said colorectal epithelial cells or fragments thereof, 
and (b) separating the complexes from the sample. 
In preferred embodiments of the present invention, the specific binding 
reagent is selected from the group consisting of an antibody or antibody 
fragment having specificity for carcinoembryonic antigen, an antibody or 
antibody fragment having specificity for epidermal growth factor receptor, 
an antibody or antibody fragment having specificity for colon specific 
antigen, an antibody or antibody fragment having specificity for MUC-1 
antigen, the antibody MA5 or an active fragment thereof, the antibody PCA 
33.28 or an active fragment thereof, an antibody or antibody fragment 
having specificity for P53 antigen or mutant forms of P53 antigen, and 
mixtures thereof. 
In other preferred embodiments of the present invention, the method further 
comprises the step of analyzing the colorectal epithelial cells or 
fragments thereof. The analyzing of the colorectal epithelial cells or 
fragments thereof may include staining the colorectal epithelial cells or 
fragments thereof and microscopically examining the cells or fragments. 
In other preferred embodiments, analyzing the colorectal epithelial cells 
or fragments thereof comprises determining the presence or absence of 
dysplastic colorectal epithelial cells or fragments thereof. Determining 
the presence or absence of dysplastic colorectal epithelial cells or 
fragments thereof may include immunostaining the colorectal epithelial 
cells or fragments thereof. The immunostaining may include contacting the 
colorectal epithelial cells or fragments thereof with a specific binding 
reagent having specificity for dysplastic colorectal epithelial cells or 
fragments thereof. 
In other embodiments of the present invention, the specific binding reagent 
may have specificity for dysplastic colorectal epithelial cells or 
fragments thereof. Such specific binding reagents may be selected from the 
group consisting of an antibody or active antibody fragment having 
specificity for MUC-1 antigen, the antibody MA5 or an active fragment 
thereof, the antibody PCA 33.28 PCA or an active fragment thereof, an 
antibody or active antibody fragment having specificity for P53 or any 
mutuant form of P53 antigen, and mixtures thereof. In this embodiment of 
the invention, the method may further comprise the step of determining the 
presence or absence of dysplastic colorectal epithelial cells or fragments 
thereof in the stool sample. Determining the presence or absence of 
dysplastic colorectal epithelial cells or fragments thereof may involve 
staining and microscopically examining the dysplastic colorectal 
epithelial cells or fragments thereof complexed with said specific binding 
reagent having specificity for dysplastic colorectal epithelial cells or 
fragments thereof. 
In preferred embodiments of the present invention, the contacting step 
comprises contacting the stool sample with a magnetic bead containing the 
specific binding reagent. In this embodiment, the separating step 
preferably comprises magnetically separating said complex from said 
sample. 
In another aspect, the present invention relates to a method of detecting 
dysplastic colorectal epithelial cells and fragments thereof in a stool 
sample. The method involves (a) contacting a stool sample with a first 
specific binding reagent having specificity for colorectal epithelial 
cells or fragments thereof to form first complexes comprising said first 
specific binding reagent and said colorectal epithelial cells and 
fragments thereof, (b) separating said first complexes from said sample, 
(c) contacting the first complexes or colorectal epithelial cells or 
fragments thereof recovered from said first complexes with a second 
specific binding reagent having specificity for dysplastic colorectal 
epithelial cells or fragments thereof to form second complexes containing 
the second specific binding reagent and the dysplastic colorectal 
epithelial cells or fragments thereof, (d) separating the second complexes 
from the sample solution, and (e) determining the presence or absence of 
dysplastic colorectal epithelial cells in the stool sample. Determining 
the presence or absence of dysplastic colorectal epithelial cells or 
fragments thereof preferably involves contacting the second complexes with 
a third specific binding reagent having specificity for the second 
specific binding reagent, separating bound third specific binding reagent 
from free third specific binding reagent, and determining the presence of 
bound third specific binding reagent. 
In another aspect the present invention relates to an article of 
manufacture containing packaging material and reagents contained within 
the packaging material, wherein the reagents are effective for recovering 
colorectal epithelial cells or fragments thereof from stool, and wherein 
the packaging material contains a label indicating that the reagents can 
be used for recovering colorectal epithelial cells or fragments thereof 
from stool, and wherein the reagents contain a specific binding reagent 
having specificity for colorectal epithelial cells or fragments thereof. 
As used herein, "colorectal epithelial cell or fragment thereof" means any 
normal or dysplastic (abnormal) cell or fragment derived from normal or 
diseased colorectal epithelium. 
As used herein, "fragment" means any degradation product (resulting from 
mechanical, chemical, or biochemical degradation) of a colorectal 
epithelial cell containing a binding partner for a specific binding 
reagent having specificity for colorectal epithelial cells, such as a 
membrane fragment. 
As used herein, "specific binding reagent" means monoclonal or polyclonal 
antibodies, a protein or peptide, or other agent that will immunologically 
bind, or specifically adhere, to a colorectal epithelial cell or fragment 
thereof. 
The invention provides an efficient and selective method of recovering 
colorectal epithelial cells or fragments thereof from stool, by exploiting 
a specific binding reaction between a specific binding reagent and 
colorectal epithelial cells or fragments thereof to segregate, or isolate, 
colorectal epithelial cells or fragments thereof from stool. The invention 
thus provides a highly desirable alternative to the currently available 
density gradient centrifugation technique of recovering colorectal 
epithelial cells, which is time-consuming, technically complex, and not a 
feasible method for large volume processing required in a screening 
technique for the asymptomatic general population. The present invention 
has additional advantages over the density gradient centrifugation 
technique described in the art. The method of this invention allows for 
the recovery of cell fragments containing antigenic markers (or other 
specific binding partners) which would not be recovered in the density 
gradient technique (but which have analytical or diagnostic significance) 
because such fragments are not isodense with whole cells. In addition, the 
use of specific binding reagents eliminates contaminating isodense debris, 
which is present with cells, in material recovered using the density 
gradient procedure. The invention thus provides a source of colorectal 
epithelial cells or fragments thereof for any type of clinical or 
laboratory testing, including the use of such cells or fragments in the 
early detection of colorectal neoplasia. 
The invention also provides methods of detecting dysplastic colorectal 
epithelial cells or fragments thereof, which cells or fragments are 
suggestive of colorectal neoplasia. These methods are amenable to large 
volume processing and could thus serve as a screen for the large 
asymptomatic population. This screening technique is markedly superior to 
fecal blood testing. 
The method provides several important advantages over fecal blood testing, 
currently the most widely used screening technique. The method of this 
invention detects cellular markers which are more specific as markers of 
colorectal neoplasia than fecal blood, and thus the method is more 
accurate, greatly reducing the incidence of false positives and the 
unnecessary and costly colonoscopic evaluation following a false positive 
result. The reduction of false positives achieved by the present invention 
is extremely important to a screening intervention aimed at a general 
population.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
This invention relates to a method of recovering exfoliated colorectal 
epithelial cells or fragments thereof from a stool sample. 
Exfoliated colorectal epithelial cells represent a potentially significant 
source of material for obtaining data regarding the status of the 
colorectal epithelium and for testing and screening for colorectal 
neoplasia. As many as one-third of cells from the epithelial surface are 
shed into the lumen each day. Because of extensive surface involutions, it 
has been estimated that an adenoma of only 1.0 cm has a surface area equal 
to about 1 percent of the colorectum; thus cells from clinically important 
neoplasms may represent a quantitatively substantial contribution to the 
exfoliated population. It is also likely that cellular fragments, 
including membrane fragments, resulting from mechanical, chemical, or 
biochemical degradation processes, appear in stool. 
The only means heretofore known of obtaining colorectal epithelial cells 
from stool involved density gradient centrifugation, a time-consuming and 
technically complex technique. Nair et al., FASEB J., 5:2856-2859 (1991). 
Accordingly, the present invention provides a method of capturing 
colorectal epithelial cells or fragments thereof from a stool sample by 
exploiting a specific binding reaction between a specific binding reagent 
and colorectal epithelial cells or fragments thereof in the sample. 
In one embodiment, the invention provides a method of recovering colorectal 
epithelial cells or fragments thereof from a stool sample. 
Stool samples suitable for use in the present invention are readily 
obtained. Such samples may be obtained by the donor individually and 
presented to a laboratory for testing, or are easily obtained in the 
clinical setting. Typically, a stool sample of several grams is placed in 
a transport medium. A suitable transport medium is typically a buffered 
solution containing an anti-bacterial agent, such as 0.5% thimerasol, and 
a mucolytic agent, such as N-acetyl cysteine. The sample is preferably 
delivered to a laboratory within 24 hours, preferably on ice. In the 
laboratory, a dispersing medium typically is added, and the sample is 
further dispersed in a laboratory blender such as a stomacher. The sample 
may then be tested. The sample may also be centrifuged, the supernatant 
discarded, and the pellet resuspended, resulting in a sample ready for 
testing. 
In accordance with the invention, the stool sample is contacted with a 
specific binding reagent having specificity for colorectal epithelial 
cells or membrane fragments thereof. Many such specific binding reagents 
are known. The invention contemplates that any specific binding reagent 
having specificity for colorectal epithelial cells or fragments thereof is 
useful in the practice of the invention. Carcinoembryonic antigen (CEA) is 
commonly expressed in non-neoplastic and neoplastic epithelium, and 
antibodies or active antibody fragments specific to CEA are useful for 
capturing exfoliated colorectal epithelial cells or fragments thereof in 
the present invention. Other non-limiting examples of specific binding 
agents useful in the capture of colorectal epithelial cells include 
antibodies or active antibody fragments specific to the epidermal growth 
factor receptor (EGFR), and antibodies or active antibody fragments 
specific to colon specific antigen (CSA). 
Certain specific binding reagents are useful in the capture or detection of 
dysplastic colorectal epithelial cells or fragments thereof because they 
have specificity for markers (binding partners) that are expressed or 
overexpressed on dysplastic cells exfoliated from colorectal neoplasms. 
Any specific finding reagent having specificity for dysplastic colorectal 
epithelial cells or fragments thereof is useful in the practice of the 
present invention. Such specific binding reagents may include: antibodies 
or active antibody fragments having specificity for MUC-1 antigen or other 
mucins that are expressed or overexpressed dysplastic colorectal 
epithelial cells, antibodies or active antibody fragments having 
specificity for P53 protein or mutants thereof, the antibody MA5 or active 
fragments thereof, and the antibody PCA 33.28 or active fragments thereof 
(PCA 33.28 described in Arlen et al., J. Tumor Marker Oncology, 5:4:p. 
313-319 (1990). 
The contacting step of the invention may be effected in a variety of 
formats known to those of skill in the art. For example, the contacting 
step may involve incubating the stool sample with a solution containing 
the specific binding reagent under conditions allowing the specific 
binding reagent to form a complex with colorectal epithelial cells or 
membrane fragments thereof from the stool sample. Such conditions, such as 
temperature, pH, and incubation time are well known to those of skill in 
this art. Preferably, the method of the present invention is performed at 
a pH of 7.2. To maximize recovery, a mixture of specific binding reagents 
may be used. This may be achieved by using beads coated with a mixed 
population of specific binding reagents, or by using a mixture of beads, 
each coated with a particular specific binding reagent. 
The contacting step may also involve incubating the stool sample with a 
solid phase containing the specific binding reagent under conditions 
allowing the specific binding reagent to form a complex containing the 
specific binding reagent and colorectal epithelial cells or fragments 
thereof from the stool sample. Such solid phase techniques are known in 
the art. Nonlimiting examples of such techniques include exposing the 
stool sample to a well, the surface of which is coated with the specific 
binding reagent, or incubating the stool sample with a suspension of beads 
coated with the specific binding reagent. In one preferred embodiment of 
the present invention, illustrated in the Examples below, the contacting 
step involves incubating the stool sample with magnetic beads coated with 
a specific binding reagent under conditions allowing binding of the 
specific binding reagent and colorectal epithelial cells or fragments 
thereof. 
In one embodiment of the present invention, the specific binding reagent is 
one of the above-described specific binding reagents having specificity 
for dysplastic colorectal epithelial cells or fragments thereof. In this 
embodiment, the invention provides a method of specifically capturing 
dysplastic colorectal epithelial cells or fragments thereof. To maximize 
recovery, a mixture of specific binding reagents may be used. 
The method of the invention further involves the step of separating the 
complexes (containing specific binding reagent and colorectal epithelial 
cells or fragments thereof) formed in the contacting step from the sample 
solution. The mode of separation will depend on the format employed in the 
contacting step. For example, in embodiments of the invention wherein the 
contacting step involves incubating the stool sample with a solubilized 
specific binding reagent, the separating step may involve capturing the 
complex containing the specific binding reagent and colorectal epithelial 
cells or fragments thereof with a second specific binding reagent, such as 
an antibody, having specificity for either the specific binding reagent in 
the complex or the colorectal epithelial cells or membrane fragments 
thereof in the complex. The second specific binding reagent may be 
attached to a solid phase such as a well or plate, or may be captured by a 
solid phase following binding to the complex. Alternatively, the complex 
may be contacted with, or coated onto, a solid phase such as a bead or 
other surface such as the surface of a well. The bead may then be 
separated from the sample solution, or the surface washed of the sample 
solution, using conventional methods known in the art. 
As described above, in embodiments of the invention wherein the contacting 
step involves incubating the stool sample with a solid phase containing a 
specific binding reagent having specificity for colorectal epithelial 
cells or membrane fragments thereof, the solid phase may consist of the 
surface of a well or a bead. Where the solid phase is the surface of a 
well, the separating step may involve simply washing the sample solution 
from the well to thereby separate the complex formed on the solid phase in 
the contacting step from the sample solution. In embodiments of the 
invention wherein the solid phase is a bead containing a specific binding 
reagent, the separating step may involve centrifuging the sample to 
separate the beads containing the complex formed in the contacting step 
from the sample solution, removing the supernatant and resuspending the 
separated beads. In a preferred embodiment of the present invention, 
illustrated in the Examples below, a magnetic bead containing a specific 
binding reagent is used in the contacting step, and thus the separating 
step may involve exposing the sample to a magnet or incubating the sample 
in a magnetic chamber, discarding the supernatant, and resuspending the 
magnetic beads containing the complexed colorectal epithelial cells or 
fragments thereof and thereby separating the complex from the sample 
solution. 
It should be recognized to those of skill in the art that any format may be 
used to separate the complex containing the colorectal epithelial cells or 
fragments thereof from the stool sample. The invention relates to the 
exploitation of a specific binding reaction to capture colorectal 
epithelial cells or fragments thereof from stool to form complexes 
containing the specific binding reagent and the cells or fragments 
thereof, and the separation of the complexes from the sample by any means 
described herein or known in the art. The invention is not limited by the 
formats used to effect the contacting or separating steps. 
In accordance with the invention, the separating of the complexes formed in 
the contacting step effects a separation of colorectal epithelial cells or 
fragments thereof from the stool sample. The colorectal epithelial cells 
or fragments thereof in the separates complexes are thus available for 
analysis. 
Accordingly, in preferred embodiments, the method of the invention further 
comprises analyzing the colorectal epithelial cells or fragments thereof. 
The analysis of the colorectal epithelial cells or fragments thereof may 
involve any testing of the captured material, including detecting the 
presence of the cells or fragments, detecting the presence of dysplastic 
colorectal epithelial cells or fragments thereof that are suggestive of 
colorectal neoplasia, quantitating the recovery of the captured material. 
The analysis may also include assaying the cells or extracts thereof for a 
genetic alteration such as a mutation, or monitoring levels of a 
therapeutic substance in captured colorectal epithelial cells to determine 
appropriate dosage levels in patients suffering from, for example, 
inflammatory bowel disease or other gastrointestinal disorders. 
In one embodiment of the invention, illustrated in the Examples below, the 
captured material, complexed with a specific binding reagent on a bead, is 
applied to a slide, stained with a staining material appropriate for 
viewing epithelial cells or membrane fragments thereofsuch as hematoxylin 
and eosin, and the stained material is viewed microscopically. 
Other methods of analysis of the colorectal epithelial cells or fragments 
thereof include immunostaining the cells or fragments. This analysis may 
be achieved, for example, by exposing the separated complex to a second 
specific binding reagent such as one of those described herein, having 
specificity for the colorectal epithelial cells or fragments thereof. The 
complex may then be exposed to a third specific binding reagent, such as 
an antibody, which may contain part of a signal detection system. One 
nonlimiting example of such a signal detection system involves a 
biotinylated antibody to which streptavidin (which has high affinity for 
biotin) coupled to horseradish peroxidase may be added. Substrate for the 
enzyme may then be added and the enzymatic reaction produces a visible 
stain and the colorectal epithelial cells or fragments thereof are 
detected. 
There are numerous methodologies for immunostaining or otherwise 
visualizing the captured colorectal epithelial cells or fragments thereof 
known in the art, and the present invention is not limited by any 
particular format or method of detecting the captured material. 
If the second specific binding reagent in the above-described analysis has 
specificity for dysplastic colorectal epithelial cells or fragments 
thereof, this analysis provides a method for detecting dysplastic 
colorectal epithelial cells or fragments thereof. 
Accordingly, the invention features a method of detecting dysplastic 
colorectal epithelial cells or fragments thereof in a stool sample. The 
method includes the steps of (a) contacting a stool sample with a first 
specific binding reagent having specificity for colorectal epithelial 
cells or fragments thereof to form first complexes comprising the first 
specific binding reagent and the colorectal epithelial cells or fragments 
thereof, (b) separating the first complexes from the sample, (c) 
contacting the first complexes, or colorectal epithelial cells or 
fragments thereof recovered from the first complexes, with a second 
specific binding reagent having specificity for dysplastic colorectal 
epithelial cells or fragments thereof to form second complexes comprising 
the second specific binding reagent and the dysplastic colorectal 
epithelial cells or fragments thereof, (d) separating said second 
complexes, and (e) determining the presence or absence of dysplastic 
colorectal epithelial cells in the stool sample. 
A third specific binding reagent may be used in a detection procedure, such 
as immunostaining, as described above. 
In accordance with the invention, the materials and conditions employed in 
this aspect of the invention may be those described above with reference 
to other preferred embodiments. 
The invention may be illustrated by way of the following examples. 
EXAMPLE 1 
Preparation of Stool Samples 
Donors were given collection kits containing a pre-weighed vial with 25 ml 
of transport medium and instructions to place approximately 2 grams (gm) 
of stool in the vial, shake gently to disperse the sample, and return the 
specimen to the laboratory on ice within 24 hours. Transport medium 
comprises a dispersing medium to which thimerosal (approximately 0.5 gm/L) 
is added as an anti-bacterial agent. Dispersing medium (pH 7.2) contains 
0.5 gm of sodium bicarbonate, 5 gm of BSA (heat shocked and fatty acid 
free), 500 mL of Puck's saline G, and 0.25 g N-acetylcysteine (Sigma 
A-9165). Puck's saline G (Puck et al., J. Exp. Med. 108:945 (1958)) 
contains, in a 10X solution, in gm/L: 0.16 calcium chloride, 4.0 potassium 
chloride, 1.5 monobasic potassium phosphate, 1.54 magnesium sulfate, 80.0 
sodium chloride, 1.54 dibasic sodium phosphate, 11.0 glucose, 0.05 phenol 
red. The 10X solution is prepared in deionized water and filter 
sterilized. To prepare the dispersing medium, the 10X solution is diluted 
with deionized water to 1X, and the sodium bicarbonate, N-acetyl cysteine, 
and bovine serum albumin are then added. Thimerosal is added to prepare 
the transport medium. pH for all solutions should be 7.2, and osmolality 
290-310 Mm/kg. 
Upon return to the laboratory, the sample was diluted to 250 mL with 
dispersing medium. The sample was then poured into a strainer bag, and 
dispersed in a Stomacher for 30 seconds at normal setting. After foaming 
settled down, the dispersed sample was poured into a 250 mL centrifuge 
tube and spun at (900.times.g) rpm for 10 minutes at 4.degree. C. The 
supernatant was poured off and the pellet resuspended in 60 mL dispersing 
medium. The pellet was disrupted with a pipette tip. Thirty mL of the 
sample was placed in each of two 50 mL tubes. To each tube 10 mL of 
Histopaue (room temperature) was added from the bottom up. The tubes were 
centrifuged at (210.times.g) rpm for 30 minutes at room temperature in a 
table top centrifuge with the brake off. Using a transfer pipette, the top 
layer was discarded until about 2-3 mL above the pellet interface (smooth 
white band). The remaining fluid and interface is then removed (nothing 
lower than interface is removed). Supernatants from both tubes were 
combined and resuspended to 150 mL with dispersing medium. The samples 
were centrifuged at (900.times.g) rpm for 10 minutes at 4.degree. C. and 
the pellet resuspended up to 10 mL in dispersing medium. The pellet was 
dispersed with an 18 gauge blunt needle prior to the next step. Five mL 
was removed from two percoll gradients with a transfer pipette, and 5 mL 
of the sample suspension was poured over each gradient. Percoll gradients 
were prepared as follows: 1 gm of sodium bicarbonate was added to 100 mL 
of distilled water. 9.6 gm of minimum essential medium, (MEM, Gibco BRL 
41500-034) was added. Ten gm of BSA (heat shocked and fatty-acid free) (BM 
100009) was mixed with 300 mL of distilled water. The BSA mixture was 
added to the bicarbonate/MEM solution. The solution was then added to 574 
mL of percoll and filled to 1000 mL with distilled water. The resulting 
solution was frozen at below -20.degree. C. for 48 hours. The percoll 
solution was thawed at 4.degree. C. for 48 hours before use. The solutions 
were usable for 2-3 weeks. 
The percoll gradients, to which 5 mL of sample suspension was added, were 
centrifuged at (800.times.g) rpm for 30 minutes at 4.degree. C. After 
centrifuging, the first 5 mL were discarded. The remaining liquid above 
the turbid band in both tubes was saved with a transfer pipette and 
resuspended together to 200 mL with cold PBS. This sample is centrifuged 
at (900.times.g) rpm for 10 minutes at 4.degree. C., the pellet was washed 
twice with PBS, and resuspended in dispersing medium at 1 mL/gm of 
original sample. 
The sample was then ready for testing. 
EXAMPLE 2 
Preparation of Immunomagnetic Beads 
Dynabeads (0.1 ml) (Dynal Dynabead M-450, 4.5 micron diameter, coated with 
sheep anti-mouse antibody) were vortexed and washed with 1 ml PBS by 
magnetically affixing beads to side of the washing tube. The beads were 
resuspended in 1 ml PBS for one minute and the wash step was repeated. The 
beads were then resuspended in 1 ml PBS and ready for use. 
EXAMPLE 3 
Preparation of Immunomagnetic Beads Coated with Antibody 
Monoclonal capture antibody was added to the resuspended Dynabeads (see 
Example 2) (6 .mu.g/25 .mu.l Dynabeads) and incubated at 4.degree. C. for 
30 minutes on a rocker device. The beads were incubated separately with 
antibodies to carcinoembryonic antigen (CEA), epidermal growth factor 
receptor (EGFR), and antibody PCA 33.28. The Dynabeads were washed to 
remove any free antibody with 1 ml of PBS 4 times as described above. The 
beads coated with the antibody were resuspended in 1 ml PBS. 
EXAMPLE 4 
Immunocapture Assay with Antibody Immobilized on Dynabeads 
A sample prepared as described in Example 1 was incubated with Dynabeads 
containing anti-CEA, anti EGFR, and PCA 33.28 prepared as described in 
Examples 2 and 3 at 4.degree. C. for 20 min. on a rocker device. The 
sample tube was placed in a magnetic chamber on ice for 15 min. The 
supernatant was discarded and the beads washed three times with 1 ml of 
PBS in a magnetic chamber (Dynal MPC-6) on ice for three minutes each. The 
Dynabeads contain the colorectal epithelial cells or fragments thereof 
complexed with the capture antibody. 
25 .mu.L of the bead suspension was smeared on a glass microscopic slide, 
air dried, and stained by an immunoperoxidase method for carcinoembryonic 
antigen as follows. The slide was incubated with a monoclonal antibody 
against CEA (Biodesign International) Catalog H45655M, clone 9201) for 30 
minutes at room temperature. The slide was washed and then incubated with 
biotin-conjugated goat anti-mouse IgG for 30 minutes at room temperature. 
After washing, the slide was incubated with peroxidase conjugated 
streptavidin for 30 minutes at room temperature. Following another washing 
step, the slide was incubated with hydrogen peroxide and amino ethyl 
carbazole for 15 minutes at room temperature. Slides were then lightly 
counterstained with hematoxylin and mounted with a coverslip, and examined 
microscopically and photographed. FIG. 1 is a photomicrograph of 
immunocaptured colorectal epithelial cells using the above-described 
technique. This confirms the epithelial lineage of the captured cells by 
virtue of the CEA immunostaining (red reaction product of the 
immunostaining procedure). 
EXAMPLE 5 
Immunomagnetic Isolation of Malignant Colorectal Epithelial Cells 
Cultured malignant colorectal epithelial cells (WIDR, available from ATCC) 
were spiked into stool from a patient without gastrointestinal disease at 
a concentration of 1.times.10.sup.6 cells/gm (approximate physiologic 
concentration). Spiking involves mixing 4.56 grams of stool with 500 .mu.L 
of a solution of 9.6 million cells/mL WIDR cells and 10 percent formalin 
to a volume of 27 mL. The spiked sample was dispersed in a Stomacher for 1 
minute at medium speed and allowed to settle at least 1 hour on ice. 
Dynabeads coated with anti-CEA, anti-EGFR, and PCA 33.28 were prepared as 
in Examples 2 and 3. Here 12 .mu.g antibody/500 .mu.L bead suspension was 
used. Dynabeads (450 .mu.L total in each tube) were added to sample 
suspension, and incubated in a rocker device for 20 minutes at 4.degree. 
C. Each sample was then diluted with 5 mL PBS. Samples were then placed in 
a magnetic chamber on ice for 15 minutes. Supernatants were discarded, 
pellets were washed 5 times with 3 mL PBS in the magnetic chamber for 3 
minutes. Supernatants were discarded and the beads resuspended in 1 mL 
PBS. Samples were smeared onto microscopic slides and stained. 
Hematoxylin and eosin staining involved: fixing in 1 percent 
paraformaldehyde for 10 minutes; washing with tap water for 2 minutes; 
exposing the hematoxylin for 2 minutes; washing with tap water for 2 
minutes; smearing with eosin (on towel--5-6 drops) and letting stand for 2 
minutes; 15 dips in 95 percent ethanol; 15 dips in absolute ethanol; 15 
dips in xylene followed by exposure to xylene for 2 minutes; and 
application of a coverslip. 
Slides were examined under a light microscope. 
FIG. 2 is a photomicrograph of immunocaptured malignant colorectal 
epithelial cells from a spiked stool sample stained with hematoxylin and 
eosin as described above. By virtue of the positive stain it shows, this 
Figure demonstrates that the contacting of a stool sample containing 
malignant colorectal epithelial cells with a specific binding reagent 
having specificity for colorectal epithelial cells in accordance with 
method of the present invention is capable of capturing the malignant (or 
dysplastic) cells from a stool sample. 
Other embodiments of the invention are within the scope of the appended 
claims.