A Mouse IgM Monoclonal Antibody Recognizes Breast and Colon Cancer

For Peer ReviewHybridoma: http://mc.manuscriptcentral.com/hybridoma

A Mouse IgM Monoclonal Antibody Recognized Breast and Colon Cancer

Journal: Hybridoma Manuscript ID: HYB-2007-0531

Manuscript Type: Original Articles Date Submitted by the

Author: 24-Aug-2007

Complete List of Authors: Abu Bakar, Ainul; UPM Alitheen, Noorjahan; Universiti Putra Malaysia, Cell and Molecular Biology Hamid, Muhajir; UPM Ali, Siti Aishah; UKM Ali, Abdul Manaf; UPM

Keyword: Antibodies, Assay/Immunoassay, Cancer (carcinoma), Hybridoma, Immunofluorescence

Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801

Hybridoma

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A Mouse IgM Monoclonal Antibody Recognized Breast and Colon Cancer

Ainul Fajariah A Bakar,1 Noorjahan B Alitheen,2* Muhajir Hamid,1,3 Siti Aishah M Ali,4 and

Abdul Manaf Ali,1, 2

1Institute of Bioscience, 2Department of Cell and Molecular Biology, 3Department of

Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra

Malaysia, 43400 Serdang, Selangor, Malaysia. 4Department of Histopathology, Faculty of Medicine, Hospital Universiti Kebangsaan

Malaysia, Cheras, Kuala Lumpur, Malaysia.

ABSTRACT

In this study, we characterized the hybridoma clone C3A8, which is a fusion product between

splenic lymphoctes of Balb/c mice immunized with MCF7 breast carcinoma cells and SP2/0

myelomas. Recloning by limiting dilutions was performed and it was found that the clone was

very stable and secreting IgM monoclonal antibody (Mab) with κ light chain. Screening by

cell-ELISA assay showed that the Mab C3A8 react strongly to the breast (MCF7) and colon

cancer cell lines (HT29) but weakly cross-reacted with cervical cancer cell line (HeLa).

Analysis by flow cytometry and immunofluorescence staining revealed that the Mab C3A8

had specific binding ability with MCF7 and HT29 but not on HeLa cell line. The Mab C3A8

reacted positively with paraffin embedded tissues of human breast and colon cancer but there

were no positive reaction on normal tissues. Through western blotting, the Mab recognized a

55kDa protein, which was present in the extract of MCF7 and HT29 cell lines. Our results

demonstrated that Mab C3A8 could be used for basic and clinical research of breast and colon

cancer.

Keywords: Monoclonal antibody, MCF7, HT29, hybridoma, flow cytometry,

immunofluorescence.

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INTRODUCTION

Breast and colon cancer ranked first and third amongst cancers affecting women

reported worldwide. (1) The diseases are believed to have multiple etiologic factors, including

genetic and life-style. Commonly used screening methods for breast cancer involves breast

self-examination or mammography to detect any breast abnormalities. Although conventional

diagnosis modalities have improved technically in recent years, there are still of limited value

for detecting and distinguishing, small, viable tumor lesions from other tissues. (2) The

development of detection method using Mab that specifically and sensitively binds to tumor

marker has overcame the above limitations. There have been several reports of their used in

the diagnosis, localization and treatment of breast and colon cancer. (3-6) The murine anti-

B72.3 indium labeled Mab was approved by U.S. FDA for radioimmunodetection of primary

and recurrent colorectal carcinoma colon cancer. (7) Apart from that, the Mab B72.3 also

being studied for used in the detection of benign breast lesion. (8) Other tumor marker which

widely being used in colon cancer detection is the carcinoembryonic antigen (CEA). Started

as a diagnostic reagent in immunohistochemistry (9), the anti-CEA antibody (Fab’ fragment)

with technetium-99m (99Tc; arcitumomab); is then being evaluated as an imaging agent for

assessing the extent of abdominopelvic colorectal cancer recurrence or metastasis. (10) In 1998,

the U.S. FDA was approved the first humanized Mab known as anti-HER2 for the treatment

of breast cancer, which offer a new hope for patient diagnosed with the disease (7).

Unfortunately, HER2-overexpression only involved one quarter of the human breast

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carcinomas. (11) Therefore, we still need great efforts to find other candidates that can be

potentially used in immunodiagnosis and immunotherapy of the disease.

In previous study (12), three hybridoma clones named C2E7, C3A8 and 10A2 were

isolated. The C2E7 clone was characterized and found that the Mab produced by this clone is

able to discriminate between breast cancer cells and a series of normal neoplastic tissues.

However, the C3A8 clone has not yet been characterized. Here, we describe the

characterization of Mab produced from C3A8 clone that specifically bind to breast and colon

cancer cells. The Mab showed a strong recognition towards paraffin embedded breast and

colon cancer tissues in immunohistochemical study.

MATERIALS AND METHODS

Cell lines and cultures

The human tumor cell lines MCF-7 (breast), HeLa (cervical) and HT-29 (colon)

[ATCC No: HTB-22, CCL-2 and HTB-38] were obtained from the American Type Culture

Collection (ATCC, Rockville, MD) and were grown in a complete growth medium [CGM:

RPMI-1640 medium (Sigma, USA), supplemented with 10% (v/v) of fetal calf serum (FCS)

(GIBCO,USA) and 1% (v/v) of 100 unit/mL of penicillin and streptomycin (GIBCO, USA)]

at 37ºC in an atmosphere of 5% CO2. The cells were subcultured by splitting the culture at 1:8

ratios with CGM after trypsinization with 0.25% of trypsin (GIBCO, USA).

Recloning of C3A8

The hybridoma clone C3A8 (from Animal Tissue Culture Lab, Universiti Putra

Malaysia) was revived from liquid nitrogen storage by transferring the thawed cells into 10

mL of CGM. After their viability became almost 100%, the hybridoma cells were subcloned

by limiting dilution. Supernatants from wells with the colonies covering more than one-third

area of the well were assayed for presence of monoclonal antibody using ELISA method.

Class and subclass determination was performed using ImmunoPure® Monoclonal Antibody

Isotyping Kit (HRP/ABTS) (Pierce, USA).

Screening for antibody-producing hybridomas by cell- ELISA

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The presence of Mab in the hybridomas supernatant were assayed by cell- ELISA

method as described by Ali et al. (1996) (12,13). MCF-7 cells were diluted with RPMI-1640 at

concentration 1x106 cells/mL. The cell suspension was dispersed at 200 µL per well in 96-

well ELISA (Nunc, Denmark) plate and maintained at 37°C in CO2 incubator. When the cells

have reached the confluent stage, the medium was removed and the monolayer cells was

washed with phosphate buffer saline (PBS) at pH 7.4 before being treated with 0.06% (v/v) of

glutaraldehyde (Sigma, USA) for 20 min at room temperature. After the glutaraldehyde was

discarded, 100 µL of 4% (w/v) of skimmed milk was added into each well as blocking

solution and the plate was incubated at 37°C for one hour. The plate was washed with PBS at

pH 7.4 for three times before a volume of a 100 µL of hybridoma supernatants from each

different clone was added into well as primary antibody. After incubation at 37°C for another

one hour, the wells were washed three times with PBS and 100 µL of the enzyme horseradish

peroxidase conjugated with goat anti-mouse immunoglobulin (KPL, USA) (1:1000) and

pipetted into each well. The plate was then incubated at 37°C for one hour. Finally, the wells

were washed with PBS and 100 µL of ready to use (ABTS) substrate (KPL, USA) was added

in the dark into each well. After 30 minutes of incubation at room temperature, 100 µL of 1%

(w/v) sodium dodecylsulphate (SDS) (Amresco, USA) was added to each well to stop the

enzymatic reaction and the optical density was read at 405 nm by using the ELISA Reader

(µQuantum). A selected C3A8 subclone, which secreted the highest Mab titer, was preceded

with in vivo Mab production.

Generation of ascites fluid (In vivo)

Six-to 8-week old female BALB/c mice, purchased from Institute for Medical

Research (IMR), Kuala Lumpur were primed with 0.5 ml pristane (2, 6, 10, 14-

tetramethylpentadecane) (Sigma, USA) into peritoneal cavity. After 14 days, the pristane

treated mice were injected with 5 x 106 of hybridoma cells per mouse. Growth may take 7 to

21 days and the mice were checked daily in the later stages of tumor growth. The tumor

growth mouse abdomen was first swabbed with 70% alcohol. A 211/2G needle (Becton

Dickinson, USA) was inserted into the swelling mouse abdomen (peritoneum) carefully and

the ascitic fluid was collected into a sterile centrifuge tube.

Antibody cross-reactivities

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The antibody cross-reactivities on HT29 and HeLa cell lines was performed using cell

ELISA method as described above. Ascites fluids containing Mab C3A8, ranging from 1/2-

1/256 was used as primary antibody.

Immunofluorescence

Cell lines for immunocytochemistry studies were grown in 8 chamber tissue culture

microscope slides (Nunc, Denmark) until almost confluent. The media was aspirated and the

adherent cells were washed gently with PBS and fixed with cold methanol (HmbG chemicals)

for 20 min at room temperature. Following fixation, the adherent cells were re-washed with

PBS, and 200 µL of diluted ascites fluid (1:100 in PBS) was added to each well. After

incubation for three hours at room temperature the cells were gently washed twice in PBS and

200 µL of fluorescein-isothiocynate (FITC)-labeled goat anti-mouse IgM (Chemicon, USA)

(1:50 in PBS) added for one hour incubation at room temperature. The adherent cells were

then washed in PBS twice. After removal of the chamber lining and sealing gasket of the

microscope slides, cover slips were applied and the slides were viewed under fluorescence

microscope (Leica) with combination of excitation filter and barrier filter of 450-490 nm and

long pass filter 520 nm.

Flow-cytometry analysis

Trypsinized MCF-7 cells were diluted into a concentration of 1 x 107 cells/mL and

washed twice with PBS. The cells were then incubated for one hour at 4°C with ascitic fluid

diluted 1:10 in PBS. After washing three times with PBS, fluorescein-isothiocynate (FITC)-

labeled goat anti-mouse IgM (Chemicon, USA) diluted 1:20 in PBS was added and further

incubated for one hour at 4°C. Finally, the cells were washed again three times with PBS, and

the cells were analyzed with a fluorescence-activated cell sorter (Coulter® Epics® AltraTM

Flow Cytometer, Coulter Corporation). In negative control experiments, ascites fluids were

omitted.

Immunoperoxidase staining

Briefly, the paraffin-embedded human tissue blocks including breast carcinoma and

colorectal carcinoma were cut into 4-µm sections and these slides supplied by the Department

of Pathology, Hospital Universiti Kebangsaan Malaysia. Immunoperoxidase staining was

carried out according to the instruction sheet issued by the manufacturer (DakoCytomation,

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Denmark). The tissue slides were deparrafinization for minimum of 60 minutes in an oven at

60°C to ensure that any moisture trapped under the tissue section is completely eliminated by

melting of the paraffin and evaporation of the water droplets. The slides then being soaked in

Xylene (Dako, Denmark) (Analytical grade) four times for five minutes each. After being

washed with rinse buffer (tris-buffered saline, TBS) pH 7.4 for five times, the slides were then

treated in 100, 70 and 30% of ethanol and deionized water consecutively for two times for one

minute each. The sections then were treated with DakoCytomation Target Retrieval Solution

(Dako, Denmark), pH 9, for 20 min at 95°C to retrieve the antigen. The slides were then

treated with Dual Endogenous Enzyme (Dako, Denmark) for 10 minutes to block endogenous

peroxidase activity, washed with rinse buffer five times and pre-incubated with Mab C3A8

from ascitic fluid diluted 1:100 for 30 minutes before being washed again as described above.

The slides were then incubated with Labelled Polymer-HRP (Dako, Denmark) for 30 minutes

and washed with rinse buffer for five times. Thereafter, the slides were reacted with 3, 3’-

Diaminobenzidine (DAB) (Dako, Denmark) chromogen solution for five minutes, washed and

counterstained with Hematoxylin (Dako, Denmark) for one minute. The slides were washed,

removed from the water before applied with mounting media (DPX) (Dako, Denmark) and

cover slipped for microscopic examination. All the procedures were carried out at room

temperature.

SDS-PAGE & Western Blot

MCF7 and HT29 cells (1x107) were lysed in 350 µL of ice-cold of 1X cell lysis buffer

(Cell Signaling Technology, USA). Lysis was achieved by gentle rotation on ice for 20

minutes. The extracts were then centrifuged at 14,000 x g, 10 min to remove cell debris.

Supernatant or lysates were collected and kept at -80°C. Sodium dodecyl sulfate

polyacrylamide gel electrophoresis (SDS-PAGE) was performed as previously described

(Laemmli, 1970). Ten mL of protein sample (10 µg) was heated on heating block at 98°C in

the same volume of 2X loading buffer (Bio Basic, USA.) for 5 min. Proteins were resolved by

12.5% SDS-PAGE gel and transferred to nitrocellulose membrane by using an electro-blotter

(Hoefer, USA) at 70mA for 45 min. Proteins transfered were monitored with prestained

protein marker (Crystalgen, USA.). The positive band was detected following the instruction

manual included in the WesternBreeze® Chromogenic Immunodetection Kit (Invitrogen,

USA) which contains complete, optimized, ready-to-use or ready-to-dilute reagents. The

membrane was blocked for one hour with blocking buffer to avoid non-specific binding.

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Ascites fluid containing Mab C3A8 at 1:10 dilution was used to incubate the membrane for

one hour. The membrane was washed with washing buffer as before. After incubated with

secondary antibody solution the membrane washed again. Immunoreactive bands were

detected by chromogenic substrate until purple to dark blue bands developed on the

membrane (1-60 min).

RESULTS

Establishment of hyrbidoma C3A8 and generation of ascites fluids

During the third time of limiting dilution, several of C3A8 subclones were isolated. A

subclone of C3A8 hybridoma which secreted the highest Mab titer was selected for ascites

production. This subclone is an IgM with kappa light chain and showed the characteristic of a

stable hybridoma clone as it could continuously produce specific Mab after being cultured for

8 months. In order to get higher yields of Mab, the hybridoma C3A8 was injected into mice

primed with pristane for in vivo production of Mab. The ascites fluids containing the Mab

C3A8 were used in all next experiments performed by this study.

Specificity of Mab C3A8 against different cancer cell lines

As shown in Fig. 1, the Mab C3A8 bound strongly to MCF7 and HT29 but weakly to

HeLa cell line. At antibody dilution 1:2, the O.D value (405nm) for MCF7, HT29 and HeLa

cell lines are 2.813, 1.986 and 1.029, respectively. The ELISA results were further confirmed

by immunofluorescence staining which showed a bright fluorescence labeling on the

cytoplasms of MCF7 and HT29 (Fig. 2A & Fig. 2B). The same experiment was performed on

HeLa cell line but there is no positive result (Fig. 2C). In order to determine the percentage of

positive cells labeled with the Mab C3A8, flow cytometry analysis was performed. From the

experiments, the MCF7 cell line presented the highest percentage of labeled cells with the

Mab C3A8 with 80.54%, followed by HT29 and HeLa cell lines with 49.03% and 2.75%,

respectively (Fig. 3).

Tissue specificity of Mab C3A8

The results for the assessment of antigen localization on all the tissue sections were

interpreted by a pathologist. A strong cytoplasmic staining for Mab C3A8 was observed in 28

of the 31 ductal breast carcinoma specimens (Fig. 4A) and 30 of the 34 fibroadenoma tissues

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(Fig. 4B). Interestingly, using the same Mab C3A8, immunohistochemical staining showed

that 4 of the 4 mucinous adenocarcinoma colon tissues were positively stained on the

cytoplasms (Fig. 4C). None of the sample from normal breast and colon tissues, ducal breast,

prostate and lung carcinoma analyzed was stained with this Mab.

Western Blotting

Through western blotting, this experiment indicated the presence of a single band of

Mr 55 kDa recognized by Mab C3A8 that appeared as single band on the membrane (Fig.

5A). Analysis with HT29 protein extracts also revealed the same molecular weight of protein

recognized by Mab C3A8 (Fig. 5B).

DISCUSSION

As was mentioned earlier, hybridoma clone C3A8 was generated against the human

breast carcinoma cell line (MCF7), isolated from pleural effusion of a patient with metastatic

mammary carcinoma. (13) According to literature (12), MCF7 was chosen as immunogen

because because it is highly immunogenic and has distinct antigens that elicit antibodies for

breast cancer. (14,15-20) The typical dome shaped cells in the MCF7 monolayer showed that they

still function as mammary epithelial cells. (21) In this study, hybridoma clone C3A8 was

revived from liquid nitrogen storage and recloned by limiting dilutions. Recloning was done

to confirm the monoclonality of the hybridoma as there is a relatively high probability of

growth on non-producer variants as a result of chromosomal loss. (16) After being cultured for

8 months, we found that the hybridoma clone C3A8 is a stable clone as it could continuously

secreting specific Mab against MCF7. The reactivity profiles of Mab C3A8 against different

types of cancer cell lines/tissues in varying assay systems were evaluated and potential

antigenic determinants were investigated. From the experiments, it showed that the Mab

C3A8 specifically binds to a common epitope present on MCF7 and HT29 cell lines. A weak

cross-reactivity also occurred against HeLa cell line when screening by cell-ELISA but not in

immunoflourescence and flow cytometric analysis. Cross-reactivities are common as in

previous studies, other Mab against the MCF7 cell lines have been shown to cross-react with

other cancer cell lines and tissues. (22-24)

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To extend the possible use of the Mab C3A8 in cancer approaches, we screened the

reactivity of the Mab C3A8 on various cancer and normal human tissues. Earlier studies

showed that the Mab C2E7 which was also rose against MCF7 cell lines, reacted to lobular

breast and fibroadenoma benign breast tissues at the cytoplasmic region. (12) The Mab C2E7

did not give any staining when tested against normal mammary tissues, stomach showing

metaplasia, ductal papilloma of the breast and cervical carcinoma. However, the epitope

recognized by Mab C2E7 is remained unknown. In this study we found that this Mab C3A8

positively reacted with fibroadenoma benign breast tissues, breast ductal carcinoma and

mucinous adenocarcinoma of colon tissues but not the normal breast and colon tissues.

Immunoperoxidase staining results on breast lobular, lung and cervical cancer tissues were

also negative. Other example of Mab generated against a human metastatic mammary

carcinoma is B72.3. This Mab recognizes a high molecular weight antigen, designated as

Tumor-Associated Glycoprotein (TAG)-72 which is rich in carbohydrate. It was reported that

Mab B72.3 has identified the (TAG)-72 in most mucin-producing adenocarcinomas and its

limited reactivity with non-malignant tissues. (25)

To determine the epitope recognized by Mab C3A8, western blotting was performed.

The Mab C3A8 recognized the same molecular weight of protein, which is 55kDa from

extracts of MCF7 and HT29 cell lines. This explained why Mab C3A8 reacted positively with

both breast and colon cancer cells/tissues in previous experiments described above. All the

results in our study, suggested that the Mab C3A8 is potentially to be used in diagnosis,

detection, monitoring and therapy of breast and colon cancer.

ACKNOWLEDGEMENTS

We thank the Ministry of Science Technology & Innovation (MOSTI), Malaysia for

supported this project under grant 54953.

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Address reprint request to: Noorjahan B. Alitheen, Ph.D. Dept. of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400, Selangor, Malaysia. Tel: + 603-8946 7471 Fax: +603-8946 7510. Email: [email protected]

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FIG. 1. Specificity of the Mab C3A8 reactivity against different of human cancer cell lines by ELISA. Note: RPMI-1640 medium without serum was used as negative control with the

O.D for negative control is 0.094. 254x190mm (72 x 72 DPI)

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FIG. 2A. Immunofluorescence reactivity of Mab C3A8 on MCF7; (400X). Note that the Mab C3A8 strongly stained only the cell cytoplasm (C).

254x190mm (72 x 72 DPI)

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FIG. 2B. Immunofluorescence reactivity of Mab C3A8 on HT29; (400X). Note that the Mab C3A8 strongly stained only the cell cytoplasm (C).

254x190mm (72 x 72 DPI)

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FIG. 3A. Staining of ductal breast carcinoma (400X) with Mab C3A8 . Note the Mab C3A8 strongly stained only the cell cytoplasm (C).

254x190mm (72 x 72 DPI)

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FIG. 3B. Staining of fibroadenoma (200X) of colon with Mab C3A8. Note the Mab C3A8 strongly stained only the cell cytoplasm (C).

254x190mm (72 x 72 DPI)

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FIG. 3C. Staining of mucinous adenocarcinoma of colon with Mab C3A8 (400X). Note the Mab C3A8 strongly stained only the cell cytoplasm (C).

254x190mm (72 x 72 DPI)

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FIG. 4A. Surface antigen expression on MCF7 cell line was examined by flow cytometry, using Mab C3A8. Note that data with positive-stained cells are shown in red line; negative

control (Mab C3A8 was omitted) are shown in black. 254x190mm (72 x 72 DPI)

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FIG. 4B. Surface antigen expression on HT29 cell line was examined by flow cytometry, using Mab C3A8. Note that data with positive-stained cells are shown in red line; negative

control (Mab C3A8 was omitted) are shown in black. 254x190mm (72 x 72 DPI)

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FIG. 4C. Surface antigen expression on HeLa cancer cell line was examined by flow cytometry, using Mab C3A8. Note that data with positive-stained cells are shown in red

line; negative control (Mab C3A8 was omitted) are shown in black. 254x190mm (72 x 72 DPI)

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FIG. 5A. Immunobloting indicate 55 kDa protein reacted with Mab C3A8 from extracts of MCF7 cell line. Lane 1: high range prestained protein marker; and lane 2, 3, 4 & 5: protein

extracted from MCF7 cell line (run in four replicate). 254x190mm (72 x 72 DPI)

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FIG. 5B. Immunobloting indicate 55 kDa protein reacted with Mab C3A8 from extracts of HT29 cell line. Lane 1 & 2: HT29 extracts (run in duplicate) and lane 3: high range

prestained protein marker. 254x190mm (72 x 72 DPI)

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LIST OF FIGURES

FIG. 1. Specificity of the Mab C3A8 reactivity against different of human

cancer cell lines by ELISA. Note: RPMI-1640 medium without serum was used as

negative control with the O.D for negative control is 0.094.

FIG. 2A. Immunofluorescence reactivity of Mab C3A8 on MCF7; (400X). Note

that the Mab C3A8 strongly stained only the cell cytoplasm (C).

FIG. 2B. Immunofluorescence reactivity of Mab C3A8 on HT29; (400X). Note

that the Mab C3A8 strongly stained only the cell cytoplasm (C).

FIG. 3A. Staining of ductal breast carcinoma (400X) with Mab C3A8 . Note the

Mab C3A8 strongly stained only the cell cytoplasm (C).

FIG. 3B. Staining of fibroadenoma (200X) of colon with Mab C3A8. Note the

Mab C3A8 strongly stained only the cell cytoplasm (C).

FIG. 3C. Staining of mucinous adenocarcinoma of colon with Mab C3A8

(400X). Note the Mab C3A8 strongly stained only the cell cytoplasm (C).

FIG. 4A. Surface antigen expression on MCF7 cell line was examined by flow

cytometry, using Mab C3A8. Note that data with positive-stained cells are shown in

red line; negative control (Mab C3A8 was omitted) are shown in black.

FIG. 4B. Surface antigen expression on HT29 cell line was examined by flow

cytometry, using Mab C3A8. Note that data with positive-stained cells are shown in

red line; negative control (Mab C3A8 was omitted) are shown in black.

FIG. 4C. Surface antigen expression on HeLa cancer cell line was examined by

flow cytometry, using Mab C3A8. Note that data with positive-stained cells are

shown in red line; negative control (Mab C3A8 was omitted) are shown in black.

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For Peer Review

FIG. 5A. Immunobloting indicate 55 kDa protein reacted with Mab C3A8 from

extracts of MCF7 cell line. Lane 1: high range prestained protein marker; and lane 2,

3, 4 & 5: protein extracted from MCF7 cell line (run in four replicate).

FIG. 5B. Immunobloting indicate 55 kDa protein reacted with Mab C3A8 from

extracts of HT29 cell line. Lane 1 & 2: HT29 extracts (run in duplicate) and lane 3:

high range prestained protein marker.

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A Mouse IgM Monoclonal Antibody Recognizes Breast and Colon Cancer

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Transcript of A Mouse IgM Monoclonal Antibody Recognizes Breast and Colon Cancer