DOI10.5012/bkcs.2009.30.12.2999
Immuno-chromatographic An
이ysis for HPV-16 and 18 E7 Proteins as a Biomarker of Cervical Cancer Caused by Human Papillomavirus
Joo-Ho Kim,1■，如 Il-Hoon Cho,:'a Sung-Min Seo,*Ji-Sook Kim,§ Kyu-HaOh,§ Heun-Soo Kang,# In-GyuKim,p andSe-HwanPaek1"*
'GraduateSchool of Life Sciences and Biotechnology, and ^Program for Bi oMi crosystem Technology, Korea University, Seoul 136-701, Korea. *E-mail:[email protected]
§R&D Center of Bio FocusCo., Ltd., 9F.,DaehyunTechnoworld, Gyeonggi-do437-753,Korea
*MetabolabInc., Cancer Research Center, Seoul National University,Seoul 110-799, Korea
Departmentof Biochemistry and Molecular Biology, CollegeofMedicine, SeoulNational University, Seoul 110-799, Korea
“Departmentof Biotechnology and Bioinformatics, KoreaUniversity, 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.
KeyWords: Rapid test, Papillomaviruses,E7protein as biomarker, Specimen stability, Cervix cancer
Introduction
Human papillomavirus (HPV), a double-stranded small DNA virus,infects basal epithelial cells of theskin and themucosal membraneof anogenitalepithelium. HPVisthought tocause the most common sexually-transmittedviral disease1 and can lead to cancers of the cervix, vulvar,vagina,and anus inwomen.
Numerousstudies havereported thatcervical cancer is mainly caused by anogenital infection with high-riskHPVtypes, in particular, 16 and 18.2Intheir viral genomes,twoearlygenes, E6and E7,areknownto act as viral oncogenes that promote tumor growth and malignant transformationin the host cells.3 Thegene products,E6 andE7 proteins,deregulate thehost cell growthcycle by binding andinactivating the tumor suppr ession protein, p53,and retinoblastoma protein (pRb) ofthehost cell, respectively. 4 TheE6protein binds and degrades p53 via ubiquitin-dependent proteolysis, whichleads toa loss ofthe cell ular protein responsible for maintaining genomic stability.5 Since the E7 protein interferes with cell-cycle checkpoint regu lators,ithas been recognized asthemost important oncogenic protein.6 Forinstance, in HPV-infectedcervicalcells,the E7 protein binds to the hypophosphorylated formof pRband func- aThese authors equally contributed to this research.
tionallyinactivates it,resulting in theliberation of thecellular transcription factor E2F-1.7 It has been reported thatthe E7 pro
tein willbe a valuable diagnostic markerfor the detection of high-risk HPVtypescausing thecancer.8 Therefore, identifi
cation of the E7-related genotype orproteinfrom HPV-16and 18 is needed for the development of in vitrodiagnosticassays.
For the past several decades papanicolaou-stained (Pap) smears have been usedas the conventionalmethodto detectab normal or cancerous cervical cells in cervical smearspecimens for theidentification ofcervical cancer. However, the Pap smear test oftenproduces a false negativeor false positive result and it has been estimated that thesefalse negative andfalsepositive results have been responsible for 10 to50% of theincorrectdiag noses.Toimprove the testperformance,liquid-basedprepara tion9 and pre-screeningsmears10 have been developed. How ever,these methodsrequiretechnologically advanced pieces of equipment and expensivereagents. DNA-basedmethods11 maycircumvent theintrinsic problems associated with achiev ing accurate diagnoses. However,the use of this technology is still limited because this method requires multiple reaction steps, long reactiontimes,well-equipped facilities, andwell- trained personnel.In addition,the DNA-based detection met hodsare not adequate for monitoring the progressstate ofcan
cer.
The aimof this study was todevelopa potentialalternative HPV detection method,the immuno-chromatographic assay, which maynot only be highly accurate, but also rapid, easy-to- use,and inexpensive. The assayinvolves a one-stepprocedure where the complexantigen-antibodyreactions aresequentially carried outbythe lateral flow alongthemembranestripupon the addition of a specimen.12 Thisconcept wasfirst demons
trated for the detection of fertilityhormones suchas the human chorionicgonadotropin13 and luteinzinghormone. More recen tly, this methodwas expandedto the analyses of various in vitro diagnostic markersfor infectious diseases,cancers,cardiac di
seases, and bio-terrorismagents.This methodology can be for
mulated as a qualitative kit using colloidal gold asatracer,which isperceivable by the nakedeye,and alsoas a quantitative sensor by merely employing a colorimetric detector.14Furthermore, in contrast totheDNA-based detection methods,thechromato graphicassaycandetect proteinsas theanalyte, whichconse
quentlyallowsoneto monitor theprogress state of illness.
In this study, we developed a chromatographic assaysystem for the rapid detectionof E7 proteinsproduced from HPV- infected cells,whichwas used as a modelof a cervical cancer specimen. To constructtheanalytical system, monoclonal anti bodies wereraised against E7 proteins prepared usingrecom
binantDNA technology. The captureanddetection binders were then selected to formsandwich-type complexes for each analyte from the host cells,Caski andHeLa celllines,which werein fectedwith the model specimens, HPV-16 and 18, respectively.
Furthermore, the conditions usedto preparethespecimen,such as thelysing agent and treatmenttemperature, wereoptimized tominimizedamage to theprotein and maximizeproteinsta bility.
Methods
Preparationof HPV E7proteins as immunogens. Piotein ex pression: To producerecombinantHPV-16 and 18E7 proteins, complementary DNAs (cDNAs) corresponding to therespective E7 oncogenewere separately cloned into the pGEX-4T-1vector (GE Healthcare,Waukesha,WI),which contained a thrombin cleavage site (Leu-Val-Pro-Arg-Gly-Ser).15 Thegene products were expressed as recombinantglutathioneS-transferase(GST)- E7proteins in Escherichia coli (E. coll)BL21(DE3), and the cellswere harvested after cultivation.They wereresuspended in20 mMTris-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 卩g/mLphenylmethylsulphonyl fluoride (PMSF) (Pierce, Rock ford, IL) and protease inhibitor cocktail (Sigma, St. Louis, MO).16 Thecell lysate wasallowedto incubate onice for30min, and centrifugedat12,000gat4 oC for 10 min. The supernatant was thenpurifiedbyaffinitychromatographyon aglutathione (GSH)-immobilized Sepharose 4B column (10 mL gel packing) (GE Healthcare,Waukesha, WI). The purified fusion proteins were cleavedby thrombin (GE Healthcare, Waukesha, WI) and theresulting E7 proteins were finallyquantified using the bicinchoninic acid (BCA)method(Pierce,Rockford, IL).
Characterization: The GST-E7 proteins before and after thrombin cleavagewere analyzed bysodiumdodecylsulfate
(SDS) polyacrylamide gelelectrophoresis(SDS-PAGE) and Western blotting.Thecrude extract (80卩g) wasboiled in 120 mM Tris-HCl, pH 7.9, containing 4% SDS, 0.02% bromophenol blue,20% glycerol, and 2%2-mercaptoethanol for 10min and thenloadedon a 15%gel for SDS-PAGE analysis. The PAGE gel wastransferredto a nitrocellulose (NC) membrane (0.45 pm poresize) (Whatman, Kent, UK) for Western blotting, and the residual surfaces were blocked in 10 mMphosphate buffer,pH 7.4, containing140 mM NaCl (PBS) and 0.5% casein (Casein- PBS).The membranewas prepared in duplicate and thenin
cubated in polyclonalanti-HPV-16E7 and anti-HPV-18E7 anti bodies(Santa Cruz, CA),respectively,diluted in Casein-PBS containing 0.1%tween-20 (Casein-PBS-TW) for 1h.Themem braneswere treatedwithanti-goat IgGantibody coupledto hor
seradishperoxidase (HRP; Invitrogen, Carlsbad, CA) in the same medium,and signals were finally producedby incubating themwiththe Western blotting substrate (Pierce, Rockford, IL).
Production of monoclonalantibodies. Antibodyproduction:
Monoclonalantibodies specific for theHPV-16 E7 and HPV-18 E7proteinswere prepared asdescribed elsewhere.16Briefly, Balb/cfemale mice(8weeksold)were immunized by intra
peritoneally injecting 10pgoftherecombinant HPV-16 and 18 E7protein after mixing with an equal volume of Complete Freund’s adjuvant (Sigma, St.Louis,MO). After 2weeks,the mice were immunizedwith thesameamount of the E7proteins emulsified with IncompleteFreund's adjuvant (Sigma,St. Louis, MO). Thesame procedure wasrepeated 2 weeks later, and the final boosting wasconducted after the same period with E7 proteins dissolved in PBS.Three days afterthe injection of the final boost,themouse splenocyteswere collected and fused with murinemyeloma cell (sp2/0-Ag14)(AmericanTypeCul tureCollection; ATCC, Manassas, VA), which was usedas a fusion partner.Fusedhybridomacells werescreened based on hypoxanthineaminopterin thymidine(HAT; Gibco BRL, Gai thersburg, MD) selection, andhybridoma clones exhibiting high titers of binding activities to each E7protein wereselectedby immunoassays using antigen-coated microtiter plates. The selected hybridoma cellswere injectedinto 2,6,10,14-tetra- methylpentadecane pristane (Sigma, St. Louis,MO)-primed femaleBalb/cmicetoproducetheantibodiesasascitic fluid, whichwas then purifiedon aprotein G column (5 mL, HiTrap protein G HP)(Amersham Biosciences,Piscataway, NJ). Each antibodyconcentration was determined by absorbance measure
mentat 280 nm.
Isotype determination:The sub-isotypes of the produced anti bodies weredetermined usinganenzyme-linkedimmunosor bent immunoassay (ELISA)-based mousemonoclonal isotyping kit(Pierce, Rockford, IL) according to the manufacturer'sins
tructions. Briefly, 25pg of thegoatanti-mouse immunoglobulin (G+A+M) antibodywas coated ontheinner surfaces of the microwellsat room temperature for 2 h, the residual surfaces were blocked, 50 pL of the monoclonal antibodysamples were reactedat 37 oC for 1 h, thesubclass-specificrabbit anti-mouse immunoglobulin antibodies were subsequently bound, and alka
line phosphatase-conjugated anti-rabbit immunoglobulin G (IgG) antibodywasfinallyadded. Thecolorimetric signalsfrom each wellwereproducedviatheenzyme-substratereaction.
Screening ofantibodies forrapid testing. Antibodylabeling
with colloidal gold: A monoclonalantibodyselected as thede tection binder was labeledwithcolloidal gold (the mean dia meter: 30nm) as previouslyreported.17Briefly,the antibodies dialyzed in 10 mMphosphate buffer were conjugated with col loidal gold that wassynthesized usingthesodium citrate met
hod.Unreacted antibodieswere removedby centrifugationand the antibody-goldconjugateswerethen resuspended in Casein- PBS such that thegolddensity was concentratedby20-fold relativetothe initial gold density.
Immuno-strip pneparation:Therapid testkit for E7proteins was constructedbycombiningdifferent functional membrane stripsasdescribed elsewhere.17 Thefour membrane pads usedin assemblingthe rapid test kit were asfollows(fromthe bottom):
a glass fibermembrane (4 乂20 mm) (Ahlstrom, Whatman, Kent, UK) for sample application, a glassfiber membrane (4 乂7 mm) (Ahlstrom, Whatman,Kent, UK) for the release of the detection antibodylabeled with colloidal gold, a NC membrane(4乂 25 mm) (Millipore, Billerica, MA) for signal generation,and a cel
lulose membrane (4 乂 50 mm) (Ahlstrom, Whatman, Kent, UK) for continuoussampleabsorption.Thesampleapplication pad was prepared byimmersing in 10 mM Tris-HCl,pH 7.5, con
taining 0.1% Triton X-100 and drying at55oC for 4 h. The signal generationpad was preparedby spotting a monoclonal antibody (1 卩L)selected as the capturebinderonthe NC membrane and then allowingit to incubateat 37 oC for 20 min.The four mem
branepads were partially superimposed in length toconstruct theimmuno-strip.
Antibody pair selection:To select antibody pairs for thean alyses ofeachE7 protein,theproduced monoclonalantibodies were used as either thecapture antibodyorthe detectionanti
body. E7protein (1卩g/mL; 100pL) in Casein-PBS was addedto each microwell containinga detection antibody(20-fold con
centrate; 2卩L) labeledwith colloidal gold and reacted for 5min.
The immuno-stripwith animmobilizedcaptureantibody was then immersed into the reaction mixture and, after 15 min, the color signal that appeared on the captureantibody sitewas stored as image in a personal computer. The signals corresponding to eachcombination of antibodies were finally used to select the best pairs.
Determination of sample preparation conditions. Antibody ladling with HRP: Since the E7 proteinmay bedenaturedby the useofHPV-hostlysing agents such as detergents, thestruc tural stability of theprotein was determinedbyELISA employ ingHRP as the tracer.The detection antibodies,clone #35 and
#1 specific toHPV-16 E7 and HPV-18E7 proteins,respectively, wereseparatelycoupledto HRPvia a chemical reaction as pre
viously described.17Briefly,theantibodies werefirstreduced usingDTT (10 mM, final concentration)andthe excess reagent was removed on a SephadexG-15gel filtration column (GE Healthcare, Waukesha, WI). HRP was activated with a 50-fold molarexcess of succinimidyl4-[N-maleimidomethyl]cyclo- hexane-1-carboxylate(SMCC;Pierce,Rockford,IL), and the excessreagent was immediatelyremoved inthesame manner.
Each antibodywas then combined witha5-fold molar excess of theactivated HRP and the conjugation was carriedout at 4 oC overnight.
C이l lysis medium conditions:TheHeLacellline (ATCC, Manassas,VA)was used as a model HPV-infectedspecimen
and grownintheculturemedia recommended by the supplier.
The cellsweremaintained in dulbecco’s modified eagle’s me dium (DMEM; Thermo scientific, Hyclone, Waltham, MA)sup plemented with 10% heat-inactivatedfetal bovineserum(FBS;
Gibco BRL, Gaithersburg,MD) and, atthe timeof analysis, the cellswere harvestedby treatment with trypsin(Gibco BRL, Gaithersburg,MD). After washing withPBS, the harvested cells werelysed for E7 protein extractionat room temperature usingdifferentmedia:Medium a: 50 mM Tris-HCl, pH7.5, con
taining150mM NaCl, 0.5% bovineserum albumin (BSA;Basic medium), 1% Triton X-100, and protease inhibitor cocktail (1:
20 dilution); Mediumb:Basicmedium supplemented with0.5%
NP-40; and Medium c: Basic medium supplemented with 0.5%
NP-40 and the protease inhibitor cocktail. Thetimebetween lysing thesample and subjectingitto ELISAanalysis for HPV- 18 E7protein detection,asdescribedbelow,was varied from 5 minto 8 h.
Lysis temperature:HeLa celllysis with Medium c was carried out separatelyat4 oC and room temperature. Thelysedcells were left at each condition for different time intervals(5minto 16h) priortoELISAanalysis for HPV-18E7 proteindetection.
ELISA: Theimmunoassay usedfor HPV-18E7 protein detec tion wascarried out using the detection antibody (clone #1) labeled with HRP and thecapture antibody(clone #36) immo bilized on thesurfaces of microwell. Thecaptureantibody (1 pg/mL; 100pL) in PBS was added into thewells and incubated in a container maintainedat 100% humidity and 37 oC for 1 h.
Afterwashing with deionizedwater, theresidual surfaces were blocked withPBScontaining3% BSA underthesame condi
tions. The test samples (100 pL) containingtheE7 protein and the labeled detectionantibody (1:1,500 dilution; 25 pL) in Casein-PBSweresequentiallyreacted in themicrowells. The incubation conditionswere identical tothoseused above and a thorough washing was conductedafter each reaction. The enzymesubstrate containing 100 pL 3,3',5,5'-tetramethylbenzi- dine (TMB; Sigma, St. Louis,MO)wasaddedinto each well and reacted for 15 min at room temperature. 1 M Sulfuric acid (50 pL)(Sigma, St. Louis, MO)was finally added to stopthe enzyme reaction, and the colors produced ineachwellwere determinedbymeasuring theabsorbanceat450nm.
An지ytic지performances ofthe rapid test kit.Rapid testkit:
Immuno-strips wereconstructed using theantibody pairsmost suitable for detection of therespective E7proteins toexamine thedetectionlimits for eachanalyte.Two monoclonal antibo dies, clone#35 and clone #1, specific to HPV-16 and 18 E7 pro
teins, respectively, were labeledwith colloidal gold as described above. Theconjugate pad was prepared by sprayingthetwo detectionantibodieson theglass fibermembrane and drying it at 35 oC overnight.Thesignal generation pad was prepared by immobilizingthefollowing threedifferent antibodieson spa
tially separatesites of the NC membrane(fromthebottom):
thecaptureantibodytoHPV-16E7(clone#119),thecapture antibody to HPV-18 E7 antibody (#36), and ananti-mouse IgGpolyclonal antibody as the control. They were dispensedon each pre-determined siteina linear patternon the NC membrane and then incubated at 37oC for 20min.Thesampleapplication padand absorption pad were prepared as described earlier,and thefour membrane pads wereassembledtofabricatetheim
muno-strip, whichwas then placedwithin the pre-designed plasticcassette.
Detection capability: Todeterminethedetection limitofthe rapidtest kit, thestandard sampleswerefirst prepared with the HPV-16 E7protein and HPV-18 E7 protein, respectively. The stock protein solutionwasseriallydiluted with Casein-PBS to variousconcentrations(1to 10ng/mL).Each samplesolution (400 卩L) wastransferred into the sample application pot of the cassette and theantigen-antibodyreactions werecarried out for 15 min. The colorsignalsproduced from the respective assay were stored as imagesin apersonal computer.
Cervical cancer celllinetest: Four celllines(ATCC, Ma
nassas, VA), Caski, HeLa, MCF7, and A549, were used as models of HPV-infectedcellspecimens.Caski cell, which is a cervical cancer cell, was infected with HPV-16 and employed as thepositivespecimen for the HPV-16 E7 protein. HeLacell, another cervical cancer cell,was infectedwith HPV-18 andused as the positive specimenfor the HPV-18 E7 protein. MCF-7 and A549,breastcancer and lung cancercell lines, respectively, were usedas thenegativecontrols. The four celllines weresepa rately cultured in DMEM media containing4 mM L-Glutamine, 4.5 g/Lglucose, and sodiumpyruvate,supplemented with hy poxanthine thymidine (HT) supplement(Gibco BRL, Gaithers burg, MD),antibiotic-antimycotic(Gibco BRL, Gaithersburg, MD),and10% FBS. Aftermaintaining the cultivations in heal thy states, eachcell was harvestedbytreatingthe adherent cells with trypsin. Thecells(approximately, 1 x 106cellseachcell line) were washed twice with PBS andthen lysedin Medium c (400 卩L; see above). Each cell lysate was centrifuged at 400g for 10minandthesupernatants were collected as the analytical samples for use in the rapidtesting kit. All testprocedures (400 卩L sample each test)werethe sameasdescribed above.
Results and Discussion
Recombinant HPV E7proteins. Recombinant protein produc tion: Since E7 proteins fromHPV-16 and18 are not currently commercially available, we neededto manufacture themto use as standard materials for screeningoptimal antibodypairs and evaluatingthesandwich immunoassay systems. Byemploying recombinant technology,18 HPV-16 and 18 E7 fusion proteins with GST were produced from microbialcellsthroughcultiva tion. The SDS-PAGEanalysis revealed that the constituents in the culture broth concentrates were heterogeneous(Figure 1, A) and, thus,the E7 fusionproteins with GST were purifiedon aGSH coupled-Sepharosegel chromatography column.19 When the purifiedfraction wassubjected toSDS-PAGEanalysis, a major band near 49 kDa was observed for theboth HPVstrains (Figure 1, B). Althoughotherminorcomponents were also observed, the purity ofthetarget proteins were greater than 98%
based ona comparison of the band areas. Since themolecular weightof GST wasabout37kDa,the E7 alonewas approxi
mately 12kDa in size.
Molecular characterization: The produced fusion proteins werecleaved using thrombin to obtainthe E7proteins for each HPV strain and thencharacterizedvia Western blotting (Figure 2). Eachcomponent was probed with thecommerciallyavailable specific polyclonal antibodies. Inthis analysis, thefusionpro-
SDS-PAGE forGST-E7 fusion protein
HPV 16 HPV 18
Figure 1. SDS-PAGE analyses of recombinant GST-HPV-16 and 18 E7 fusion proteins expressed in E. coli BL21 (A) and then purified by GSH-Sepharose gel chromatography (B). The GST-E7 protein band was detected at a molecular weight of approximately 49 kDa after frac
tionation.
Westernblot for E7 protein
Figure 2. Western blotting analyses of HPV-16 and 18 E7 proteins be
fore and after thrombin cleavage. Prior to cleavage, the fusion 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.
teins (GST-E7) werefoundto be approximately thesamein the molecular size as mentioned above. However, after cleavage, therewere slight differences in thesize of theE7protein (E7) from each strain. Even thoughall E7geneswererecombined with thesame GSTgene, they maybe expressedatdifferent sizesdepending on theincluded genesize of thevectorwhen translated. According to a previous report, 18 thebacteriallyex pressedHis-tagged HPV-16E7proteinappeared as a predo minant band at 16 kDa even though thetheoretical molecular weight of the E7 His6 monomer was12.1kDa.Underthecon-
Detectionantibody toHPV16-E7
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 labeled 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.
ditions usedin thisstudy, the free HPV-16 and18 E7 proteins were foundto be about 16 and 12kDa in size, respectively,and had the appropriatemolecularstructuresincetheywere able tobind to thecorrespondingspecificantibody.
Optimalimmuno-analytical conditions. Incervical cancer di
agnosis, monoclonal antibodies have been used toimmunolo gically staincancer cells,20 whichmayovercome some of the drawbacksassociated with the conventional histochemical met hod, the Pap smear. Although immuno-staining has the potential tospecifically detect cancer with a single antibody, it often pro
duces a false positive result due to thenon-specific binding of the labeled-secondary antibody employed forsignalgeneration. To alleviatesuch problems, we developed a sandwich-type binding system, the immuno-chromatographic assay,whichis based on the lateralflowof the membrane and hasan additional advantage in regardsto point-of-care testing.17
Optimal antibodypairing: Amongthe monoclonal antibody pools previously established for HPV-16 and 18 detection,16we selected antibodypairs thatcould simultaneously bindtothe res pective E7 protein molecule.Toscreen bymeans ofimmuno
chromatography, anantibody to theHPV-16E7protein was used as thecapturebinder byimmobilizing it on apre-deter
mined site of the NC membrane and theotherantibodieswere used for detection bylabeling them withcolloidal gold.After carryingoutthe chromatographic assays,the color signals that appeared on the capture antibody sites werethen compared with eachother to select theantibody pair (#119asthecaptureand
#35 as the detection) that yielded the highestcomplexformation with theanalyte (Figure 3). The same procedure wasused to select the mostoptimal antibody pair for theHPV-18E7 protein assay (antibody #36as the capture and #1 as the detection) (results not shown). Furthermore,theselected monoclonal anti bodies were subtyped using a commerciallyavailablekitand antibodies#119 and#1 weredetermined to betheIgG2b type, and #35 and #36weretheIgG2a.
Detectioncapability forE7 protein: The antibodypairs for therespectiveanalytewere usedto construct therapidtestkit and the experimental conditionswereoptimizedtomaximize the detectioncapability of the kit. The majorfactors,including theconcentration of thecapture and detection antibodies,and
Table 1. Determination of detection capabilities of the immuno-chro
matographic assays for HPV-16 and 18 E7 proteins under optimal conditions
E7 protein concentration in standard sample
Test results 10 8 6 4 2 1
ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL
For HPV-16 E7 ++ ++ ++ ++ +-
For HPV-18 E7 ++ ++ ++ ++ +-
++:Strong signal; +: weak signal; and -: no signalproduced.
the lateral flow rate alongthe immuno-strip, were optimized as described in ourpreviousreports.17 Standard samples of the targetproteins were prepared andused to analyzethe immuno chromatographic assay according to thepre-determinedpro tocol.The results of this experiment revealed that the produced signals were proportional tothe analyte concentrationand detect
able by thenakedeyes at ananalyte concentration aslowas2 ng/mL for the bothof HPV-16 and 18E7proteins (Table 1).
Although therehavebeenno clearreports regarding the levels of E7 proteinin cervicalcancer cells infectedby HPV,they couldbe presentin traceamounts inreal samples.21 Thissug geststhatthedetectioncapability of the immuno-chromato graphic assay may not besufficient for clinicalapplications, anda more sensitive analyticalmethodsuch as ELISA-based rapid testing22 mayneed tobeemployedin the future. Further more, variations inthelevel oftheE7proteinas a functionof the progress of the virally infected diseaseshouldalsobe clinically determined.
Cervical cancer cell line tests. The E7 protein produced from cells infected byHPV-16 or 18has been reported tobe unstable during storage.23 This instabilitycouldbeevenworse after the cells have beentreated witha lysing agent, such as detergents, duringprotein extraction.Thus, the lysis conditions, such as the detergent type and temperature,were testedinorder to deter
mine the effect ofthese factors on protein stabilization. The resi dual activitiesof the extractedproteins weredeterminedby microtiter plate-basedimmunoassays (e.g.,ELISA)using the same capture and detectionantibodypairs as selected above for each E7 protein.
A) Effect of lysis medium
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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 in test A: ‘Medium a’ con
tained Triton X-100 as the lysing agent, and ‘Media b and c’ contained NP-40 as the lysing agent. Furthermore, ‘Media a and c’ included a pro
tease inhibitor, but ‘Medium b’ did not. In test B, the E7 protein inte
grity was examined at two different temperatures, 4 oC and room tem
perature.
Lysis medium composition: Fortesting in a realsample pre
paration, weselectedthe cervical cancercell line, HeLa cells, as a model host forHPV-18 infection. Among the cell linesderived from thesamesource,theHeLa celllinewas the mostappro
priate for this study since the cells werecultivated fairly fast in normal medium(e.g., DMEM) and reproducibly producedthe E7 protein.6HeLacellshavealsobeen used for studyingthe roleof E7 protein in HPV-inducedcancer.24 The cultivated HeLa cellswere lysedusingthree different typesof mediaeach containing a different composition of lysingagentand protease inhibitor, and theproteinintegrityunder each condition were quantitatively monitored as a functionof treatmenttime (Figure 4, A). For the protein integrity test,an ELISA system wasde vised by chemically couplingthe detection antibody to theen zyme,HRP, and by coatingthe capture antibody on the surfaces of the microwells. Under these conditions,thesystemwas able to detect aminimum of 1.0 ng/mL of E7 protein.
Whenthelysismediumcontained Triton X-100asthecell breakage agent (Medium a), the proteinactivitywasinitiallyso low that only asmall portion of the applied moleculesseemed tobe intact (Figure4, A). Thestructuraldegradation rate, there-
HPV16E7L HPV18 E7L*
Cancercells
Caski HeLa MCF-7 A549
Control
Figuie 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.
after,was quite slow as a functionofincubation time. When NP- 40 was usedas the lysingagent (Media band c), the initialpro teinintegrity was significantly improvedand was 3.5 times higher than that when Triton X-100 was used, but abruptly decreased 10 min afteronsetof treatment.Suchenhancement efl&t of NP-40 has alsobeenpreviously reported.25In contrast, the effect of theprotease inhibitor contained in ‘Media aand c’ was relatively insignificant.Nevertheless, no matter what cell lysis media were used, treatment times longer 100 min seemed to completelydegrade theprotein integrity. These data indicate that the detergent, Triton X-100, which is conventionally used to lyse cells, shouldbe replaced with NP-40 for E7 protein extraction and thelysistimeshould be short priortoanalysis.
Temperatuie effect: Further testswere performedto deter
minethe optimal temperature for E7 protein extraction from the HeLacell culture. Cell lysis was carried out usingthe optimal medium(i.e., ‘Mediumc’ in Figure4,A)attwodifferenttem peratures,4 oC androomtemperature.HPV-18 E7 proteinacti vity was determined as a function of time using ELISA asdes
cribed above (Figure 4, B). These experiments showed that theprotein integrityin thecelllysatewasmaintainedat4 oC for 2 h, whileitwasonlymaintained for 30min when kept at roomtemperature. This result indicated that upon treatment, even with NP-40 as detergent,it is essentialto maintainthe celllysateatlowtemperatureprior toanalysis.
Rapid testpeifoimance: To detect the presence ofE7 proteins viaimmuno-chromatography,we first prepared HPV-infected cell lines that closelymimicked clinical samples usedfor cervi cal cancer diagnosis. These model HPV-infected cellslineswere HPV-16-infected cervical cancer cell line, Caskicell, and a HPV-18-infectedcell line, HeLa cell. In addition, two HPV-ne- gative cells, MCF-7 andA549, were alsoused as controls.After culturing the celllines under optimal conditions, analytical sam ples were prepared by lysis andthen subjected to immunoassays for detection of E7 proteins using therapid testkit (Figure5).
Theimmuno-strip was made todetectHPV-16 and 18 E7 pro
teins at thesametime byimmobilizing eachcaptureantibody (as indicated above) atspatially separate sites ontheNC mem
brane. Identical detectionantibodies labeled with colloidalgold werealsoemployed.In addition,thecontrol line,which gener
ated a colorsignal regardlessof theanalytedose,waslocated on the top oftheanalyte linesby coating a secondaryantibody thatbinds tothemouse IgG.
From the test resultsshowninFigure5,the twoHPV-infec- tion positive cell samples, Caski cell and HeLa cell,were detect ed oneach analyte line, HPV-16E7 and HPV-18E7, respec
tively, onthe immuno-strip. The infection-free samples, MCF-7 cell and A549 cell, didnotexhibitanysignalsattheanalyte lines. These results demonstrated that therapidtestingwas not onlyhighlyspecific, but alsocapableof detectingE7 proteins producedfrom cancer cellsinfected with HPV.
Conclusions
The E7 protein,anoncogene product ofHPV,was detected from hostcells usingan immunoassay,indicating that this pro tein can be used asa diagnosticmarker forcervical cancercaus edby HPV infection.Toobtain immunogens, high-risk HPV types 16and18 E7proteins were produced via DNA recombi nation atmolecular sizes of approximately 16 and 12 kDa, res
pectively. Themonoclonal antibodiesspecifically raised against the recombinant proteins were suitable for constructing the rapid testkit based on immuno-chromatography,which couldsimul taneously measurebothanalytes. In considerationofclinical testsforcancerdiagnosis,wealsodeterminedthe optimal con
ditions forhandlingthe host cell such that theextractedE7 protein could be stable at 4oC for 2 h. Although this rapidtesting kitshowed a cleardiscrimination betweentheHPVinfected and normal cells, thedetection capability maynotbesufficient for futureclinicalapplications. We,therefore,plan toreplace the colloidalgold tracer inthe test kit with a moresensitive label such as anenzyme and also to determine therelationship between the E7 concentrationand the progress of cervicalcan cer.
Acknowledgments.This work wassupportedby the Korea ResearchFoundation Grant fundedbythe KoreanGovernment (MOEHRD) (KRF-2005-041-D00261).
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