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IRMET-11850; No. of Pages 9

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Contents lists available at SciVerse ScienceDirect

Journal of Virological Methods

jou rn al h om epage: www.elsev ier .com/ locate / jv i romet

mmunodiagnosis of foot-and-mouth disease using mutated recombinant 3ABColyprotein in a competitive ELISA

aurav K. Sharma, Jajati K. Mohapatra, Laxmi K. Pandey, Sonalika Mahajan, Basavaraj S. Mathapati,niket Sanyal, Bramhadev Pattnaik ∗

roject Directorate on Foot and Mouth Disease, Mukteswar, India

rticle history:eceived 4 October 2011eceived in revised form 28 May 2012ccepted 30 May 2012vailable online xxx

eywords:oot and mouth diseaseifferentiation of infected from vaccinated

a b s t r a c t

Differentiation of infected from vaccinated animals (DIVA) is essential for effective control of foot-and-mouth disease (FMD) by vaccination. The antibody response against FMD viral non-structural proteins(NSPs) has been used widely for this purpose. Among all the NSPs, the 3ABC polyprotein has been rec-ognized as the most appropriate indicator for DIVA. In this study, mutated full-length 3ABC polyproteinwas expressed in a prokaryotic system and monoclonal antibody against the recombinant protein wasdeveloped. A competitive ELISA (C-ELISA) for DIVA was standardized for different species of livestockanimals using recombinant 3ABC and monoclonal antibodies. The diagnostic sensitivity and specificity ofthe assay were estimated by testing a panel of known serum samples consisting of sera from naive, vac-

nimals (DIVA)ompetitive ELISAABCOC

cinated and infected animals as 86.9% with 66.4–97.2 (95%) confidence interval and 97% with 89.6–99.6(95%) confidence interval respectively at 40% inhibition cut-off. The assay was validated further by testingsera from different livestock species collected at random from different parts of the country. The assaywill provide a common method for testing sera from different species of livestock and wild animals. TheC-ELISA is a sensitive and specific DIVA assay for FMD and can be used as a method for FMD controlprogramme with vaccination.

. Introduction

Foot-and-mouth disease (FMD) is a highly infectious and conta-ious disease of cattle, buffalo, pig, sheep, goat and wild ruminantpecies including elephants. There are seven distinct serotypes ofMD virus (FMDV), designated as O, A, C, South African TerritoriesSAT) 1–3 and Asia1. The economic impacts of FMD reflect mainlyn production losses while the indirect losses are associated withhe restrictions of international and regional trade. To date, thedopted policies by FMD free nations are to prevent this infection byestroying “infected and in-contact” animals together with a bann the import of animal and animal products from the endemicountries. Slaughtering large number of animals is not economicalence the consensus for emergency vaccination using inactivatediral antigens in preference to testing and slaughter is increasing.

Please cite this article in press as: Sharma, G.K., et al., Immunodiagnosis of foin a competitive ELISA. J. Virol. Methods (2012), http://dx.doi.org/10.1016/

ccordingly “vaccinate-to-live” policy is now emerging as a real-stic alternative to large scale slaughter for control of the diseaseParida, 2009), at the same time in endemic countries, control by

∗ Corresponding author at: Project Directorate on Foot and Mouth Disease, Muk-eswar 263138, District Nainital, Uttrakhand, India. Tel.: +91 5942286004; fax: +91942286307.

E-mail address: pattnaikb@gmail.com (B. Pattnaik).

166-0934/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jviromet.2012.05.029

© 2012 Elsevier B.V. All rights reserved.

vaccination is a practical option. However, vaccination of suscep-tible herds raises some other critical issues such as differentiationof infected from vaccinated animals (DIVA) and the developmentof carrier animals due to vaccination (Uttenthal et al., 2010) henceactive surveillance of the disease is required to control the disease.Serology helps in retrospective diagnosis and surveillance of FMDwhere animals are screened with very sensitive enzyme linkedimmunosorbent assay (ELISA) which detects antibodies againstthe structural proteins of FMDV. Vaccination with inactivated vac-cines elicits antibody response against the structural proteins ofthe virus similar to the infection, making discrimination of infectedand vaccinated herd difficult. To overcome this problem, differ-ent methods have been proposed such as virus isolation, reversetranscriptase-polymerase chain reaction (RT-PCR), virus neutral-ization test, mucosal IgA ELISA and non-structural proteins (NSPs)based ELISAs. Among all these methods, NSPs based ELISAs are themost promising because of their high sensitivities and specifici-ties. Purified preparations of vaccines consist of inactivated virionswhich almost exclusively induce antibodies against the structuralproteins and not to the NSPs (Clavijo et al., 2004a). These purified

ot-and-mouth disease using mutated recombinant 3ABC polyproteinj.jviromet.2012.05.029

FMD vaccines can be considered as the marker vaccines for DIVA(Parida, 2009). Among all the NSPs, detection of antibodies against3ABC polyprotein of FMDV has been shown to be early and reli-able indicator of infection, since this polyprotein is cell associated

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Table 1List of primers with restriction enzyme site reorganization for amplification of whole3ABC gene along with inserted site directed mutagenesis.

S. No. Primer name Primer sequence No. of bases

1 3ABCF 5′ TTC AAG GAT CCA ATT CCTTCC CAA AAG

27

2 3ABCR 5′ TGT CAG CGG CCG CGT GGTGTG GTT CGG

27

3 3C163R 5′ CTC CTC CGC CGT AGC CAGCCT TA

23

4 3C163F 5′ TAA GGC TGG CTA CGG CGGAGG AG

23

5 3C46F 5′ CGT ACC TCG TTA TCT TTT 22

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nd does not come in antigen fragment during vaccine purificationBergmann et al., 1993; Mackay et al., 1998b).

The aim of this study was to develop and validate a compet-tive ELISA (C-ELISA) for DIVA using recombinant mutated 3ABColyprotein and monoclonal antibodies (mAbs) which can be usedor wide range of species.

. Materials and methods

.1. Serum samples

A panel of the sera was prepared as per the standard guidelinesor standardization and statistical validation of immunodiagnosticssays (Jacobson, 1998; Parida et al., 2007). The serum samples fromMD naive, vaccinated and infected animals were obtained fromhe serum repository maintained at Project Directorate on Foot and

outh Disease (PD-FMD), Mukteswar, India. A total of 3465 bovine,28 pig, 42 sheep and 159 goat serum samples were included in thetudy. The status of 120 bovine samples was confirmed by testing inABC antigen based commercial indirect ELISA kit (Svanovir FMDVABC-Ab ELISA Kit, Svanova Biotech, Sweden), while rest of the seraere tested in house recombinant 3AB indirect ELISA (Mohapatra

t al., 2011) and in house liquid phase blocking ELISA (LPBE). Thistudy complied with the international standards for animal welfare.

.1.1. FMD negative bovine seraBovine sera of known negative status (n = 55) were included in

he panel. Sera were collected at zero day from 27 animals used inMD vaccine potency testing. Four adult bovine cattle sera of FMDree origin were procured commercially (Hyclone Laboratories,ogan, UT, USA; Sigma–Aldrich, St. Louis, MO, USA; Life Technolo-ies, NY, USA and PAA Laboratories, Ontario, Canada). Another 24era were collected from naive animals from a farm having no his-ory of FMD in last five years and located at an isolated geographicalocation.

.1.2. FMD positive bovine seraBovine sera of known to be positive for FMD (n = 65) were

ncluded in the panel. Bovine sera (n = 23) from cattle and buf-alo with clinical disease were obtained from the serum repository.hese animals were found to be infected with either FMDVerotypes O, A or Asia1 as confirmed by virus isolation and neutral-zation assays. Serum samples (n = 42) were collected from cattle

ith a recent history of FMD and included in the panel. Sera fromhallenged animals were obtained from the commercial organiza-ion engaged in FMD vaccine potency testing. These samples wereollected from four cattle on 0 days post-infection (DPI), 3 DPI, 5 DPI,4 DPI and 21 DPI to assess the earliest detection limit of the ELISA.

.1.3. FMD pre- and post-vaccinated bovine seraBovine sera (n = 1150) were obtained from the areas where

MD control programme (FMD-CP) with vaccination haseen implemented and animals were immunized (at least0 vaccinations/animal) with different commercial vaccines avail-ble in the country. Out of these, 694 samples were collectedmmediately before the vaccination and another 456 samples afterne month post vaccination. Bovine sera (n = 2175) collected atandom from different parts of the country were included in theanel for validation of the assay.

.1.4. Sera from other livestock animalsSera from goats (n = 159) and sheep (n = 42) were obtained from

Please cite this article in press as: Sharma, G.K., et al., Immunodiagnosis of foin a competitive ELISA. J. Virol. Methods (2012), http://dx.doi.org/10.1016/

he FMD endemic regions where small ruminants are usually notaccinated. Samples were classified as FMD negative or positiveased upon the FMDV structural protein and NSP responses in LPBEnd recombinant 3AB3 indirect ELISA respectively (Ranabijuli et al.,

CGC6 3C46R 5′ TGC GAA AAG ATA ACG AGG

TAC G22

2010). Pig sera (n = 128) were obtained from the serum repositoryof which 22 samples were collected from a farm which had recenthistory of FMD outbreak. The clinical samples collected from thefarm were confirmed for FMDV by viral antigen trapping ELISA andmultiplex PCR. The remaining of the samples were collected fromhealthy animals with unvaccinated status.

2.2. Virus strain and 3ABC cDNA template

The supernatant from baby hamster kidney (BHK-21) cell mono-layers infected with IND 491/1997 (Asia1 serotype) FMDV isolatewas used for RNA extraction for 3ABC gene amplification. RNA wasextracted using Qiagen RNA easy kit (Qiagen, Hilden, Germany)according to the manufacturers’ instructions. The extracted RNAwas quantified in Nanodrop (Thermo, Wilmington, DE, USA) andreversed transcribed using oligo dT reverse primer (Qiagen, Hilden,Germany) and M-MLV reverse transcriptase enzyme (Promega,Madison, USA) as described earlier (Mohapatra et al., 2011). Thesynthesized cDNA was stored at −20 ◦C till further use.

2.3. Primers

A set of six primers were designed for amplification of the fulllength 3ABC gene with substitution of two key amino acids in theprotease active sites of 3CPro. The amino acid cystedine-163 (GGC)and histidine-46 (CAT) were substituted by glycine-163 (TGC) andtyrosine-46 (TAT), respectively in 3CPro gene by site directed muta-genesis. The details of primers along with their amplification targetare listed in Table 1. Primers 3ABCF and 3ABCR were modified at5′ ends to insert recognition sites for restriction enzymes BamH 1and Not 1 respectively. All the primers were synthesized by theMetabion (Martinsried, Germany).

2.3.1. In vitro amplification of the 3ABC geneThe full length 3ABC gene with amino acid substitutions was

obtained by an overlap PCR technique as described previously (Hoet al., 1989). The 3ABC gene was amplified in three segments des-ignated as segment I, II and III. Three fragments were combined inthe subsequent fusion reactions by PCR to obtain the full length3ABC gene. Briefly, segment I was amplified using 2 �l cDNA, 5 mMeach of 3ABCF and 3C46R primers, 1.5 mM MgCl2, 5 mM dNTPs, and1 U Pfu polymerase enzyme (Fermentas, Ontario, Canada) in PCRbuffer. The reaction mixture was placed under initial denaturationat 95 ◦C for 2 min followed by 35 cycles of 95 ◦C for 30 s, 58 ◦C for30 s, 72 ◦C for 2 min. The DNA extension was continued for another10 min. Segment II was amplified from the cDNA using the 3C46F

ot-and-mouth disease using mutated recombinant 3ABC polyproteinj.jviromet.2012.05.029

and 3C163R primers and reaction was placed under initial denat-uration of 95 ◦C for 2 min followed by 35 cycles of 95 ◦C for 30 s,60 ◦C for 30 s, 72 ◦C for 1 min and final extension for 10 min. Simi-larly, segment III was amplified using 3C163F and 3ABCR primers

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t 95 ◦C for 2 min followed by 35 cycles of 94 ◦C for 30 s, 58 ◦C for0 s, 72 ◦C for 1 min and final extension for 10 min. All the amplifiedroducts were gel purified using gel purification kit (Qiagen, Hilden,ermany) according to the manufacturers’ instructions. Fused seg-ents I and II were obtained by overlap PCR using 1 �l each of

el purified segment I and II as DNA template, 5 mM of 3ABCF andC46R primers, 1.5 mM MgCl2, 5 mM dNTPs, 1 U Pfu polymerasenzyme in PCR buffer and the reaction was placed under touch-upCR. The reaction mixture was denaturated at 95 ◦C for 2 min thennderwent 10 cycles of 95 ◦C for 30 s, 50 ◦C for 30 s, 72 ◦C for 2 minollowed by 25 cycles of 94 ◦C for 30 s, 60 ◦C for 30 s, 72 ◦C for 2 min.he final extension was continued for 10 min. Full length 3ABC geneas obtained by fusion of segments I, II and III. Gel purified segment

II and fused segments I and II as DNA template, 3ABCF and 3ABCRrimers, 1.5 mM MgCl2, 5 mM dNTPs, 1 U Pfu polymerase enzymeere mixed in the PCR buffer and reaction was placed under the

ouch-up PCR. The reaction mixture was placed under the initialenaturation of 95 ◦C for 2 min then 10 cycles of 95 ◦C for 30 s, 50 ◦Cor 30 s, 72 ◦C for 2 min followed by 25 cycles of 95 ◦C for 30 s, 60 ◦Cor 30 s, 72 ◦C for 2 min. The final PCR product was gel purified andtored at −20 ◦C till further use.

.3.2. Molecular cloning of the mutated 3ABC geneStandard molecular cloning techniques (Sambrook and Russell,

001) were followed for cloning the 3ABC gene into the prokary-tic expression vector pET45(b)+ (Novagen, Darmstadt, Germany).riefly, gel purified full length mutated 3ABC gene and vectorET45(b)+ were digested with BamH 1 and Not 1 (Promega, Madi-on, USA) restriction enzymes as prescribed by the manufacturer.he gel purified double digested vector and insert were ligatedogether using DNA ligation kit (Novagen, Darmstadt, Germany)s per the manufacturers’ instructions. The ligated insert and vec-or were transformed in competent Escherichia coli JM109 cellssing calcium chloride method (Sambrook and Russell, 2001) andransformed cells were selected on ampicillin LB agar plates. Theacterial colonies were screened for 3ABC gene amplification inolony PCR using insert specific primers. The plasmid was isolatedrom positive clone using plasmid purification kit (G-Biosciences,

O, USA) according to the manufacturers’ instructions.The purified recombinant plasmid was retransformed into

rokaryotic expression host cell strain E. coli BL21 (pLysS DE3)Novagen, Darmstadt, Germany). Transformed cells were selectedn LB agar plate containing ampicillin and chloramphenicol. Posi-ively transformed cells were selected by colony PCR as describedbove.

.3.3. Confirmation of molecular cloningThe purified cloned plasmid was double digested with BamH 1

nd Not 1 restriction enzymes and resolved on 1% agarose gel toetermine the release of the gene insert. Nucleotide sequence of

nsert was determined using the T7 terminator primer providedlong with the pET45(b)+ vector in an ABI 3130 DNA automatedequencer (Applied Biosystems, CA, USA) to check the orientation ofhe insert and substitutions of nucleotide bases at specific locations.

.3.4. Expression of mutated 3ABC polyproteinDifferent combinations of time, temperature and IPTG concen-

ration were optimized to maximize the expression. Overnightulture of transformed E. coli BL21 (pLysS DE3) cells was inoculatedt 1:100 dilutions into the fresh LB broth containing ampicillin andhloramphenicol and incubated in the shaker incubator at 37 ◦C and80 rpm for approximately 3 h (until OD reaches approximately

Please cite this article in press as: Sharma, G.K., et al., Immunodiagnosis of foin a competitive ELISA. J. Virol. Methods (2012), http://dx.doi.org/10.1016/

.6). The final concentration of 1 mM IPTG was added for induc-ion and culture was incubated in the shaker incubator at 30 ◦C and20 rpm for 6 h followed by centrifugation to pellet down the cells.

nduced culture supernatant was concentrated by adding 5% PEG

PRESSal Methods xxx (2012) xxx– xxx 3

8000 and 20% NaCl and incubating it at 4 ◦C overnight followed bycentrifugation at 12,000 × g for 15 min to collect the concentratedpellet. Both the bacterial pellet and the medium supernatant con-centrate were treated with sample treatment buffer for SDS-PAGEanalysis (Sambrook and Russell, 2001).

2.3.5. Detection, purification and concentration of expressedrecombinant 3ABC protein

Induced bacterial proteins, pre-stained protein marker (Fer-mentas, Ontario, Canada), un-induced and untransformed E. coli celllysate were resolved on 12.5% SDS-PAGE gel. The gel was stainedwith Coomassie brilliant blue stain for 2 h in the shaker followedby destaining. For Western blot analysis, resolved SDS-PAGE gelwas blotted on the 0.4 �m nitrocellulose membrane (Pierce, IL,USA) using an electrophoresis apparatus (Atto, Tokyo, Japan) atconstant volt of 90 V for 1 h. Blotted nitrocellulose membrane waspre-blocked in the blocking buffer [3% BSA in 0.1 M Tris-Base, 0.5 MNaCl, Tween-20 (0.1%) Sigma–Aldrich, St. Louis, MO, USA] at 4 ◦Covernight and anti-His conjugated monoclonal antibodies (Qiagen,Hilden, Germany) diluted to 1:2000 in a blocking buffer were addedfor 1 h. After three washings, the reaction was developed by addingsubstrate solution containing hydrogen peroxide (H2O2) and DAB(Sigma–Aldrich, St. Louis, MO, USA) until the band of desired inten-sity appeared. The reaction was stopped by adding deionized water.Similarly, reactivity of the expressed recombinant protein was esti-mated using FMD bovine convalescent serum diluted to 1:20,000in blocking buffer by Western blot.

The expressed recombinant 3ABC polyprotein was purified frombacterial lysate using HisPur Ni-NTA resin kit (Pierce, IL, USA) withminor modifications. Briefly, induced transformed E. coli cells werepelleted by centrifugation at 3000 × g for 15 min. The media super-natant was discarded and pellet was homogenized in the lysisbuffer provided in the kit with a help of 20 G needle and syringe.For complete lysis, re-suspended culture was sonicated (Soniprep,Sanyo, Japan) at 15 mAmp for 20 s with 10 s of interval in the icechamber until solution became clear. The bacterial lysate was cen-trifuged at 12,000 × g for 5 min to settle down cell debris. Theclarified supernatant was loaded on to the nickel NTA column andallowed to pass through. The flow-throw from the column wascollected and preserved for SDS-PAGE analysis. The column waswashed twice by passing wash buffer-I then with wash buffer-II, provided in the kit. The bound proteins were eluted using theelution buffer. The purified protein was concentrated using 10 kDacut-off membrane centrifugation column filter (PAL Life Sciences,NY, USA).

The recombinant 3ABC polyprotein was purified in addition bySDS-PAGE passive gel elution method, adopted from Thermo Sci-entific technical tips with modifications. The concentrated elutionfraction from nickel NTA metal chromatography was treated withthe sample treatment buffer and resolved on 12.5% SDS-PAGE ina single large well. A negative control of untransformed E. coli celllysate and prestained protein marker were also resolved along withtarget protein in small wells. A strip of gel containing negativecontrol, prestained marker and thin strip of the 3ABC protein wasincised and stained with Coomassie brilliant blue stain for 1 h inshaker followed by destaining. During staining, the reminder ofthe gel was kept soaked in deionized water. The destained gel stripwas realigned with rest of the gel and desired protein band wasincised with scalpel blade and chopped into small fractions. Theresolved protein was eluted passively from the polyacrylamide gelunder denaturating conditions by adding passive gel elution buffer

ot-and-mouth disease using mutated recombinant 3ABC polyproteinj.jviromet.2012.05.029

(0.1% SDS in 0.25 M Tris–HCl), vortexed and incubated at 37 ◦Cfor 2 h. The protein elution buffer was collected using a pipettein 2 ml micro-centrifuge tube. Eluted protein was concentratedusing cut-off membrane and preserved by adding the protease

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ocktail inhibitor set III (Novagen, Darmstadt, Germany) and storedt −40 ◦C.

.4. Monoclonal antibody production

Anti-3ABC mAbs were developed against gel purified recom-inant 3ABC polyprotein by out-sourcing to a commercialrganization (Merck-Bangalore Geni, Bangalore, India). Hybridomaulture supernatants were received from the manufacturer andcreened for reactivity against the 3ABC protein by indirect ELISA.he media supernatants from the selected hybridoma clones werelso screened for non-specific reactivity with E. coli lysate, BSA, andHK-21 cell lysate in ELISA. Specific reactive mAb producing clonesere selected. Isotype specificities of the selected mAbs were car-

ied out by the commercial organization (Merck-Bangalore Geni,angalore, India).

.5. Optimization of the C-ELISA

Different combinations of antigen concentration, mAb dilution,est serum dilution and conjugate dilution were optimized for stan-ardization of the C-ELISA. Affinity purified and whole anti-mousentibodies conjugated with HRPO were evaluated for tracing of theAbs bound to the antigen in the C-ELISA. The optimized proto-

ol is described in brief. Wells of ELISA plate (Nunclone, Roskilde,enmark) were coated with approximately 50 ng of the 3ABC

ecombinant purified protein in 50 �l of carbonate bi-carbonateoating buffer (pH 9.2) and incubated at 4 ◦C overnight. The coatedells of the ELISA plates were washed with the wash buffer (0.05%

ween-20 PBS) and were preblocked with 50 �l of 1.5% skimmedilk powder (SMP, Merck, NJ, USA) in 0.05% Tween-20 phosphate

uffer saline (PBS) for 1 h at 37 ◦C. In a low protein binding perspexlate, sera from bovine sera panel were diluted to 1:7.5 in 0.025%ween-20 PBS. Pre-blocked ELISA plates were washed three timesith wash buffer (0.05% Tween-20 PBS) for 3 min and 50 �l fromiluted sera were transferred in duplicate to the wells. mAbs fromve selected hybridoma clones (3B3A5, 3B3A10, 3B3H7, 3E7F8A10,nd 3E7E3G10) were pooled in equal proportion to produce anntibody pool for use. 50 �l from the pool of mAbs was added toach well of the ELISA plate. For antigen and mAb control, 100 �lf pools of mAbs diluted at 1:1 in PBS was added in duplicate inhe ELISA plate. A positive and a negative bovine serum samplesere added with the pools of mAb to serve as positive and neg-

tive controls, respectively. One bovine negative and one positiveiluted serum were also added without mAb to serve as a neg-tive mAb control. The plate was incubated at 37 ◦C for 1 h withntermittent gentle shaking followed by three times washing with

ash buffer. Affinity purified anti-mouse antibodies conjugatedith HRPO (Sigma, MO, USA) was diluted to 1:1000 in blocking

uffer (1.5% SMP in 0.05% Tween-20 PBS) and dispensed into eachell in 50 �l volume. The plate was incubated at 37 ◦C for 1 h with

ntermittent gentle shaking followed by washing with the washuffer. A color reaction was developed by adding the substrateolution [H2O2 (Merck, NJ, USA) and O-phenylenediamine dihy-rochloride (OPD) (Sigma–Aldrich, St. Louis, MO, USA) in substrateuffer, pH 4.2]. The reaction was stopped after 15 min of incuba-ion by adding 1 M H2SO4 and reading was taken at a wavelengthf 492 nm with a reference wavelength of 620 nm in an ELISA readerTecan, Kawasaki-shi, Japan).

.6. Cut-off estimation and statistical validation of the C-ELISA

Please cite this article in press as: Sharma, G.K., et al., Immunodiagnosis of foin a competitive ELISA. J. Virol. Methods (2012), http://dx.doi.org/10.1016/

Bovine sera (n = 120) of known status from the serum panelSection 2.1) were tested by the optimized ELISA for estimationf the test parameters. OD values were normalized by calculatinghe percentage inhibition (PI) with reference to the OD of antigen

PRESSal Methods xxx (2012) xxx– xxx

controls. Normalized data were converted into Medcalc spread-sheet (trial version). In column “A” the sample numbers wereentered, in column “B” the PI values of sera were entered and in col-umn “C” status of animals was entered as “0” for negative and “1”for positive status. Receiver operating characteristic curve (ROC)analysis was performed by the Medcalc software following DeLonget al. (1988) procedure available in the software. Graphs and resultswere exported to MS-excel spreadsheet for analysis. The diagnosticsensitivity and diagnostic specificity of the assay were estimated atdifferent cut-off values using ROC analysis. Area under curve (AUC)along with confidence interval and standard errors for DSs and DSpwere estimated using ROC procedure.

Vaccinated bovine sera collected from FMD-CP area (n = 1150)and random bovine sera (n = 2175) (Section 2.1) were tested withthe optimized C-ELISA and classified as positive or negative basedupon the calculated cut-off value. The results of these samples werecompared with that of in house 3AB3 indirect ELISA (Mohapatraet al., 2011) for evaluation of diagnostic sensitivity and specificity.Sera collected during FMD vaccine potency testing exercise weretested with the optimized C-ELISA. Sera from vaccinated, non-vaccinated and infected goat, sheep and pigs (Section 2.1) weretested by the optimized C-ELISA and results were compiled.

3. Results

3.1. Molecular cloning of the mutated 3ABC gene

The full-length 3ABC gene with desired amino acid substitu-tions was obtained by in vitro amplification of IND 491/1997 (Asia1serotype) cDNA using overlap PCR technique. The first three reac-tions were assembled to amplify segments I, II and III of 819 bp,372 bp, and 162 bp amplicon size respectively. Another two PCRswere assembled to fuse segments I and II initially then with seg-ment III with amplicon size of 1191 bp and 1353 bp respectively.

The full-length 3ABC gene with amino acid substitutions wasthen gel purified for cloning. The full-length 3ABC gene was ligatedinto the pET45b(+) prokaryotic expression vector. The recombi-nant plasmid with the 1353 bp 3ABC gene was transformed intothe cloning followed by expression host. Cloning was confirmed bycolony PCR for amplification of full length 3ABC gene amplicon andrestriction enzyme double digestion to check the release of 1353 bpinsert. Nucleotide sequencing confirmed the correct frame of the3ABC gene fused with histidine tag in the expression vector. Sub-stitutions of histidine at position 3C46 with tyrosine and cystidineat position 3C163 with glycine were also confirmed by nucleotidesequencing (Fig. 1).

3.2. Expression of the recombinant mutated 3ABC gene in theE. coli expression host

The mutated 3ABC polyprotein with histidine tag was expressedsuccessfully in E. coli BL21 (pLysS DE3) cells, while the full length3ABC gene cloned into the selected expression system could notbe expressed. Maximum expression of the mutated 3ABC recom-binant protein was found at 30 ◦C at 220 rpm when induced with1 mM of IPTG for 6 h. SDS-PAGE analysis revealed a protein bandof approximately at 55 kDa in induced cells, which was absent inuntransformed and un-induced cells (Fig. 2). The expressed 3ABCprotein reacted with anti-His mAb-conjugated with HRPO in West-ern blot confirming the presence of poly histidine residues as tag.The 3ABC protein also reacted with bovine convalescent serum in

ot-and-mouth disease using mutated recombinant 3ABC polyproteinj.jviromet.2012.05.029

the Western blot under denaturating conditions (Fig. 3). Fair level ofpurity, sufficient for the immunodiagnostic purposes, was obtainedwith nickel-NTA affinity chromatography, however for the devel-opment of the mAb, greater level of purity was required, which

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AGT GGC GCC CCA CCG ACC GAC TTG CAA AAG ATG GTC ATG GGC AAC ACC AAG CCC GTT GAG

CTC ATA CTC GAC GGG AAG ACA GTA GCC ATC TGC TGT GCT ACC GGA GTG TTT GGC ACT GCT

TAC CTC GTA CCT CGT TAT CTT TTCGCA GAG AAG TAT GAC AGG ATC ATg CTG GAC GGT AGA

GCC ATG ACA GAC AGT GAC TAC AGA GTG TTT GAG TTT GAG ATT AAA GTA AAA GGa CAG GAC

ATG CTC TCA GAc GCC GCC CTC ATG GTG CTT CAC CGT GGG AAC CGC GTG AGA GAC ATC ACG

AAA CAC TTT CGT GAT ACA GCA AGA ATG AAG AAA GGC ACC CCC GTC GTT GGC GTG GTC AAC

AAC GCC GAT GTT GGG AGA CTG ATT TTC TCT GGT GAG GCC CTT ACC TAC AAG GAT ATT GTA

GTG TGC ATG GAT GGA GAC ACC ATG CCT GGC CTC TTT GCC TAC AAA GCC GCC ACT AAG GCT

GGC TAC GGC GGA GGA GCC GTT CTC GCG AAG GAC GGA GCT GCG GGT GGC AAC GGA GTT

GGA TAC TGC TCA TGC GTT TCC AGG TCC ATG CTG CTG AAA ATG AAG GCA CAC GTT GAC CCC

GAA CCA CAC CAC GCG GCC GC

Natural CAT

Natural TGC

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Fig. 1. Nucleotide sequence of mutated 3C clone in pET45b(+). Insertion of mutation is depicted in bold.

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ig. 2. SDS-PAGE gel analysis of induced 3ABC protein. 1, negative control of BL2uninduced transformed cells); 3, induced 3ABC transformed BL21 (pLysS DE3) cells

as obtained by the passive polyacrylamide gel elution method.ecovery of the protein from the gel by passive diffusion was foundptimum only under denaturating conditions and not in nativeonditions (Fig. 2). The purified protein was concentrated up to

Please cite this article in press as: Sharma, G.K., et al., Immunodiagnosis of foin a competitive ELISA. J. Virol. Methods (2012), http://dx.doi.org/10.1016/

mg/ml using a 10 kDa cut-off membrane filter. The concentratedroteins were preserved by addition of protease cocktail inhibitorsnd storing at −80 ◦C without losing the reactivity up to 6 months.

ig. 3. Western blot analysis of expressed 3ABC recombinant protein. 1–3 and 7, positiveL21 (pLysS DE3) cells (untransformed cells); 6, negative control of BL21 (pLysS DE3) cell

sS DE3) cells (untransformed cells); 2, negative control of BL21 (pLysS DE3) cellspurified passive gel eluted 3ABC proteins; 8, Prestained Protein Ladder (Fermentas).

3.3. Monoclonal antibody development

The gel purified recombinant 3ABC protein, reactive only to thebovine convalescent sera in Western blot, was used for the devel-

ot-and-mouth disease using mutated recombinant 3ABC polyproteinj.jviromet.2012.05.029

opment of anti-3ABC mAb. The mAb clones were developed infive phases by a commercial organization. Ten fused hybridomamedium supernatants were received from the manufacturer for

3ABC expressed confirmed clones; 4, weak 3ABC producer; 5, negative control ofs (uninduced transformed cells); 8, Prestained Protein Ladder (Fermentas).

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gainst the different criterion values. (c) Interactive dot diagram (0 = negative; 1 = poxes indicating the differentiation of positive and negative sera samples with decifferent serum samples.

creening of the clone reactive to 3ABC protein. Three clones wereelected for the generation of single cell clones reactive with recom-inant 3ABC protein only. The mAbs reactive to other protein like. coli lysate, BSA or SMP were discarded.

Nine hybridoma clones were finally selected in the laboratory.ive from nine clones, based on strong reactivity with the 3ABCrotein, were selected for standardization of the C-ELISA. Isotypingevealed that all clones were of IgG1 isotype. The selected clonesere slowly adapted on the serum free media to eliminate fetal

ovine serum that may cross react in the C-ELISA. Media super-atants of these five clones were pooled in equal quantity for use

n the C-ELISA.

.4. Optimization of the C-ELISA

The C-ELISA for DIVA was optimized in such a way that maxi-um discrimination between positive and negative samples could

e made based on the PI values. The test was optimized to obtainhe OD of mAb/antigen control near to 1.0 while OD value of theegative mAb control remains below 0.1. The optimum concentra-ions of 3ABC protein per well, test serum dilution, and anti-mouseonjugate dilutions were estimated as 50 ng, 1:7.5 and 1:1000espectively. Whole anti-mouse HRPO conjugated antibodies cross-eact with bovine sera that as ODs of the controls containing only

Please cite this article in press as: Sharma, G.K., et al., Immunodiagnosis of foin a competitive ELISA. J. Virol. Methods (2012), http://dx.doi.org/10.1016/

ovine serum were consistently high, while the affinity purifiednti-mouse HRPO conjugated antibodies did not cross-react withhe bovine sera as the OD in the mAb controls, containing onlyovine sera remained below 0.1. Two different protocols were

); the data of the negative and positive groups are displayed as dots on two verticalut-off. (d) Wishker plot; indicating the frequency distribution of the PP values of

evaluated for the detection of free 3ABC antigens. In one proto-col, diluted test sera were transferred to the wells of ELISA plateand incubated at 37 ◦C for 1 h to allow the binding of sera with thecoated antigens followed by addition of mAb to detect the unboundantigens. While in another protocol, the test sera were diluted andtransferred along with the pool of mAbs in the wells of ELISA plateas a single step and incubated at 37 ◦C for 1 h. In both the protocols,similar results were found. These two protocols were comparedstatistically by testing 450 bovine sera collected at random fromthe FMD infected and vaccinated animals in both the protocols.Out of 450 samples, 110 samples were found positive with boththe protocols and only two more samples were found positive withthe former protocol. These two samples were faintly positive bythe C-ELISA. Paired samples t-test showed that these two meth-ods were statistically insignificant at the 5% confidence interval(P > 0.05). Hence simultaneous addition of the test serum with mAbwas adopted to minimize the total time required for testing.

3.5. Cut-off estimation and statistical validation of the C-ELISA

PI values of 120 bovine sera from the panel were used for theestimation of cut-off values for C-ELISA. On ROC analysis by Med-calc software, different cut-off values along with their DSn and

ot-and-mouth disease using mutated recombinant 3ABC polyproteinj.jviromet.2012.05.029

DSp were obtained. Among these, 40% PI value was accepted ascut-off for C-ELISA at which DSn was 86.9% with 66.4–97.2 (95%)confidence interval and DSp of 97% with 89.6–99.6 (95%) confi-dence interval was observed. Area under curve in the C-ELISA was

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Fp

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ig. 5. Reactivity of serum samples with 3ABC NSP after 0, 3, 5, 10, 14 and 21 daysost infection.

etermined to be 0.851 (SE = 0.0409) and 0.760–0.917 confidencenterval (Fig. 4a).

The standardized C-ELISA was validated by testing bovine seraollected from the vaccinated and infected animals. Only 12 (1.7%)ut of 694 pre-vaccinated bovine sera and 9 (2%) out of 456 post-accinated bovine sera that were collected from different parts ofndia where the FMD control programme has been implemented,

ere found positive by the C-ELISA. On testing these sera by theAB3 indirect ELISA, 2% of pre-vaccinated and 2.1% post-vaccinatedera were found positive (Table 2). Bovine sera collected at randomn = 2175) were classified into reactors or non-reactors to the 3ABCrotein, based on the estimated C-ELISA cut-off values. Out of these175 samples, 518 (23.81%) serum samples were positive while restf them were negative at 40% inhibition, while by the 3AB3 indirectLISA, 27% of the above samples were positive.

For validation of the assay for use in other domestic animals, aotal of 462 sera samples collected from the vaccinated, infected orollected at random from the sheep, goat and pigs were tested byhe C-ELISA and classified as reactors or non-reactors to the 3ABCrotein (Table 2). Animals challenged during vaccine potency test-

ng showed no significant rise in reactivity up to 5 days, but after0 days reactivity increased steeply in all the four animals (Fig. 5).

. Discussion

Foot-and-mouth disease poses a serious threat to the inter-ational animal trade. Annual direct losses alone in India werestimated to be about US$ 4.5 billion in the year 2010, whileosses due to trade barriers could be greater (Annual Report, Theroject Directorate on Foot and Mouth Disease, Mukteswar, India).eing endemic country, preventive vaccination followed by sero-urveillance has been adopted for the control of FMD in India. Afterhe launch of the Government endorsed ‘National FMD Controlrogramme’ (FMD-CP) in 2003, livestock animals are systemati-ally vaccinated with a trivalent vaccine consisting of serotype O,, Asia1 inactivated antigens resulting in increase in the numbersf multiple vaccinated animals and reducing the clinically affectednimals.

The disease is strictly monitored in the FMD free countriesy measuring the antibody response against viral structural pro-

Please cite this article in press as: Sharma, G.K., et al., Immunodiagnosis of foin a competitive ELISA. J. Virol. Methods (2012), http://dx.doi.org/10.1016/

eins as an indicator. Since the vaccine induces antibody responseshich are similar to those in infected ones, hence vaccination in

hese countries is not practised. However, in May 2002, the OIEpdated its International Animal Health Code in light of advances

PRESSal Methods xxx (2012) xxx– xxx 7

in diagnostic tests, allowing countries to vaccinate in the face ofan FMD outbreak, and subsequent differentiation of vaccinatedfrom infected or convalescing animals (OIE code; Council Directive2003/85/EEC). Hence, monitoring virus activity by a discrimina-tory assay in FMD endemic regions practising vaccination is of theutmost importance (Mohapatra et al., 2011). The advantage of theDIVA strategy over test and slaughter policy has been previouslydescribed (Parida, 2009).

Identification of convalescent or infected animals is basedmainly on detecting the antibodies against NSPs of the FMDV byimmunoassays. NSP ELISAs for DIVA have gained wide use in therecent past due to high level of precision and high throughput.These DIVA assays have been described extensively using eithera panel of NSPs or the individual 2C and 3ABC proteins. Otherrecombinant NSPs such as 3A, 3B and 3AB have also been vali-dated with different levels of sensitivity and specificity for DIVA(He et al., 2010; Mohapatra et al., 2011). Still 3ABC has been con-sidered to be highly effective for early identification of infectedin vaccinated herds (Bergmann et al., 2000; De Diego et al., 1997;Brocchi et al., 1998; Shen et al., 1999; Bruderer et al., 2004; Clavijoet al., 2004b; Lee et al., 2006). Additionally, tests for antibodies tothe polyprotein 3ABC are most promising in terms of sensitivityand specificity (Sorensen et al., 1998, 2005; Mackay et al., 1998a;Brocchi et al., 2006). With improvement in the vaccine purificationmethods, occurrence of NSP or live virus particle in the vaccines hasbeen limited that could interfere in NSPs based DIVA test (Clavijoet al., 2004a; Mohapatra et al., 2011). A wide range of cloven-hoofedlivestock and wild animals are susceptible to FMD, an individualindirect format of ELISA has to be optimized for diagnosis for each ofthe species. On the other hand, the C-ELISAs using mAb are knownfor their high specificity and provide a common method for testingall the species.

The present study was undertaken with an objective of devel-oping a C-ELISA using mutated recombinant 3ABC polyproteinand mAb against 3ABC protein. The assay was validated using aserum panel consisting of infected and vaccinated serum samplesobtained from different species of livestock.

Initial attempts to express the full-length native 3ABC polypro-tein in prokaryotic expression system failed. Expression inprokaryotic system of 55 kDa (Fig. 2) could only be obtainedafter insertion of point mutations to substitute two out of threeactive protease sites (Fig. 1) (histidine-46 and cysteine-163 posi-tion replaced with tyrosine-46 and glycine-163, respectively).The FMDV 3CPro protein is a major viral protease that cleaves10 of 13 cleavage sites within the viral polyprotein (Clarke andSangar, 1988; Vakharia et al., 1987). The 3CPro has cysteine-163-histidine-46-asparagine/glutamine-84 catalytic triad in theactive site (Birtley and Curry, 2005; Grubman et al., 1995). Thischymotrypsin-like cysteine protease may cleave completely theexpressed protein (Curry et al., 2007). Substitutions of these aminoacids probably deactivated the catalytic nucleophile suppressingthe proteolytic activity resulting in the high protein yield. The thirdamino acid residue in active motif that is asparagine/glutamine-84left unchanged. Similarly, Sariya et al. (2011) substituted cysteine-142 and cysteine-163 to serine-142 and glycine-163 respectivelyto inactivate proteolytic activity leading to expression of the 3ABCpolyprotein in a prokaryotic system. This mutated 3ABC polypro-tein could be used effectively for the development of DIVA assayfor FMD (Sariya et al., 2011).

The expressed protein purified under denaturating conditionsusing Ni-NTA columns followed by dialysis which showed opti-mum reactivity in the ELISA. Satisfactory level of purity with minor

ot-and-mouth disease using mutated recombinant 3ABC polyproteinj.jviromet.2012.05.029

contaminants was achieved by this method. However, for thedevelopment of mAb clones, more purified protein was requiredthan was achieved by passive elution of protein from the SDS-PAGE gel (Fig. 2). Purification by this method, although slow and

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Table 2Test result of C-ELISA for different species of livestock tested.

Species No. of samples 3AB3 I-ELISA 3ABC C-ELISA

No. of animals positive (%) No. of animals negative (%) No. of animals positive (%) No. of animals negative (%)

Goat 159 61 (38.3%) 98 (61.6%) 54 (33.9%) 105 (66%)Sheep 141 49 (34.7%) 92 (65.2%) 41 (29%) 100 (70.9%)Pig 128 15 (11.7%) 113 (88.2%) 14 (0.03%) 107 (99.9%)Bovine (pre-vaccinated) 694 14 (2%) 680 (98%) 12 (1.72%) 1118 (97.2%)Bovine (post-vaccinated 456 13 (2.8%) 443 (97.1%) 9 (1.97%) 447 (98%)

86 (72

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umbersome, results in high level of purity. Elution under denatu-ating conditions (0.5% SDS) greatly enhances the diffusion processesulting in faster and complete elution of protein from the gel.inely chopped gel increased the surface area and hastened thelution process. The eluted protein was refolded by dialysis in theative buffer (PBS) and there were no effects of presence of residualDS in subsequent ELISA applications. Electro-elution of recombi-ant 3ABC protein for antibody production was also demonstratedy Clavijo et al. (2004b). The addition of protease cocktail inhibitors

ncreased the shelf-life when stored at −80 ◦C or below for up to sixonths without loss in reactivity by the ELISA.The ELISA protocol in solid phase competition format was opti-

ized using a panel of known bovine serum samples to obtainufficient discrimination between positive and negative PI values.he optimal OD of antigen control with mAb was standardizedpproximately to 1.0 with background not more than 0.1. The opti-um serum dilution was fixed to 1:7.5 where sufficient antibodies

an compete with mAb without increasing the background OD.ompetition of mAbs and test serum antibodies was allowed inhe solid phase as a single step rather than in two steps. There wereo significant differences in the results while adding mAb simul-aneously with test serum or added 1 h after the addition of theest serum. Hence, mAb was added with the test serum as a singletep unlike many other commercial C-ELISA kits to reduce the totalime required for testing without affecting adversely the sensitiv-ty and specificity of the assay. Use of the pool of mAbs against theABC polyprotein increases the sensitivity (Uttenthal et al., 2010)s different epitopes of full length 3ABC could be targeted. For trac-ng bound mAbs in the presence of other species antibodies, onlyffinity purified anti-mouse goat antibodies conjugated with HRPOere found suitable and whole anti-mouse antibodies cross-reactith bound bovine antibodies and did not show inhibition in terms

f the OD.The cut-off value for the C-ELISA was determined by ROC

ethod as 40% of PI with reference to negative control at whichiagnostic sensitivity and diagnostic specificity were 86.9% and7%, respectively (Fig. 4a). While the diagnostic sensitivity andiagnostic specificity of the 3AB3 indirect ELISA for bovineseveloped in this laboratory were estimated to 96% and 96.4%,espectively (Mohapatra et al., 2011). The C-ELISAs when comparedo the indirect ELISA have near perfect test specificity but compro-

ised sensitivity (Bronsvoort et al., 2004) that also occurred in thistudy. The whisker plots (Fig. 4c and d) demonstrated that most ofhe negative samples are well below the 40% PI cut-off value, how-ver some of the positive samples were also below this limit. Theower sensitivity could be due to weak antibody response in thease of mild infections or later phase of infections which may gondetected by the C-ELISA.

The validity of the C-ELISA for discrimination of infected from

Please cite this article in press as: Sharma, G.K., et al., Immunodiagnosis of foin a competitive ELISA. J. Virol. Methods (2012), http://dx.doi.org/10.1016/

accinated animals was estimated by examining bovine sera whichere vaccinated repeatedly with different commercial vaccinessed commonly in India. On average, 10 doses of vaccine weredministered to these animals. Out of 694 pre-vaccinated samples

.9%) 518 (23.8%) 1657 (76.2%)

only 1.7% samples were positive, that corroborates with significantdrop in occurrence of the disease in regularly vaccinated areas. Inpost vaccinated samples (collected 1 month post-vaccination), thepositive reactors increased to nearly 2%. The result showed that thelevel of 3ABC NSP in purified commercial FMD vaccines used com-monly in India is sufficiently low to produce detectable antibodyresponse. On the other hand, 23.8% bovines were positive in ran-dom samples collected throughout the India. This figure is lesserthan national prevalence of 27.9% in bovine population estimatedduring 2008–2010 (Project Directorate on Foot and Mouth Disease,Annual Report-2009) and that could be due to lower sensitivitylimits of the C-ELISA. The developed assay could detect infection inall the four cattle on 10th day post-virus challenge. These animalswere challenged after vaccination and none of the animals showedany clinical symptoms. The 3ABC antibody response can persist inanimals for approximately 2 years, which is considered as safe forimport and export serology (Lu et al., 2007). However, to ascer-tain the detection limits of this assay further experimental kineticstudies are required.

The protocol standardized for testing bovine species was usedsuccessfully for sheep, goat and pigs and it has also been previouslyreported that antibody response against the FMDV 3ABC polypro-tein in majority of the livestock species is more or less similar(Clavijo et al., 2004b), hence it was anticipated that the assay willbe equally suitable for testing sera from other susceptible speciesincluding wild animals. However, further evaluation and valida-tion is required by examining more number of sera from otherlivestock species and wild animals. Till now small ruminants inIndia are usually not vaccinated but strong emphasis is now paid tocover small ruminants under FMD control programme, where theC-ELISA will be highly useful for providing a common method forsero-surveillance studies in susceptible domestic and wild animalspecies and reduce the dependence on species-specific conjugatedantibodies. Taking into account the hundreds of millions of serawhich will have to be tested during probably the next two decadesin order to eradicate FMD or at least limit it to a few pockets of dis-ease in remote areas, this kit will be an economical alternative tothat of expensive commercial tests. The in-house assay will be suit-able for serological screening for long term use in a cost effectivemanner.

Acknowledgments

The authors are grateful to the Indian Council of AgriculturalResearch (ICAR) for arranging the necessary facility for the work.The authors are also thankful to Mr. N.S. Singh and Mr. D.S. Deoliafor assistance in sample collection and serological testing.

References

ot-and-mouth disease using mutated recombinant 3ABC polyproteinj.jviromet.2012.05.029

Anon., 2003. Council directive 2003/85/EC on community measures for the con-trol of foot and mouth disease repealing directive 85/511/ECC and decisions89/531/EEC and 96/665/EEC and amending directive 92/46/EEC. Official Journalof European Union 46.

ING Model

V

rologic

B

B

B

B

B

B

B

C

C

C

C

D

D

G

H

ARTICLEIRMET-11850; No. of Pages 9

G.K. Sharma et al. / Journal of Vi

ergmann, I.E., de Mello, P.A., Neitzert, E., Beck, E., Gomes, I., 1993. Diagnosis of per-sistent aphthovirus infection and its differentiation from vaccination responsein cattle by use of enzyme-linked immunoelectrotransfer blot analysis withbioengineered nonstructural viral antigens. American Journal of VeterinaryResearch 54, 825–831.

ergmann, I.E., Malirat, V., Neitzert, E., Beck, E., Panizzutti, N., Sanchez, C., Falczuk,A., 2000. Improvement of a serodiagnostic strategy for foot-and-mouth diseasevirus surveillance in cattle under systematic vaccination: a combined systemof an indirect ELISA-3ABC with an enzyme-linked immunoelectrotransfer blotassay. Archives of Virology 145, 473–489.

irtley, J.R., Curry, S., 2005. Crystallization of foot-and-mouth disease virus 3Cprotease: surface mutagenesis and a novel crystal-optimization strategy. ActaCrystallographica 61, 646–650.

rocchi, E., Bergmann, I.E., Dekker, A., Paton, D.J., Sammin, D.J., Greiner, M., Grazioli,S., De Simone, F., Yadin, H., Haas, B., Bulut, N., Malirat, V., Neitzert, E., Goris, N.,Parida, S., Sorensen, K., De Clercq, K., 2006. Comparative evaluation of six ELISAsfor the detection of antibodies to the non-structural proteins of foot-and-mouthdisease virus. Vaccine 24, 6966–6979.

rocchi, E., De Diego, M.I., Berlinzani, A., Gamba, D., De Simone, F., 1998. Diagnos-tic potential of Mab-based ELISAs for antibodies to non-structural proteins offoot-and-mouth disease virus to differentiate infection from vaccination. TheVeterinary Quarterly 20 (Suppl. 2), S20–S24.

ronsvoort, B.M., Sorensen, K.J., Anderson, J., Corteyn, A., Tanya, V.N., Kitching, R.P.,Morgan, K.L., 2004. Comparison of two 3ABC enzyme-linked immunosorbentassays for diagnosis of multiple-serotype foot-and-mouth disease in a cat-tle population in an area of endemicity. Journal of Clinical Microbiology 42,2108–2114.

ruderer, U., Swam, H., Haas, B., Visser, N., Brocchi, E., Grazioli, S., Esterhuysen, J.J.,Vosloo, W., Forsyth, M., Aggarwal, N., Cox, S., Armstrong, R., Anderson, J., 2004.Differentiating infection from vaccination in foot-and-mouth-disease: evalu-ation of an ELISA based on recombinant 3ABC. Veterinary Microbiology 101,187–197.

larke, B.E., Sangar, D.V., 1988. Processing and assembly of foot-and-mouth dis-ease virus proteins using subgenomic RNA. The Journal of General Virology 69,2313–2325.

lavijo, A., Wright, P., Kitching, P., 2004a. Developments in diagnostic techniques fordifferentiating infection from vaccination in foot-and-mouth disease. VeterinaryJournal 167, 9–22.

lavijo, A., Zhou, E.M., Hole, K., Galic, B., Kitching, P., 2004b. Development and useof a biotinylated 3ABC recombinant protein in a solid-phase competitive ELISAfor the detection of antibodies against foot-and-mouth disease virus. Journal ofVirological Methods 120, 217–227.

urry, S., Roque-Rosell, N., Sweeney, T.R., Zunszain, P.A., Leatherbarrow, R.J., 2007.Structural analysis of foot-and-mouth disease virus 3C protease: a viable targetfor antiviral drugs? Biochemical Society Transactions 35, 594–598.

e Diego, M., Brocchi, E., Mackay, D., De Simone, F., 1997. The non-structuralpolyprotein 3ABC of foot-and-mouth disease virus as a diagnostic antigen inELISA to differentiate infected from vaccinated cattle. Archives of Virology 142,2021–2033.

eLong, E.R., DeLong, D.M., Pearson, C., 1988. Comparing the areas under two or morecorrelated receiver operating characteristic curves: a nonparametric approach.Biometrics 44, 837–845.

rubman, M.J., Zellner, M., Bablanian, G., Mason, P.W., Piccone, M.E., 1995. Identifi-

Please cite this article in press as: Sharma, G.K., et al., Immunodiagnosis of foin a competitive ELISA. J. Virol. Methods (2012), http://dx.doi.org/10.1016/

cation of the active-site residues of the 3C proteinase of foot and mouth diseasevirus. Virology 213, 581–589.

e, C., Wang, H., Wei, H., Yan, Y., Zhao, T., Hu, X., Luo, P., Wang, L., Yu, Y., 2010. Arecombinant truncated FMDV 3AB protein used to better distinguish betweeninfected and vaccinated cattle. Vaccine 28, 3435–3439.

PRESSal Methods xxx (2012) xxx– xxx 9

Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K., Pease, L.R., 1989. Site-directed muta-genesis by overlap extension using the polymerase chain reaction. Gene 15,51–59.

Jacobson, R.H., 1998. Validation of serological assays for diagnosis of infectious dis-eases. Revue Scientifique et Technique 17, 469–526.

Lee, F., Jong, M.H., Yang, D.W., 2006. Presence of antibodies to non-structural pro-teins of foot-and-mouth disease virus in repeatedly vaccinated cattle. VeterinaryMicrobiology 115, 14–20.

Lu, Z., Cao, Y., Guo, J., Qi, S., Li, D., Zhang, Q., Ma, J., Chang, H., Liu, Z., Liu, X., Xie, Q.,2007. Development and validation of a 3ABC indirect ELISA for differentiationof foot-and-mouth disease virus infected from vaccinated animals. VeterinaryMicrobiology 125, 157–169.

Mackay, D.K., Forsyth, M.A., Davies, P.R., Berlinzani, A., Belsham, G.J., Flint, M., Ryan,M.D., 1998a. Differentiating infection from vaccination in foot-and-mouth dis-ease using a panel of recombinant, non-structural proteins in ELISA. Vaccine 16,446–459.

Mackay, D.K., Forsyth, M.A., Davies, P.R., Salt, J.S., 1998b. Antibody to the nonstruc-tural proteins of foot-and-mouth disease virus in vaccinated animals exposedto infection. The Veterinary Quarterly 20 (Suppl. 2), S9–S11.

Mohapatra, J.K., Pandey, L.K., Sanyal, A., Pattnaik, B., 2011. Recombinant non-structural polyprotein 3AB-based serodiagnostic strategy for FMD surveillancein bovines irrespective of vaccination. Journal of Virological Methods 177,184–192.

Parida, S., 2009. Vaccination against foot-and-mouth disease virus: strategies andeffectiveness. Expert Review of Vaccines 8, 347–365.

Parida, S., Fleming, L., Gibson, D., Hamblin, P.A., Grazioli, S., Brocchi, E., Paton, D.J.,2007. Bovine serum panel for evaluating foot-and-mouth disease virus non-structural protein antibody tests. Journal of Veterinary Diagnostic Investigation19, 539–544.

Ranabijuli, S., Mohapatra, J.K., Pandey, L.K., Rout, M., Sanyal, A., Dash, B.B., Sarangi,L.N., Panda, H.K., Pattnaik, B., 2010. Serological evidence of foot and mouthdisease virus infection in randomly surveyed goat population of Orissa, India.Transboundary and Emerging Diseases 57, 448–454.

Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual, 3rd edi-tion. Cold Spring Harbor Laboratory Press.

Sariya, L., Thangthumniyom, N., Wajjawalku, W., Chumsing, W., Ramasoota, P.,Lekcharoensuk, P., 2011. Expression of foot and mouth disease virus non-structural polyprotein 3ABC with inactive 3C(pro) in Escherichia coli. ProteinExpression and Purification 80, 17–21.

Shen, F., Chen, P.D., Walfield, A.M., Ye, J., House, J., Brown, F., Wang, C.Y., 1999. Differ-entiation of convalescent animals from those vaccinated against foot and mouthdisease by a peptide ELISA. Vaccine 17, 3039–3049.

Sorensen, K.J., de Stricker, K., Dyrting, K.C., Grazioli, S., Haas, B., 2005. Differentiationof foot-and-mouth disease virus infected animals from vaccinated animals usinga blocking ELISA based on baculovirus expressed FMDV 3ABC antigen and a 3ABCmonoclonal antibody. Archives of Virology 150, 805–814.

Sorensen, K.J., Madsen, K.G., Madsen, E.S., Salt, J.S., Nqindi, J., Mackay, D.K., 1998.Differentiation of infection from vaccination in foot-and-mouth disease by thedetection of antibodies to the non-structural proteins 3D, 3AB and 3ABC in ELISAusing antigens expressed in baculovirus. Archives of Virology 143, 1461–1476.

Uttenthal, A., Parida, S., Rasmussen, T.B., Paton, D.J., Haas, B., Dundon, W.G., 2010.Strategies for differentiating infection in vaccinated animals (DIVA) for foot-and-mouth disease, classical swine fever and avian influenza. Expert Review of

ot-and-mouth disease using mutated recombinant 3ABC polyproteinj.jviromet.2012.05.029

Vaccines 9, 73–87.Vakharia, V.N., Devaney, M.A., Moore, D.M., Dunn, J.J., Grubman, M.J., 1987. Pro-

teolytic processing of foot-and-mouth disease virus polyproteins expressedin a cell-free system from clone-derived transcripts. Journal of Virology 61,3199–3207.