间接ELISA检测⽅法
ARTICLE
Development of an indirect ELISA based on a truncated S protein of the porcine epidemic diarrhea virus
Yufeng Li,Fangyuan Zheng,Baochao Fan,Hassan Mushtaq Muhammad,Yao Zou,and Ping Jiang
Abstract:Porcine epidemic diarrhea (PED)is a highly contagious,enteric disease of swine caused by the porcine epidemic diarrhea virus (PEDV).To ?nd a suitable ELISA method to assess the infection of PEDV and the effective-ness of vaccines,we developed and evaluated an indirect enzyme-linked immunosorbent assay (iELISA)based on a truncated recombinant spike (S)protein expressed in Escherichia coli .The parameters of the iELISA were opti-mized,and the cutoff value determined as 0.259by analyzing optical density (OD)values of 80PEDV negative sera con?rmed by western
blot.Repeatability tests revealed that the coef?cients of variation of positive sera within and between runs were both less than 10%.Cross-reactivity assays demonstrated that iELISA was PEDV-speci?
c.A virus neutralization test with sera of 7different OD values showed a positive correlation between the OD values and virus neutralization.The results suggest this iELISA is speci?c,sensitive,and repeatable.F
urther studies should focus on the relationship between OD values of sera and its virus neutralization.Key words:indirect ELISA,PEDV,truncated S protein,virus neutralization.
Résumé:La diarrhée épidémique porcine (DEP)est une maladie entérique du porc hautement contagieuse qui est
causée par le virus de la diarrhée épidémique porcine (PEDV).A?n de concevoir une méthode ELISA apte a
`évaluer l’infection au PEDV et l’ef?cacitédes vaccins,on a élaboréet mis a
`l’essai un test enzymatique indirect par immunoadsorption (iELISA)basésur une forme recombinante et tronquée de la protéine de spicule (S)exprimée
chez Escherichia coli .On a optimiséles paramètres de l’iELISA et établi la valeur seuil a
`0,259en vertu d’une analyse des valeurs de DO de 80sérums a
`PEDV négatif con?rmépar immunobuvardage de type western.Les tests de répétabilitéont révéléque les coef?cients de variation des sérums positifs intraessais et interessais se situaient tous deux sous l
a barre des 10%.Les essais de réactivitécroisée ont démontréque l’iELISA était spéci?que aux PEDV.Un test de neutralisation du virus d’après 7valeurs de DO a mis en évidence une corrélation positive entre les valeurs de DO et la neutralisation virale.Les résultats indiquent que cet iELISA est spéci?que,sensible et
reproductible.Les analyses ultérieures devront s’attarder a
`la relation entre les valeurs de DO des sérums et la neutralisation virale correspondante.[Traduit par la Rédaction]
Mots-clés :ELISA indirect,PEDV,protéine S tronquée,neutralisation virale.
Introduction
Porcine epidemic diarrhea virus (PEDV)is an envel-oped virus possessing an approximately 28kb,positive-sense,single-stranded RNA genome with a 5=cap and a 3=polyadenylated tail (Pensaert and Debouck 1978).The virus possesses glycosylated spike (S),Poll (P1),en-velope (E),glycosylated membrane (M),and an unglyco-sylated RNA-binding nucleocapsid (N)proteins (Song and Park 2012).The S protein of PEDV is a type I mem-brane glycoprotein composed of 1383amino acids and is
predicted to contain a signal peptide (1–24aa),a large extracellular region,a single transmembrane domain (1334–1356aa),and a short cytoplasmic tail (Lee et al.2010).The S protein plays important roles in induction of neutralizing antibodies,speci?c receptor binding,and cell membrane fusion (Chang et al.2002;Sun et al.2008).The S1-GST protein (S1D,aa 636–789)is essential for in-duction of neutralizing antibodies (Sun et al.2007).The N-terminal region of the S protein is especially impor-tant for the receptor binding,and the 25–225aa region is indispensable to bind with amino peptidase N (receptor of PEDV)(Lee 2011).To date,most of the reported PEDV vaccines belong to attenuated vaccines and subunit vac-cines (Kweon et al.1999;Kang et al.2005;Song et al.2007;
Received 8April 2015.Revision received 31July 2015.Accepted 4August 2015.
Y.Li,F.Zheng,B.Fan,Y.Zou,and P.Jiang.Key Laboratory of Bacteriology,Ministry of Agriculture,College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,People’s Republic of China.
H.M.Muhammad.Department of Epidemiology and Public Health,Faculty of Veterinary Science,University of Veterinary and Animal Sciences,Lahore,Pakistan.
Correspondence author:Yufeng Li (e-mail:yufengli@www.doczj/doc/597e2535d5bbfd0a785673aa.html ).
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Meng et al.2013).To date,M protein-based enzyme-linked immunosorbent assay (ELISA)and double anti-body sandwich ELISA have been developed to detect PEDV (Sozzi et al.2010;Ren et
al.2011).N protein and viral protein have been used as coating antigens to estab-lish ELISA methods to detect antibodies against PEDV (Oh et al.2005;Hou et al.2007).Viral proteins have been used as a coating antigen of PEDV ELISA,and ELISA has been compared with virus neutralization.An overall agreement of 84.2%was generated between the serum neutralization and ELISA (Oh et al.2005).
In the present study,an indirect ELISA (iELISA)based on a truncated recombinant protein S (tSc,N-terminal region 25–225aa of PEDV)was developed and assay conditions were optimized.The relationship between the iELISA results and virus neutralization titers was assessed.
Materials and methods
Sera and antibody
Eighty-six sow sera were obtained from farms with the outbreaks of porcine epidemic diarrhea (PED)and health farms without clinical signs of PED.Among of them,6and 80sera were con?rmed as positive and negative,respectively,with western blot (WB)and immuno?uo-rescence (IFA)and were then stored at –80°C.The blood was drawn from the precaval vein of anesthetized pigs.Animal experiments were approved by the Institutional Animal Care and Ethics Committee of Nanjing Agricul-tural University (Approval No.IACECNAU20100902).
Cloning and sequencing of the recombinant tSc protein gene
The tSc gene was ampli?ed from plasmids pFast-S (constructed by our laboratory)using a forward primer (5=-ATAGGATCCAGGTGCCAGTCTACT-3=)and a reverse primer (5=-GCGCTCGAGTTAAGGTTCATAGTAAAT-3=)(positions 20682–21284;DR13:GenBank accession No.JQ023162.1)containing restriction enzyme sites Bam HI and Xho I,underlined).The PCR product was cloned into the prokaryotic expression vector pET-28a(+)on corresponding restriction enzyme sites.This vector was designated as pET-28a/tSc,and its sequence was con-?rmed by sequencing analysis by Invitrogen (Shanghai,China).
Expression and puri?cation of tSc protein
The recombinant plasmid was transformed into Escherichia coli BL-21.The tSc protein was induced by addi-tion of 1mmol/L isopropyl-?-D -thiogalactopyranoside (IPTG).Four hours after IPTG induction,the cells were harvested and lysed by sonication.According to the man-ufacturer’s instructions,under the native condition,the recombinant protein was puri?ed by af?nity chro-matography with Ni-nitrilotriacetic acid agarose.The expressed protein was analyzed by sodium dodecyl sulfate –polyacrylamide gel electrophoresis (SDS–PAGE)with Coomassie brilliant blue R250and con?rmed by WB
analysis using His Tag monoclonal antibody (Boster,China)and PEDV polyclonal antibody prepared by immu-nizing rabbits with tSc protein that containing tSc.
The puri?ed tSc protein was separated by SDS–PAGE using 12%separating gel and 5%stacking gel.Gels were stained with Coomassie brilliant blue R250,followed by immunoblotting.Brie?y,after transferring the proteins to nitrocellulose membrane,the membrane blocking was performed with 10%skimmed milk and incubated overnight at 4°C.The membranes were incubated with His Tag monoclonal antibody and PEDV polyclonal anti-body in blocking solution at 1:5000and 1:500dilution,respectively,for 2h at room temperature.After 1h of incubation,the membrane was extensi
vely washed with PBS containing 0.05%Tween 20(PBST)and incubated for 45min with goat anti-mouse IgG (Boster,Wuhan).After washing,the membranes were incubated with horserad-ish peroxidase (HRP)-tagged goat anti-mouse IgG second-ary antibody (Boster,Wuhan),and the colorimetric reaction was developed using chemiluminescence lumi-nal reagents (Super Signal West Pico Trial
Kit,Pierce).
iELISA
A microtiter plate (96-well;Nunc,Nunclon,China)was coated with 100?L of tSc in 0.05mol/L bicarbonate–carbonate buffer (pH 9.6)and incubated for 2h at 37°C.Following 3washes with PBST,the plates were blocked for 2h at 37°C with 5%skimmed milk in PBST and then incubated for 1h at 37°C with 100?L serum samples in 5%skimmed milk in PBST.All samples were analyzed in triplicate.After
5washes with PBST,the plates were fur-ther incubated with 100?L of HRP-conjugated protein A in PBST at 37°C.Then,the plates were washed again,and the colorimetric reaction was developed using 100?L of chromogenic substrates (0.4mol/L tetramethylbenzidine and 1mmol/L H 2O 2in 100mmol/L acetate buffer,pH 5.6)at 37°C.Color development was stopped with 2mol/L H 2SO 4,and the optical de
nsity at 450nm (OD 450)was recorded using an ELISA plate reader (BioTek,USA).
Optimization of tSc iELISA working conditions
Based on the procedure described above,the optimal antigen concentration and sera dilutions were deter-mined through standard checkerboard titration proce-dures with triplicates.Brie?y,the tSc protein was coated on 96-well microtiter plates in serial 2-fold dilutions from 8?g to 250ng/well.Correspondingly,standard swine PEDV-positive serum and PEDV-negative control serum were also diluted in serial 2-fold dilutions from 1:25to 1:800for optimization.After the antigen and anti-serum dilutions were analyzed,HRP-labeled protein A was added to the plate with dilutions from 1:5000to
1:20000to determine the optimal conjugate dilution.The conditions that gave the highest OD 450ratio between the positive and negative serum (P/N value)and an OD 450value for positive serum close to 1.0(with the OD 450value
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of negative serum ≤0.2)were scored as optimal working conditions.
After the conditions mentioned above were deter-mined,the coating conditions were optimized from at 37°C for 2h to 4°C for overnight.Then,the blocking buffers with 1%BSA,1%gelatin,5%and
10%skimmed milk were used and blocked for 1,2,and 3h.The dilu-tions and incubation time of serum sample and HRP-conjugate staphylococal protein A were optimized with 15,30,45,60,90,or
truncated value120min.The reactions were stopped and optimized by assessing 5,10,15,and 20min.Validation of tSc iELISA
To establish a negative–positive cutoff value for this assay,80negative serum samples collected from healthy pig farms without clinical signs of PED (con?rmed by WB)were tested in duplicate using the tSc iELISA.The cutoff value of the ELISA was calculated from the mean OD values of the 80sera negative samples plus 2or 3standard deviations (SDs).This calculation gives 95%or 99%con? dence,respectively,that all negative values will fall within the de?ned range.
To determine the speci?city of the tSc iELISA,positive sera against porcine circovirus type 2(PCV2),classical swine fever virus (CSFV),pseudorabies virus (PRV),foot-and-mouth disease virus (FMDV),porcine reproductive and respiratory syndrome virus (PRRSV),and transmissi-ble gastroenteritis virus (TGEV)were tested using the tSc iELISA protocol.Each sample was tested in triplicate,and the mean OD values were calculated.At the same time,6positive and another 30negative sera con?rmed by WB and IFA were detected by tSc iELISA to determine the sensitivity of this assay.
Reproducibility experiments
Evaluation of assay reproducibility within and between runs was performed as previously proposed (Jacobson 1998).Six ?eld serum samples (5WB-positive samples and 1WB-negative sample)were selected for reproduc-ibility experiments.For intra-assay reproducibility,3replicates of each serum sample were assigned to the same plate.For inter-assay (between-run)reproducibil-ity,3replicates of each sample were run on different plates.The mean OD values,standard deviation (SD),and coef?cient of variation (CV)were calculated.
Neutralization test
Vero-E6cells (1×105cells/well)were seeded in a 96-well cell culture plate,and plates were incubated ov
ernight at 37°C with 5%CO 2until a con?uent monolayer formed.Seven serum samples with different OD values (1.531,1.294,0.980,0.784,0.661,0.378,and 0.078)were heated at 56°C for 30min and used for testing.Sera were diluted 2-fold and incubated with 200TCID 50of strain DR13at 37°C for 1h.After incubation,100?L of the serum+virus mixture was transferred from each well of the incuba-tion plate to a 96-well cell culture plate containing Vero-E6cells.After 1h of incubation at 37°C,the serum+virus mixtures were replaced by complete Dul-becco’s Modi?ed Eagle Medium with 10%fetal calf se-rum.The plates were incubated at 37°C with 5%CO 2for 5days,and the numbers of wells with cells with cyto-pathic effect were counted under an inverted micro-scope and neutralization titers were evaluated.
Statistics
PASW Statistics for Windows,Version 18(SPSS Inc.,Chicago,Illinois,USA),was used to analyze standard de-viation,CV,and correlation analysis.
Results
Expression,puri?cation,and identi?cation of tSc protein
SDS–PAGE and Coomassie brilliant blue staining dem-onstrated the successful expression of the tSc protein li (Fig.1a ).Compared with the expression of li /pET-28a li culture,tSc with a molecular mass of approximately 28kDa was observed in li /pET-28a-Sc (Fig.1a ).The tSc protein was expressed in large amounts and puri?ed using nickel af?nity chro-matography.SDS–PAGE indicated that the protein was of high purity (Fig.1a ).In addition,WB showed that the tSc protein could be detected with an anti-His antibody and PEDV-positive sera (Fig.1b ). Fig.1.Identi?cation of the truncated recombinant protein S (tSc)protein by SDS–PAGE (a )and western blot (b )by His tag monoclonal antibody and porcine epidemic diarrhea virus (PEDV)polyclonal antibody.
(a )Lanes:M,unstained protein molecular mass marker;1,pET-28a-Sc induced with IPTG;2,pET-28a-Sc not induced with IPTG;3,supernatant of cell lysates;4,precipitation of cell lysates;5,puri?ed Sc protein.(b )Cell lysates were incubated with His tag monoclonal antibody (lanes 1and 2)and PEDV polyclonal antibody (lanes 3and 4).Lanes:1and 3,pET-28a-Sc induced with IPTG;2and 4,pET-28a-Sc not induced with
IPTG.
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Optimization of iELISA working conditions
Using a checkerboard ELISA,the optimal antigen concentration and serum sample dilution were set at 1?g/well and 1:100,respectively,based on the following standards:the OD 450value of positive serum was close to 1.0,the OD 450ratio between positive and negative serum (P/N value)was highest,and the background was www.doczj/doc/597e2535d5bbfd0a785673aa.html ing the same standards,the optimal blocking condi-tion was at 37°C for 2h,and the optimal dilution of the conjugate was de?ned as 1:20000.After the above-mentioned conditions were determined,it was found that 5%skimmed milk in PBST was the optimal block-ing buffer,and the best blocking time was 2h at 37°C.For the optimal exposure time of serum samples and conjugate,it was determined that incubations of 60and 45min for the serum samples and the conjugate,respectively,were suf?cient and time-saving in the tSc ELISA,as the P/N value was relatively stable and high.Finally,the optimal stopping time was determined to be 15min (Fig.2).
Cutoff value of iELISA
The OD values of 80negative serum samples varied from a minimum of 0.052to a maximum of 0.201.Thus,samples with OD values of ≤0.223were considered neg-ative,and those with values of
≥0.259were considered positive (Supplemental Table 11).
After the cutoff was determined,the speci?city of the tSc iELISA was evaluated by testing the reactivity of antibodies against other porcine viruses.The OD values of standard positive sera against
PCV2,CSFV,PRV,FMDV,TGEV,and PRRSV are shown in Table 1.The OD values of all anti-sera were smaller than the cutoff value.These data revealed that there is no cross-reactivity between the PEDV tSc antigen and antibodies against other por-
cine viruses,proving that the tSc antigen is speci?c for antibodies against PEDV.Diagnostic sensitivity tests showed that 6positive sera and another 30negative sera identi?ed by WB and IFA were still positive and negative in ELISA results.
Repeatability of the tSc iELISA
The repeatability of tSc iELISA was determined by comparing the OD value of each ?eld serum sample.As shown in Table 2,the intra-assay CV of 5positive serum samples ranged from 0.019to
0.094,whereas the inter-assay CV of these positive serum samples ranged be-tween 0.038and 0.088.These data indicate that the tSc iELISA is repeatable and yields low and acceptable varia-tion (
DeSilva et al.2003).
Neutralization tests
The results showed that 6positive sera had different levels of virus neutralization activity.Interestingly,OD values are positively correlated with virus neutralization activity (Fig.3).More positive sera are needed to further con?rm the relationship between OD values and virus neutralization titers.
Discussion
In China,PEDV was ?rst con?rmed in 1984,and since 2010,it has caused enteric disease with a devastat-ing impact on swine industry.This disease is character-ized by high morbidity and mortality in preweaning piglets,causing serious economic losses to the swine industry in China (Gao et al.2013;Li et al.2014).On the basis of the gene sequence of PEDV,many methods had been developed to detect the infection of PEDV,such as RT-PCR (Ishikawa et al.1997),duplex RT-PCR (Kim et al.2001),immunohistochemistry,in situ hy-bridization (Kim and Chae 2002),and RT-LAMP (Ren and Li 2011).Recombinant N protein had been used to establish an ELISA method to detect antibodies against PEDV (Hou et al.2007).To date,the recombi-nant S protein as a coating antigen for the ELISA
method has not been reported.
1Supplementary data are available with the article through the journal Web site at www.doczj/doc/597e2535d5bbfd0a785673aa.html /doi/suppl/10.1139/cjm-2015-0213.
Table 1.Speci?city of the truncated S pro-tein indirect ELISA to antibodies against some swine viruses.
Virus antisera X ¯±SD PEDV
1.037±0.012PRV 0.054±0.030PCV20.046±0.016FMDV 0.066±0.028PRRSV 0.055±0.035CSFV 0.047±0.023TGEV
0.086±0.013Negative serum
0.063±0.008
Note:Data are the mean ±standard deviation of 3replicate measurements.PEDV,porcine epi-demic diarrhea virus;PRV,pseudorabies virus;PCV2,positive sera against porcine circovirus type
2;FMDV,foot-and-mouth disease virus;PRRSV,porcine reproductive and respiratory syndrome vi-rus;CSFV,classical swine fever virus;TGEV,trans-missible gastroenteritis virus.
Table 2.Intra-assay and inter-assay repeatability of the in-direct ELISA.Serum sample Intra-assay variability Inter-assay variability X
¯±SD CV X
¯±SD CV 1 1.095±0.0210.019 1.087±0.0510.0472 1.066±0.0770.0720.987±0.0560.0573 1.076±0.0750.070 1.100±0.0970.08840.955±0.0560.0590.912±0.0350.0385
0.730±0.0640.0880.690±0.0660.096Negative
0.096±0.009
0.094
0.100±0.009
0.090
Note:Data are the mean ±standard deviation of 3replications.
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Recent studies reported that the N-terminal region (25–225amino acids)of PEDV is associated with the binding of the virus to the receptor aminopeptidase N (Lee 2011).So,we postulated that antibody against this protein fragment may be associated with virus neu-tralization.In this study,the tSc protein (25–225ami-no acids)was expressed and an iELISA method was constructed.Cross-reactivity assays revealed that the tSc iELISA was PEDV-speci?c,and repeatability tests demonstrated that the assay is repeatable.Thus,the tSc iELISA is simpler to produce and perform,time-saving,and may be more suitable for large-scale surveys of PEDV www.doczj/doc/597e2535d5bbfd0a785673aa.html bining virus neutral-ization results with 7different OD values sera,we ?nd that the OD values obtained by iELISA established by tSc are positively correlated with virus neutralization.The preliminary results showed that the sensitivity of this assay is 100%;however,more positive sera samples should be used to further con?rm the sensitivity.
Conclusion
The tSc iELISA established in the present study was repeatable and speci?c for PEDV antibody detection,simple and economical to produce and perform,and time-saving.The present report may facilitate the devel-opment of a reliable tool for the large-scale detection of PEDV neutralization antibodies and assess the effective-ness of vaccines.
Acknowledgements
We thank Jiang Ping for helpful comments.The au-thors declare no potential con?icts of interest with re-spect to the research,authorship,and (or)publication of this article.This study was supported by public welfare grants from the Ministry of Agriculture,the People’s Re-public of China (201203039),and Priority Academic Pro-gram Development (PAPD)of Jiangsu Higher Education Institutions. References
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