Vol.1, No.4, 325-329 (2009)
SciRes Copyright © 2009 Openly accessible at http://www.scirp.org/journal/HEALTH/
Enhanced immuno-detection of shed extracellular
domain of HER-2/neu
Sinang Chourb, Brian C. Mackness, Leslie R. Farris, Melisenda J. McDonald
Biochemistry Program, Chemistry Department, University of Massachusetts, Lowell, USA; Melisenda_McDonald@uml.edu.
Received 10 October 2009; revised 20 November 2009; accepted 1 December 2009.
HER-2/neu oncogene is over-expressed and
amplified in patients associated with metastatic
breast cancer. An increased level (>15 ng/mL) in
the shed extracellular domain (sECD-HER 2/neu)
is indicative of the potential presence and as-
sociated progression of this disease. A fluo-
rescent ELISA incorporating the newly devel-
oped ALYGNSA antibody-orientation system
revealed a 10-fold increase in sensitivity (0.63
ng/mL) of sECD-HER 2/neu when compared to a
control standard ELISA kit (7.5 ng/mL). This
enhanced mode of detection has the potential to
not only address breast and other cancers per
se but also permit an in depth evaluation of
“shed extracellular domains”, in general, and
the role of these “proteolytic derived factors” in
physiological signalling at normal levels.
Keywords: HER-2/neu; Shed Extracellular Domain;
C-erbB2; Breast Cancer Marker; ELISA; Fluores-
cent immunoassay; ALYGNSA
Breast cancer, which is the most diagnosed form of can-
cer in women, will account for 27% of cancer cases in
2009 according to the American Cancer Society. The
human epidermal growth factor HER-2 oncogene (c-
erbB-2) has been implicated in this disease [1-4]. The
HER-2/neu oncoprotein is amplified and over-expressed
in 25 to 30 percent of patients with aggressive breast
cancer. Further, the full-length receptor (p185HER2)
undergoes a proteolytic cleavage resulting in the release
of the soluble shed ECD/HER2 (sECD-HER 2/neu)
fragment. The resultant truncated intracellular form con-
taining the kinase domain is associated with enhanced
signaling activity and consequently contributes to me-
tastatic breast cancer [5]. The sECD/HER 2/neu frag-
ment from the surface of breast cancer cells once shed
into the blood of individuals can be quantified, making a
useful breast cancer biomarker.
Measured levels of the sECD-HER 2/neu protein
greater than 15 ng/ml were indicative of the potential
presence and the associated progression of primary tu-
mors to metastatic breast cancer [4,6,7]. Most commer-
cially available assays use this concentration as a focal
point leaving smaller amounts undetected. Since the over
expression of HER-2/neu oncogene is a useful tool as a
prognostic and predictive marker for breast cancer, de-
velopment of a more sensitive biomarker assay appeared
ideal for monitoring the progression, the early recurrence
of metastatic breast cancer, and the response to therapy
[5,8,9]. A more sensitive assay would also allow for the
establishment of an individual’s baseline level of sECD-
HER-2/neu protein through which more accurate detec-
tion of unexpected increase might be achieved. Finally,
an improved assay may be useful to monitor the normal
signaling activity of the Her-2/neu pathway.
A current, rapid method to detect Her-2/neu protein in
biological fluids, such as serum, is the Enzyme-Linked
Immunosorbant Assay (ELISA) [10,11]. A typical com-
mercial HER-2/neu ELISA assay uses the “sandwich”
principle, where a capture antibody is directly adsorbed
onto a substrate. The detector antibody is labeled with an
enzyme, which upon addition of the substrate, produces
a colored product quantifiable by absorbance analysis.
In this study, an assay was developed for the detection of
the sECD-HER-2/neu protein utilizing the ALYGNSA
system consisting of a protein biolinker (Protein G’) and
poly (methyl methacrylate) or PMMA, a thermoplastic
polymer. The unique interaction of Protein G’ with
PMMA has been demonstrated to improve human IgG
capture antibody alignment/orientation [12] and provide
greater sensitivity in detection of cancer biomarkers
CA-125 [13] and PSA [14].
In this report, this system has been shown to detect an
order of magnitude lower level of (sECD-HER 2/neu)
than the commercial ELISA kit counterpart.
2.1. ELISA Assay Method
Microwells coated with mouse monoclonal anti-HER-2/-/
S. Chourb et al. / Natural Science 1 (2009) 325-329
SciRes Copyright © 2009 Openly accessible at http://www.scirp.org/journal/HEALTH/
Siemens ELISA
y = 0.0640x + 0.240
= 0.999
0510 1520 25 3035 40
sECD-HER 2/neu [ng/mL]
Figure 1. Siemens sECD-HER 2/neu ELISA: Re-
sults for assay (closed squares) and baseline (open
squares) points. The standard deviation values are
presented in Table 1. The LOD was determined by
the blank value +2 times the standard deviation.
The intra-assay variability (%CV) or [(standard
deviation / mean)*100] was calculated based on
the difference in duplicate measurements. The
highly linear relationship between the standards
was confirmed by the coefficient of determination
(R2) value of 0.998. Each data point represents the
average of eight replicates.
y = 1010x + 7370
= 0.997
051015 20 25
sECD-HER 2/neu [ng/mL]
Relative Fluorescent Units
Figure 2. sECD-HER 2/neu ALYGNSA: Results for
assay (closed squares) and baseline (open squares)
points. The standard deviation values are presented in
Table 2. As with the Her-2/neu ELISA, the
ALYGNSA method showed a highly linear response
as indicated by the R2 value of 0.997. Each data point
represents the average of six replicates.
neu protein antibody, HER-2/neu reference standards,
sample diluent, detector antibody, substrate diluent,
conjugate diluent, conjugate concentrate, substrate tablet,
substrate diluent and stop solution were used in the
HER-2/neu colorimetric ELISA (Siemens). The ELISA
assay was performed according to manufacturer’s in-
Table 1. Siemens ELISA kit detection of sECD-HER
[ng/mL] Absorbance ±SD %CV
35 2.50 0.25 10
25 1.80 0.17 9.4
15 1.20 0.10 8.3
7.5* 0.740 0.12 16
2.5 0.410 0.24 59
0 0.220 0.18 82
* Level of Detection (LOD) was 7.5 ng/mL and was defined
as the blank + 2 standard deviations (SD) [9]. The intra-assay
variability (%CV) or [(standard deviation / mean) x 100] was
calculated based on the difference in duplicate measurements.
Table 2. SECD-HER 2/neu detection by ALYGNSA.
[ng/mL] Fluorescence ±SD %CV
20 27900 1100 3.9
10 17100 790 4.6
5 11900 2300 19
2.5 10000 1300 13
1.25 9080 105 1.2
0.63 8330 58 0.7
0.31 7450 190 2.5
0 4870 1400 29
* Level of Detection (LOD) was 0.63 ng/mL as defined above.
The %CV was also calculated; value and SD of blank were con-
sistent with previously published reports [12,13].
structions. First, the HER-2/neu antigen reference sam-
ple was diluted to 9.21 ng/mL with sample diluent. The
standards and control were dispensed into the appropri-
ate wells at 100 L/well and incubated at 37oC for 3
hours. The plate was rinsed three times with 350 L/well
volume of plate wash buffer. Then, 100 L of detector
antibody was dispensed into each well (except for the
substrate blank wells) and incubated at 37oC for 1 hour.
After washing, 100L of working conjugate solution
was applied to all wells except for the substrate blank
wells. Following incubation at room temperature for 30
minutes, 100 L of working substrate solution was ap-
plied to all wells, including the substrate blank wells.
After incubation at room temperature for 45 minutes in
the dark, 100 L/well of stop solution was applied to all
the wells and the plate was read for absorbance at 498
nm on a BioRad plate reader.
2.2. ALYGNSA Assay
Protein and antibody reagents used in the sECD-
HER-2/neu ALYGNSA assay were recombinant Protein
G’ (Sigma), C-erbB-2 monoclonal antibody (Labvision,
S. Chourb et al. / HEALTH 1 (2009) 325-329
SciRes Copyright © 2009 Openly accessible at http://www.scirp.org/journal/HEALTH/
clone# c-erbB-2/HER-2/neu Ab 20) and HER-2/neu an-
tigen, reference sample (Siemens). Poly (methyl metha-
crylate) (PMMA) (Sigma) was used to coat polypropyl-
ene plates (Corning). The coating buffer was phosphate
buffered saline (PBS); pH 7.4. A PMMA solution in ace-
tone prepared to a concentration of 10 mg/mL was solu-
tion cast onto polypropylene plates and incubated over-
night at room temperature in a desiccator. After drying,
the plates were washed with one volume of 1% SDS in
PBS, pH 7.4 and 3 volumes of PBS. The PMMA plates
were coated with recombinant Protein G’ (1 g/mL at 50
L/well) and incubated overnight at 4°C. After washing,
the plates were blocked with 5% NFDM at 150 L/well
for 2 hours at room temperature, and then re-washed.
The plates were coated with the primary HER-2 anti-
body at 2 g/mL at 50 L/well and incubated overnight
at 4oC. After washing, the plates were re-blocked and
washed. The reference sample (antigen) was diluted to
20 ng/mL and 100 L applied to the top rows of each
plate. A 1:1 serial dilution in PBS was performed and
carried out to 0.31 ng/mL. The plates were incubated for
1 hour at room temperature, and then washed. The HER-
2/neu secondary antibody was labeled using DyLight
488 from Pierce. First, 8 L of the Borate Buffer (0.67
M) was added to 100 L of 1 mg/mL HER-2/neu anti-
body in PBS. Then, 100 L of the prepared protein was
added to the vial of DyLight Reagent, briefly centrifuged
and incubated for 60 minutes at room temperature in the
dark. One hundred microliters of the purification resin
was added into the spin column and centrifuged for 1
minute at ~1,000 x g. In a new tube 100 L of the label-
ing reaction was added to the spin column, mixed with
the purification resin and centrifuged for 1 minute at
~1,000 x g to collect the purified protein. This fluores-
cent-labeled secondary HER-2 antibody was diluted to 2
g/mL, and applied at 50 L/ well. Following incubation
for 1 hour at room temperature and washing, the plates
were read for fluorescence on a BioTek Microplate
3.1. ELISA Assay
An ELISA kit (Siemens) served as a model for a com-
mercially available method to detect sECD-Her-2/neu
protein in breast cancer. A standard curve of absorbance
versus concentration of sECD-HER 2/neu (ng/mL) was
generated after multiple runs of the Siemens ELISA kit
(Figure 1). The limit of detection (LOD) for sECD-
HER-2/neu protein, in our hands, was determined to be
7.5 ng/mL (Table 1).
3.2. ALYGNSA Assay
The ALYGNSA assay for sECD-HER 2/neu detection
employs a sandwich assay protocol similar to the ELISA
method, except for the protein biolinker, Protein G’, and
the fluorescently labeled detector antibody; Fluorescence
detection analysis is a more sensitive method of analysis
than colorimetric detection employed in the ELISA
method. Multiple determinations revealed an assay LOD
of 0.63 ng/mL for sECD-HER 2/neu (Figure 2, Table 2)
and, hence, a 10-fold greater sensitivity than the Siemens
ELISA kit.
4.1. sECD-HER 2/neu Detection: Importance
in Breast Cancer Treatment
HER-2/neu is over-expressed and amplified in patients
with metastatic breast cancer. An increase serum level
(>15 ng/mL) of its shed extracellular domain (sECD-
HER 2/neu) is indicative of the potential presence and
associated progression of this disease. In this report a
newly developed ALYGNSA system has been shown to
detect an order of magnitude lower level of sECD-HER
2/neu than the commercial ELISA kit counterpart. Pre-
vention of HER-2/neu overexpression has been exten-
sively demonstrated using novel HER-2/neu-blocking
agents [8,15,16]. Monoclonal therapeutic antibodies
known to bind to extracellular domains of HER-2/neu
have been well characterized [17 and references therein];
they include: pertuzumab/Omnitarg which acts upon
Domain II and is believed to hinder receptor dimeriza-
tion; cetuximab/Erbitux which blocks essential struc-
tural transitions of Domain III; and perhaps the most
well known and of greatest interest here, trastuzu-
mab/Herceptin which binds to Domain IV blocking
the action of sheddase and inhibiting sECD-HER 2/neu
formation. Trastuzumab was the first FDA approved
HER-2/neu monoclonal antibody used in the treatment
of HER2/neu overexpression in breast cancer patients.
sECD-HER 2/neu (>15 ng/mL) is currently used to se-
lect patients for therapy with trastuzumab.
4.2. sECD-HER 2/neu Quantitation:
Detection and Treatment of Additional
Numerous reports have focused on evaluation of serum
sECD-HER 2/neu levels in patients not only in breast
cancer but also in several other tumor types including
but not limited to ovarian [18] prostate [19], and lung
cancer [20]. Each condition has a unique set of labora-
tory, pathological, and clinical factors essential to the
detection, treatment, and prognosis. Greater detection,
evaluation and standardization of sECD-HER 2/neu lev-
els in serum, as well as other cancer biomarkers, may
provide a universal approach to epidermal growth factor
receptor (EGFR) related cancers [21,22].
S. Chourb et al. / Natural Science 1 (2009) 325-329
SciRes Copyright © 2009 Openly accessible at http://www.scirp.org/journal/HEALTH/
4.3. sECD-HER 2/neu Detection:
Assessment of Normal Physiological
EGFR and the other three homologous members of the
EGFR family of receptor tyrosine kinase (also referred
to as the ErbB or HER family) are comprised of four
members: EGFR, HER2, HER3, and HER4. Under nor-
mal physiological conditions, the EGFR family governs
vital processes including cell growth, cell differentiation,
and cellular migration (processes which are altered in
cancerous states). Disruption of signaling from the re-
ceptors can result in aggressive diseases including many
cancers (epithelial tumors) which could possibly lead to
poorer outcomes. A recent review [16] has focused on
the structural/functional aspects of ligand-induced ErbB
receptor structural transitions and dimerization, and
mechanistic modes of activation and inhibition. Further,
a key part of the EGFR story involves sECD-HER 2/neu
[23] and its proteolytic processing via protein ectodo-
main shedding by the ADAM family of proteins [24].
Post-translational processing of extracellular membrane
components is a major mode of regulation [25]. The
ALYGNSA assay, with its enhanced sensitivity, may be
the vehicle that could drive these investigations.
This work utilized a fluorescent ELISA incorporating the
newly developed ALYGNSA antibody-orientation sys-
tem which revealed a 10-fold increase in sensitivity
(0.63 ng/mL) of sECD-HER 2/neu when compared to a
control standard ELISA kit (7.5 ng/mL). The
ALYGNSA assay could aid in evaluation and detection
of sECD-HER 2/neu under normal conditions and may
provide additional insight into its role in disease states.
This experimental success with analytical (buffer-based)
samples should encourage future work with clinical (se-
rum-based) samples.
This work was supported by NSF award # 0425826 and a University of
Massachusetts System-Wide CVIP award (MJM). S.C. participated in
the NSF Research Experience for Undergraduates (REU) at UMass
Lowell. Special thanks to Lisa-Jo Ann Clarizia and Davin Sok for their
helpful discussions and support in the early stages of this work, and to
Peter S. Chiev and Adrianna Morris for critical reading of this manu-
[1] Slamon,D.J., Clark, G.M., Wong, S.G., Levin,W.J., Ull-
rich, A. and McGuire, W.L. (1987) Human breast cancer:
correlation of relapse and survival with amplification of
the HER-2/neu oncogene. Science, 235, 177-182.
[2] Ménard, S., Pupa, S.M., Campiglio, M. and Tagliabue, E.
(2003) Biologic and therapeutic role of HER2 in cancer.
Oncogene, 22, 6570-6578.
[3] Ross, J.S., Fletcher, J.A., Linette, G.P., Stec, J., Clark, E.,
Ayers, M., et al. (2004) Targeted therapy in breast cancer:
The HER-2/neu gene and protein. Mol. Cell. Proteomics,
3, 379-398.
[4] Carney, W.P., Leitzel, K., Ali, S., Neumann, R. and Lip-
ton, A. (2007) A HER-2/neu diagnostics in breast cancer.
Breast Cancer Res., 9, 207-217.
[5] Bramwell, V.H.C., Doig, G.S., Tuck, A.B., Wilson, S.M.,
Tonkin, K.S., Tomiak, A., et al. (2009) Changes over
time of extracellular domain of HER2 (ECD/HER2) se-
rum levels have prognostic value in metastatic breast
cancer. Breast Cancer Res. Treat., 11 4, 503-511.
[6] Lipton, A., Ali, S.M., Leitzel, K., Demers, L., Chinchilli,
V., Engle, L., et al. (2002) Elevated serum HER-2/neu
level predicts decreased response to hormone therapy in
metastatic breast cancer. J. Clin. Oncology, 20, 1467-
[7] Fornier, M.N., Seidman, A.D., Schwartz, M.K. and
Ghani, F. (2005) Serum HER2 extracellular domain in
metastatic breast cancer patients treated with weekly
trastuzumab and paclitaxel: association with HER2 status
by immunohistochemistry and fluorescence in situ hy-
bridization and with response rate. Ann. Oncology, 16,
[8] Slamon, D.J., Jones, B.L., Shak, S., Fuchs, H., Paton, V.,
Bajamonde, A., et al. (2001) Use of chemotherapy plus a
monoclonal antibody against Her2 for metastatic breast
cancer that over expresses Her2. N. Engl. J. Med., 344,
[9] Payne, R.C., Allard, J.W., Anderson-Mauser, L., Hum-
phreys, J.D., Tenney, D.Y. and Morris, D.L. (2000)
Automated assay for HER-2/neu in serum. Clin. Chem.,
46, 175-182.
[10] Meenakshi, A., Kumar, R.S. and Kumar, N.S. (2002)
ELISA for quantitation of serum c-erbb-2 oncoprotein in
breast cancer patients. J. Immunoassay Immunochem., 23,
[11] James, R., Thriveni, K., Ramaswamy, G., Krishnamoor-
thy, L., Mukherjee, G., Deshmane,P.P.V., et al. (2008)
Evaluation of immunohistochemistry and enzyme linked
immunosorbent assay for Her-2/Neu expression in breast
carcinoma. Indian J. Clin. Biochem., 23, 345-351.
[12] Clarizia, L-J.A., Sok, D., Wei, M., Mead, J., Barry, C.
and McDonald, M.J. (2009) Antibody orientation en-
hanced by selective polymer–protein noncovalent inter-
actions. Anal. Bioanal. Chem., 393, 1531-1538.
[13] Sok, D., Clarizia, L-J.A., Farris, L.R. and McDonald,
M.J., (2009) Novel fluoroimmunoassay for ovarian can-
cer biomarker CA-125. Anal. Bioanal. Chem., 393,
[14] Mackness, B.C., Chourb, S., Farris, L.R. and McDon-
ald, M.J. Polymer-protein enhanced fluoroimmunoassay
for prostate-specific antigen. Anal. Bioanal. Chem., DOI
S. Chourb et al. / HEALTH 1 (2009) 325-329
SciRes Copyright © 2009 http://www.scirp.org/journal/HEALTH/Openly accessible at
[15] Cho, H.S., Mason, K., Ramyar, K.X., Stanley, A.M.,
Gabelli, S.B., Denney, D.W., et al. (2003) Structure of
the extracellular region of HER2 alone and in complex
with the Herceptin Fab. Nature, 421, 756-760.
[16] Zhang, H., Berezov, A., Wang, Q., Zhang, G., Drebin, J.,
Murali, R., et al. (2007) Erbb receptors: from oncogenes
to targeted cancer therapies. J. Clin. Invest., 117, 2051-
[17] Ferguson, M.K. (2008) Structure-based view of epider-
mal growth factor receptor regulation. Annu. Rev. Bio-
phys., 37, 353-73.
[18] de Graeff, P., Crijns, A.P.G., ten Hoor, K.A., Klip, H.G.,
Hollema, H., Oien, K., et al. (2008) The erbb signalling
pathway: protein expression and prognostic value in
epithelial ovarian cancer. Br. J. Cancer, 99, 341-349.
[19] Osman, I., Mikhail, M., Shuch, B., Clute, M., Cheli, C.D.,
Ghani, F., et al. (2005) Serum levels of shed Her2/Neu
protein in men with prostate cancer correlate with disease
progression. J. Urology, 174, 2174-2177.
[20] Salam, I.A. Gaballa, H.E. and Wahab, N.A. (2009) Se-
rum levels of epidermal growth factor and HER-2Neu in
non small-cell lung cancer: prognostic correlation. Med.
Oncology, 26,161-166.
[21] Hartwell, L., Mankoff, D., Paulovich, A., Ramsey, S. and
Swisher, E. (2006) cancer biomarkers: a systems ap-
proach. Nature Biotech, 24, 905-908
[22] Jain, K.K. (2008) Innovations, challenges and future
prospects of oncoproteomics. Mol. Oncology, 2, 153-160.
[23] Yuan, C.-X., Lasut, A.L., Wynn, R., Neff, N.T., Hollis,
G.F., Ramaker, M.L., et al. (2003) Purification of Her-2
extracellular domain and identification of its cleavage
site. Protein Expression Purif, 29, 217-222.
[24] Blobel, C.P. (2005) ADAMs: key components in EGFR
signalling and development. Nature Rev. Mol. Cell. Biol.,
6, 32-44.
[25] Murphy, G., Murthy, A. and Khokha, R. (2007) Clipping,
shedding and RIPping keep immunity on cue. Trends
Immunol, 29, 75-82.