Recombinant UL16 antigen-based indirect ELISA for serodiagnosis of duck viral enteritis

Journal of Virological Methods 189 (2013) 105–109

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Recombinant UL16 antigen-based indirect ELISA for serodiagnosis of duck viral enteritis Qin He a,c , Anchun Cheng a,b,c,∗ , Mingshu Wang a,b,c,∗∗ , Dekang Zhu b,c , Yi Zhou c , Renyong Jia a,b,c , Shun Chen a,b,c , Zhengli Chen c , Xiaoyue Chen b,c a

Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, Sichuan, China Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Ya’an 625014, Sichuan, China c Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, Sichuan, China b

a b s t r a c t Article history: Received 26 May 2012 Received in revised form 2 November 2012 Accepted 22 January 2013 Available online 31 January 2013 Keywords: Duck enteritis virus UL16 Indirect ELISA

In this study, a recombinant fusion antigen of duck enteritis virus (DEV) UL16 protein was expressed in Escherichia coli Rossetta (DE3). This target protein was used as a coating antigen to establish an indirect ELISA for detecting anti-DEV antibodies in serum samples from ducks. In the optimal method for the UL16-ELISA, the fusion protein was coated at 1.25 ␮g/ml and duck serum samples were diluted at 1:160. The endpoint cut-off value of this assay was 0.598. The inter-assay and intra-assay coefficients of variation (CVs) were both lower than 10%. There was no cross-reaction with duck positive sera of either DHBV, DHV, RA, E. coli, Salmonella anatum, H5N1 or DSHDV. The assay was applied successfully to examine the suspected duck serum samples and showed 95.5% (73/76) identity with the serum neutralization test (SNT). The results showed that recombinant DEV UL16 protein could be used as a coating antigen and the developed UL16-ELISA approach was rapid, specific, sensitive and repetitive. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Duck viral enteritis (DVE) is a highly lethal, acute and contagious disease caused by duck enteritis virus (DEV) that exerts a serious impact on the duck industry worldwide (Sandhu and Metwally, 2008). The clinical characteristics of DVE are fever, depression, nasal discharge, adherent eyelids, photophobia, ataxia, tremors, diarrhea and reduced egg production. In addition, DVE can cause vascular lesions, nervous impairments and tissue hemorrhage (Campagnolo et al., 2001; Shawky et al., 2000). Currently, no methods or drugs are available for controlling or curing this disease. The traditional diagnosis of DVE was based on the clinical and pathological characteristics. Final diagnosis can be made by viral isolation and identification. However, routine examination may be inaccurate and time consuming. In recent years, loopmediated isothermal amplification (LAMP) (Ji et al., 2009), electron microscopy negative staining (Guo et al., 2008), polymerase chain

reaction (PCR) (Cheng et al., 2004), and antigen-capture enzymelinked immunosorbent assay (AC-ELISA) (Jia et al., 2009) have been developed. These methods are sensitive and quick. Recently, an indirect ELISA assay was established with the whole DEV virion as coating antigen for detection of antibodies against DEV (Qi et al., 2007). The assay was specific, repetitive and the kit could be stored for 10 months at −20 ◦ C. However, the virion is very difficult to purify. An indirect ELISA based on recombinant thymidine kinase (TK) protein as coating antigen to detect anti-DEV antibodies was developed (Wen et al., 2010). The TK-ELISA is simple, specific, sensitive, and repetitive. In this study, based on the expressed and purified recombinant UL16 protein, an indirect ELISA (UL16-ELISA) method for serodiagnosis of duck viral enteritis was developed and evaluated. 2. Materials and methods 2.1. Strains, sera and reagents

∗ Corresponding author at: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, 211# Huimin Road, Wenjiang, Chengdu 611130, China. Tel.: +86 835 2885774; fax: +86 835 2885774. ∗∗ Corresponding author at: Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, 46# Xinkang Road, Ya’an 625014, Sichuan, China. Tel.: +86 835 2885774; fax: +86 835 2885774. E-mail addresses: [email protected] (A. Cheng), [email protected] (M. Wang). 0166-0934/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2013.01.007

Escherichia coli DH5a, Rossetta (DE3), pET32b (+) vector, DEV CHv strain, rabbit anti-DEV, and sera positive and negative for DEV were all kept in the Key Laboratory of Animal Disease and Human Health of Sichuan Province. The Ni2+ -chelating column, DEAE–Sepharose column, 96-well ELISA microplates, and Bio-Rad model 860 plate reader were purchased from Bio-Rad (USA). HindIII, XhoI, and T4 ligase were purchased from TaKaRa

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Biotechnology Company (China). Horseradish peroxidase-labeled goat anti-duck IgG (HRP-goat anti-duck IgG) and 3,3 ,5,5 tetramethylbenzidine (TMB) were purchased from KPL (USA). Horseradish peroxidase-labeled goat anti-rabbit IgG (HRP-goat anti-rabbit IgG) was purchased from Zhongshan. Plasmid purification kits and agarose gel DNA purification kits were purchased from Axgene Company. Bovine serum albumin (BSA) was purchased from Promega. 2.2. Recombinant protein expression and purification Expression and purification were performed as described previously (He et al., 2011). The recombinant plasmid pET32b–UL16 was transformed into E. coli Rossetta (DE3). The positive single clone was cultured in LB medium with 100 ␮g/ml ampicillin at 37 ◦ C to an optical density (OD600 ) of 0.6. The cells were then induced with 0.2 mM IPTG and allowed to grow further for 6 h at 37 ◦ C. Cells were harvested, freeze thawed, lysed, purified and renatured. The recombinant UL16 protein was obtained and used for western-blot analysis. 2.3. Western-blot assay To confirm antigenicity and immunogenicity, the recombinant fusion DEV UL16 protein was subjected to 12% SDS-PAGE and electro-transferred onto PVDF membrane as described previously (Kano et al., 2008; Towbin et al., 1979). The membrane was then blocked with 3% BSA in PBST (0.2% Tween-20 in PBS, PH 7.4) at 37 ◦ C for 1.5 h. Subsequently, the membrane was incubated with rabbit anti-DEV IgG at a dilution of 1:100 with 0.5% BSA in PBST at 4 ◦ C overnight. The membrane was incubated further with HRP-goat anti-rabbit IgG at a dilution of 1:3000 at 37 ◦ C for 1 h. The reaction was developed with diaminobenzidine substrate buffer and terminated by washing with distilled water.

2.5. Repeatability of the UL16-ELISA Three serum samples and two batches of UL16 protein were selected to validate the test and evaluate the repeatability of the assay. With the optimal working concentration of the coating antigen, the three serum samples were detected by the positive serum and HRP-goat anti-duck IgG. Each serum sample was tested in eight different replicates to calculate intra-assay variation. The other batch of UL16 protein was coated and the OD450 values of the three serum samples were measured. Each serum sample was tested in eight different plates to calculate inter-assay variation. 2.6. Sensitivity of the UL16-ELISA In order to determine the sensitivity of this method, sera were diluted 1:320, 1:640, 1:1280, 1:2560, 1:5120 and 1:10,240. The other operating conditions were performed as the optimal working procedure. 2.7. Specificity of the UL16-ELISA The cross-reaction test and the inhibition-reaction test were performed to determine the specificity of the UL16-ELISA method as described previously (Jia et al., 2009). Based on the cut-off value, duck antisera of duck hepatitis B Virus (DHBV), duck hepatitis virus (DHV), Riemerella anatipestifer (R.A), influenza virus (H5N1), Salmonella anatum (S. anatum), E. coli and duck swollen head hemorrhagic disease virus (DSHDV) were detected in two replicates according to the method above. According to the optimal concentration, an equal volume of fusion UL16 protein and anti-DEV (or negative) sera were mixed and incubated at 37 ◦ C for 1 h to perform the inhibition-reaction test. These mixtures were tested with the UL16-ELISA. Furthermore, negative and positive sera without any treatment were used as control sera. The percentage of inhibition was calculated as reported previously (Ko et al., 2009). The fusion protein was regarded as positive for DEV and the UL16-ELISA was specific when the percentage of inhibition was higher than 50%.

2.4. Development of the UL16-ELISA 2.8. Comparison of UL16-ELISA and serum neutralization A checkerboard titration was performed to determine the optimal working dilution of the coating antigen, serum and HRP-goat anti-duck IgG using a 96-well ELISA plate. A 96-well microtiter plate was coated with 100 ␮l of purified UL16 protein and incubated at 4 ◦ C overnight. The plates were then blocked for 1 h with 1% BSA/PBST at 37 ◦ C and washed three times with PBST. Subsequently, 100 ␮l of duck sera were added and incubated at 37 ◦ C for 1 h. The samples were washed, and then incubated for 1 h with 100 ␮l of HRP-goat anti-duck IgG diluted 1:5000 in 0.1% BSA/PBST at 37 ◦ C, washed again, and detected with 100 ␮l of TMB/H2 O2 for 30 min at room temperature (RT) and away from light. The reaction was then stopped by the addition of 50 ␮l of 2 M H2 SO4 . Optical density (OD) values were measured at 450 nm. Test sera included positive, negative and blank-sample controls. The dilutions that gave the maximum between positive and negative sera (P/N) with the lowest levels of background readings by absorbance at 450 nm were selected for the optimal antigen-coating concentration and serum dilutions. The optimal antigen-coating concentration and serum dilutions to determine the optimal HRP-goat anti-duck IgG dilutions were performed. Twenty-four negative sera from duck were used to determine the cut-off value according to the optimization of the ELISA procedure. The cut-off value was determined by titration as the mean OD450 value plus 3 SDs of the antibody levels of the negative controls. The serum sample was regarded as positive if the OD450 value was higher than the cut-off value; or, it was considered to be negative.

Ninety suspected serum samples were collected from ducklings from Sichuan Province and kept in the Key Laboratory of Animal Disease and Human Health of Sichuan Province. These serum samples were examined using the developed UL16-ELISA assay as described above. In addition, these serum samples were tested by the traditional serum neutralization test (SNT) as described previously (Shaoying et al., 2006; Wolf et al., 1974). The correlation was carried out by comparing results of the two methods (Chaudhuri et al., 2010). 3. Results 3.1. Recombinant protein expression and purification The UL16 gene was subcloned successfully into the prokaryotic expression vector pET32b (+) and induced by IPTG to produce recombinant UL16 protein. The size of the purified fusion protein was about 60 kDa by SDS-PAGE analysis. The results of westernblot analysis suggested that the recombinant fusion UL16 protein possessed high levels of antigenicity and immunogenicity. 3.2. Development of an UL16-ELISA By checkerboard titration tests, the OD450 value gave the maximal difference between the positive sera and negative sera (P/N value, data not shown) when the dilutions of antigen and serum

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Table 1 The results of the repeatability assay for the UL16-ELISA. Samples No.

A B C

Intra-assay

Inter-assay

Mean (X)

Standard deviation (SD)

Coefficients of variation (C.V%)

Mean (X)

Standard deviation (SD)

Coefficients of variation (C.V%)

1.276 1.239 1.331

0.0358 0.0671 0.0776

2.81 5.42 5.83

1.277 1.262 1.244

0.0337 0.0417 0.0740

1.64 3.30 5.95

Table 2 The determination of specificity for the newly developed UL16-ELISA. Sera samples

DEV

DHBV

S. anatum

DSHDV

H5N1

DHV

R.A

E. coli

OD value Results

0.962 P

0.147 N

0.170 N

0.243 N

0.201 N

0.112 N

0.154 N

0.146 N

P, positive; the OD450 value was higher than the cut-off value; N, negative, the OD450 value was lower than the cut-off value. Table 3 The detection results of the inhibition test for UL16-ELISA. Sera samples

OD450 of normal sera a

Mean OD450 of normal sera

OD450 of positive control serab

Mean OD450 of positive control sera

Percentage of inhibition (%)c

1

1.832 1.798

1.815

0.601 0.625

0.613

66.2

2

1.689 1.702

1.700

0.541 0.523

0.532

68.7

3

1.366 1.384

1.375

0.382 0.388

0.385

72.0

a b c

The positive for DEV sera without any treatment was used as normal control sera. The mixtures of an equal volume of recombinant UL16 protein and anti-DEV sera were used as positive control sera. The percentage of inhibition = (OD450 of normal sera − OD450 of positive serum)/OD450 of normal sera.

were 1:80 (1.25 ␮g/ml) and 1:160, respectively; and the optimal dilution of the HRP-goat anti-duck IgG was 1:10,000. Twenty-four sera samples from ducks uninfected with DEV were then selected randomly in order to calculate cut-off values. The mean (X) of the OD450 nm values for these sera was 0.442, with a standard deviation (SD) of 0.052 (data not shown). The cut-off value of the UL16-ELISA was calculated (X + 3SD = 0.598). If the OD value of the sample was ≥0.598, the result was positive; if less it was considered to be negative. 3.3. Repeatability of the UL16-ELISA For the three selected sera and the two batches of UL16 protein, the intra-assay CV% and the inter-assay CV% were both lower than 10% (Table 1). The results showed that the UL16-ELISA assay was highly reproducible and stable. 3.4. Sensitivity of the UL16-ELISA The sensitivity of a panel of diluted sera was evaluated, and a minimal detection limit of 1:5120 (OD450 nm = 0.625) was obtained according to the endpoint cut-off value (0.598), but the blank control did not yield a positive result (data not shown). 3.5. Specificity of the UL16-ELISA For the results of the cross-reaction test, there was no evidence of cross-reactivity with known positive sera from ducks infected with either DHBV, DHV, R.A, E. coli, H5N1, S. anatum, or DSHDV (Table 2). In addition, the OD values at 450 nm in the inhibition test were decreased after mixing the recombinant UL16 protein with the anti-DEV sera, and the percentage of inhibition was more than 50% (Table 3). All these results suggested that the recombinant

Table 4 Relation between the results of the UL16-ELISA and the sera neutralization test. UL16-ELISA

Positives (+) Negatives (−) Total

Sera neutralization test Positives (+)

Negatives (−)

Total

73 3 76

7 7 14

80 10 90

UL16 protein was positive for DEV and the method of UL16-ELISA was specific for the anti-DEV antibody. 3.6. Comparison of the UL16-ELISA and SNT methods Ninety suspected serum samples were collected from ducklings and tested using the UL16-ELISA. The results showed that there were 80 true positives and 10 true negatives. Compared with that of the sera neutralization test, the proportions of these two groups that were correctly diagnosed by the UL16-ELISA were 73/76 (95.5%) and 6/14 (42.8%), respectively (Table 4). The results indicated that the UL16-ELISA was specific and sensitive for the detection of anti-DEV antibodies. 4. Discussion Due to its high morbidity and mortality, DVE has caused tremendous economic loss in the poultry industry since DEV was first recorded in domestic ducks in Holland in 1923, and then was reported in China in 1957 (Baudet, 1923; Huang, 1959). Various diagnostic assays have been developed to detect the antibodies against DEV, but most of these assays were inaccurate, timeconsuming, expensive, or difficult to utilize. A rapid, sensitive, specific and accurate diagnostic method for serological detection of DEV is urgent to develop to detect this disease.

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The UL16 protein, known as one of the viral structural proteins, is conserved throughout the herpesvirus family (Wu et al., 2012). It plays an important role in viral DNA packaging, virion assembly, budding, and egress according to interaction with the C-capsid and other viral proteins (Baines and Roizman, 1991; Klupp et al., 2005; Meckes and Wills, 2007; Nalwanga et al., 1996; Oshima et al., 1998; Wagenaar et al., 2001; Yeh et al., 2008). In the absence of pseudorabies virus UL16, the viral titer and plaque size were reduced relative to the corresponding wild-type titer and plaque diameter (Klupp et al., 2005). The tegument protein ORF45 and the 65-kDa form of glycoprotein B were not recruited into the assembled viral particle by the infected murine gammaherpesvirus 68 ORF33 (UL16 homologue) deletion mutant (Guo et al., 2009). Previous research has shown that anti-UL16 antibodies could be induced in organisms. The gene ORF33 (UL16 homologue) of Kaposi’s sarcoma-associated herpesvirus was a frequently recognized CD4 target causing a T-cell immune response (Robey et al., 2009). The human cytomegalovirus UL94 epitope (UL16 homologue) shows homology with NAG-2, a surface molecule of human endothelial cells that triggers postadhesion signaling events. The UL94 epitope induced anti-UL94 IgG and caused apoptosis of endothelial cells (Lunardi et al., 2000, 2005; Namboodiri et al., 2004). Previous studies have indicated that the DEV UL16 protein shared high sequence identity with UL16 homologs (He et al., 2011). Importantly, DEV from different strains exhibited the same immunogenicity due to only a single antigenic type that has been recognized (Shawky and Sandhu, 1997). Therefore, we believe that the recombinant UL16 antigen-based indirect ELISA for serological detection of DEV is theoretical and practical for epidemiological surveys and monitoring of antibody levels. In this study, recombinant UL16 protein was first expressed in E. coli and purified; and an indirect ELISA based on the UL16 protein as coating antigen was developed. The western-blot analysis showed that the recombinant UL16 protein possessed a high level of antigenicity and immunogenicity, and could be used as a candidate antigen for detection of anti-DEV antibodies raised in the infected animal. Unfortunately, concentrations of coating antigen, sera and HRP-labeled secondary antibody dilutions lead to the non-specificity and high background readings (Li et al., 2010). To overcome these problems, the UL16-ELISA was conducted on serial dilutions of the coating antigen, sera and HRP-labeled secondary antibody to determine optimal conditions for the UL16ELISA. False-negatives and false-positives are two crucial factors in detecting variable antibody titers of different sera using an indirect ELISA test (Wu et al., 2007). To eliminate the false-negatives and false-positives, an optimal cut-off value was created. The most widely used method is the “mean + 3SD” value of antibody levels for negatives (Pinto et al., 2000). Based on the cut-off value, the antibodies against DEV could be detected by using the UL16-ELISA when the serum was diluted in 1:1520, which implied a high sensitivity for this assay. It was reported that using the fusion protein expressed by E. coli as coating antigen to detect antibody might produce a non-specific result because host E. coli protein may be incorporated into the sample serum and coating antigen (Alonso et al., 1990). In the specificity assay, duck-positive antisera of E. coli (originating from duck) were chosen, and no cross-reactivity was observed. Moreover, there was no serum cross-reactivity with known positives to DHBV, DHV, R.A, H5N1, S. anatum, or DSHDV. Ninety suspected clinical sera samples showed that there were seven true negative and 73 true positive sera identified by the two assays, UL16-ELISA and SNT. These results suggested that the UL16-ELISA exhibited a high degree of specificity and sensitivity, and could be used as a candidate antigen for clinical detection of anti-DEV antibodies. Furthermore, as the inter-assay and intraassay CVs were all less than 10%, it could be concluded that the assay possessed good repeatability and could be used in a practical context.

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