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Immuno-chromatographic Analysis for HPV-16 and 18 E7 Proteins as a Biomarker of Cervical Cancer Caused by Human Papillomavirus



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 technology. For each E7 protein, the optimal antibody pair was selected using the immuno-chromatographic sandwichtype 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 commonly 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 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.
Rapid Testing for HPV E7 Protein Bull. Korean Chem. Soc. 2009, Vol. 30, No. 12 2999
DOI 10.5012/bkcs.2009.30.12.2999
atographic Analysis for HPV-16 and 1 8 E7 Proteins as a B io
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, 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:
§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
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
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-
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-
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
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
Figure 1. SDS-PAGE analyses of recombinant GST-HPV-16 and 18 E7
fusion proteins expressed in E. coli BL21 (A) and then purified
GSH-Sepharose gel chromatography (B). The GST-E7 protein
was detected at a molecular weight of approximately 49 kDa after frac-
Western blot for E7 protei
170 K
100 K
70 K
55 K
40 K
35 K
25 K
15 K
10 K
HPV 16 HPV 18
170 K
100 K
70 K
55 K
40 K
35 K
25 K
15 K
10 K
HPV 16 HPV 18
170 K
100 K
70 K
55 K
45 K
35 K
25 K
15 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
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
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
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
Test r esu lts
E7 protein concentration in standard sample
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
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
Residual activity, A
Medium b
Medium c
Medium a
B) Effect of lysis temperature
At room
At 4°C
At room
At 4°C
At room
At 4°C
1 10 100 1000 10000
Lysis time, min
At room
At 4 oC
Residual activity, A
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
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-
Caski HeLa MCF-7 A549
Cancer cells
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.
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-
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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... Protein E6 dihitung menggunakan metode Bicinchoninic Acid (BCA). Langkah tersebut diulang untuk menghasilkan antigen protein E6 HPV-18 hingga didapatkan keseluruhan protein rekombinan yang dibutuhkan (Yetisen et al., 2013;Kim et al., 2009). Untuk mendapatkan antibodi monoklonal digunakan mencit sebagai hewan coba. ...
... Pada test line di NC membrane diletakkan antibodi yang telah disiapkan sebanyak 1 μL. Antibodi protein E6 HPV-16 diletakkan pada T1 dan antibodi protein E6 HPV-18 pada T2 (Kim et al., 2009). Control line mengandung antibodi spesifik terhadap konjugat partikel emas. ...
Full-text available
Cervical cancer is still become main health problem especially in development country. This cancer is the second most common cancer in Indonesian women. It commonly caused by infection of Human Papillomavirus (HPV) type 16 and 18. Actually if cervical cancer was detected earlier, it shows better respond therapy than other cancers, but this opportunity is not supported by appropiate screening methods. Nowadays, pap smear and IVA are commonly used for detect cervical cancer but they only recommend for women who have had sexual intercourse. Both of them also require medical expertise and pap smear is highly cost. Based on these problem, this paper describes novel method for cervical cancer screening by using rapid immunochromatography kit which is more applicative, noninvasive, and affordable. This paper was a literature review (library research). Data obtained through the search engine using keywords: "Cervical Cancer", "HPV", "Urine", "Screening", “Lateral flow test”, and "Rapid immunochromatography". The references were taken from reliable journal in the range of 2009 to 2019. Data was also taken from textbooks and the health institution official sites which support the analysis. This method detect the presence of oncoprotein E6 & E7 of HPV-16 and HPV-18 in urinusing antigen-antibody binding principle. Sensitivity and specificity test done by comparing the results of the kit with the results of pap smear tests as a standard screening method and PCR HPV test on urinsamples. We conclude that with further protocol development and standardization to achieve clinical sensitivity, this kit is a solution for noninvasive detection of high risk cervical cancer so that treatment can be do immediately and the mortality rate due to cervical cancer can be reduced as much as possible.
... As known, E6 and E7 transforming proteins are consistently expressed in HPV life cycle [39][40][41]. Several studies have shown the induction of antibody responses against E6 and E7 oncoproteins in patients with cervical cancer higher than in healthy subjects [39,42,43]. ...
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Background Among different types of human papillomavirus (HPV), types 16 and 18 were known to be high-risk agents causing mainly cervical cancer. Up to now, the potential of HPV E7 protein has been proved as a diagnostic marker of cervical cancer. Moreover, the levels of anti-heat shock protein (Hsp) and anti-high mobility group box-1 (HMGB1) antibodies in cancer patients have been useful in tumor diagnosis. The goal of the present study was to determine the efficiency of the potential serologic markers including HPV E7, Hsp20, Hsp27 proteins and Hp91 peptide in Iranian HPV-exposed women, for the first time. Methods At first, the recombinant HPV E7, Hsp20 and Hsp27 proteins were expressed in E. coli system, and purified by affinity chromatography under native conditions. Then, antibody responses were detected against the recombinant proteins as well as Hp91 peptide as potential markers in 49 Iranian women who were seropositive for HPV-16 and 18 L1 capsids (i.e., HPV-exposed women) and 49 controls using indirect ELISA. Results Our data indicated that the seroreactivities of women exposed to HPV16, HPV18 and both of them against the recombinant E7, Hsp20, Hsp27 proteins and Hp91 peptide were significantly higher than those in control group (p < 0.05 for HPV16 or HPV18; p < 0.01 for both of them versus all markers). HPV-exposed women with high antibody responses to HPV-16 and 18 L1 capsids as a commercial biomarker had significant seroreactivity to HPV-16 and 18 E7 and Hsp27 (p < 0.05). The recombinant E7 and Hsp27 proteins showed higher efficiency than Hsp20 and Hp91 for detection of individuals exposed to HPV infections (p < 0.05). Conclusion Generally, the levels of serum E7 and Hsp27 were increased in HPV-16 and 18 L1- seropositive women suggesting their potential value as a diagnostic marker for HPV infections.
... The signal generation membrane was prepared by dispensing a monoclonal antibody and recombinant HBsAg on the NC membrane as the test line and control line, respectively, and then allowing it to incubate at 37 °C for 1 h. The four membrane pads were partially superimposed in length to construct the immunochromatography strip [11], [13]. ...
Full-text available
An immunochromatographic assay for the detection of hepatitis B surface antigen (HBsAg) was developed using Fe3O4 nanoparticles as signal materials. Anti-HBsAg antibodies were labeled with carboxylated Fe3O4 particles with an average diameter of 153.6 nm, and an immunochromatography strip was constructed. The detection capability of the assay developed here is 1 ng/mL HBsAg.
Human papillomavirus (HPV) types 16 and 18 are known to be high-risk viruses that cause cervical cancer. An HPV rapid testing kit that could help physicians to make early and more informed decisions regarding patient care is needed urgently but not yet available. This study aimed to develop a multiplex nested polymerase chain reaction-immunochromatographic test (PCR-ICT) for the rapid identification of HPV 16 and 18. A multiplex nested PCR was constructed to amplify the HPV 16 and 18 genotype-specific L1 gene fragments and followed by ICT which coated with antibodies to identify rapidly the different PCR products. The type-specific gene regions of high-risk HPV 16 and 18 could be amplified successfully by multiplex nested PCR at molecular sizes of approximately 99 and 101 bp, respectively. The capture antibodies raised specifically against the moleculars labeled on the PCR products could be detected simultaneously both HPV 16 and 18 in one strip. Under optimal conditions, this PCR-ICT assay had the capability to detect HPV in a sample with as low as 100 copies of HPV viral DNA. The PCR-ICT system has the advantage of direct and simultaneous detection of two high-risk HPV 16 and 18 DNA targets in one sample, which suggested a significant potential of this assay for clinical application. (C) 2014 Published by Elsevier B.V.
A recent report that 93 per cent of invasive cervical cancers worldwide contain human papillomavirus (HPV) may be an underestimate, due to sample inadequacy or integration events affecting the HPV L1 gene, which is the target of the polymerase chain reaction (PCR)‐based test which was used. The formerly HPV‐negative cases from this study have therefore been reanalysed for HPV serum antibodies and HPV DNA. Serology for HPV 16 VLPs, E6, and E7 antibodies was performed on 49 of the 66 cases which were HPV‐negative and a sample of 48 of the 866 cases which were HPV‐positive in the original study. Moreover, 55 of the 66 formerly HPV‐negative biopsies were also reanalysed by a sandwich procedure in which the outer sections in a series of sections are used for histological review, while the inner sections are assayed by three different HPV PCR assays targeting different open reading frames (ORFs). No significant difference was found in serology for HPV 16 proteins between the cases that were originally HPV PCR‐negative and ‐positive. Type‐specific E7 PCR for 14 high‐risk HPV types detected HPV DNA in 38 (69 per cent) of the 55 originally HPV‐negative and amplifiable specimens. The HPV types detected were 16, 18, 31, 33, 39, 45, 52, and 58. Two (4 per cent) additional cases were only HPV DNA‐positive by E1 and/or L1 consensus PCR. Histological analysis of the 55 specimens revealed that 21 were qualitatively inadequate. Only two of the 34 adequate samples were HPV‐negative on all PCR tests, as against 13 of the 21 that were inadequate ( p < 0·001). Combining the data from this and the previous study and excluding inadequate specimens, the worldwide HPV prevalence in cervical carcinomas is 99·7 per cent. The presence of HPV in virtually all cervical cancers implies the highest worldwide attributable fraction so far reported for a specific cause of any major human cancer. The extreme rarity of HPV‐negative cancers reinforces the rationale for HPV testing in addition to, or even instead of, cervical cytology in routine cervical screening. Copyright © 1999 John Wiley & Sons, Ltd.
A chemiluminometric biosensor system for point-of-care testing has been developed using an immuno-chromatographic assay combined with an enzyme (e.g., horseradish peroxidase) tracer that produces a light signal measurable on a simple detector. Cross-flow chromatography, a method previously investigated by our laboratory, was utilized in order to accomplish sequential antigen-antibody binding and signal generation. This enzyme-linked immunosorbent assay (ELISA) was effectively carried out on a plastic chip that was redesigned to simplify the fabrication process. To enhance the sensitivity, biotin-streptavidin capture technology was employed in preparing an immuno-strip that was then incorporated onto the chip in order to generate the ELISA-on-a-chip (EOC) biosensor. Samples containing cardiac troponin I (cTnI) were analyzed using the EOC. A chemiluminescent signal proportional to the analyte concentration was produced by adding a luminogenic substrate to the tracer enzyme complexed with the analyte on the chip. The luminescent signal was detected in a dark chamber mounted with a cooled charge-coupled device and the signal was converted to optical density for quantification. This EOC biosensor system was capable of detecting cTnI present in serum at concentrations as low as 0.027 ng mL(-1), 30 times lower than those measured using the conventional rapid test kit with colloidal gold as the tracer. In addition, the final data was acquired within 30s after the addition of the enzyme substrate, which was faster than the detection time required when using a colorimetric substrate with the same tracer enzyme.
ELISA capture assays were established for the E7 transforming proteins of HPV16 and HPV18, based on a range of previously characterised polyclonal and monoclonal antibodies. No cross-reactivity was observed in the ELISAs between HPV18 E7 and HPV16 E7. Immunoreactive E7 protein (iE7) was measured in a series of HPV-transformed cell lines, and ranged from 0.6 to 17.7 ng iE7/mg cell protein. iE7 was labile at 22 degrees C (t1/2 = 37 min) but relatively more stable at 4 degrees C (t1/2 = 210 min). HPV16 E7 protein at concentrations from 0.10 to 0.69 ng iE7/mg cell protein was detected in 5 of 13 smears from women with abnormal cervical cytology. Assay of E7 protein may play a role in the detection of HPV-induced cervical lesions with malignant potential.
Industry has been intimately involved in the development of products used in the monitoring of fertility at home. The perspective of industry and the development of such tests including their rationale are presented. The methods taken by industry to utilize current technology in the development of reliable, rapid, one-step tests for urinary human chorionic gonadotropin and luteinizing hormone and the subsequent expansion into other areas are covered. PIP Home tests to monitor fertility are discussed in terms of the evolution of home tests and recent advances. The conception of home testing has been around for at least a century, but home diagnosis is limited to 2 product types: blood glucose monitoring and home pregnancy testing, which were the 1st immunoassays to be sold over the counter in the mid 1970s. These tests were difficult to use. In 1984, a market research report identified the needs as: rapid (30 minutes), sensitive (as early as the 1st day to the missed period), easy to use, easy to read, hygienic. The development of monoclonal antibodies in 1975 lead the way for quicker test results and greater sensitivity. When monoclonal antibodies were merged with dipstick enzyme immunoassay, ease of use and reading were improved. Pregnastick was 1 of the 1st such tests, and required only 30 minutes. Clearblue was developed and added the improvement of including a sampling device and all solutions were premixed. The quest for faster and easier testing lead to the exploration of a membrane based enzyme immunoassay product. In 1986, Hybritech Icon was marketed with reduction of testing time to 5 minutes. The speed was achieved by the membrane surface area being saturated with antibodies and a high surface area to volume ration within the membrane. However, it was multistep and required use of droppers. Another technique was enzyme immunochromatography, which had limited sensitivity and required liquid reagents. The requirements in 1986 were for a product that was rapid (5 minutes), required no manipulation by the user, used sample collection and hygienic handling like Clearblue, and had a clearly readable result. In June, 1988, Clearblue 1 Step by Unipath was introduced as a test which reduced manipulations. The user simply holds the sampler in the morning urine stream, replaces the cap, and waits until it shows the test to be complete (3 minutes). The essential features of the test are described. The recent advances in technology include 1 step tests for HG, streptococcus A, and Chlamydia under the tradename Clearview, which for Chlamydia has reduced culture time to 30 minutes from 3-5 days. Other tests which measure semiqualitative values rather than a simple yes/no include the Clearplan 1 step for determining the peak level of luteinizing hormone (LH) in the preovulatory surge.
Deletions or mutations of the retinoblastoma gene, RB1, are common features of many tumors and tumor cell lines. Recently, the RB1 gene product, p105-RB, has been shown to form stable protein/protein complexes with the oncoproteins of two DNA tumor viruses, the adenovirus E1A proteins and the simian virus 40 (SV40) large T antigen. Neither of these viruses is thought to be associated with human cancer, but they can cause tumors in rodents. Binding between the RB anti-oncoprotein and the adenovirus or SV40 oncoprotein can be recapitulated in vitro with coimmunoprecipitation mixing assays. These assays have been used to demonstrate that the E7 oncoprotein of the human papilloma virus type-16 can form similar complexes with p105-RB. Human papilloma virus-16 is found associated with approximately 50 percent of cervical carcinomas. These results suggest that these three DNA viruses may utilize similar mechanisms in transformation and implicate RB binding as a possible step in human papilloma virus-associated carcinogenesis.
α-Thrombin cleavage of 30 polypeptide hormones and their derivatives were analysed by quantitative amino-terminal analysis. The polypeptides included secretin, vasoactive intestinal polypeptide, cholecystokinin fragment, dynorphin A, somatostatins, gastrin-releasing peptide, clacitonins and human parathyroid hormone fragment. Most of them were selected mainly on the ground that they contain sequence structures homologous to the well known tripeptide substrates of α-thrombin. All selected polypeptides have one single major cleavage site and both Arg-Xaa and Lys-Xaa bonds were found to be selectively cleaved by α-thrombin. Under fixed conditions (1 nmol polypeptide/0.5 NIH unit α-thrombin in 20 μl of 50 mM ammonium bicarbonate at 25°C), the time required for 50% cleavage ranges from less than 1 min to longer than 24 h. Heparin invariably enhanced thrombin cleavage on all polypeptide analysed. The optimum cleavage site for α-thrombin has the structures of (a) P4-P3-Pro-Arg-P1'-P2', where P3 and P4 are hydrophobic amino acid and P1',P2' are nonacidic amino acids and (b) P2-Arg-P1', where P2 or P1' are Gly. The requirement for hydrophobic P3 and P4 was further demonstrated by the drastic decrease of thrombin cleavage rates in both gastrin-releasing peptide and calcitonins after chemical removal of hydrophobic P3 and P4 residues. The requirement for nonacidic P1' and P2' residues was demonstrated by the drastic increase of thrombin cleavage rates in both calcitonin and parathyroid hormone fragments, after specific chemical modification of acidic P1' and P2' residues. These findings confirm the importance of hydrophobic P2 - P4 residues from thrombin specificity and provide new evidence to indicate that apolar P1' and P2' residues are also crucial for thrombin specificity. It is concluded that specific cleavage of polypeptides by α-thrombin can be reasonably predicted and that chemical modification can be a useful tool in enhancing thrombin cleavage.
In many cervical cancers, human papillomavirus type 16 (HPV-16) DNA genomes are found to be integrated into the host chromosome. In this study, we demonstrate that integration of HPV-16 DNA leads to increased steady-state levels of mRNAs encoding the viral oncogenes E6 and E7. This increase is shown to result, at least in part, from an increased stability of E6 and E7 mRNAs that arise specifically from those integrated viral genomes disrupted in the 3' untranslated region of the viral early region. Further, we demonstrate that the A+U-rich element within this viral early 3' untranslated region confers instability on a heterologous mRNA. We conclude that integration of HPV-16 DNA, as occurs in cervical cancers, can result in the increased expression of the viral E6 and E7 oncogenes through altered mRNA stability.
The authors compared the accuracy of conventionally prepared smears and smears prepared by an automated, fluid-based, thin-layer processing device in the detection of cytologic abnormalities. A total of 3218 patients from five centers took part in this study, in which a single cervical sample was split into a matched pair. The conventional smear was made in the routine fashion; the remainder of the cells on the sampling device were rinsed into a transport-fixation fluid. A slide was then prepared from the solution using the thin-layer processor. Diagnostic findings identified on the two preparations were compared in a blinded fashion, and a discrepancy resolution procedure was used to eliminate screening differences. Overall, there was a high correlation in the diagnoses of the two methods. For low-grade or more severe disease, the thin-layer method resulted in a 13% increase in the rate of detection, as compared with the conventional Papanicolaou smear technique.
The ubiquitin-dependent proteolytic pathway plays a major role in selective protein degradation. Ubiquitination of proteins requires the sequential action of the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (E2), and in some cases ubiquitin-protein ligases (E3s). The oncogenic human papillomavirus (HPV) types 16 and 18 utilize this cellular proteolytic system to target the tumor suppressor protein p53. The HPV E6 oncoprotein binds to a cellular protein of 100 kd, termed E6-associated protein (E6-AP). The E6-E6-AP complex specifically interacts with p53, resulting in the rapid ubiquitin-dependent degradation of p53. Here we report the purification and identification of the factors necessary for the E6-E6-AP-mediated ubiquitination of p53. The ubiquitination of p53 requires the E1 enzyme and a novel E2 in mammalian cells, while E3 activity is conferred by the E6-E6-AP complex. Furthermore, E6-AP appears to have ubiquitin-protein ligase activity in the absence of E6.
To determine the effectiveness of AutoPap QC System rescreening of all qualified negative cervical cytologic smears with selection of approximately 10% for cytotechnologist rescreening as compared to a random selection of 10% of negative cases. The study included 1,141,913 consecutive smears. In February 1997 an AutoPap-based QC program was implemented to select a population for rescreening. Negative cases were studied prospectively by the AutoPap QC System. Detection rates by diagnostic classification of false negative cases were compared to rates obtained using a randomly selected 10% rescreening of negative cases from the immediately preceding time period. The AutoPap QC System was 5.2-fold more effective for the detection of high grade squamous intraepithelial lesion + false negatives, 2.0-fold for low grade squamous intraepithelial lesions, 2.8-fold for atypical squamous cells of undetermined significance and 5.1-fold for atypical glandular cells of undetermined significance. In a large reference laboratory a QC program utilizing the AutoPap QC System was significantly more effective in the detection of false negative smears as compared to a QC program utilizing 10% random rescreening of negative smears.