Phosphoprotein Detection

Bio-Plex suspension array system®

tech note 5590

Detection of Genetic Abnormalities in Cancer Cells
Using Bio-Plex Immunoassays
Andrew Romano, Brad Smith, and Reggie Prioli, Cell Signaling
Technology, Inc., 3 Trask Lane, Danvers, MA 01923 USA

Introduction
Receptor tyrosine kinases initiate a tightly controlledcell-signaling network that regulates multiple biologicalprocesses. Aberrant activation of such networks plays
significant roles in both development and treatment of avariety of cancers (Amann et al. 2005). Genetic abnormalities
can further deregulate these networks and confer alteredresponses to treatment drugs. For example, mutations in theepidermal growth factor receptor (EGFR) gene can lead toeither increased sensitivity or resistance to EGFR-targeteddrugs. Likewise, mutations within the Bcr-Abl gene abolishresponsiveness to Gleevec (imatinib mesylate, Novartis)
treatment in chronic myelogenous leukemia (CML). Assaysthat can detect such mutations in patient samples would
facilitate selection of the optimal treatment regimen andenable monitoring of the cancer during therapy.

Streptavidin-PEBiotinNL858R

L858R
detection antibody

CBead

Total EGFR
capture antibody

Streptavidin-PE

We developed two sets of capture and detection antibodies
specific to the L858R point mutation in EGFR and to
Bcr-Abl translocations. In this study, we demonstratespecificity and, for Bcr-Abl, limits of detection of theseaberrations in cancer cells. We use Bio-Plex bead-based
immunoassays and western blotting for comparison.

BiotinTotal c-Abl
detection antibody

Methods
Bio-Plex Assays

Bio-Plex immunoassays detecting the L858R point mutationin EGFR and fusion proteins resulting from translocations ofthe Bcr-Abl genes were developed. The EGFR mutation assay
was developed using a capture antibody reactive with EGFR
regardless of its mutation status, and a detection antibody
specific for the aberrant form of the receptor (Figure 1).
Bcr-Abl was captured with an antibody to Bcr and detectedwith anti-c-Abl antibody. Cell Signaling Technology, Inc.
provided all antibodies.

BeadTotal Bcr
capture antibody

Fig. 1. Method of detection of mutated EGFR (top) and translocated
Bcr-Abl (bottom) using Bio-Plex bead-based immunoassays. In eachcase, detection requires two specific antibodies: one to capture total proteinon the bead, the other to detect the specific abnormality. Antibody pairs weredeveloped to detect EGFR mutation at L858R and Bcr-Abl fusion proteins ofvarying lengths. Figure is not drawn to scale.

Reactions were performed in 96-well, filter-bottomed
plates according to the Bio-Plex phosphoprotein detection
instruction manual. Briefly, 2,500 beads coated with theappropriate capture antibody were added to each well in
the plate and incubated overnight with lysates from oneof eight cell lines obtained from American Type Culture
Collection (ATCC) (Table 1). Cells were homogenized bysonication in cell lysis buffer. Following washing, detection
antibodies were added and the plates incubated for 30 min.
The beads were then washed and incubated for 5 min in
streptavidin R-conjugated phycoerythrin. The beads werewashed again and analyzed using the Bio-Plex system array
reader. Western blot analyses of identically treated cell lysates
were performed following protein separation by SDS-PAGE
and transfer to nitrocellulose membranes.

Table 1. Cell lines used to test detection of EGFR point mutation andBcr-Abl translocation.
Trait

Cell LineEGFR		
Wild type
NCI-H358
L858R point mutation
NCI-H1975
Bcr-Abl		No translocation
THP-1
b2a2**
T1
b3a2**
K-562
e1a2**
SUP-B15c-Abl positive
A-204
EGFR-wild; Bcr-Abl-normal HeLa

Cancer TypeNSCLC*
NSCLC
AML*
CML*
CML
CML
Rhabdomyosarcoma
Cervical cancer* 	NSCLC, non-small cell lung cancer; AML, acute myelogenous leukemia;
CML, chronic myelogenous leukemia.
** See Figure 2.

BcrChromosomal abnormalities can create a variety of Bcr-Abltranslocations. Depending on translocation length, this resultsin Bcr-Abl fusion proteins of different composition (Figure 2),
which can present a challenge to detection by antibodies. To
test our antibody performance, we examined three cell lines
expressing differing fusion proteins: e1a2, b2a2, and b3a2,
as well as c-Abl alone and Brc-Abl negative as controls (seeTable 1 for names of cell lines).

Ablb2a2b3a2
e1a2
Bcr-AblResults and Discussion
EGFR Mutation

The L858R mutant-specific antibody pair preferentiallyrecognized the L858R point mutation in EGFR (Figure 3).
The results corroborated western blotting analysis using theL858R mutant-specific antibody.

Fig. 2. Bcr-Abl fusion protein isoforms tested in this study.
Translocation can result in fusion proteins containing differing Bcr andAbl lengths. Three such isoforms, b2a2, b3a2, and e1a2, were tested
using a single antibody pair.

18,000

Bcr-Abl Chromosomal Translocations

The antibody pair used in the Bio-Plex immunoassay detected
all three Bcr-Abl isoforms tested (Figure 5). Western blot
analysis using a Bcr-specific antibody yielded similar results.

14,00012,000MFI

Limit of detection, as determined by the minimum detectable
number of cells/ml, was calculated using K-562 lysates.
The Bio-Plex assays reliably detected Bcr-Abl at levels as
low as 165 cells/ml (Figure 4A), about 4 orders of magnitude
more sensitive than our results for western blotting(~2 x 106 cells/ml, Figure 4B). The Bio-Plex assay detected
Bcr-Abl at densities from 165 cells/ml to 5 x 106 cells/ml.

16,00010,000
8,000
6,000
4,000
2,0000

Wash buffer		

NCI-H1975(L858R mutation)

NCI-H358(Wild type)

HeLa(Wild type)Fig. 3. Detection of the L858R point mutation in EGFR protein in lysatesfrom three cell lines. Line NCI-H1975 harbors the mutation; NCI-H358
and HeLa lines are wild type. Bio-Plex assays (top) and western blotting (bottom)
yielded similar results.

© 2007 Bio-Rad Laboratories, Inc.

Bulletin 5590

Conclusions
Antibody pairs reactive with EGFR point mutation
demonstrated suitable specificity for use in diseasemanagement and monitoring.

A2,500R2 = 0.9848

2,000

The Bio-Plex immunoassay for the detection of Bcr-Abltranslocations was more sensitive than western blotting by
about 4 orders of magnitude. The Bcr-Abl specific antibody
pair recognized all three Bcr-Abl isoforms tested.

MFI

1,500

1,000

500
394.5

Limit of detection (wash buffer + 2 standard deviations)

00200

400

600

800
1,000
Number of cells

1,200

1,400

1,600

B

Reference

16,000

Amann J et al., Aberrant epidermal growth factor receptor signaling
and enhanced sensitivity to EGFR inhibitors in lung cancer, Cancer Res
65, 226–235 (2005)

14,00012,00010,000ATCC is a trademark of American Type Culture Collection. Cell Signaling
Technology and the Cell Signaling Technology logo are trademarks ofCell Signaling Technology, Inc. Gleevec is a trademark of Novartis AG.
The Bio-Plex suspension array system includes fluorescently labeled
microspheres and instrumentation licensed to Bio-Rad Laboratories, Inc.
by the Luminex Corporation.

MFI

8,000
6,000
4,000
2,0000We successfully developed assays capable of detecting
genetic abnormalities in cancer cells using specific antibodies
and the Bio-Plex platform. The ability to multiplex genetic
analysis will enable the precise and rapid evaluation of themutational status of research and clinical tissue samples.

0123456Information in this tech note was current as of the date of writing (2007)
and not necessarily the date this version (rev A, 2007) was published.

Number of cells/ml (x 106)Fig. 4. Limits of detection for Bcr-Abl translocations in K-562 cells.
A, Bio-Plex assays detected translocations at concentrations as low as
165 cells/ml; B, upper limit of detection for Bio-Plex assays (upper scan) was
5 x 106 cells/ml; western blotting (lower image) lower limit of detection was
2 x 106 cells/ml.

CST antibodies exclusively developed
and validated for Bio-Plex phosphoprotein
and total target assays.

10,000
9,000
8,000
7,000
MFI

6,000
5,000
4,000
3,000
2,000
1,0000

Wash buffer				

SUP-B15(e1a2fusion)

CML-T1(b2a2fusion)

K-562(b3a2fusion)

A-204(c-Ablpositive)

THP-1HeLa(Bcr-Abl (Bcr-Ablnegative) negative)

Fig. 5. Bio-Plex assay (top) and western blot (bottom) detection of three
Bcr-Abl isoforms. The isoforms (see Figure 2) were detected in three CML
cell lines and absent from three control cell lines.

© 2007 Bio-Rad Laboratories, Inc.

Bulletin 5590

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Life ScienceGroup

Bulletin 5590

US/EG

Rev A

07-03490807

Sig 1106