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Direct Human Papillomavirus E6 Whole-Cell Enzyme-Linked Immunosorbent Assay for Objective Measurement of E6 Oncoproteinsin Cytology Samples

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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

http://cvi.asm.org/content/19/9/1474

Updated information and services can be found at:

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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 25␮l 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 100␮l 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 (50␮l/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 50␮l of Streptavidin conjugated with horse-radish peroxidase (HRP) (1:600; Vector Laboratories) for 45 min at RT, washed, and incubated with 50␮l of 3,3=,5,5=-tetramethylbenzidine sub-strate (BD Bioscience, San Jose, CA) for 10 min. The reaction was stopped by addition of 25␮l 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 4␮g/ml of PBS) overnight at 4°C. Blocking of each well was performed on the second day with 100␮l 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

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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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Insights 1:215–225.

Direct HPV E6 Whole-Cell ELISA

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TABLE 2 Paired-test results of the HPV E6 whole-cell ELISA and HR-HPV DNA HC2 DNA

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