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Rapid Testing for HPV E7 Protein Bull. Korean Chem. Soc. 2009, Vol. 30, No. 12 2999
DOI 10.5012/bkcs.2009.30.12.2999
I
m
m
uno-c
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ro
m
atographic Analysis for HPV-16 and 1 8 E7 Proteins as a B io
m
a
rker
of Cervical Cancer Caused by Human Papillomavirus
Joo-Ho Kim,†,§,a Il-Hoon Cho,‡,a Sung- Min S eo,‡ Ji-Sook Kim,§ Kyu-Ha Oh,§
Heun-Soo Kang,# In-Gyu Kim,ℙ and S e- Hwan Paek†,‡,¶,*
†Graduate School of Life Sciences and Biotechnology, and ‡Program for BioMicrosystem Technology, Korea University,
Seoul 136-701, Korea. *E-mail: shpaek@korea.ac.kr
§R&D Center of Bio Focus Co., Ltd., 9F., Daehyun Technoworld, Gyeonggi-do 437-753, Korea
#Metabolab Inc., Cancer Research Center, Seoul National University, Seoul 110-799, Korea
ℙ
Department of Biochemistry and Molecular Biology, College of Medicine, Seoul National University, Seoul 110-799, Korea
¶Department of Biotechnology and Bioinformatics, Korea University, Jochiwon, Choongnam 339-800, Korea
Received October 16, 2009, Accepted November 3, 2009
Among the more than 120 different types of human papillomavirus (HPV), types 16 and 18 have been known to be high
risk agents that cause cervical cancer. We examined, in an immuno-chromatographic analysis, the potential of using
the early gene product, E7 protein, as a diagnostic marker of cervical cancer caused by HPV. We developed monoclonal
antibodies specific to HPV-16 and 18 E7 proteins that were produced from bacterial cells using gene recombinant tech-
nology. For each E7 protein, the optimal antibody pair was selected using the immuno-chromatographic sandwich-
type binding system based on the lateral flow through membrane pores. Under these conditions, this rapid testing assay
had a detection capability as low as 2 ng/mL of E7 protein. Furthermore, since viral analysis required the host cell to
be lysed using chemicals such as detergents, it was possible that the E7 protein was structurally damaged during this
process, which would result in a decrease in detection sensitivity. Therefore, we examined the detrimental effects
caused by different detergents on the E7 protein using HeLa cells as the host. In these experiments, we found that the
damage caused by the detergent, nonylphenylpolyethylene glycol (NP-40), was minimal relative to Triton X-100 com-
monly used for the cell lysis. Temperature also affected the stability of the E7 protein, and we found that the E7 protein
was stabilized at 4 oC for about 2 h, which was 4 times longer than at room temperature. Finally, a HPV-infected cervical
cancer cell line, which was used as a real sample model, was treated using the optimized conditions and the presence of
E7 proteins were analyzed by immuno-chromatography. The results of this experiment demonstrated that this rapid
test could specifically detect HPV-infected samples.
Key Words: Rapid test, Papillomaviruses, E7 protein as biomarker, Specimen stability, Cervix cancer
Introduction1
Human papillomavirus (HPV), a double-stranded small DNA
virus, infects basal epithelial cells of the skin and the mucosal
membrane of anogenital epithelium. HPV is thought to cause
the most common sexually-transmitted viral disease1 and can
lead to cancers of the cervix, vulvar, vagina, and anus in women.
Numerous studies have reported that cervical cancer is mainly
caused by anogenital infection with high-risk HPV types, in
particular, 16 and 18.2 In their viral genomes, two early genes,
E6 and E7, are known to act as viral oncogenes that promote
tumor growth and malignant transformation in the host cells.3
The gene products, E6 and E7 proteins, deregulate the host
cell growth cycle by binding and inactivating the tumor suppr-
ession protein, p53, and retinoblastoma protein (pRb) of the host
cell, respectively. 4 The E6 protein binds and degrades p53 via
ubiquitin-dependent proteolysis, which leads to a loss of the cell-
ular protein responsible for maintaining genomic stability.5
Since the E7 protein interferes with cell-cycle checkpoint regu-
lators, it has been recognized as the most important oncogenic
protein.6 For instance, in HPV-infected cervical cells, the E7
protein binds to the hypophosphorylated form of pRb and func-
aThese authors equally contributed to this research.
tionally inactivates it, resulting in the liberation of the cellular
transcription factor E2F-1.7 It has been reported that the E7 pro-
tein will be a valuable diagnostic marker for the detection of
high-risk HPV types causing the cancer.8 Therefore, identifi-
cation of the E7-related genotype or protein from HPV-16 and
18 is needed for the development of in vitro diagnostic assays.
For the past several decades papanicolaou-stained (Pap)
smears have been used as the conventional method to detect ab-
normal or cancerous cervical cells in cervical smear specimens
for the identification of cervical cancer. However, the Pap smear
test often produces a false negative or false positive result and
it has been estimated that these false negative and false positive
results have been responsible for 10 to 50% of the incorrect diag-
noses. To improve the test performance, liquid-based prepara-
tion9 and pre-screening smears10 have been developed. How-
ever, these methods require technologically advanced pieces
of equipment and expensive reagents. DNA-based methods11
may circumvent the intrinsic problems associated with achiev-
ing accurate diagnoses. However, the use of this technology is
still limited because this method requires multiple reaction
steps, long reaction times, well-equipped facilities, and well-
trained personnel. In addition, the DNA-based detection met-
hods are not adequate for monitoring the progress state of can-
cer.
3000 Bull. Korean Chem. Soc. 2009, Vol. 30, No. 12 Joo-Ho Kim et al.
The aim of this study was to develop a potential alternative
HPV detection method, the immuno-chromatographic assay,
which may not only be highly accurate, but also rapid, easy-to-
use, and inexpensive. The assay involves a one-step procedure
where the complex antigen-antibody reactions are sequentially
carried out by the lateral flow along the membrane strip upon
the addition of a specimen.12 This concept was first demons-
trated for the detection of fertility hormones such as the human
chorionic gonadotropin13 and luteinzing hormone. More recen-
tly, this method was expanded to the analyses of various in vitro
diagnostic markers for infectious diseases, cancers, cardiac di-
seases, and bio-terrorism agents. This methodology can be for-
mulated as a qualitative kit using colloidal gold as a tracer, which
is perceivable by the naked eye, and also as a quantitative sensor
by merely employing a colorimetric detector.14 Furthermore, in
contrast to the DNA-based detection methods, the chromato-
graphic assay can detect proteins as the analyte, which conse-
quently allows one to monitor the progress state of illness.
In this study, we developed a chromatographic assay system
for the rapid detection of E7 proteins produced from HPV-
infected cells, which was used as a model of a cervical cancer
specimen. To construct the analytical system, monoclonal anti-
bodies were raised against E7 proteins prepared using recom-
binant DNA technology. The capture and detection binders were
then selected to form sandwich-type complexes for each analyte
from the host cells, Caski and HeLa cell lines, which were in-
fected with the model specimens, HPV-16 and 18, respectively.
Furthermore, the conditions used to prepare the specimen, such
as the lysing agent and treatment temperature, were optimized
to minimize damage to the protein and maximize protein sta-
bility.
Methods
Preparation of HPV E7 proteins as immunogens. Protein ex-
pressi on: To produce recombinant HPV-16 and 18 E7 proteins,
complementary DNAs (cDNAs) corresponding to the respective
E7 oncogene were separately cloned into the pGEX-4T-1 vector
(GE Healthcare, Waukesha, WI), which contained a thrombin
cleavage site (Leu-Val-Pro-Arg-Gly-Ser).15 The gene products
were expressed as recombinant glutathione S-transferase (GST)-
E7 proteins in Escherichia coli (E. coli) BL21(DE3), and the
cells were harvested after cultivation. They were resuspended
in 20 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 0.5%
nonylphenylpolyethylene glycol (NP-40) (Sigma, St. Louis,
MO), 0.5 mM dithiotheritol (DTT) (Sigma, St. Louis, MO), 2
µg/mL phenylmethylsulphonyl fluoride (PMSF) (Pierce, Rock-
ford, IL) and protease inhibitor cocktail (Sigma, St. Louis,
MO).16 The cell lysate was allowed to incubate on ice for 30 min,
and centrifuged at 12,000 g at 4 oC for 10 min. The supernatant
was then purified by affinity chromatography on a glutathione
(GSH)-immobilized Sepharose 4B column (10 mL gel packing)
(GE Healthcare, Waukesha, WI). The purified fusion proteins
were cleaved by thrombin (GE Healthcare, Waukesha, WI)
and the resulting E7 proteins were finally quantified using the
bicinchoninic acid (BCA) method (Pierce, Rockford, IL).
Characte rizatio n: The GST-E7 proteins before and after
thrombin cleavage were analyzed by sodium dodecyl sulfate
(SDS) polyacrylamide gel electrophoresis (SDS-PAGE) and
Western blotting. The crude extract (80 µg) was boiled in 120
mM Tris-HCl, pH 7.9, containing 4% SDS, 0.02% bromophenol
blue, 20% glycerol, and 2% 2-mercaptoethanol for 10 min and
then loaded on a 15% gel for SDS-PAGE analysis. The PAGE
gel was transferred to a nitrocellulose (NC) membrane (0.45 µm
pore size) (Whatman, Kent, UK) for Western blotting, and the
residual surfaces were blocked in 10 mM phosphate buffer, pH
7.4, containing 140 mM NaCl (PBS) and 0.5% casein (Casein-
PBS). The membrane was prepared in duplicate and then in-
cubated in polyclonal anti-HPV-16 E7 and anti-HPV-18 E7 anti-
bodies (Santa Cruz, CA), respectively, diluted in Casein-PBS
containing 0.1% tween-20 (Casein-PBS-TW) for 1 h. The mem-
branes were treated with anti-goat IgG antibody coupled to hor-
seradish peroxidase (HRP; Invitrogen, Carlsbad, CA) in the
same medium, and signals were finally produced by incubating
them with the Western blotting substrate (Pierce, Rockford, IL).
Production of mono clonal antibodie s. A ntibody producti on:
Monoclonal antibodies specific for the HPV-16 E7 and HPV-18
E7 proteins were prepared as described elsewhere.16 Briefly,
Balb/c female mice (8 weeks old) were immunized by intra-
peritoneally injecting 10 µg of the recombinant HPV-16 and
18 E7 protein after mixing with an equal volume of Complete
Freund’s adjuvant (Sigma, St. Louis, MO). After 2 weeks, the
mice were immunized with the same amount of the E7 proteins
emulsified with Incomplete Freund's adjuvant (Sigma, St. Louis,
MO). The same procedure was repeated 2 weeks later, and the
final boosting was conducted after the same period with E7
proteins dissolved in PBS. Three days after the injection of the
final boost, the mouse splenocytes were collected and fused
with murine myeloma cell (sp2/0-Ag14) (American Type Cul-
ture Collection; ATCC, Manassas, VA), which was used as a
fusion partner. Fused hybridoma cells were screened based on
hypoxanthine aminopterin thymidine (HAT; Gibco BRL, Gai-
thersburg, MD) selection, and hybridoma clones exhibiting high
titers of binding activities to each E7 protein were selected by
immunoassays using antigen-coated microtiter plates. The
selected hybridoma cells were injected into 2,6,10,14-tetra-
methylpentadecane pristane (Sigma, St. Louis, MO)-primed
female Balb/c mice to produce the antibodies as ascitic fluid,
which was then purified on a protein G column (5 mL, HiTrap
protein G HP) (Amersham Biosciences, Piscataway, NJ). Each
antibody concentration was determined by absorbance measure-
ment at 280 nm.
Isotype determination: The sub-isotypes of the produced anti-
bodies were determined using an enzyme-linked immunosor-
bent immunoassay (ELISA)-based mouse monoclonal isotyping
kit (Pierce, Rockford, IL) according to the manufacturer’s ins-
tructions. Briefly, 25 µg of the goat anti-mouse immunoglobulin
(G+A+M) antibody was coated on the inner surfaces of the
microwells at room temperature for 2 h, the residual surfaces
were blocked, 50 µL of the monoclonal antibody samples were
reacted at 37 oC for 1 h, the subclass-specific rabbit anti-mouse
immunoglobulin antibodies were subsequently bound, and alka-
line phosphatase-conjugated anti-rabbit immunoglobulin G
(IgG) antibody was finally added. The colorimetric signals from
each well were produced via the enzyme-substrate reaction.
Sc reening of antibodie s for rapid testing. Antibody labeli ng
Rapid Testing for HPV E7 Protein Bull. Korean Chem. Soc. 2009, Vol. 30, No. 12 3001
with colloidal gold: A monoclonal antibody selected as the de-
tection binder was labeled with colloidal gold (the mean dia-
meter: 30 nm) as previously reported.17 Briefly, the antibodies
dialyzed in 10 mM phosphate buffer were conjugated with col-
loidal gold that was synthesized using the sodium citrate met-
hod. Unreacted antibodies were removed by centrifugation and
the antibody-gold conjugates were then resuspended in Casein-
PBS such that the gold density was concentrated by 20-fold
relative to the initial gold density.
Immuno-strip preparation: The rapid test kit for E7 proteins
was constructed by combining different functional membrane
strips as described elsewhere.17 The four membrane pads used in
assembling the rapid test kit were as follows (from the bottom):
a glass fiber membrane (4 × 20 mm) (Ahlstrom, Whatman, Kent,
UK) for sample application, a glass fiber membrane (4 × 7 mm)
(Ahlstrom, Whatman, Kent, UK) for the release of the detection
antibody labeled with colloidal gold, a NC membrane (4 × 25
mm) (Millipore, Billerica, MA) for signal generation, and a cel-
lulose membrane (4 × 50 mm) (Ahlstrom, Whatman, Kent, UK)
for continuous sample absorption. The sample application pad
was prepared by immersing in 10 mM Tris-HCl, pH 7.5, con-
taining 0.1% Triton X-100 and drying at 55 oC for 4 h. The signal
generation pad was prepared by spotting a monoclonal antibody
(1 µL) selected as the capture binder on the NC membrane and
then allowing it to incubate at 37 oC for 20 min. The four mem-
brane pads were partially superimposed in length to construct
the immuno-strip.
Antibody pair selec tion: To select antibody pairs for the an-
alyses of each E7 protein, the produced monoclonal antibodies
were used as either the capture antibody or the detection anti-
body. E7 protein (1 µg/mL; 100 µL) in Casein-PBS was added to
each microwell containing a detection antibody (20-fold con-
centrate; 2 µL) labeled with colloidal gold and reacted for 5 min.
The immuno-strip with an immobilized capture antibody was
then immersed into the reaction mixture and, after 15 min, the
color signal that appeared on the capture antibody site was stored
as image in a personal computer. The signals corresponding to
each combination of antibodies were finally used to select the
best pairs.
Determinatio n of sample preparation conditions. Antibody
labeling with HRP: Since the E7 protein may be denatured by
the use of HPV-host lysing agents such as detergents, the struc-
tural stability of the protein was determined by ELISA employ-
ing HRP as the tracer. The detection antibodies, clone #35 and
#1 specific to HPV-16 E7 and HPV-18 E7 proteins, respectively,
were separately coupled to HRP via a chemical reaction as pre-
viously described.17 Briefly, the antibodies were first reduced
using DTT (10 mM, final concentration) and the excess reagent
was removed on a Sephadex G-15 gel filtration column (GE
Healthcare, Waukesha, WI). HRP was activated with a 50-fold
molar excess of succinimidyl 4-[N-maleimidomethyl] cyclo-
hexane-1-carboxylate (SMCC; Pierce, Rockford, IL), and the
excess reagent was immediately removed in the same manner.
Each antibody was then combined with a 5-fold molar excess
of the activated HRP and the conjugation was carried out at 4
oC overnight.
Cell lysis medium conditions: The HeLa cell line (ATCC,
Manassas, VA) was used as a model HPV-infected specimen
and grown in the culture media recommended by the supplier.
The cells were maintained in dulbecco’s modified eagle’s me-
dium (DMEM; Thermo scientific, Hyclone, Waltham, MA) sup-
plemented with 10% heat-inactivated fetal bovine serum (FBS;
Gibco BRL, Gaithersburg, MD) and, at the time of analysis, the
cells were harvested by treatment with trypsin (Gibco BRL,
Gaithersburg, MD). After washing with PBS, the harvested
cells were lysed for E7 protein extraction at room temperature
using different media: Medium a: 50 mM Tris-HCl, pH 7.5, con-
taining 150 mM NaCl, 0.5% bovine serum albumin (BSA; Basic
medium), 1% Triton X-100, and protease inhibitor cocktail (1:
20 dilution); Medium b: Basic medium supplemented with 0.5%
NP-40; and Medium c: Basic medium supplemented with 0.5%
NP-40 and the protease inhibitor cocktail. The time between
lysing the sample and subjecting it to ELISA analysis for HPV-
18 E7 protein detection, as described below, was varied from
5 min to 8 h.
Lysis temperature: HeLa cell lysis with Medium c was carried
out separately at 4 oC and room temperature. The lysed cells
were left at each condition for different time intervals (5 min to
16 h) prior to ELISA analysis for HPV-18 E7 protein detection.
ELISA: The immunoassay used for HPV-18 E7 protein detec-
tion was carried out using the detection antibody (clone #1)
labeled with HRP and the capture antibody (clone #36) immo-
bilized on the surfaces of microwell. The capture antibody (1
µg/mL; 100 µL) in PBS was added into the wells and incubated
in a container maintained at 100% humidity and 37 oC for 1 h.
After washing with deionized water, the residual surfaces were
blocked with PBS containing 3% BSA under the same condi-
tions. The test samples (100 µL) containing the E7 protein and
the labeled detection antibody (1:1,500 dilution; 25 µL) in
Casein-PBS were sequentially reacted in the microwells. The
incubation conditions were identical to those used above and
a thorough washing was conducted after each reaction. The
enzyme substrate containing 100 µL 3,3',5,5'-tetramethylbenzi-
dine (TMB; Sigma, St. Louis, MO) was added into each well
and reacted for 15 min at room temperature. 1 M Sulfuric acid
(50 µL) (Sigma, St. Louis, MO) was finally added to stop the
enzyme reaction, and the colors produced in each well were
determined by measuring the absorbance at 450 nm.
Analy ti cal pe rfo rmances of the rapid te st kit. Rapid test ki t:
Immuno-strips were constructed using the antibody pairs most
suitable for detection of the respective E7 proteins to examine
the detection limits for each analyte. Two monoclonal antibo-
dies, clone #35 and clone #1, specific to HPV-16 and 18 E7 pro-
teins, respectively, were labeled with colloidal gold as described
above. The conjugate pad was prepared by spraying the two
detection antibodies on the glass fiber membrane and drying it
at 35 oC overnight. The signal generation pad was prepared by
immobilizing the following three different antibodies on spa-
tially separate sites of the NC membrane (from the bottom):
the capture antibody to HPV-16 E7 (clone #119), the capture
antibody to HPV-18 E7 antibody (#36), and an anti-mouse
IgG polyclonal antibody as the control. They were dispensed on
each pre-determined site in a linear pattern on the NC membrane
and then incubated at 37 oC for 20 min. The sample application
pad and absorption pad were prepared as described earlier, and
the four membrane pads were assembled to fabricate the im-
3002 Bull. Korean Chem. Soc. 2009, Vol. 30, No. 12 Joo-Ho Kim et al.
SDS-PA GE for GST-E7 fusio n protei
n
170 K
130 K
100 K
70 K
55 K
45 K
35 K
170 K
130 K
100 K
70 K
55 K
45 K
35 K
B A Marker
HPV 16 HPV 18
Marker A B
HPV 16 HPV 18
B A Marker Marker A B
170 K
130 K
100 K
70 K
55 K
45 K
35
K
Figure 1. SDS-PAGE analyses of recombinant GST-HPV-16 and 18 E7
fusion proteins expressed in E. coli BL21 (A) and then purified
b
y
GSH-Sepharose gel chromatography (B). The GST-E7 protein
b
an
d
was detected at a molecular weight of approximately 49 kDa after frac-
tionation.
Western blot for E7 protei
n
170 K
100 K
70 K
55 K
40 K
35 K
25 K
15 K
10 K
HPV 16 HPV 18
GST-E7 E7 GST-E7 E7
170 K
100 K
70 K
55 K
40 K
35 K
25 K
15 K
10 K
HPV 16 HPV 18
GST-E7 E7 GST-E7 E7
GST-E7 E7 GST-E7 E7
170 K
100 K
70 K
55 K
45 K
35 K
25 K
15 K
10
K
Fig ure 2 . Western blotting analyses of HPV-16 and 18 E7 proteins be-
fore and after thrombin cleavage. Prior to cleavage, the fusio n proteins
(GST-E7) were monitored using a polyclonal antibody to GST. The
cleaved products (E7) were also probed using polyclonal antibodies
specific to each E7 protein. Based on this analysis, the molecular
weights of HPV-16 E7 and HPV-18 E7 were approximately16 kDa and
12 kDa, respectively.
muno-strip, which was then placed within the pre-designed
plastic cassette.
De tection c apabil ity : To determine the detection limit of the
rapid test kit, the standard samples were first prepared with the
HPV-16 E7 protein and HPV-18 E7 protein, respectively. The
stock protein solution was serially diluted with Casein-PBS to
various concentrations (1 to 10 ng/mL). Each sample solution
(400 µL) was transferred into the sample application pot of the
cassette and the antigen-antibody reactions were carried out
for 15 min. The color signals produced from the respective assay
were stored as images in a personal computer.
Cervical cancer cell line test: Four cell lines (ATCC, Ma-
nassas, VA), Caski, HeLa, MCF7, and A549, were used as
models of HPV-infected cell specimens. Caski cell, which is a
cervical cancer cell, was infected with HPV-16 and employed
as the positive specimen for the HPV-16 E7 protein. HeLa cell,
another cervical cancer cell, was infected with HPV-18 and used
as the positive specimen for the HPV-18 E7 protein. MCF-7 and
A549, breast cancer and lung cancer cell lines, respectively,
were used as the negative controls. The four cell lines were sepa-
rately cultured in DMEM media containing 4 mM L-Glutamine,
4.5 g/L glucose, and sodium pyruvate, supplemented with hy-
poxanthine thymidine (HT) supplement (Gibco BRL, Gaithers-
burg, MD), antibiotic-antimycotic (Gibco BRL, Gaithersburg,
MD), and 10% FBS. After maintaining the cultivations in heal-
thy states, each cell was harvested by treating the adherent cells
with trypsin. The cells (approximately, 1 × 106 cells each cell
line) were washed twice with PBS and then lysed in Medium
c (400 µL; see above). Each cell lysate was centrifuged at 400 g
for 10 min and the supernatants were collected as the analytical
samples for use in the rapid testing kit. All test procedures (400
µL sample each test) were the same as described above.
Results and Discussion
Recombinant HPV E7 proteins. Rec ombinant protein produc -
tion: Since E7 proteins from HPV-16 and 18 are not currently
commercially available, we needed to manufacture them to use
as standard materials for screening optimal antibody pairs and
evaluating the sandwich immunoassay systems. By employing
recombinant technology,18 HPV-16 and 18 E7 fusion proteins
with GST were produced from microbial cells through cultiva-
tion. The SDS-PAGE analysis revealed that the constituents
in the culture broth concentrates were heterogeneous (Figure 1,
A) and, thus, the E7 fusion proteins with GST were purified on
a GSH coupled-Sepharose gel chromatography column.19 When
the purified fraction was subjected to SDS-PAGE analysis, a
major band near 49 kDa was observed for the both HPV strains
(Figure 1, B). Although other minor components were also
observed, the purity of the target proteins were greater than 98%
based on a comparison of the band areas. Since the molecular
weight of GST was about 37 kDa, the E7 alone was approxi-
mately 12 kDa in size.
Molecular characterization: The produced fusion proteins
were cleaved using thrombin to obtain the E7 proteins for each
HPV strain and then characterized via Western blotting (Figure
2). Each component was probed with the commercially available
specific polyclonal antibodies. In this analysis, the fusion pro-
teins (GST-E7) were found to be approximately the same in the
molecular size as mentioned above. However, after cleavage,
there were slight differences in the size of the E7 protein (E7)
from each strain. Even though all E7 genes were recombined
with the same GST gene, they may be expressed at different
sizes depending on the included gene size of the vector when
translated. According to a previous report, 18 the bacterially ex-
pressed His-tagged HPV-16 E7 protein appeared as a predo-
minant band at 16 kDa even though the theoretical molecular
weight of the E7 His6 monomer was 12.1 kDa. Under the con-
HPV 16 HPV 18
Rapid Testing for HPV E7 Protein Bull. Korean Chem. Soc. 2009, Vol. 30, No. 12 3003
Detection antibod
y
to HPV 16-E7
#42 #119 #130 #41 #35 #17 #42 #119 #130 #41 #35 #17 #42 #119 #130 #41 #35 #17
Mab #42 Mab #41 Mab #35
Capture
antibody
AnalyteAnalyte
Figure 3. Selection of an optimal antibody pair that could simultaneously bind to the HPV-16 E7 protein molecule via immuno-chroma-
tographic assay. One of the produced antibodies was immobilized as the capture binder on the NC membrane surfaces and the other was labele
d
with colloidal gold and used as the detection antibody. The assays were carried out by systematically combining the capture and detection
antibodies and the signals resulting from the sandwich complex formation were compared.
Table 1. Determination of detection capabilities of the immuno-chro-
matographic assays for HPV-16 and 18 E7 proteins under optimal
conditions
Test r esu lts
E7 protein concentration in standard sample
10
ng/mL
8
ng/mL
6
ng/mL
4
ng/mL
2
ng/mL
1
ng/mL
For HPV-16 E7 ++ ++ ++ ++ + -
For HPV-18 E7 ++ ++ ++ ++ + -
++: Strong signal; +: weak signal; and -: no signal produced.
ditions used in this study, the free HPV-16 and 18 E7 proteins
were found to be about 16 and 12 kDa in size, respectively, and
had the appropriate molecular structure since they were able
to bind to the corresponding specific antibody.
Optimal immuno-analytical conditions. In cervical cancer di-
agnosis, monoclonal antibodies have been used to immunolo-
gically stain cancer cells,20 which may overcome some of the
drawbacks associated with the conventional histochemical met-
hod, the Pap smear. Although immuno-staining has the potential
to specifically detect cancer with a single antibody, it often pro-
duces a false positive result due to the non-specific binding of the
labeled-secondary antibody employed for signal generation. To
alleviate such problems, we developed a sandwich-type binding
system, the immuno-chromatographic assay, which is based on
the lateral flow of the membrane and has an additional advantage
in regards to point-of-care testing.17
Optimal antibody pairing: Among the monoclonal antibody
pools previously established for HPV-16 and 18 detection,16 we
selected antibody pairs that could simultaneously bind to the res-
pective E7 protein molecule. To screen by means of immuno-
chromatography, an antibody to the HPV-16 E7 protein was
used as the capture binder by immobilizing it on a pre-deter-
mined site of the NC membrane and the other antibodies were
used for detection by labeling them with colloidal gold. After
carrying out the chromatographic assays, the color signals that
appeared on the capture antibody sites were then compared with
each other to select the antibody pair (#119 as the capture and
#35 as the detection) that yielded the highest complex formation
with the analyte (Figure 3). The same procedure was used to
select the most optimal antibody pair for the HPV-18 E7 protein
assay (antibody #36 as the capture and #1 as the detection)
(results not shown). Furthermore, the selected monoclonal anti-
bodies were subtyped using a commercially available kit and
antibodies #119 and #1 were determined to be the IgG2b type,
and #35 and #36 were the IgG2a.
De te c ti o n c apabili ty f o r E7 protein: The antibody pairs for
the respective analyte were used to construct the rapid test kit
and the experimental conditions were optimized to maximize
the detection capability of the kit. The major factors, including
the concentration of the capture and detection antibodies, and
the lateral flow rate along the immuno-strip, were optimized
as described in our previous reports.17 Standard samples of the
target proteins were prepared and used to analyze the immuno-
chromatographic assay according to the pre-determined pro-
tocol. The results of this experiment revealed that the produced
signals were proportional to the analyte concentration and detect-
able by the naked eyes at an analyte concentration as low as 2
ng/mL for the both of HPV-16 and 18 E7 proteins (Table 1).
Although there have been no clear reports regarding the levels
of E7 protein in cervical cancer cells infected by HPV, they
could be present in trace amounts in real samples.21 This sug-
gests that the detection capability of the immuno-chromato-
graphic assay may not be sufficient for clinical applications,
and a more sensitive analytical method such as ELISA-based
rapid testing22 may need to be employed in the future. Further-
more, variations in the level of the E7 protein as a function of the
progress of the virally infected disease should also be clinically
determined.
Cervical cancer cell line tests. The E7 protein produced from
cells infected by HPV-16 or 18 has been reported to be unstable
during storage.23 This instability could be even worse after the
cells have been treated with a lysing agent, such as detergents,
during protein extraction. Thus, the lysis conditions, such as the
detergent type and temperature, were tested in order to deter-
mine the effect of these factors on protein stabilization. The resi-
dual activities of the extracted proteins were determined by
microtiter plate-based immunoassays (e.g., ELISA) using the
same capture and detection antibody pairs as selected above for
each E7 protein.
3004 Bull. Korean Chem. Soc. 2009, Vol. 30, No. 12 Joo-Ho Kim et al.
A) Effect of lysis medium
Medium a
Medium b
Medium c
Medium a
Medium b
Medium c
Medium a
Medium b
Medium c
1 10 100 1000
Lysis time, min
0.8
0.6
0.4
0.2
0
Residual activity, A
450
Medium b
Medium c
Medium a
B) Effect of lysis temperature
At room
temperature
At 4°C
At room
temperature
At 4°C
At room
temperature
At 4°C
1 10 100 1000 10000
Lysis time, min
2.5
2
1.5
1
0.5
0
At room
temperature
At 4 oC
Residual activity, A
450
Figure 4. Effect of lysis medium (A) and temperature (B) on the inte-
grity of the E7 protein produced from HeLa cells by HPV-18 infection.
Three different lysis media were employed i n test A: ‘Medi um a’ con-
tained Triton X-100 as the lysing agent, and ‘Media b and c’ contained
N
P-40 as the lysing agent. Furthermore, ‘Media a and c’ included a pro-
tease inhibitor, but ‘Medium b’ did not. In test B, the E7 protein inte-
grity was examined at two different temperatures, 4 oC and room tem-
perature.
Caski HeLa MCF-7 A549
Cancer cells
Control
HPV 16 E7
HPV 18 E7
Figure 5. Rapid test results for HPV-E7 proteins produced from dif-
ferent cervical cancer cell lines. Positive indicators for the protein em-
ployed here were Caski cell with HPV-16 infection and HeLa cell with
HPV-18 infection. MCF-7 and A549 cells were used as negative con-
trols. The cells under cultivation were lysed and used as samples for the
rapid testing. The rapid test immuno-strip was prepared to simultan-
eously detect the two different E7 proteins on distinct analyte lines
marked as HPV-16 E7 and HPV-18 E7, respectively. The control line
was also made on the top position of the analyte lines.
Lysis medium composition: For testing in a real sample pre-
paration, we selected the cervical cancer cell line, HeLa cells, as
a model host for HPV-18 infection. Among the cell lines derived
from the same source, the HeLa cell line was the most appro-
priate for this study since the cells were cultivated fairly fast in
normal medium (e.g., DMEM) and reproducibly produced the
E7 protein.6 HeLa cells have also been used for studying the
role of E7 protein in HPV-induced cancer.24 The cultivated
HeLa cells were lysed using three different types of media each
containing a different composition of lysing agent and protease
inhibitor, and the protein integrity under each condition were
quantitatively monitored as a function of treatment time (Figure
4, A). For the protein integrity test, an ELISA system was de-
vised by chemically coupling the detection antibody to the en-
zyme, HRP, and by coating the capture antibody on the surfaces
of the microwells. Under these conditions, the system was able
to detect a minimum of 1.0 ng/mL of E7 protein.
When the lysis medium contained Triton X-100 as the cell
breakage agent (Medium a), the protein activity was initially so
low that only a small portion of the applied molecules seemed
to be intact (Figure 4, A). The structural degradation rate, there-
after, was quite slow as a function of incubation time. When NP-
40 was used as the lysing agent (Media b and c), the initial pro-
tein integrity was significantly improved and was 3.5 times
higher than that when Triton X-100 was used, but abruptly
decreased 10 min after onset of treatment. Such enhancement
effect of NP-40 has also been previously reported.25 In contrast,
the effect of the protease inhibitor contained in ‘Media a and
c’ was relatively insignificant. Nevertheless, no matter what cell
lysis media were used, treatment times longer 100 min seemed
to completely degrade the protein integrity. These data indicate
that the detergent, Triton X-100, which is conventionally used
to lyse cells, should be replaced with NP-40 for E7 protein
extraction and the lysis time should be short prior to analysis.
Temperature effect: Further tests were performed to deter-
mine the optimal temperature for E7 protein extraction from the
HeLa cell culture. Cell lysis was carried out using the optimal
medium (i.e., ‘Medium c’ in Figure 4, A) at two different tem-
peratures, 4 oC and room temperature. HPV-18 E7 protein acti-
vity was determined as a function of time using ELISA as des-
cribed above (Figure 4, B). These experiments showed that
the protein integrity in the cell lysate was maintained at 4 oC
for 2 h, while it was only maintained for 30 min when kept at
room temperature. This result indicated that upon treatment,
even with NP-40 as detergent, it is essential to maintain the
cell lysate at low temperature prior to analysis.
Rapid te st performance: To detect the presence of E7 proteins
via immuno-chromatography, we first prepared HPV-infected
cell lines that closely mimicked clinical samples used for cervi-
cal cancer diagnosis. These model HPV-infected cells lines were
HPV-16-infected cervical cancer cell line, Caski cell, and a
HPV-18-infected cell line, HeLa cell. In addition, two HPV-ne-
gative cells, MCF-7 and A549, were also used as controls. After
culturing the cell lines under optimal conditions, analytical sam-
ples were prepared by lysis and then subjected to immunoassays
for detection of E7 proteins using the rapid test kit (Figure 5).
The immuno-strip was made to detect HPV-16 and 18 E7 pro-
Rapid Testing for HPV E7 Protein Bull. Korean Chem. Soc. 2009, Vol. 30, No. 12 3005
teins at the same time by immobilizing each capture antibody
(as indicated above) at spatially separate sites on the NC mem-
brane. Identical detection antibodies labeled with colloidal gold
were also employed. In addition, the control line, which gener-
ated a color signal regardless of the analyte dose, was located
on the top of the analyte lines by coating a secondary antibody
that binds to the mouse IgG.
From the test results shown in Figure 5, the two HPV-infec-
tion positive cell samples, Caski cell and HeLa cell, were detect-
ed on each analyte line, HPV-16 E7 and HPV-18 E7, respec-
tively, on the immuno-strip. The infection-free samples, MCF-7
cell and A549 cell, did not exhibit any signals at the analyte
lines. These results demonstrated that the rapid testing was not
only highly specific, but also capable of detecting E7 proteins
produced from cancer cells infected with HPV.
Conclusions
The E7 protein, an oncogene product of HPV, was detected
from host cells using an immunoassay, indicating that this pro-
tein can be used as a diagnostic marker for cervical cancer caus-
ed by HPV infection. To obtain immunogens, high-risk HPV
types 16 and 18 E7 proteins were produced via DNA recombi-
nation at molecular sizes of approximately 16 and 12 kDa, res-
pectively. The monoclonal antibodies specifically raised against
the recombinant proteins were suitable for constructing the rapid
test kit based on immuno-chromatography, which could simul-
taneously measure both analytes. In consideration of clinical
tests for cancer diagnosis, we also determined the optimal con-
ditions for handling the host cell such that the extracted E7
protein could be stable at 4 oC for 2 h. Although this rapid testing
kit showed a clear discrimination between the HPV infected
and normal cells, the detection capability may not be sufficient
for future clinical applications. We, therefore, plan to replace
the colloidal gold tracer in the test kit with a more sensitive
label such as an enzyme and also to determine the relationship
between the E7 concentration and the progress of cervical can-
cer.
Acknowledgments. This work was supported by the Korea
Research Foundation Grant funded by the Korean Government
(MOEHRD) (KRF-2005-041-D00261).
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