Recombinant hexon antigen based single serum dilution ELISA for rapid serological profiling against fowl adenovirus-4 causing hydropericardium syndrome in chickens

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ARTICLE IN PRESSG ModelIRMET 12558 1–7

Journal of Virological Methods xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Virological Methods

j o ur na l ho me pa ge: www.elsev ier .com/ locate / jv i romet

ecombinant hexon antigen based single serum dilution ELISA forapid serological profiling against fowl adenovirus-4 causingydropericardium syndrome in chickens

. Rajasekhar, Parimal Roy ∗

epartment of Veterinary Microbiology, Tamil Nadu Veterinary and Animal Sciences University, Madhavaram Milk Colony, Chennai 600007, India

rticle history:eceived 17 December 2013eceived in revised form 19 June 2014ccepted 20 June 2014vailable online xxx

eywords:hickensydropericardium syndrome

nclusion body hepatitisowl adenovirus (FAdV)-4ecombinant antigeningle serum dilution ELISAot ELISAaccination

mmunity

a b s t r a c t

A recombinant hexon antigen based single serum dilution enzyme linked immunosorbent assay (ELISA)was developed to measure the specific antibody in sera of chickens against fowl adenovirus-4 (FAdV) caus-ing hydropericardium syndrome (HPS). An immunodominant partial hexon gene of 737 bp was clonedinto pRSET vector and expressed in Escherichia coli strain BL21 DE3 pLys S. Expression was tested byWestern Blot test. The purified recombinant protein antigen was used in coating ELISA plate for FAdV-4serology. A linear relationship was found between the predicted antibody titres at a single working dilu-tion of 1:100 and the corresponding observed serum titres as determined by the standard serial dilutionmethod. Regression analysis was used to determine a standard curve from which an equation was derivedthat allowed the demonstration of this correlation. The equation was then used to convert the correctedabsorbance readings of the single working dilution directly into the predicted ELISA antibody titres. Theassay proved to be sensitive, specific and accurate as compared to Q-AGID test. Recombinant antigen wasalso used in Dot ELISA. In an experimental vaccination of broiler chicken at 10 days old age, the geometricmean (GM) antibody titres as measured by ELISA ranged from 5.006 ± 0.11 log 10 to 4.526 ± 0.04 log 10and by Dot ELISA titre were from 2.240 ± 0.08 log 10 to 0.180 ± 0.04 log 10 during 5th–8th weeks of age,
results were compared with Q-AGID results. Field samples were collected randomly from breeder flocks,found to have antibody titre by both ELISA and Dot ELISA at 10th and only 75% samples were positiveat 14th weeks of age. After revaccination at 16th weeks of age, all sera samples were found have con-siderably high antibody titre on 24th week but all samples were negative at 32nd weeks. Advantages ofrecombinant hexon antigen based ELISA and Dot ELISA in FAdV-4 serology have been discussed.
© 2014 Published by Elsevier B.V.

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. Introduction

Hydropericardium syndrome (HPS) is an important disease ofroiler chickens, characterized by sudden death with hydroperi-ardium, mottling of liver and nephritis. Most often, chickens of–6 weeks old age groups are affected and mortality varies from0% to 80% in natural field outbreaks (Khawaja et al., 1988; Anjumt al., 1989; Cheema et al., 1989; Asrani et al., 1997). The disease isaused by fowl adenovirus serotype 4 (FAdV-4) (Jadhao et al., 1997;azaheri et al., 1998; McFerran, 2003) and reported from many

Please cite this article in press as: Rajasekhar, R., Roy, P., Recomrapid serological profiling against fowl adenovirus-4 causing hydrhttp://dx.doi.org/10.1016/j.jviromet.2014.06.017

ountries including Pakistan (Khawaja et al., 1988), India (Gowdand Satyanarayana, 1994), Mexico, Peru, Chile (Voss et al., 1996;owen et al., 1996; Shane, 1996), Russia (Borisov et al., 1997), Japan

∗ Corresponding author. Tel.: +91 4425551581; fax: +91 442555157.E-mail addresses: [email protected], [email protected] (P. Roy).

ttp://dx.doi.org/10.1016/j.jviromet.2014.06.017166-0934/© 2014 Published by Elsevier B.V.

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(Abe et al., 1998) and Bangladesh (Biswas et al., 2002). Subsequentlythe disease has also been recorded in Japanese quails (Roy et al.,2004a). The disease is causing greater economic loss to the broilerindustry (Ganesh et al., 2001). An inactivated vaccine prepared byAfzal and Ahmad (1990) was found less effective but in anotherstudy an oil adjuvanted HPS vaccine evoked immune response of2 log 2 to 1.8 log 2 by 3–5 week post vaccination as assessed by AGIDtest, withstood challenge and field trial was also effective (Roy et al.,1999). Assessment of post vaccinal immunity poses a challenge assuitable serological test is not available for large scale sampling.

FAdV-4 virion causing HPS has eight structural polypeptides(Park et al., 2011). Hexon is the major capsid protein of aden-oviruses responsible for virus neutralizing and serotype specificity(Norby, 1969; Norby and Wadell, 1969; Toogood et al., 1989).

binant hexon antigen based single serum dilution ELISA foropericardium syndrome in chickens. J. Virol. Methods (2014),

Earlier an indirect ELISA was developed based on recombinanthexon protein of FAdV-2 expressed in Escherichia coli which showedspecificity and sensitivity of 80% and 80%, respectively (Junnuet al., 2014). This paper describes cloning and expression of

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dx.doi.org/10.1016/j.jviromet.2014.06.017


http://www.sciencedirect.com/science/journal/01660934

http://www.elsevier.com/locate/jviromet

mailto:[email protected]

mailto:[email protected]


IN PRESSG ModelV

2 irological Methods xxx (2014) xxx–xxx

it(r

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Fig. 2. Colony PCR to confirm cloning of partial hexon gene of FAdV-4. Lanes 1–3:amplification of partial hexon gene of 737 bp from recombinant colonies; Lane M:

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ARTICLEIRMET 12558 1–7

R. Rajasekhar, P. Roy / Journal of V

mmunodominant partial hexon gene of FAV-4 in prokaryotic vec-or and development of an enzyme linked immunosorbent assayELISA) for assessment of post vaccinal immune response usingecombinant hexon gene expressed protein as antigen.

. Materials and methods

.1. DNA extraction from FAdV-4 and polymerase chain reactionPCR) of the hexon gene

FAdV-4 was adapted in chicken embryo liver (CEL) culture andurified virus was used for DNA extraction using DNA isolation kitBangalore Genie, India; Cat No. 107822) following manufacturer’snstructions. PCR amplification of partial hexon gene of FAdV-4

as done using the primers designed for an immunodominantoding sequence of hexon gene (737 bp) spanning 1792–2485 bprom the total hexon gene sequence (2916 bp; Gene Bank Acces-ion No. AJ554049) using DNA star software (Laser gene), restrictionndonuclease sites (Pst1 and BamH1) were incorporated for direc-ional cloning. The primer sequences were as follows: F: 5′ AAA CCTGA TCC CGA CGG CGC CAA CAT CAT CTA 3′; R: 5′ GAT AAC TGC AGGGT TGT TGC GAA CGG CTT CCT 3′. PCR amplification was carriedut in a thermocycler (AB System, USA) with an initial denaturationt 94 ◦C for 5 min followed by 30 cycles of denaturation at 94 ◦C for

min; annealing at 57 ◦C for 1 min and extension at 72 ◦C for 2 minith a final extension of 72 ◦C for 10 min.

.2. Cloning of the PCR products

Purification of the amplified hexon gene product was carriedut using Pure LinkTM gel extraction kit (Invitrogen, USA) as peranufacturer’s protocol. The purified PCR products were cloned

irectionally into the bacterial expression vector pRSET B (Invitro-en, USA) plasmid (Fig. 1). For transformation, E. coli strain BL21 DE3Lys S (Invitrogen, USA) was used. The recombinant clones were

nitially identified by colony PCR using hexon gene specific primerFig. 2) and verified by plasmid isolation and restriction enzymeigestion for the release of the insert (Fig. 3). PCR amplified prod-ct was sequenced to observe correct orientation and submitted toenBank (Accession No. EU 747350).


.3. Expression and purification of the recombinant hexon protein

Isopropyl-B-d-thiogalactopyranoside (IPTG; 1 mM final concen-ration, Bangalore Genei, India) was added to log phase cultures

Fig. 1. Cloning site of pRSET vector.

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100 bp Ladder DNA.

of E. coli BL21 DE3 pLys S cells transformed with pRSET B recom-binant plasmid for expression of His6 fusion recombinant hexonprotein. The optimum expression of protein was found out bypilot expression. Optimum expression of protein was 4–5 h afterIPTG induction. At this point cells were harvested by centrifuga-tion (3000 × g for 10 min at 4 ◦C) and pellet was collected. Thepellet was resuspended in 20 ml of binding buffer (Bangalore Genie,India; Cat No. PC-137) and after 3 cycles of freezing and thawingthe expressed protein was taken for purification. The recombinanthistidine tagged hexon protein was purified by affinity chromatog-raphy containing Ni-NTA (Bangalore Genie, Bangalore, India; CatNo. PC-137). The recombinant histidine tagged fusion protein waseluted with elution buffer of different pH as per the manufacture’smanual. The purity of the eluted protein was analysed on SDS-PAGE(Fig. 4), characterized by Western Blot (Fig. 5), quantified usingspectrophotometer (Warberg and Christian, 1991) and used in bothELISA and Dot ELISA.


Fig. 3. Restriction enzyme digestion recombinant plasmid. Lane M: 1 kbp LadderDNA; Lane 1: restriction enzyme digested product showing the release of 737 bpinsert. Lane 2: undigested recombinant plasmid.


ARTICLE IN PRESSG ModelVIRMET 12558 1–7

R. Rajasekhar, P. Roy / Journal of Virological Methods xxx (2014) xxx–xxx 3

Table 1Details of experimental vaccination and sample collection.

Sl. no. Age of vaccination Age of the birds No. of sera samples

Vaccinated Unvaccinated

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Fig. 4. SDS-PAGE analysis of purified recombinant protein. Lane 1: recombinantprotein after elution with buffer of pH 5.8. Lane 2: recombinant protein after elutionw3

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ith buffer of pH 4.8. Lane 3: recombinant protein after elution with buffer of pH.8. Lane M: molecular weight marker.

.4. Experimental vaccination

Thirty specific antibodies negative (SAN) chickens were dividednto two groups consisting of 15 in treatment group and 15 age

atched unvaccinated chickens in control group. The treatmentroup was vaccinated with 0.3 ml of a commercial inactivatedFAdV-4) oil adjuvanted vaccine at the age of 10 days, subcuta-eously at the back of neck. Ten sera sample each were collectedandomly at zero day of vaccination (10 days old chickens) from


oth vaccinated (before vaccination) and control groups. Further0 sera samples were collected at random from each of both vacci-ated and control groups at 5th, 6th, 7th and 8th weeks old age

ig. 5. Western Blot analysis of recombinant protein. Lane M: molecular weightarker; Lanes 1 and 2: recombinant protein.

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(Table 1). Sera samples were screened by ELISA, Dot ELISA andresults were compared with AGID test.

2.5. Collection of sera from the field

Sera were collected at random from four different breeder flocks(Table 2) of Tamil Nadu which were vaccinated subcutaneouslyagainst IBH/HPS with a commercial oil adjuvanted inactivatedvaccine (FAdV-4) at 2nd week of age (dose 0.3 ml) and booster vac-cination was done at 16th weeks age (dose 0.5 ml). Twenty serasamples were collected randomly from 16th weeks old, 24th weeksold and 32 weeks old flocks but only 19 sera samples were collectedfrom 14th weeks old flock. Sera samples were screened by ELISA,Dot ELISA and results were compared with AGID test results.

2.5.1. ELISA with recombinant hexon proteinThe ELISA for the detection of antibodies to fowl adenovirus

was done as described by Dawson et al. (1979) with some mod-ifications. Prior to performing ELISA checkerboard titration wasperformed to arrive at the optimum dilutions of antigen and conju-gate to be used as described earlier (Rose et al., 1997). The wells inpolystyrene microtitre plates (Nunc, USA) were coated with 100 �lof 3.0 ng/�l of recombinant protein in carbonate–bicarbonate coat-ing buffer, pH 9.6. The coated plates were incubated overnight at4 ◦C in refrigerator. After the overnight incubation, the plates werewashed three times with PBS containing Tween-20 0.05% (PBST)and each well was added with 100 �l of blocking buffer [PBST with5% (w/v) skim milk powder] and incubated at 37 ◦C for 1 h. Theplates were washed with PBST for three times and 100 �l of eachserum dilution (1:100, 1:500, 1:1000, 1:2000, 1:5000, 1:10,000 and1:50,000) was added to wells in duplicates and the plates wereincubated for 1 h at 37 ◦C. The plates were washed with PBST againfor three times. Then, predetermined 100 �l of 1:2000 dilution ofanti-chicken IgY-HRP conjugate (Sigma, USA) was added to all wellsand incubated for 1 h at 37 ◦C. The unreacted conjugate was washedout with PBST for three times and finally 100 �l of freshly preparedsubstrate (ABTS-2.2 mg/10 ml of substrate buffer containing 6 �l ofhydrogen peroxide) was added in subdued light. The plates wereincubated in the dark for 15–30 min at 37 ◦C for colour develop-ment. The colour reaction was stopped by adding 100 �l of 1% SDS.The OD readings were taken at 405 nm.

2.5.2. Positive negative threshold and standard curve anddetermination of titre

The positive negative threshold line to find out the titre wascarried out following the procedure of Snyder et al. (1983). Fif-teen serum samples from SAN (specific antibody negative) chickensthat were negative by Dot ELISA were selected. The sera samplewere diluted (1:100, 1:500, 1:1000, 1:2000, 1:5000, 1:10,000 and1:50,000) in serum dilution buffer and ELISA was done as describedabove. The resultant OD values (mean OD + 3SD for 15 sera sample)were plotted on Y-axis against dilutions in X-axis in logarithmic


graph. The resultant line is referred as PNT line and used for find-ing out the observed titre (OT) values for the known positive serasamples, observed titre (OT) was estimated using the subtractionmethod (OD value for sample − OD value of conjugate control) as

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Table 2Details of random sample collection from vaccinated flocks in the field.

Flock no. Age of vaccination Age of the birds Flock no. No. of sera samples

12 weeks

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pecified earlier (Snyder et al., 1983). The resultant OD values werelotted on Y-axis against dilutions in X-axis. The point where theample line cuts the PNT line is taken as titre of the sample. Positiveegative threshold baseline with different positive serum samples

s presented diagrammatically (Fig. 6).The formula to find out the titre from single serum dilution was

eveloped by applying the principle of linear regression. The ODalues obtained for every logarithmic dilution was compared withbserved titre (OT) and correlation coefficient was obtained. Theilution that gives highest positive correlation was selected for pre-icting the titre from that dilution. The other constants like slopend intercept were calculated by plotting a scatter chart with ODalues in Y-axis and OT in X-axis. The linear regression formula

= ax + b is converted as x = (y − b)/a to find out the log titre andntilog of titre is referred as predicted titre of the sample. Where

= corrected absorbance; x = log 10 titre; a = slope; b = intercept.

.6. Dot ELISA with recombinant hexon protein

Detection of antibodies to FAdV-4 in serum sample by DotLISA was done as described earlier (Roy and Venugopalan, 1999;anoharan et al., 2004) with modifications. In short, the recombi-

ant hexon antigen of FAdV-4 (50 ng/�l) was separately spotted at �l volume onto nitrocellulose membrane immunocomb strips.he antigen spotted combs were incubated at 37 ◦C for 1 h in aumid chamber. After 1 h of incubation the combs were washedhree times with PBST and incubated for 60 min in blocking buffer.he combs were again washed three times with PBST and then incu-ated at 37 ◦C for 45 min with sera samples from the field or seraample collected from experimentally vaccinated birds at a dilutionf 1:25, 1:50, 1:100, 1:200 and 1:400 in serum dilution buffer. Afterncubating with sera sample the strips were washed three times in


BST and incubated at 37 ◦C for 30 min with anti-chicken IgY HRPOonjugate diluted 1:500 in conjugation dilution buffer. After wash-ng, the combs were exposed to the substrate (5 mg of DAB in 10 ml

ig. 6. Diagrammatic representation of positive negative threshold baseline withifferent positive serum samples. The dotted line represents PNT line and solidepresents five different positive serum samples with different antibody titres.

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of distilled water and 10 �l of hydrogen peroxide) for 10 min. Theenzymatic reaction was stopped by washing the combs with tapwater. The positive reaction was indicated by the appearance of abrown dot.

2.7. Quantitative agar gel immunodiffusion (Q-AGID) test

The serum collected from the vaccinated breeder chickens fromthe field and experimentally vaccinated chicken under laboratorycondition were diluted serially in two fold dilutions from neat(undiluted) to 1 in 32 and subjected to Q-AGID as described ear-lier (Roy et al., 1999). Geometric mean (GM) QAGID results havebeen presented graphically for comparison.

2.8. Diagnostic sensitivity, specificity and accuracy

The test results were analysed for the sensitivity, specificityand percentage of agreement by using kappa statistics (Kelseyet al., 1986). The diagnostic sensitivity, specificity and accu-racy of the single dilution ELISA for the detection of FAdV-4antibodies were determined in comparison to the Q-AGID. Sensi-tivity = a/(a + c) × 100 where ‘a’ is the number of sera positive bysingle dilution ELISA and Q-AGID, ‘c’ is the number of sera posi-tive by Q-AGID but negative by ELISA. Specificity = d/(b + d) × 100where ‘d’ is the number of sera negative by single dilution ELISAand Q-AGID, ‘b’ is the number of sera negative by Q-AGID but pos-itive by recombinant antigen ELISA. Accuracy is calculated usingthe formula, (a + d)/(a + b + c + d) × 100. The results obtained fromthe tests were analysed for the percentage of agreement by kappastatistics: k = (a + d − P)/(1 − P) where P = (a + b)(a + c) + (c + d)(b + d).A kappa value greater than 0.81 indicates perfect agreement.

3. Results

3.1. Recombinant hexon protein antigen

The immunodominant coding sequence (cds) of hexon gene wascloned into pRSET B vector (Fig. 1). The vector was transformed intochemically competent BL 21 DE3 pLys S E. coli cells. The cells weregrown in LB agar containing ampicillin. The hexon gene insert inthe plasmid was confirmed by touch colony PCR using hexon genespecific primers which yielded expected amplicon of size 737 bp(Fig. 2). The recombinant cloned plasmid DNA was isolated by alka-line lysis method and subjected to the restriction enzyme digestionusing BamHI and PstI to release the insert from the plasmid. Thedigestion yielded two products of size 2.9 kbp and 737 bp respec-tively (Fig. 3). The recombinant hexon gene was expressed in BL21DE3 pLys S cells as a fusion protein with 6× His tag was foundto be of expected size 31 kDa approximately as analysed by SDS-PAGE (Fig. 4). Western Blot analysis with FAdV-4 specific serumfurther confirmed its immunodominant identity (Fig. 5). Averageyield of the recombinant protein were 1.4 mg/ml of the culture.Epitope prediction of the expressed protein with ABCpred software


and analysis with DNASTAR software revealed presence of 18 epi-topes. When epitope score was considered, 90.27% was found to beunique for FAdV-4 and remaining 9.27% was found to be overlap-ping or cross reacting epitopes. Major epitopes with score 0.90 and

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R. Rajasekhar, P. Roy / Journal of Virological Methods xxx (2014) xxx–xxx 5

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ig. 7. Comparison of antibody titres in experimentally vaccinated chickens byLISA, Dot ELISA and AGID. chickens were vaccinated at 10 days old.

bove were observed to be unique for FAdV-4. Thus the expressedrotein was considered as specific antigen for FAdV-4 serology. Inddition, control FAdV-4 specific positive serum samples were usedo construct positive negative threshold baseline.

.2. ELISA

The optimum working range with regard to antigen and conju-ate was determined by checkerboard titration. By this titration,oating antigen of recombinant hexon protein was found to be

ng/�l and the conjugate dilution is 1 in 2000. The PNT curveor the calculation of the OT was manually obtained. ELISA usingingle serum dilution method was performed. For prediction ofitre values, a formula was developed using 78 serum samples.he correlation coefficient at 1:100 was 0.92, and which was morehan the correlation coefficient calculated from all other dilutions.ence, 1:100 was preferred to predict the titre. The other two con-

tants required predicting titres from serum dilution of 1:100 viz.,lope (a) and intercept (b) were estimated by drawing a scatterhart. The slope and intercept were 0.1158 and 0.2702 respec-ively. Using these two constants, by applying the linear regressionquation, the formula for calculating titre was obtained which isead as follows x = (y − b)/a, where log 10 titre (x) = [absorbancey) − intercept]/slope. Using this formula all the sera samples werecreened and results are presented in Tables 2 and 3 respectively.

In experimentally vaccinated at 10 days old chickens, ELISAalues ranged from 5.006 ± 0.11 log 10 at 5 weeks of age to.526 ± 0.04 log 10 at 8 weeks of age (Fig. 7). For sera samples col-

ected from the field, ELISA tire ranged between 4.431 ± 0.12 log 10o 3.716 ± 0.04 log 10 during 10 weeks to 32 weeks of age chickensFig. 7). Results have been compared with QAGID (Figs. 7 and 8).

.3. Dot ELISA


The Dot ELISA titre for experimentally vaccinated birds rangedrom 2.240 ± 0.08 log 10 at 5 weeks of age to 0.180 ± 0.04 log 10 at

weeks of age (Fig. 7). For sera samples collected from the field,

able 3elative sensitivity, specificity and accuracy values of recombinant antigen ELISA and QA

Cell ELISA AGID Number Sensit

a + + 83 100%

b + − 12c − + 0d − − 93

Fig. 8. Comparison of antibody titres in field sera samples by ELISA, Dot ELISA andAGID.

the titres ranged between 1.968 ± 0.08 log 10 to 1.445 ± 0.02 log 10during 10 weeks to 32 weeks of age (Fig. 8).

3.4. Diagnostic sensitivity, specificity and accuracy

The sensitivity, specificity and accuracy of the assay relative tothe methods of ELISA and Q-AGID are shown in Table 3. Calculatedsensitivity – 100%, specificity – 88.6%, accuracy – 93.6% and k-value = 0.9901.

4. Discussion

HPS is an emerging disease of primarily 3–6 weeks old broilerchickens, characterized by hydropericardium, mottling of liver andnephritis (Qureshi, 1988; Khawaja et al., 1988; Anjum et al., 1989;Roy et al., 2004b). The disease causes 20–80% mortality in broilerchickens during natural disease outbreaks (Khawaja et al., 1988;Anjum et al., 1989; Cheema et al., 1989; Asrani et al., 1997). Fur-ther, disease and death among Japanese quails (Roy et al., 2004a)and immunosuppression in chickens (Naeem et al., 1995; Deepak,1998) have been reported. Since it is a disease of young chickens,breeder vaccination and passive immunization of young chicks areimportant. Vaccination of commercial you chicks at second week ofage and breeder vaccination is being practiced in the Indian poultryindustry but suitable serological test is not available for large scaleflock profiling. AGID has been used to assess immunity followingvaccination and challenge studies (Roy et al., 1999) but AGID is not asensitive test and difficult for large scale seromonitoring. In presentstudy attempt was made to develop an ELISA test using recombi-nant hexon gene of FAdV-4 expressed protein as antigen. Same wayDot ELISA was also done and results were compared with existingQAGID test results.

Hexon is the major capsid protein of adenoviruses responsi-


ble for virus neutralizing and serotype specificity (Norby, 1969;Norby and Wadell, 1969; Toogood et al., 1989). Hexon gene playsan important role in the immunogenicity of FAdV. Recently, con-siderable laboratory investigation has been directed towards the

GID for FAdV-4 serology.

ivity Specificity Accuracy Kappa-value

88.6% 93.6% 0.99

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evelopment of ELISAs for sero-epidemiological detection of expo-ure to various etiological agents. Recombinant protein-basederological tests may have higher sensitivity and specificity as thearget antigen is immuno-dominant and devoid of any non-specific

oieties present in whole cell preparations. The recombinant anti-en is safe as it is non-infectious too.

In the present study immunodominant partial hexon geneelected and amplified by PCR, subsequently cloned in pRSET vec-or and expressed in BL 21 DE3 p Lys S E. coli cells to produceecombinant hexon protein which should specifically react withAV-4 specific antibody. The recombinant protein produced was ofxpected size and evaluated by sequence analysis of the hexon genend also by Western Blot. Based on epitope analysis, the expressedrotein was found to be specific for FAdV-4 serology. The recom-inant hexon protein was used as a coating antigen for ELISA andot ELISA. Single serum dilution technique was preferred as in sin-le serum dilution ELISA, 35–40 samples can be tested for antibodyetection with sufficient accuracy, whereas in serial dilution ELISAnly 8–10 samples can be examined in a single microtitre plate.his assay reduces reagent costs, time and minimizes error whichs inherent to serial dilutions. This is a preferred method for rapidnd efficient screening of a large number of serum samples espe-ially during assessment of post vaccinal immune response in aarge flock. By using the regression equation developed and test-ng sera samples at a single working dilution of 1:100, a linearelationship was found to exist between the predicted antibodyitres and corresponding observed antibody titres. Thus, the pre-icted antibody titres could be obtained which reflect closely thectual observed titres. These results are in accordance with theesults obtained by Snyder et al. (1983) who developed a singleerum dilution ELISA using whole cell virus as the coating antigenor rapid serological profiling in chickens exposed to Newcastleisease virus. The single serum dilution ELISA developed in this

tudy with recombinant antigen had a sensitivity of 100% relativeo the Q-AGID titres and specificity of 88.6% (Table 3). The closeorrelation obtained between ELISA and Q-AGID titres is a goodndication that both the tests measured similar trends in FAdV-4erology. But ELISA appears to be more sensitive than Q-AGID testnd is able to detect antibody levels against FAdV-4 in field sam-les that is not detectable by the Q-AGID test (Fig. 8). The newlyeveloped ELISA test could detect all the experimentally vaccinatedhicken as positive and the all control unvaccinated chickens asegative indicating that the developed test is more specific andensitive.

The positive and negative sera samples as determined by theerial dilution ELISA were selected for standardization of singleilution ELISA in order to increase the confidence of the regressionquation so that unknown serum samples with different antibodyitres could be predicted correctly by this assay. The similarity inhe antibody titres obtained by the serial and single dilution ELISAevealed that the results of the two tests were similar statisticallynd thus increased the confidence of the regression equation asescribed in earlier studies by Briggs and Skeeles (1984), Manujat al. (2001) and Dey et al. (2004) for detecting antibodies to otherathogens like Pastuerella multocida, Theileria annulata and Lep-ospira.

In the present study of experimental vaccination, GM ELISAitre ranged from 5.0 ± 0.11 log 10 to 4.52 ± 0.04 log 10 during 5theek to 8th weeks of age and matching GM Dot ELISA titres were

.24 ± 0.08 log 10 and 0.18 ± 0.04 log 10 respectively. QAGID tiresere 1.9 ± 0.23 log 2 and 0.5 ± 0.15 log 2 respectively (Fig. 7). In an

arlier experimental challenge study, HPS disease was found to be


ge dependent, an AGID titre of 0.6 log 2 at 7th week was foundo 100% protective (Roy et al., 1999). In the present study, alwaysLISA showed higher titre and found to be a preferred test to assessost vaccinal immune response. Dot ELISA was also found to be a

PRESSical Methods xxx (2014) xxx–xxx

better test than AGID except at 8th week in experimental vaccina-tion where AGID titre were high than Dot ELISA.

When field sera samples were evaluated, the titre values werehigh at 10th weeks of age but not at 14th weeks of age (Fig. 8). Butafter booster vaccination at 16th weeks of age, again titre valueswere considerably high at 24 weeks of age but not at 32 weeks of age(Fig. 8). Since progeny chicks to be passively immunized to provideprotection in early age of life, the breeder should have sufficientantibody but in the present study the results were not encouraging.The breeder chickens should be monitored regularly and vaccinatedbased on seromonitoring results. Further studies are required toformulate a breeder vaccination schedule.

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Recombinant hexon antigen based single serum dilution ELISA for rapid serological profiling against fowl adenovirus-4 causing hydropericardium syndrome in chickens