Use of a BCR/ABL Fusion Protein
Specific Antibody in Cytometry
www.cellsignal.com
Randy Wetzel, Carrie Brown, Lori Lebel, Valerie Goss, and Bradley L. Smith • Cell Signaling Technology, Beverly MA 01915
9q+
9
INTRODUCTION: The BCR/ABL fusion protein that results from
the Philadelphia chromosome translocation drives chronic
myelogenous leukemia (CML) and subsets of other leukemias.
In this poster we describe the development and use of a mouse
monoclonal antibody that is specific for the p210 BCR/ABL
fusion protein. The antibody may be used with flow cytometry
as well as image cytometry to detect expression of the protein
in cell lines or patient samples.
22
22q
Reciprocal
Translocation
“Philadelphia Chromosome”
Abl
Bcr
Bcr-Abl
Antibody Epitope
CML
CNL (rare)
Bcr-Abl
Bcr-Abl
Bcr
Bcr-Abl
Bcr
Bcr-Abl
Bcr
Bcr-Abl
Bcr
Bcr-Abl
Bcr
Bcr-Abl
Bcr
Bcr-Abl
Bcr
Bcr-Abl
Bcr
Bcr
Bcr-Abl
p190
Bcr-Abl
p210
Bcr-Abl
p230
ALL, AML
Abl
Mab
METHODS: Peptide synthesis, monoclonal antibody development
and testing were perfomed at CST using standard techniques.
Phospho- specific and total antibodies were obtained from CST.
Flow cytometric analysis was performed on a FC500 flow cytometer.
3T3
A431
HeLa
Jurkat
LNCaP
SUPB15
3T3-Abl
49
K562
Western blot analysis of BCR/ABL anttibody versus BCR-=specific
antibody on eight cell lines. Left lane was probed with the BCR/ABL
antibody while the right lane was probed with the BCR antibody.
SUPB15 cell line expresses the p190 BCR/ABL protein.
Events
Normal Blood
CML Blood
Flow Cytometric Analysis of Bcr and Bcr-Abl in Cell Lines
250
Flow cytometric analysis
of Bcr-Abl antibody stain
in normal (orange) and CML
(blue) blood samples.
1.2
200
Bcr-Abl
Ratio
1
0.8
150
0.6
100
0.4
50
Bcr-Abl/Bcr Ratio
Mean Fluorescence Intensity
Bcr
FL2 Log
0.2
0
0
Jurkat
K562
SUP-B15
Ramos
HeLa
HIH3T3
Flow cytometric analysis of Bcr (blue) and Bcr-Abl (orange) antibody
stain in various cells lines. Red bars indicate ratio of Bcr-Abl to Bcr.
Highest ratio is in K562 cells, which express p210Bcr-Abl.
Jurkat
Ramos
K562
Immunohistochemical analysis of paraffin-embedded cells stained with the
BCR/ABL antibody.
SUMMARY: The BCR/ABL antibody does not detect the wild type BCR or c-ABL protein and may be used with flow cytometry as well
as image cytometry to detect expression of the protein. This antibody allows for a very fast, simple and cost effective method to identify
cells expressing the fusion protein. This method may help clinicians diagnose CML in patients, evaluate therapuetic effectiveness, and
monitor successfully treated patients for recurrance.