Published Ahead of Print 18 July 2012.
10.1128/CVI.00388-12.
2012, 19(9):1474. DOI:
Clin. Vaccine Immunol.
Cheng
Guo, Tiea Kesler, Alicia Carter, Philip E. Castle and Shuling
Yvonne Lai, Ju-Hwa Lin, Ting-Chang Chang, Hsiao-Yun
Yi-Shan Yang, Karen Smith-McCune, Teresa M. Darragh,
in Cytology Samples
Objective Measurement of E6 Oncoproteins
Enzyme-Linked Immunosorbent Assay for
Direct Human Papillomavirus E6 Whole-Cell
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Immunosorbent Assay for Objective Measurement of E6 Oncoproteins
in Cytology Samples
Yi-Shan Yang,aKaren Smith-McCune,bTeresa M. Darragh,cYvonne Lai,aJu-Hwa Lin,dTing-Chang Chang,eHsiao-Yun Guo,e
Tiea Kesler,fAlicia Carter,fPhilip E. Castle,gand Shuling Chenga
OncoHealth Corporation, Fremont, California, USAa; Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, San
Francisco, California, USAb; Department of Pathology, University of California, San Francisco, San Francisco, California, USAc; Department of Biological Science and
Technology, China Medical University, Taichung, Taiwand; Department of Obstetrics and Gynecology, Chang-Gung Memorial Hospital Linkou Medical Center, Taoyuan,
Taiwane; Laboratory Corporation of America Holdings, Burlington, North Carolina, USAf; and American Society for Clinical Pathology Institute, Washington, DC, USAg
A novel, whole-cell enzyme-linked immunosorbent assay (ELISA) based on a non-type-specific anti-human papillomavirus
(HPV) E6 antibody was tested on 182 residual cytological specimens. For samples with a designation of more severe than cervical
intraepithelial neoplasia grade 3 (CIN3
ⴙ), 83% tested positive for E6; in a subset with paired testing for E6 ELISA and HPV
DNA, 72% tested E6 positive and 92% tested high-risk (HR)-HPV DNA positive (P
ⴝ 0.2). Among the women with a less than
CIN3 diagnosis, 31% and 47% tested positive for E6 and HR-HPV DNA, respectively (P
ⴝ 0.0006).
C
ervical cancer is the second most common cause of cancer
deaths in women worldwide. Routine screening and
treat-ment have substantially decreased the cervical cancer mortality
rate in the United States. However, according to the World Health
Organization, there are approximately 500,000 new cases and
250,000 deaths from cervical cancer every year worldwide.
Al-though Pap tests and colposcopy have contributed tremendously
to the decreased mortality of cervical cancer, they are subjective
tests, prone to human error, and not always conclusive. Therefore,
it is extremely important to develop more objective screening
tools that can identify patients who are most at risk for developing
cervical cancer (
8
).
Human papillomavirus (HPV) infection is a major cause of
virtually all invasive cervical cancers (
2
,
4
,
26
,
28
,
33
). Of the 40
HPV types that infect the genital tract, only a subset of HPV
sub-types are classified as “high-risk” HPV (HR-HPV) sub-types that were
found in cancers (
33
). Most of these HPV infections are transient,
are resolved by the body’s immune system, and have no major
clinical consequences. However, persistent HPV infections are
found in 5 to 10% of infected women and represent a high risk
factor for progression to cervical cancer (
3
,
25
). Thus, it is
impor-tant to identify the small percentage of women with HPV
infec-tions who are truly at risk for developing cervical cancer.
Unfor-tunately, current screening tests cannot accurately predict the risk
of dysplasia or cancer. Therefore, there is a significant need to
develop a test that could better predict progression to these
out-comes.
The current paradigm for cervical cancer screening is based on
the Pap test, which is a cytologically based test using cells scraped
from the cervix that are examined microscopically to detect
dys-plastic lesions (
9
,
15a
,
20
,
23
). There are approximately 4 million
abnormal Pap tests each year in the United States. Under current
practice guidelines, most of these patients are referred for
colpos-copy and cervical biopsy to identify the subset that has clinically
significant high-grade precancers, such as cervical intraepithelial
neoplasia grade 2/3 (CIN2/3) (
15a
). However, the Pap test is
sub-jective, with significant interobserver variability, and is limited by
low sensitivity. In addition, high false-positive rates, defined as a
positive Pap result with no clinically significant disease by
subse-quent biopsy (i.e., histology results of less than CIN2/3), were
observed in two-thirds of patients with abnormal Pap smears (
6
,
7
,
18
,
23
,
34
). As a result, approximately 3 million colposcopic
ex-aminations performed each year, at a cost exceeding $2 billion
dollars annually, may not be necessary.
In the last few years, HR-HPV DNA testing has been included
in routine screening to increase the sensitivity and negative
pre-dictive value of the Pap test. While these tests can detect the
pres-ence of HPV DNA, they cannot differentiate a true precancerous
state from self-limited HPV infection (
1
,
8
,
22
,
24
), which
repre-sents the majority of infections. The low specificity of HPV DNA
testing potentially results in overdiagnosis and inefficient disease
management (
26
). In addition to DNA tests, a number of host
cellular proteins, including p16
INK4a, Ki67, and ProExC, have
also been identified as biomarkers for cervical cancer diagnosis.
However, they are considered surrogate markers and not
spe-cific to HPV.
HPVs are DNA viruses that code for several functional (E1 to
E7) genes and two late structural (L1 and L2) genes. When
high-risk HPV types integrate into the host genome, loss of
negative-feedback control results in increased expression of viral E6 and E7
oncogenes, which in turn inactivate tumor suppressor genes that
operate at key cell cycle checkpoints (
10
–
12
,
19
,
29
). Since these
two oncogenes are integral to the development of cervical cancer,
their gene products could potentially serve as highly specific
bio-markers to identify high-grade precancerous lesions that may
progress to cervical cancer if left untreated. Indeed, evidence
sug-gests that elevated levels of the E6 and E7 oncoproteins are better
indicators of increased risk for cervical cancer than the presence of
Received 21 June 2012 Accepted 6 July 2012 Published ahead of print 18 July 2012
Address correspondence to Shuling Cheng, slcheng@oncohealthcorp.com. Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/CVI.00388-12
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HPV DNA (
6
,
15
). The recent FDA approval of the Aptima HPV
E6/E7 RNA test is a significant milestone for the application of
E6/E7 as specific biomarkers for cervical cancer screening.
How-ever, RNA is prone to degradation, and its detection requires
ex-pensive instrumentation and cumbersome procedures; tests based
on the detection of E6/E7 mRNA may have limited clinical
appli-cation in routine gynecological practice. Diagnostic tests based on
the direct detection of the E6/E7 oncoproteins may have
advan-tages over detection of HPV DNA or HPV E6/E7 mRNA. Until
recently, most of the antibodies developed against the HPV E6 or
E7 protein used either peptides or denatured proteins, as it has
been difficult to purify recombinant nondenatured E6 and E7
proteins suitable for antibody production (
31
). Antibodies
pro-duced using these denatured proteins do not have sufficient
sen-sitivity for clinical use (
16
). We have recently overcome the
tech-nical hurdles and have purified recombinant HPV E6 and E7
proteins in their native form to generate monoclonal antibodies
that recognize HPV E6 and E7 from many high-risk HPV types.
In this study, we describe a simple whole-cell enzyme-linked
immunoabsorbent assay (ELISA) using a pan-HPV anti-E6
monoclonal antibody to detect HPV E6 protein in previously
col-lected and frozen cytology samples. Using cervical biopsy results
as the gold standard, ELISA results are compared to HPV DNA
test results. The ability to detect the E6 oncoprotein in clinical
samples is a critical advance that will facilitate the development of
diagnostic testing to distinguish benign HPV infections from
pre-cancers, thus preventing unnecessary colposcopies and biopsies.
MATERIALS AND METHODS
Purification of recombinant HPV proteins. HPV E6 and E7 cDNAs
con-taining the respective coding regions were produced by PCR amplification and cloned into a histidine tag expression vector. The proteins were then expressed in Escherichia coli BL21(DE3) using isopropyl--D -thiogalacto-pyranoside (IPTG)-driven induction. In order to produce HPV E6 and E7 proteins in soluble, nondenatured form, full-length HPV type 18 (HPV18) E6 and E7 were expressed at 25°C and purified at low concen-tration using affinity chromatography without denaturation and refold-ing (Amersham and New England BioLabs). Recombinant HPV18 E6 protein, estimated to be⬎90% pure based on PAGE analysis, was used as an immunogen for generation of polyclonal and monoclonal antibodies.
Mouse monoclonal anti-HPV E6 antibody. Anti-HPV E6 antibodies
were generated using the purified native forms of recombinant E6 protein as immunogens in the BALB/c mouse strain at 1-mg/ml concentration using Freund’s adjuvant. Monoclonal antibodies were screened by ELISA using HPV-related or non-HPV-related protein. To obtain non-HPV type-specific monoclonal antibodies, HPV16 and HPV18 E6 proteins were used to screen hybridoma cell lines. Monoclonal antibodies pro-duced from ascites fluid were purified on a protein G column (Thermo-Scientific, IL).
Western blot analysis of cell lines. Human cervical epithelial cell
lines, HeLa (ATCC CCL-2), SiHa (ATCC HTB-35), and C33A (ATCC HTB-31), were purchased from ATCC and were used within 10 passages of purchase. Proteins from these cell extracts were prepared using 3% NP-40 lysis buffer. The protein concentration was determined by Brad-ford protein analysis. Proteins were separated by SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad) previously blocked with 5% (wt/vol) bovine serum albumin (BSA). The primary antibodies, mouse monoclonal anti-HPV E6 (1:1,000 dilution; NeoDiagnostic Laboratories Inc.) and anti-actin (1:5,000 dilution; Chemicon), were incubated overnight at 4°C, followed by secondary an-tibody (horseradish peroxidase-conjugated anti-mouse from Biobasic Inc., Canada; 1:5,000) and detected with an ECL detection kit (Biobasic Inc.).
HPV E6 whole-cell ELISA. To test the hypothesis that E6 protein can
serve as a valuable biomarker for HPV disease progression, we developed a whole-cell ELISA in which the residual cells from liquid-based cytology samples are directly immobilized onto 96-well microtiter plates. This whole-cell ELISA allows objective measurement of the HPV E6 oncopro-tein expression level in cervical cancer cell lines or clinical specimens. Cells from cell lines or from cervical scrapes were immobilized by passive ad-sorption on 96-well plates for 30 min at room temperature (RT). Each plate was then washed 3 times with phosphate-buffered saline (PBS) for 5 min following each incubation, unless otherwise specified. After the washes, the cells were fixed with 25l 100% ethanol and air blow dried at RT, followed by cell permeabilization with chilled (⫺20°C) 90% metha-nol. To decrease the background signal and to block endogenous hydro-gen peroxidase, the wells were incubated with 3% H2O2for 20 min at RT, washed, and blocked with 100l of 10% normal goat serum for 2 h at RT. A proprietary anti-E6 monoclonal antibody developed by OncoHealth (diluted 1:200 in 10% normal goat serum) was added, and the plate was incubated for 1 h at RT, washed, and then incubated with biotinylated secondary antibody (50l/well; 1:500 in 5% normal goat serum; Vector Laboratories, Burlingame, CA) for 30 min at RT. After further washes, the wells were incubated with 50l of Streptavidin conjugated with horse-radish peroxidase (HRP) (1:600; Vector Laboratories) for 45 min at RT, washed, and incubated with 50l of 3,3=,5,5=-tetramethylbenzidine sub-strate (BD Bioscience, San Jose, CA) for 10 min. The reaction was stopped by addition of 25l of acid stop solution, and the signal intensity was measured as the optical density at 450 nm (OD450) using a plate reader.
To determine the detection limits of the whole-cell ELISA and to gen-erate standard curves using recombinant HPV E6 protein, a high-binding 96-well microtiter plate was directly coated with serial titrations of puri-fied HPV E6 protein (0.4 pg to 4g/ml of PBS) overnight at 4°C. Blocking of each well was performed on the second day with 100l of 10% normal goat serum for 2 h at RT. Assay procedures were performed as described above to generate a standard curve for HPV18 E6.
Clinical samples. The cervical samples used in this study were
ob-tained from collaborative clinical laboratories from Asia and North Amer-ica in compliance with institutional review board (IRB)-approved proto-cols. These clinical laboratories performed colposcopy/biopsy and HR-HPV testing and provided us the cytological diagnosis and HR-HPV DNA results. The histology diagnosis was categorized as benign, CIN grade 1, 2, or 3, or cancer. CIN3 and the more severe CIN3⫹ (n ⫽ 42), the precancerous and cancer stage where treatment takes place in current clinical practice, were analyzed as one group. Clinical samples with diag-noses of less than CIN3 (n⫽ 140) were analyzed as another group. CIN2 was not analyzed separately from CIN1, since there were only five CIN2 cases in the study. The HR-HPV DNA test results (assigned as either positive or negative) were based on the presence of the HR-HPV DNA according to the protocol used and validated at the clinical sites provided by the manufacturer of the test kits. Samples (182) were collected in liq-uid-based Pap test vials (preserved in ThinPrep from Hologic, Inc., Mar-lborough, MA, or SurePath from BD Diagnostics, Burlington, NC) and used in the current study. Specimens were normalized to the cellular vol-ume of a 25-l cell pellet per ml of solution, and all 182 specimens were tested in duplicate by E6 whole-cell ELISA. Of these samples, the clinical laboratories had results on HR-HPV DNA testing (using Hybrid Capture 2 from Qiagen) for 165 specimens. The 17 cases that were not tested for HR-HPV DNA were cancer cases (Tables 1and2). The results of our E6 ELISA were compared to those of the previously performed HR-HPV DNA test using histology diagnoses as a gold standard. An exact version of the McNemar chi-square test was used to test for differences between testing positive for CIN3⫹ and less than CIN3 for paired-test results.
Data analysis. Purified recombinant HPV18 E6 protein was used as a
positive control. Pap-negative PreservCyt liquid-based cytology (LBC) samples were pooled to be used as a negative control. Blank, empty wells were used as assay background controls. The OD of the blank well was subtracted from the OD450raw data collected from the plate reader
Direct HPV E6 Whole-Cell ELISA
September 2012 Volume 19 Number 9 cvi.asm.org 1475
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(BioTek ELX800) to obtain an average signal intensity as a readout for data analysis. Absorbance of individual samples obtained from whole-cell ELISA using anti-E6 antibody, as well as the average absorbance from each group, was determined. E6 protein levels were compared to the severity of the histologic grade using a Kruskal-Wallis test. Receiver operating char-acteristic (ROC) analysis (ROC curve) (data not shown) with a prelimi-nary cutoff threshold of 0.33 was used to obtain the optimal sensitivity and specificity of the ELISA. The sensitivity was calculated as follows: number of true positives/(number of true positives⫹ number of false negatives). The specificity was calculated as follows: number of true negatives/(num-ber of true negatives⫹ number of false positives).
RESULTS
Pan-HPV E6 antibody recognizes the E6 protein in cell lines
in-fected with different high-risk HPV types and in clinical
sam-ples. Currently available anti-HPV antibodies were produced
against either small synthetic peptides or denatured recombinant
proteins. These antigens often do not react with naturally
oc-curring HPV antibodies (
13
,
14
,
27
,
30
,
32
). In order to obtain
antibodies that recognize HPV E6 proteins in clinical samples,
antibodies were generated using the purified native form of
recombinant E6 protein as the immunogen. The mouse anti-E6
monoclonal antibody recognized recombinant HPV E6 proteins
from HPV16 and -18, but not recombinant E7 proteins from
HPV16 or -18 or the N-terminus of the HPV16 recombinant
ma-jor capsid protein, L1 protein (
Fig. 1A
). The limit of detection for
this anti-E6 antibody on recombinant HPV18 E6 protein ranged
from 10 to 100 pg/ml (0.5 to 5 pg) (
Fig. 1B
) in an ELISA. The
antibody also recognized E6 protein from HPV18-positive
cervi-cal-cancer-derived HeLa cells, but not that from HPV-negative
cervical-cancer-derived C33A cells, in immunoblot analysis (
Fig.
1C
). Using whole-cell ELISA, the anti-E6 antibody detected E6
protein from SiHa and HeLa cells expressing HPV16 and HPV18,
respectively, with signal strengths dependent on cell density
(9,000 to 36,000 cells/well) (
Fig. 1D
). The E6 protein was
mini-mally detected in HPV-negative C33A cells (
Fig. 1D
). The anti-E6
antibody examined in the current study possesses a unique
bind-ing site recognizbind-ing a common epitope of the E6 proteins that are
accessible in clinical samples (S. Cheng, U.S. patent application
US 2010/033944). In addition, the antibody is not HPV type
spe-cific and detects E6 proteins from the high-risk types HPV16, -18,
-31, -33, -45, -51, -52, -58, and -59 (S. Cheng, U.S. patent
appli-cation 2010/0003704 A1), which were available in the cases tested
by immunohistochemistry and genotyping on formalin-fixed,
paraffin-embedded (FFPE) cervical tissues (S. Cheng, U.S. patent
application 2010/0003704 A1).
HPV E6 detection in whole-cell ELISA correlates with the
disease grade. To determine whether this whole-cell ELISA could
detect HPV E6 protein in clinical samples and whether the level of
HPV E6 detected by the assay correlated with the disease grade, we
tested 182 previously collected liquid-based cytology specimens
(
Fig. 2
). We compared the results of the E6 whole-cell ELISA to the
following categories defined by histology and HR-HPV results: (i)
histology and HPV DNA negative (n
⫽ 62), (ii) histology negative
and HPV DNA positive (n
⫽ 27), (iii) CIN1/2 (n ⫽ 51), and (iv)
CIN3
⫹ (n ⫽ 42; 15 CIN3 and 27 cancers). The absorbance of
individual samples and the average absorbance obtained from
each group are shown in
Fig. 2A
and
B
, respectively. E6 whole-cell
ELISA detected significantly more E6 protein in cases of CIN3⫹
than the low absorbance levels observed from clinical samples that
were less than CIN3 (P
⬍ 0.0001) (
Fig. 2B
). We set a positive
threshold for absorbance of 0.33 for subsequent analyses.
The E6 whole-cell ELISA and HPV DNA test results were
com-pared (
Tables 1
and
2
). Thirty-five of the 42 (83%) CIN3
⫹ cases
tested E6 positive. Among the 25 cases of CIN3
⫹ with paired
testing by HR-HPV DNA and E6 whole-cell ELISA, 23 (92%)
tested HR-HPV DNA positive and 18 (72%) tested E6 positive
(P
⫽ 0.2). Among the 140 women without CIN3⫹ (less than
CIN3), 31% tested positive for E6 and 48% tested positive for
HR-HPV DNA (P
⫽ 0.0006).
Since degradation of E6 protein in archived samples may be
responsible for the lower positive rate of E6 whole-cell ELISA than
HPV DNA testing (
Tables 1
and
2
), we conducted a post hoc
anal-ysis on a subset of 45 samples that were tested within 1 to 2 weeks
of collection using the same positive cutoff (OD
⫽ 0.33) (
Fig. 2C
).
Of the 9 women diagnosed with CIN3⫹, 100% tested positive for
TABLE 1 Single-test results of the HPV E6 whole-cell ELISA and HR-HPV DNA
HC2 DNA result
E6 ELISA result
All CIN3⫹ ⬍CIN3
No. tested No. positive % Positive No. tested No. positive % Positive No. tested No. positive % Positive
Positive Negative 165 89 49 25 23 92 140 66 47
Negative Positive 182 78 42 42 35 83 140 43 31
TABLE 2 Paired-test results of the HPV E6 whole-cell ELISA and HR-HPV DNA
HC2 DNA result
E6 ELISA result
All CIN3⫹ ⬍CIN3
No. positive % Cola No. positive % Col No. positive % Col
Positive Positive 49 36 16 38 33 24 Positive Negative 40 21 7 17 33 24 Negative Positive 12 7 2 5 10 7 Negative Negative 64 35 0 0 64 46 NAc Positive 17b 9 17b 40 0 NA Negative 0 0 0
a% Col, column percentage. b
All cervical cancers.
cNA, not applicable.
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HPV E6 whole-cell ELISA and 89% tested positive for HR-HPV
DNA. For the 36 women with less than CIN3, 28% tested positive
for HPV E6 whole-cell ELISA and 58% tested positive for
HR-HPV DNA.
DISCUSSION
We generated a pan-HPV E6 monoclonal antibody that is
ca-pable of binding a common epitope among different high-risk
HPV types. Based on that monoclonal antibody, we developed
a whole-cell ELISA that was more specific but nonsignificantly
less sensitive for CIN3⫹ than HR-HPV DNA detection. This is
the first report demonstrating that a pan-HPV E6
anti-body used in a whole-cell ELISA can detect cervical precancers
and cancer sensitively and specifically. The E6 whole-cell
ELISA presented here incorporates a simple detection method
that can potentially be used in diagnostic laboratories
through-out the world.
In this study, the sensitivity and specificity of our E6
whole-cell ELISA were compared with the results of HPV DNA tests
previously performed by the clinical laboratories that provided
us with the samples for whole-cell ELISA testing. Both E6
whole-cell ELISA and HPV DNA testing were sensitive in
iden-tifying clinical cytologic samples with biopsy-proven CIN3 or
cancer (
Tables 1
and
2
). Post hoc analysis of samples tested
within 1 to 2 weeks of collection suggests that testing of more
recent samples improves sensitivity (
Fig. 2C
). For clinical
sam-ples with histology results graded less than CIN3, E6 whole-cell
ELISA detected a lower percentage of samples than HPV DNA
testing (
Tables 1
and
2
). This result is consistent with published
reports showing that HPV DNA testing has poor specificity (or
high false-positive rates) despite the fact that it is highly
sensi-tive (
5
,
21
). Thus, E6 whole-cell ELISA is significantly more
specific than HPV DNA testing while retaining similar
sensi-tivity for detecting clinically significant disease. This improved
specificity is important, since false-positive rates often result in
excessive referrals and diagnostic procedures.
One of the limitations of this study is the fact that clinical
samples were collected retrospectively. Our E6 whole-cell ELISA
detects HPV E6 protein in CIN3 and cancer samples collected in
ThinPrep or SurePath. In this 182-case study, it is noted that the
E6 whole-cell ELISA is less sensitive for specimens from ThinPrep
than for those from SurePath (data not shown). This could be due
to the different preservative agents and/or the sample age. A
pro-spective study with larger sample size using both ThinPrep and
SurePath for collection from each patient is under way to
deter-mine the effect of the medium and sample age on the detection of
HPV E6 protein by whole-cell ELISA.
Currently, HPV E6 and E7 expression can be measured only at
the RNA level, which requires a more expensive instrument for
analysis and sample processing. Direct measurement of HPV E6
protein expression may avoid the technical challenges associated
with RNA testing. The E6 whole-cell ELISA presented in this study
uses a simple detection method routinely used in diagnostic
lab-oratories and thus may have advantages over RNA detection.
Clinical samples are applied directly to 96-well plates without cell
lysis or protein extraction. HPV E6 protein is detected using a
monoclonal antibody that recognizes an epitope common to the
E6 proteins from most high-risk HPV types. Thus, a single assay
FIG 1 Anti-E6 antibody binds specifically to HPV E6 protein. (A) ELISA results showing that anti-E6 antibody recognizes recombinant E6 proteins from HPV16
and -18 but not recombinant E7 proteins from HPV16 or -18, recombinant HPV16 L1 protein, or the 6⫻His tag. (B) Titration curves of recombinant HPV18 E6 protein detected by anti-E6 antibody in ELISA showing that the limits of detection range from 10 to 100 pg/ml. The data are presented as means and standard errors. (C) Immunoblot using cervical cancer cell line lysates. The top blot shows that anti-E6 antibody recognizes recombinant HPV18 E6 protein (right lane) and E6 protein from cell lysates from HeLa (HPV-positive) but not C33A (HPV-negative) cells. At the bottom is an immunoblot using anti--actin for the corresponding cell lysates. (D) Whole-cell ELISA using anti-E6 antibody detected E6 proteins from HeLa and SiHa cells expressing HPV18 and HPV16, respectively, with signal strengths dependent on the cell density. E6 protein was minimally detected in HPV-negative C33A cells.
Direct HPV E6 Whole-Cell ELISA
September 2012 Volume 19 Number 9 cvi.asm.org 1477
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using anti-E6 antibody can detect the presence of HPV E6 protein
from liquid-based cytology samples.
An essay that detects both HPV E6 and E7 proteins may further
improve assay specificity and sensitivity. Prospective studies are
under way to validate and determine the clinical utility of the assay
using a larger set of recently collected clinical samples. The results
will be compared side by side with HR-HPV16/18 DNA testing.
Such a whole-cell ELISA (using anti-E6, anti-E7, or a combination
of the two antibodies) may be a useful tool to screen for cervical
precancer and cancer, either as a primary screening tool with or
without Pap testing or as a triage test following an abnormal Pap
and/or an HPV-positive result. It may offer an effective tool for
physicians to differentiate precancerous lesions from benign HPV
infections, thus reducing unnecessary repeat testing and invasive
diagnostic procedures.
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FIG 2 Whole-cell ELISA using anti-E6 antibody. (A) Scatter dot plot of the
individual absorbance signals of clinical samples in a whole-cell ELISA using anti-E6 antibody. E6 whole-cell ELISA was performed on cells from 182 cer-vical scrapes that were categorized into the following 4 groups: group 1, his-tology negative and HPV DNA negative (Neg/HPV⫺) (n ⫽ 62); group 2, histology negative and HPV DNA positive (Neg/HPV⫹) (n ⫽ 27); group 3, CIN1/2 (n⫽ 51); and group 4, CIN3⫹ (n ⫽ 42). Lines show means and standard errors of the means (SEM). (B) Average absorbances obtained from the ELISA in panel A graphed as means and SEM. (C) Results from E6 whole-cell ELISA using 45 SurePath fresh samples obtained within 1 to 2 weeks of collection. The data are presented as percent positive rates for histological designations of CINⱕ 1 (36 samples), CIN3 (6 samples), and cervical cancer (3 samples) and compared to the positive rates for HPV DNA.
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