2015 Recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibody to turkey corona

2015·0.58 MB·English

JournalofVirologicalMethods217(2015)36–41

ContentslistsavailableatScienceDirect

Journal of Virological Methods

journal homepage: www.elsevier.com/locate/jviromet

Recombinant nucleocapsid protein-based enzyme-linked

immunosorbent assay for detection of antibody to turkey coronavirus

MohamedAbdelwahab,ChienChangLoa∗,ChingChingWu,TsangLongLin

DepartmentofComparativePathobiology,PurdueUniversity,WestLafayette,IN47907,UnitedStates

a b s t r a c t

Articlehistory: Nucleocapsid(N)proteingeneofturkeycoronavirus(TCoV)wasexpressedinaprokaryoticsystemand
Receiv ed15October2014 usedtodevelo pa nenzym e-link ed immu nosorbentas say(EL ISA) fordetectio n of antibodyto TCoV.A nti-
AReccceepivteedd i2n5 r Feevbisreuda rf yor2m0 1255 February 2015 TCoV hy perimm un e turkey serum a nd normal turke y seru m were use d as positi ve or negati ve contro ls for
optimizationoftheELISA.Goatanti-turkeyIgG(H+L)conjugatedwithhorseradishperoxidasewasused
Availableonline5March2015
asdetectorantibody.Threehundredandtwentytwoturkeyserafromthefieldwereusedtoevaluatethe

performanceofELISAanddeterminethecut-offpointofELISA.TheestablishedELISAwasalsoexamined
Keywords:
withserumsamplesobtainedfromturkeysexperimentallyinfectedwithTCoV.Thoseserumsamples
Coronavirus
werecollectedatvarioustimeintervalsfrom1to63dayspost-infection.Theoptimumconditionsfor
Enteritis
NEnuzcylemoec-alpinsikded immunosorbent assay TdCifofeVreNntpiraotitoenin bceotnwceeenntr aantitoin-TaCto2V0 h(cid:2)ygp/emrilm,smeruunme dsielruutmion aantd1 n:8o0rm0,aaln tdurckoenyju sgeartuemd iwluetiroen reacto1m:1b0i,n0a0n0t.

Recombinantprotein Ofthe322serafromthefield,101werepositiveforTCoVbyimmunofluorescentantibodyassay(IFA).The

Turkey sensitivityandspecificityoftheELISArelativetoIFAtestwere86.0%and96.8%,respectively,usingthe

optimumcut-offpointof0.2asdeterminedbylogisticregressionmethod.Reactivityofanti-rotavirus,

anti-reovirus,anti-adenovirus,oranti-enterovirusantibodieswiththerecombinantNproteincoatedon

theELISAplateswasnotdetected.Theseresultsindicatedthattheestablishedantibody-captureELISA

inconjunctionwithrecombinantTCoVNproteinasthecoatingproteincanbeutilizedfordetectionof

antibodiestoTCoVinturkeyflocks.

©2015ElsevierB.V.Allrightsreserved.

1. Introduction Immunofluorescent antibody assay (IFA) has been developed

andsuccessfullyusedintheserologicaldiagnosisofturkeycoron-

Turkeycoronavirus(TCoV)hasbeenrecognizedasanimportant aviralenteritis(PatelandPomeroy,1976).TheIFAassayissimple

pathogenofyoungturkeys.TCoVinfectioncausessignificanteco- toperformandhighlyeffectiveindetectingantibodiestoTCoVin

nomic losses in turkey industry due to uneven growth and poor turkeyflocksinfectedwithTCoVorrecoveringfromTCoVinfection.

feed conversion. The turkey coronaviral enteritis was the most However,theIFAproceduresaretimeconsumingandlaborinten-

costlydiseaseofturkeysinMinnesotaintheearly1960s(Panigrahy siveforhandlinglargenumbersofsamples.Inaddition,IFArequires

etal.,1973).Outbreaksofasimilarentericdiseaseinturkeypoults well-trainedpersonneltopreparefrozensectionsofTCoV-infected

occurred in Indiana and North Carolina in the early and middle turkeyembryointestinesorslideswithepithelialcellsexfoliated

1990sandremainsasathreattotheturkeyindustry. fromtheinfectedbursaofFabricuisaswellastoperformfluores-

centmicroscopy.

InordertohaveanearlyandrapiddiagnosisofTCoVinfection

and effective control of TCoV-induced enteritis in turkey flocks,

developmentofanantibody-captureenzymelinkedimmunosor-
Abbreviations: BCoV,bovinecoronavirus;BSA,bovineserumalbumin;ELISA,
bent assay (ELISA) for detecting antibodies to TCoV in serum is
enzyme-linkedimmunosorbentassay;HRPO,horseradishperoxidase;IBV,infec-
tiousbronchitis virus;IFA,imm unoflu orescen tantibodya ssay;Mab,m ono clonal essential. ELISA has high specificity and sensitivity and can be
antib ody;NC,ne gative cont rol;PBS,phosphateb ufferedsa line;PC ,posi tivecontrol; appliedto large num berso fsampless imul taneously.
TCoV,turk ey coronavir us;TGEV ,tra nsmissible gastroen teritis viru s;TMB, tetram- Deve lop men tofELISA fo rdetecti onofTCoVinfectionrequires
ethylb enzidi ne.
∗ largeamountsofTCoVantigen.AttemptstopropagateTCoVincell
Correspondingauthor.Currentaddress:3711CollinsFerryRoad,Morgantown,
cultureinmanylaboratorieshavenotbeensuccessful.Whenthe
WV26505,UnitedStates.Tel.:[phone];fax:[phone].
E-maila ddress: chienc hang .loa @m ylan .com( C.C. Loa ). viralantigenpreparedfromtheintestinesandintestinalcontents

http://dx.doi.org/10.1016/j.jviromet.2015.02.024
0166-0934/©2015ElsevierB.V.Allrightsreserved.
M.Abdelwahabetal./JournalofVirologicalMethods217(2015)36–41 37

wasusedasthecoatingantigen,astrongnon-specificreactionwas thecolumnwith5mlofelutebuffer(1Mimidazle,0.5NaCl,20mM

observedintheELISA(Loaetal.,unpublisheddata). Tris–HCl,pH7.9)containing6Murea.Theconcentrationofpuri-

Nucleocapsid(N)proteinofTCoVappearstobethemostabun- fiedNproteinwasdeterminedbyProteinAssayreagent(Bio-Rad,

dantviralpolypeptideincoronavirusinfectedcellsduringallstages Hercules,CA,USA).

of infection. The only known post-translational modification of

coronavirus N proteins is phosphorylation (Garwes et al., 1984). 2.3. SDS-PAGEandWesternblotting

The coronavirus N protein is immunodominant (Ignjatovic and

McWaters, 1991). Carboxyl-terminal 119 amino acids of nucleo- Electrophoresiswasperformedwiththediscontinuousbuffer

capsidproteinofIBVexpressedbyFowlpoxVirushasbeenshown system (Laemmli, 1970). For Western blotting, proteins on the

toinducecross-protectiveimmunityagainstinfectionsamongdif- acrylamide gel were transferred onto nitrocellulose membrane

ferent IBV strains (Yu et al., 2001). The N proteins from various (Millipore, Bedford, MA, USA) with transfer buffer contain-

RNAviruses,suchasmumps,rabies,measles,andIBVhavebeen ing 50mM Tris, 384mM glycine, and 20% (v/v) methanol, pH

used as coating antigens in antibody-capture ELISA for the diag- 8.3. Membranes were incubated with turkey anti-TCoV anti-

nosisofinfections(Reid-sandenetal.,1990;Hummeletal.,1992; serum at a dilution of 1:500 for 1h at room temperature.

Ndifuna et al., 1998). Subcloning of the N gene to a prokaryotic Membranes were washed three times with PBS buffer contain-

expressionsystemhasresultedinahigh-levelinductionofrecom- ing 0.05% Tween-20 (PBS-T) and incubated with horseradish

binantproteininourlaboratory(Loaetal.,2004).Therecombinant perioxidase-conjugated goat anti-turkey IgG (Kirkegard & Perry

TCoVNproteinexpressedfromtheprokaryoticsystemisexpected Laboratories,Gaithersburg,MD,USA)atadilutionof1:500for1h

to maintain its antigenic integrity because it is not glycosylated. atroomtemperature.ThemembranewaswashedwithPBS-Tand

The purpose of the present study was to develop a recombinant covered with 10ml of perioxidase substrate, 0.05% diaminoben-

TCoVNprotein-basedELISAfordetectionofantibodiestoTCoVin zidine (Kirkegard & Perry Laboratories) solution. The blot was

turkeysera. developed and washed with distilled water to stop the reac-

tion.
2. Materialsandmethods
2.4. Serumsamplesandantibodiestodifferentviruses

2.1. Animalwelfarestatement
Positive control (PC) serum was the hyperimmune serum

of turkeys experimentally infected with TCoV. Negative control
TurkeypoultsusedforexperimentalinfectionwithTCoVwere
(NC) serum was collected from normal healthy turkeys grown
obtainedfromacommercialoperation(PerdueFarm,Thorntown,
in the isolation rooms. Three hundred and twenty two turkey
IN,USA).Turkeyswerehousedintheisolationunitsintheanimal
serum samples, positive or negative for TCoV by IFA, from the
facility at Purdue University (West Lafayette, IN, USA). Food and
turkey flocks in the field were used to evaluate the perfor-
waterweresuppliedadlibitum.Allturkeyswerehandledwiththe
manceoftherecombinantNprotein-basedantibody-captureELISA.
guidelinesestablishedbytheUnitedStatesDepartmentofAgricul-
ChickenantiseratoIBV,avianreovirus,orrotaviruswereobtained
ture(USDA)and the protocolfor usingturkeysfor experimental
from SPAFAS (Storrs, CT, USA). Turkey antiserum to avian ade-
infectionwithTCoVinthepresentstudywasapprovedbyPurdue
novirus was obtained from SPAFAS. Bovine antiserum to bovine
UniversityAnimalCareandUseCommittee.
coronavirus (BCoV) and porcine antiserum to transmissible gas-

troenteritisvirus(TGEV)wereobtainedfromNationalVeterinary

2.2. PreparationofNprotein Services Laboratory (Ames, IA, USA). Monoclonal antibody to

enteroviruswasobtainedfromDr.J.S.Guy(NorthCarolinaState

TherecombinantplasmidcontainingtheentireNproteingene University, Raleigh, NC, USA). Dilution factors and other infor-

(pTri-N)wastransformedtocompetentEscherichiacolistrainTuner mation of the antibodies used in the present study are listed in

(DE3)pLacIorOrigami(DE3)pLacI(Novagen,Madison,WI,USA). Table1.
Transf orman ts were gr own i n LB medium c ontaining 50(cid:2) g/ml
ampicillin, 34(cid:2) g/ml chloram ph en icol, and 1% glucose. A s tarter 2.5. OptimizationofELISA

culture of bacteria cells containing recombinant plasmid pTri-N
waspre pa redin3m lofL Bmedium. Cellswerein cubateda t37◦C ThePCandNCserumsampleswereusedtooptimizethecondi-

with shaking at 250rpm overnight. The entire 3ml culture was tionsofELISAfordetectionofantibodytoTCoVusingrecombinant

added to 100ml of fresh medium and incubated until the OD TCoVNprotein.TheELISAwasoptimizedbycheckerboardtestsof
reache d0 .5at6 00 nm wave length.On emi lliliterofcu lturew ass ep- recom b inantTC oVN prote inc oncentratio ns from5to160 (cid:2)g/m l,

aratedasun-inducedcontrol.Theremainingcultureswereinduced dilutionsofserumfrom1:200to1:1600,anddilutionsofconju-

byadditionofIPTGtoafinalconcentrationof1mM.Theinduced gateantibodyfrom1:5000to1:20,000.TheELISAplatecoatedwith
cul tureswe rei ncub ate d foran other4h.Theb ac te rialc ultu reswere reco mbinantN pro teinwa sin cubated at4 ◦Cfor 16h. Thewe llsof
harveste dand thecellp elle twasre s us pend edcomp letelyin 5ml the ELISA pla te were w ash ed with PB S- T fo r 3 tim es and 150 (cid:2)l

ofBugBusterreagent(Novagen)pergramofwetcellweight.The of PBS solution containing 1% bovine serum albumin was added
nu clease reag ent Ben zonase (No vag en) w as adde d w ith 1(cid:2)l (25 to each well.Fo llowinginc uba tionat 37◦Cf or1h,th epla tewas

units)foreverymlofBugBusterreagentused.Aftercentrifugation washed with PBS-T for 3 times as described above. The diluted
at16,0 00 ×gfo r20 m inat4◦C, thesolu blesu pern atantwasdis- serums ample sforPC an d NCwer ep reparedan d100(cid:2) lwa sadded
ca rdedan d th ein sol uble fr action (in clusion bodies)wasd isso lved toeach respecti vew el l.Th ep latew asincuba teda t37 ◦C for1 hand

in 10ml of binding buffer (5mM imidazole, 0.5M NaCl, 20mM washed with PBS-T for 5 times. Goat anti-turkey IgG (H+L) con-
Tr is–H Cl ,pH 7.9)con tainin g6 Mu rea.Therec omb in antN pro tein jugated with horser adis h peroxi dasew asdiluted and 100(cid:2)l was
inthedis solv edin clusionbo di es wasp urifi edbyHis-Bin d column addedto each well.Incuba tionat37◦C for 1handw ash with P BS-T

(Novagen)equilibratedwith10mlofbindingbuffer.Thecolumn for5timeswerefollowedbyadditionofsubstratetetramethylben-
waswashe dsequentiall ywit h10 m lo fbinding buffer and 10mlof zid in e(Kir kegar d&Perry La boratori es) at100(cid:2) l/eachwell.A fter
was hbuffer( 20mMimida zole, 0.5 M Na Cl,20m MTris– HCl ,pH 7. 9) incuba tioninthe da rkfor 30min,100(cid:2) lo f2N HClwa sadd edto

containing6Murea.TherecombinantNproteinwaselutedfrom eachwell.Theabsorbanceofeachwellat450nmwasdetermined.
38 M.Abdelwahabetal./JournalofVirologicalMethods217(2015)36–41

Table1 3. Results
Listof antibodiesagainstavianvirusesanddifferentcoronavirusesandtheirsources

usedinthisstudy.
3.1. ExpressionandpurificationofrecombinantNproteinin

Antibodya Conjugate Sourceb Dilutionc E.coli

Chickenanti-IBV(Mass41) None SPAFAS 1:800

Chickenanti-rotavirus None SPAFAS 1:800 The recombinant N protein from TCoV was induced success-
Chicken anti-reovirus None SPAFAS 1:800 fully as a fusion pro tei n in E. c oli st rains T uner or Orig ami cells
Turkeya nti-TCoV-IN None C.C.Loa 1:800
as revealed by SDS-PAGE and Western blotting (Fig. 1). The size
Turkeyanti-adenovirus None SPAFAS 1:800
ofinducedNproteinisapproximately57kDa.TherecombinantN
ABonvtii-neenatenrtoi-vBirCuosV Mab NNoonnee JN.SV. SGLuy 11::880000 pr oteinwa sp urified by Nickel-chelatin ga ffini tych romatograph y.

Porcineanti-TGEV None NVSL 1:800 TheyieldofpurifiedNproteincouldbe10mgfroma100-mlcul-
Goat an ti-mouse IgG (H+L) HRPO KPL 1:10,000 ture ofTu ne rcells.In co ntrast,2 mgof rec om bin antN p roteinc ould
Goatanti-turkeyIgG(H+L) HRPO KPL 1:10,000
berecoveredfromthesamevolumeofOrigamicellculture.Thus,
Rabbitanti-chickenIgG(H+L) HRPO Sigma 1:10,000
Rabbit anti-porcine IgG (H+L) HRPO Sigma 1:10,000 therecombinantNproteinexpressedfromTunercellswasusedfor
Rabbit anti-bovineI gG( H+L) HRPO Sigma 1:10,000 the development o fantibo dy-capture ELISA fora ntibo dies toTC oV

a BCoV, bovine coronavirus; IBV, infectious bronchitis virus; TCoV-IN, turkey coro- due to the yield being higher than that from Origami cells.
navirusIndianaisolate;TGEV,transmissiblegastroenteritisvirus;Mab,monoclonal

antibody;IgG(H+L),immunoglobulinG(heavypluslightchains);HRPO,horseradish

peroxidase. 3.2. ELISAdevelopmentandoptimization
b SPAFAS,Storrs,CT;Dr.C.C.Loa,PurdueUniversity,WestLafayette,IN;Dr.J.S.

Guy.NorthCarolinaStateUniversity,Raleigh,NC;NVSL,NationalVeterinarySer-
vices Labora tory,Am es,IA; KPL,Kirkeg aard&P erry Labora tories,Ga ithersburg, MD; When the dilution of conjugate was 1:5000, the maximum
Sigm a,St.Louis,M O. PC/NCrat ioo f27was obt ainedwith TCo VNprot eina t40(cid:2)g/ml
c Un -lab eleda ntibodiesweredilutedat1:800andHRPOconjugatedantibodies andse rumd ilu tio nat1 :1600.W hent hedil uti onofco nju ga tewas
weredilutedat 1:10,000. Those dilutio nf actors wer einlin ewiththe optimized
1:10,000,themaximumPC/NCratioof92wasobtainedwithcoat-
conditions of serum sample and conjugate antibody for the ELISA. ingconcen tra tionofTCoV Npro teina t2 0(cid:2) g/m landseru mdil ution

at1:800(Fig.2).Whenthedilutionofconjugatewas1:20,000,the

maximumPC/NCratioof65wasobtainedwithcoatingconcentra-
The ratio of PC to NC was calculated for different combinations tionofTCo VNpro teina t2 0(cid:2) g/m landseru mdi lutionat 1:200.The
of recombinant TCoV N protein concentration, serum dilutions, com bin ation o f recom bi nan t TCoV N protein at 20(cid:2) g/ ml, ser um
and conjugate dilutions. The combination that consistently pro-
dilutionat1:800andconjugatedilutionat1:10,000waschosenas
duced the highest ratio of PC to NC was selected as the optimal
thebestandoptimalconditionforantibody-captureELISAbased
condition for detecting antibodies to TCoV by antibody-capture
onthemaximumPC/NCratio.
ELISA.
2.6. EvaluationofELISA 3.3. ELISAevaluation

Three hundred and twenty two turkey serum samples from Amongthe322serumsamplescollectedfromthefield,101were

turkeyflocksinthefieldwereusedasthetestserumsamplesto positive and 221 were negative for TCoV by IFA. The ELISA val-

evaluatetheELISA.Twowellscontainingallthereagentsexcept ues(S/Pratio)ofIFApositiveserumsamplesrangedfrom1.0882

serumwereleftasblankreferencesineachplate.ThePC,NC,and to 0.0132 and that of IFA negative serum samples ranged from
test se rum s amp le s were analyzed in dupl icate. The ELIS A v alue 0.2 762 to −0.0 04. T he dis tribution of ELIS A values (S/P rat io) of

(S/Pratio)ofeachtestserumsamplewascalculatedas(absorbance serumsamplespositiveforTCoVbyIFAandthosenegativeforTCoV

valueoftestserumsampleminusabsorbancevalueofNC)divided by IFA are shown in Fig. 3. The logistic regression model at the
by (ab so rban ce val ue of P C minu s absorban ce val ue of N C). For op timu mc ut-offp oin tw as 0=(ˇ 1)X+ˇ0 ,whereˇ1 andˇ0 w ere
det ermination o f the op tim um cut -off point o f the E LIS A va lue, −20.1502 and4.2 067,re spec ti ve ly.Th e opt imumc ut- offpo int (X)is
logisticregress ion ana lysiswasu sedtod isting uis hse rumsa mples 0.2(−4.20 67/− 20.150 2).Thesensit ivity andspeci ficityo fthee stab -

positiveornegativeforTCoVbyIFA(ShoukriandPause,1999).The lishedELISArelativetoIFAwere86.0%and96.8%,respectively.The

relativesensitivityoftheELISAwascalculatedasthepercentage agreementbetweenELISAandIFAassaywas95.6%(Table2).

ofserumsamplespositiveforTCoVbyIFA,whichwerepositivein TheELISAresultsofserumsamplescollectedfromtheturkey

ELISA. The relative specificity of the ELISA was calculated as the poults experimentally infected with TCoV showed that the anti-

percentageofserumsamplesnegativeforTCoVbyIFA,whichwere body response was detected initially at 14 days after infection

negativeinELISA.Statisticalcomputationswereperformedbythe andincreasedgraduallyuntil28daysafterinfection.Theantibody

SASprogram(SASinstitute,Inc.,Cary,NC,USA). response was detectable up to 63 days after infection when the

TheestablishedELISAwerefurtherevaluatedwiththeserum experimentwasterminated(Fig.4).TheELISAresponsesofnor-

samplescollectedfromexperimentallyinfectedturkeypoults.One malturkeyserawerenegativethroughouttheentireexperimental

group of 40 turkey poults were inoculated with turkey embryo- period(Fig.4).

propagatedTCoV.Theothergroupof40turkeypoultsofthesame Cross reactivity of recombinant TCoV N protein coated on

agewereinoculatedwithPBSandusedasthenon-infectedcontrol. theELISAplatewithbovineanti-BCoVorporcineanti-TGEVwas

Fiveturkeyswererandomlyselectedfromeachgroupandsacrified notdetectedwithspecies-specificconjugateantibodies.Thecross

at1,3,7,14,21,28,42,and63daysafterinoculation.Thecollected reactivity with chicken anti-IBV antibodies was observed with

serumsamplesweretestedbyELISAusingELISAplatescoatedwith chicken-specific conjugate antibody (Table 3). The reactivity of

recombinantTCoVNproteinpreparedfromthepresentstudy. recombinant TCoV N protein on the ELISA plate with bovine

Forevaluationofcross-reactivity,theNproteincoatedonthe anti-BCoV,pocineanti-TGEV,turkeyanti-adenovirus,chickenanti-

ELISAplateswasreactedwithantibodiestoIBV,BCoV,TGEV,avian rotavirus, chicken anti-reovirus, or anti-enterovirus monoclonal

rotavirus, reovirus, adenovirus, or monoclonal antibody to avian antibodywasnotdetectablewithturkey-specificconjugateanti-

enterovirus. body(Table4).ThecrossreactivityofrecombinantTCoVNprotein
M.Abdelwahabetal./JournalofVirologicalMethods217(2015)36–41 39

Fig.1. Sodiumdodecylsulfate-polyacrylamidegelelectrophoresis(A)andWesternblottingwithantiserumtoturkeycoronavirus(B)ofpurifiedrecombinantturkey

coronavirus(TCoV)nucleocapsid(N)proteinbyHis-Bindcolumnchromatography.Lane1,Insoluablefraction(inclusionbodies);lane2,flowthroughofloadingtheinclusion

bodiestoacolumn;lane3,thefiltratewith10mlbindingbuffer;lane4,thefiltratewithwashbuffer;lane5,theelutedrecombinantprotein;lane6,molecularweight

marker.
100 1.2
90
1
80
70 200 0.8
NC 5600 400 atio 0.6
PC/ 3400 8106000 S/P r 0.4
0.2
20
10 0
0
-0.2
1.25 2.5 5 10 20 40
IFA + IFA -

Concentration of N protein (ug/ml)
Fig.3. Distributionofenzyme-linkedimmunosorbentassay(ELISA)values(S/P
Fig. 2. Checkerboard tests for optimizing coating concentration of recombi- ratio )o fserumsamp les thatwereposit ive(IFA+)ornega tive(IF A−)fort urkeyc oro-
nan ttu rkeycoronavir us(TC oV) nucleocapsi d(N)pro teinandserum d ilutionfor naviru s (TCoV) byimm unofl uore cencean tibody as say(IFA) .Thea ssa ycond itions
antib ody-cap tureenzym e-linked immunosorb ent assay(E LISA ).Each linerep re- werere combin ant turkeycoronavirus(T CoV)nuc leocap sid(N )pro teina t20(cid:2)g/ml,
sentsadilutionfa ctorofserum.T heconjugatedilu tionw as1:10 ,000. Theh ighest serum dilutionat1 :800,a ndconjugate dilutio nat1:10,000 .
PC/NC r atioof9 2waso bt ainedw ithc oatingcon centratio nof Nprotein at2 0(cid:2)g/ml

andserumdilutionat1:800.

constructionofthisparticularfunctioninOrigamicellsmightaffect

theirexpressioncapacity,becausetheexpressionlevelofrecombi-
coatedontheELISAplatewithchickenanti-IBVantibodieswasstill
nantTCoVNproteinwasapparentlyhigherinTunercellsthanthat
detectedwhenturkey-specificconjugateantibodywasused.
inOrigamicells.

It was reported that a recombinant TCoV N protein from

4. Discussion baculovirus-expressionsystemwasusedforthedevelopmentofa

competitiveantibody-captureELISA.Therewereatleast2proteins

Accordingtothemanufacturer,Tunercellswereusedforregular of different sizes in the purified N protein from the baculovirus

expressionofpTri-EXsystemwhileOrigamicellswereespecially expressionsystem(Breslinetal.,2001).Incomparison,therecom-

designedtoenhancetheformationofdisulfidebondsintherecom- binantTCoVNproteinexpressedfromtheprokaryoticsystemcould

binantprotein.Theresultsofthepresentstudysuggestedthatthe be purified to be a homogenous preparation containing only a
40 M.Abdelwahabetal./JournalofVirologicalMethods217(2015)36–41

Table2

Comparisonofantibody-captureenzyme-linkedimmunosorbentassay(ELISA)utilizingrecombinantturkeycoronavirus(TCoV)nucleocapsid(N)proteinascoatingantigen

withimmunofluorescentantibodyassay(IFA)fordetectionofantibodytoTCoV.

Totalno.samples IFA ELISAc Relative Relative Agreementf

+a −b + − Sensitivityd Specificitye

322 101 87 14 86.0% 95.6%

221 7 214 96.8%

a Serumsamplepositiveforantibodytoturkeycoronavirus(TCoV)inIFA.
b Serum sample negative for antibody to TCoVi nIFA.
c Theop timium cutoffEL ISA value(S/ Pr atio)o f0 .20wasobtainedbylogisticregressionmethodasdescribedinmaterialsandmethods.SerumsamplehadELISAvalue
highero rlowertha nthe cutoff value was positiv e (+)or neg ative(−), res pectivel y,inELISA.
d Rel ati vesen sitivi ty= 87/(87 +14) ×100 %.
e Relative specificity= 214/(7 + 214 )× 100%.
f Agreeme nt=(87+21 4)/(87 + 14+7 + 214)×100%.

3 Table4

Reactivityofrecombinantturkeycoronavirus(TCoV)nucleocapsid(N)proteinwith
2.5 antibodies sp ecificfordiff erentc oronaviruse sandav ianvirusesa sde termine dby

enzyme-linkedimmunosorbentassayandusinggoatanti-turkeyhorseradishperi-
2 oxidase(HRPO) conjugate.

450 1.5 Normal Antibodies Absorbance value S/P ratioa Result
OD 1 Inf ected CChhiicckkeenn aannttii--IRBoVtavirus 30..474999 10..100028 −+
0.5 Chichen anti-Reovirus 0.353 0.083 −

Normalchickenserum 0.038
0
Bovineanti-BCV 0.038 0.0009 −
-0.5 0 20 40 60 80 Norma lbovines erum 0.034

Days PI Porcineanti-TGEV 0.037 0.001 −

Normalporcineserum 0.035

Fig. 4. Evaluation of antibody responses in serum samples of turkeys experimen- Turkeyanti-adenovirus 0.048 0.001 −
tallyinfectedwithturkeycoronavirus(TCoV)bytheestablishedenzyme-linked
Normalturkeyserum 0.037
immunosorbentassayutilizingrecombinantturkeycoronavirus(TCoV)nucleocap-
sid(N)proteinas coati ngantige n.Turkeypou ltswer einfectedw ithTCoV at10days Anti-enterovirusMab 0.037 0.001 −

ofage.Fivebirdswererandomlyselectedandsacrificedat1,3,7,14,21,28,42and63 Cellculturemedium 0.037

daysafterinfection.Serumsampleswerecollected,dilutedat1:800,andanalyzed
byth eesta blishedEL ISAme thodwit hreco mbinantT CoVNp ro teinat 20(cid:2) g/mland a S/P ratio was calculated as absorbance value of serum sample minus absorbance
valueofnegativecontroldividedbyabsorbancevalueofpositivecontrolminus
conjugateantibodydilutionat1:10,000.
absorbancevalueofnegativecontrol.

Table3

Reactivityofrecombinantturkeycoronavirus(TCoV)nucleocapsid(N)proteinwith

antibodiesspecificfordifferentcoronavirusesasdeterminedbyenzyme-linked
Expressionlevelofthecellculture-basedbaculovirusexpres-
immunosorbent assay and using species-specific conjugate antibody. sion system is usua lly low er th an that of pro karyotic exp ression
Antibodiesa Absorbance value S/Nb Result syste m.Itisc he aperan dmore conv enien tt opreparelar geamounts

Turkeyanti-TCoV 3.986 90.590 + of pure recombinant TCoV N protein by the prokaryotic system
Normal turkeyser um 0.038 th anby thecellcultu re-bas ed baculov irus sys tem.Theexp ression

Chickenanti-IBV 3.826 112.529 + ofprokaryoticsystemasdemonstratedinthepresentstudyfrom

Normalchickenserum 0.034 preparation of starter for a 100-ml culture to the production of

Bovineanti-BCoV 0.035 1.093 − 10mgofpureNproteincouldbefinishedin20h.Fortheexpres-
Norma lbovinese rum 0.032 sio n o f b aculo vi rus syste m, it too k 52–54 h for in cub atio n of Sf9

cellsafterinfectionwiththerecombinantbaculovirus.Theyieldof
NPoorrcminael apnotric-iTnGeEsVe rum 00..003229 1.103 Npro tein bytheba culov irus systeminth efinalprepa ratio nsw as

notreported(Guyetal.,2002).
a BCoV,bovinecoronavirus;IBV,infectiousbronchitisvirus;TCoV,turkeycoro-
navirus;T GEV,tra nsmissiblega stro enteritisvi rus. Based on the optimum coating concentration of recombinant
b S/Nw ascal culatedasabso rbancevalueof antibodytesteddividedbyabsorbance TCoVNpr ote ina t20(cid:2)g/ml ,thepro ductionofreco mb inantTCoVN

valueofnormalcontrolserum. proteinfroma100-mlcultureisenoughforcoating50ELISAplates.

Theoptimumdilutionofconjugateat1:10,000ishigherthanthat

singleproteinasshowninthepresentstudy.Althoughaprotein (1:1600)inIBV-basedantibody-captureELISAasshowninourpre-

bandbelow50kDawithreactivitytoaTCoVantiserumwasnoted viousstudy(Loaetal.,2000).Theresultsfromthepresentstudy

intheinclusionbodyandflowthroughsolutions.Theproteinwas indicatedthatELISAusingrecombinantTCoVNproteinascoating

likelyadegradationproductoftherecombinantNproteinthrough antigenismoresensitivethanthatusingIBVantigenandrequires

dissolutionandextractionstepsofinclusionbodypellets.Expres- lessamountofconjugateantibodyinthereaction.Consequently,

sionoftwodifferentforms(52and43kDa)ofTCoVNproteinwas thecostofELISAusingrecombinantTCoVNproteinforeverysam-

observedinpreviouspublications(Guyetal.,2002;Gomaaetal., pletobetestedisreduced.Theoptimumdilutionoftestingserum

2008).Theproteinsizeasnotedinthepresentstudyisapproxi- samplesat1:800indicatedthattheestablishedELISAisconvenient

matelyintherangeofsmallerformofthosepreviousobservations. to use and only requires a very small amount of serum for test-
Thispro te ino fsmal ler sizewa snot fo undin thefinal elutionsolu- ing . Th eore ticall y, 0.25(cid:2) l o f ser um sa mple is eno ugh fo r te sting

tionofpurifiedNproteinand,subsequently,wasnotinvolvedin in duplicate wells in the ELISA. In addition, in order to alleviate

theELISAreactivity. variationinthepreparationofserumdilutions,2-stepsequential
M.Abdelwahabetal./JournalofVirologicalMethods217(2015)36–41 41

dilutionsat1:100and1:8wereperformedfortheserumsamples Acknowledgements

inthepresentstudy.

The relative specificity (96.8%) of the established ELISA using We thank Dr. Tom Bryan, Mr. Tom Hooper and Ms. Donna

recombinantTCoVNproteinfromE.coliissimilartothat(96.2%) SchraderfromIndianaAnimalDiseaseDiagnosticLaboratory(West

using recombinant TCoV N protein from baculovirus-expression Lafayette, Indiana, USA) for their assistance in collecting turkey

(Guyetal.,2002)orthat(96.7%)usingIBVantigen(Loaetal.,2000). serumsamplesfromsuspectedturkeycoronaviralenteritiscases

Therelativesensitivityseemedlowerinthepresentstudythanthat orperformingimmunofluorescentantibodytestfordetectinganti-

fromdifferentstudiesanddifferentlaboratories.Itispossiblethat bodytoturkeycoronavirusinserum.WethankDr.DavidHermes

decayofspecificantibodiesintheserumsamplesduringthetime fromPerdueFarm(Thorntown,Indiana,USA)forsupplyingturkey

afterIFAtestandbeforeELISAmaycontribute,atleastinpart,to eggsandturkeypoultsforexperimentalinfection.Wealsothank

thisfinding.Therelativesensitivity(86.0%)oftheestablishedELISA USDA (Grant No. 2001-34413-10421) for providing the financial

inthepresentstudyiscomparabletothat(92.9or93.1%)fromthe support.

other2studies.However,suchslightvariationmightbeduetodif-

ferentpoolsofserumsamplesindifferentstudies.Theresultsof References

lowersensitivityandhigherspecificityinthesestudiessuggested

thatthepredictivequalityofpositiveresultsismorereliablethan Akin,A.,Lin,T.L.,Wu,C.C.,Bryan,T.A.,Hooper,T.,Schrader,D.,2001.Nucleocap-
si dp rote inge nese quen ceanal ysisr evealscl ose genomic rela tionsh ipbetween
that of negative results in the ELISA. This is true to IFA test with the tur keycoro navir usandav ianinfec tiousbr onchi tisvirus. ActaVirol.45 ,31–38.
knownlowsensitivenature. Breslin,J.J. ,Smith,L.G., Full er,F.J. ,Guy,J.S., 1999.Sequ ence analy sisof the turkey
Itsh ould benoted thattheELISAusinganotherrecombinantN coro nav irus nu cleo capsid pro tein g ene and 3 untransl ated reg ion ide ntifies
prote in (Gom aa et al. , 200 8) w as ev aluate d with t he serum sam - 1th8e7 –v1i9ru3s. a s a close rela tive of infect ious br onchitis viru s. Virus Res. 65,
plescollectedfromexperimentallyinfectedandnegativecontrol Breslin,J.J.,Smith,L.G.,Guy,J.S.,2001.Baculovirusexpressionofturkeycoronavirus
turk eys.Asdem onst ratedinthepres entstudy ,the negative control nuc leoc apsidp rote in.Av ian Dis.4 5,136–143.
group co ns istently got low b ack ground readin gs in ELISA th rough- Garwpoersc, iDn.eJ.,t rBaonus nmtiifsfs, iLb.l,e M giallsstoron e, nGt.e Cr.,i tEi sllevmiruasn,i nC.aJ., c1o9n8ti4n. uDoeufseccteivllel irneep.liAcdatvi.oEnx opf.
outtheentireexperimentalperiod.Incontrast,theserumsamples Med.Bio l.173,79–93.
neg ativ eforT CoVbyIFAfr omthe fie ldsgoth ighe rread ingsand Gomaa,M .H.,Y oo, D.,Ojkic,D.,Barta,J.R.,2008.Seroprevalenceofturkeycoronavi-
variation sin ELISA .Th eref ore,th ecu t-offp oin tbased onevalua tion rus inNor thAm er icatur ke ysdete rmi nedby anewlydevelo pe denzym e-linked
imm u nosorb entassa ybased onrecombin an t antige n.Clin.Vac cineImmunol.,
of ELISA performance with those two experimental groups tends to 1839–1844.
belower.Thecut-offvalueasODreadingwasat0.18inthatreport. Hummel,K.B.,Erdman,D.D.,Heath,J.,Bellini,J.W.,1992.Baculovirusexpressionof
Th e cut-o ff p oint as ELISA va lue (S/P ra tio) at 0.2 i n the p resent thenu cleo capsidge neof measl es virusan dut ilityo ftherecomb inantprote in
ind iagnosticenzy meim m unoassa ys.J. Clin. Microb io l.30 ,2874–2880.
dstiuludtyio unsfuaacltloyr sreoffesrse rtuom OsDa mrepaldeiantg1s :a2b5o0vaen 0d.5co. nFjuurgthateermato1r:e2, 0t0h0e Ignjatetoi nvsic,o Jf., aMvicaW naitnefresc, tG io.Pu.s, 1b9r9o1n. cMhiotniso cvl iornuas:l acnhtaibraocdtieersi ztaot it ohnreoe fsterpuicttoupreasl parnod-
inthats tudyw ere lower thanth os e(seru ms ampleat1 :8 00and antig eni cdiffer entiationo fAustralian strain s.J.Gen.Virol.72 ,29 15–2922.
Laemmli,U.K. ,1970.Cleavage o fstructural protein s durin gthea sse mblyofthehead
conjugate at 1:10,000) in the present study. Those differences in ofbac terio phage T4.Natu re 227,680– 685.
theELISAsuggestedlessamountoftestingserumandlowercost Loa,C. C.,Lin,T.L.,Wu ,C. C.,Brya n,A.T .,Thacker,H.L.,Hooper,T.,Schrader,D.,2000.
ofc onjuga tereagent sare require db ytheE LISAfo ran tibodie sto D etec tion ofa ntibo dyto turkey coro navirusb yan tibody-ca pt ureenzym e-l inked
TC oV establis hed in th e pr esent stu dy. 4im98m–u5n0o6s.o rb ent assay ut ilizing infectious br on chitis virus antige n. Avian Dis. 44,
The observation of anti-TCoV antibody response that was Loa,C.C.,Lin,T.L.,Wu,C.C.,Bryan,T.A.,Hooper,T.,Schrader,D.,2004.Expression
induced in experim ent ally infecte d turkeys from 2 w eeks after a ndp urifi catio nof turke ycoro navir usnucle oca psidprote in inEsch erichiacoli.
infection to the end of the e xperime nt in the prese nt study using NdifJu. nVai r,oAl.. ,MWeathteordss, K1.1A 6.,, Z1h6o1u– ,1M67.,.Collisso n,W.E.,1998 .Recom bin antnucleo cap-
recombinantTCoVNproteininELISAwasinlinewiththatusingIBV sidp rot einispo tent iallya nin expensive effec tivese rodiagnostic reagentfor
ascoatingan tigen (L oaetal. ,20 00).T heE LI SAr esults from exp eri- infe ctiousb ro nchitisvirus .J. Virol.Method s70,37– 44.
me ntallyi nfected turke ys we recom para bleto those ofIFA results Panigrahy,B., Naqi,S.A., Hall, C. F.,197 3.Isolati ona ndcharacterizationofviruses
associa ted with tran smiss ible enterit is(bluec omb )ofturkeys.Avia n Dis.17,
as demonstrated previously (Loa et al., 2000). 430–438.
Thecross-reactivitybetweentherecombinantTCoVNprotein Patel, B.L., Pomeroy, S.B., 1976. Indirect fluorescent antibody test for the diag-
andant iserumtoIBVwa sexpecte ddu etothehighs imilar ity (>90%) no sis o f coronav iral enterit is of tur keys (Bluec omb). Am . J. Vet . Re s. 37,
1111– 11 12.
attheaminoacidsequencesbetweenTCoVNproteinandIBVN
pr otei n(Bres linet al.,1999; Akineta l.,200 1) .Inaddi tion, the re Reid1-9S9an0d.Renab, Fie.Ls.,d Siuagmnnoesrt,i cWre.Ja., gSemntitshp, rSe.Jp.,a Freekdafdruom, Ma., rSahbaidesdoNckg,e Hn.eJ.,r Beceollminbi,i nJ.Wan.t,
iscross -antigen icit yb etween turk ey and chick en immunog lobu- expre ssedin baculovirus .J.Clin.M icrobiol. 28,85 8 –863.
Shoukri,M.M., Pa use,C.A.,199 9 .Stati sticalMeth ods forHealthSciences,2nded.CRC
lins (Stephensen et al., 1999). Therefore, both antibody to TCoV in press LLC, BocaRa ton, FL,14 1pp.
turkeyserumandantibodytoIBVinchickenserumweredetectable Stephense n,C .B.,Ca sebolt, D.B .,Ga ngopadhyay,N.N.,1999.Phylogeneticanalysisof
bythe establi shed ELISAas sh ow n inthepr esents tudy .Thepres- ahighly cons ervedreg iono fthepolymeras egen efrom 11coronav irusesan d
en ce o f antibody to IBV in turke y s eru m is no t likely be cause d1 8ev1e–l1o8p9m .ent of a c onsens us pol ymerase chi an re action as say. Virus Res . 60,
infectionofIBVtoturkeyshasneverbeenestablishedorreported. Yu,L.,Liu,W.,Schnitzlein,W.M.,Tripathy,D.N.,Kwang,J.,2001.Studyofprotection
Intheab sen ceo fI BVinfec tion ,antib odyt oIBVisnot ex pectedto by rec omb inantfowl poxvir usexpres sing C-termi na lnucl eocaps id proteinof
be ind ucedint ur key serum.Ift herewere IB Vin fe ction inturkey s, inf ectious bronc hitis v irus again st challeng e. Avian Dis . 45, 340–348 .

itcouldbediagnosedbytheestablishedELISAmethod.