Journal of Virological Methods 204 (2014) 1–5

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Development of a blocking ELISA for detection of antibodies against avian hepatitis E virus Baoyuan Liu a,b , Qin Zhao a,b , Yani Sun a,b , Xinjie Wang a,b , Jinan Zhao a,b , Taofeng Du a,b , Chengbao Wang a,b , Shuqi Xiao a,b , Yang Mu a,b , Gaiping Zhang a,c , Jianxun Luo d , Walter H. Hsu e , En-Min Zhou a,b,∗ a

Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, China Ministry of Agriculture, Yangling, Shaanxi 712100, China c Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan 450002, China d State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu 730046, China e Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA b

a b s t r a c t Article history: Received 16 December 2013 Received in revised form 27 March 2014 Accepted 28 March 2014 Available online 13 April 2014 Keywords: Avian hepatitis E virus Blocking ELISA Monoclonal antibody ORF2 protein

A blocking enzyme-linked immunosorbent assay (bELISA) was developed for the detection of immunoglobulin G antibodies against avian hepatitis E virus (HEV). In the bELISA, the coating antigen was a truncated protein containing C-terminal 268-amino acid region of ORF2 from an avian HEV strain isolated in China (CaHEV) and blocking antibody was a monoclonal antibody (mAb) 1H5 recognizing the epitope within amino acids 384–414 in the C-terminal 268-amino acid region. The concentration of blocking mAb 1H5 was determined as that yielded an OD450 nm value of 1.0 for binding to the coating antigen and the antigen concentration and serum dilution were optimized using a checkerboard titration. A cut-off value of 20.7% at the mean percent inhibition plus 3 standard deviations was determined by testing 265 negative sera. The bELISA had a sensitivity of 98.3% by testing 116 positive sera from chickens infected experimentally with CaHEV and had no cross-reaction with other anti-avian virus antibodies. The compliance rates of the bELISA with indirect ELISA and Western blot were 83.7% and 93.3%, respectively, by testing 300 field chicken sera. These results suggested that the bELISA developed in this study can be used for detection of antibodies against avian HEV and showed high reproducibility compared with indirect ELISA and Western blot methods. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Big liver and spleen disease or hepatitis-splenomegaly syndrome causes mainly an increase in mortality, a decrease in egg production and an enlarged liver and spleen in broiler breeder and laying hens aged 30–72 weeks (Ritchie and Riddell, 1991). In 1999, a hepatitis E virus (HEV)-related virus named big liver and spleen virus (BLSV) was detected from the chickens with big liver and spleen disease in Australia based on the amplification of a 523 bp fragment (Payne et al., 1999). Avian HEV being genetically and antigenically related to human HEV was identified from bile samples

∗ Corresponding author at: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China. Tel.: +86 29 87091280; fax: +86 29 87091032. E-mail address: [email protected] (E.-M. Zhou). http://dx.doi.org/10.1016/j.jviromet.2014.03.023 0166-0934/© 2014 Elsevier B.V. All rights reserved.

of chickens with hepatitis-splenomegaly syndrome in USA in 2001 and might represent variants of the same virus with BLSV strain (Haqshenas et al., 2001). To date, avian HEV infection in chickens has been documented in Canada (Agunos et al., 2006), United States (Haqshenas et al., 2001), Europe (Bilic et al., 2009; Peralta et al., 2009; Marek et al., 2010), Korea (Kwon et al., 2012) and China (Zhao et al., 2010). In addition, antibodies against avian HEV have been detected in healthy chickens suggesting that avian HEV can cause subclinical infection in chickens (Huang et al., 2002; Sun et al., 2004; Peralta et al., 2009). Avian HEV, a non-enveloped, positive-sense and singlestranded RNA virus, belongs to the family Hepeviridae along with human and swine HEVs. Its 6.6 kb genome is approximately 600 bps shorter than mammalian HEV genome (Huang et al., 2004). However, similar to mammalian HEV, avian HEV also contains three open reading frames (ORFs) and two noncoding regions (Huang et al., 2004). The ORF1 encodes a non-structural protein with

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B. Liu et al. / Journal of Virological Methods 204 (2014) 1–5

multiple functional domains, ORF2 encodes the capsid protein and ORF3 that overlaps partially with ORF2 encodes a small phosphoprotein (Huang et al., 2004). The capsid protein contains the primary epitopes of the viral particles and is used universally as the antigen for detection of antibodies against avian HEV (Haqshenas et al., 2002; Guo et al., 2006; Dong et al., 2011). Two indirect enzyme-linked immunosorbent assays (iELISAs) using ORF2 protein as the coating antigens have been developed for the detection of chicken IgG antibodies against avian HEV (Huang et al., 2002; Zhao et al., 2013). Serological prevalence of avian HEV infection has been investigated in USA, Spain, China and Korea using these iELISAs (Huang et al., 2002; Peralta et al., 2009; Kwon et al., 2012; Zhao et al., 2012). The iELISAs have the limitations of the requirement of highly purified coating antigen and non-specific background signals. In contrast, monoclonal antibody-based blocking ELISA (bELISA) can overcome these limitations and therefore it has been developed for the detection of antibodies, not only IgG isotype but also other immunoglobulin isotypes, to various viruses (Oem et al., 2007; Shearera et al., 2009; Heo et al., 2010; Huang et al., 2011; Sotelo et al., 2011). Monoclonal antibodies (mAbs) specifically recognize epitopes located in the C-terminal 268-amino acid region of avian HEV ORF2 protein have been produced in our laboratory (Dong et al., 2011). To develop a bELISA for detecting antibodies against avian HEV, a mAb that showed highest affinity with the truncated ORF2 protein was selected from the six mAbs as the blocking antibody and the truncated ORF2 protein was used as the coating antigen. Then, the concentration of coated antigen and serum dilution were optimized using a checkerboard titration; the cut-off value, sensitivity, specificity and cross reaction with other anti-avian virus antibodies were determined. The results demonstrated that the bELISA developed in the study can detect the specific IgG antibody against avian HEV and showed high reproducibility and more specificity than iELISA and more convenient than Western blot. 2. Materials and methods 2.1. Antigen and mAbs The truncated ORF2 protein of avian HEV isolated from China (CaHEV) expressed by a bacterial system was used as the coating antigen in the bELISA. The protein contains the C-terminal 268-amino acid region of CaHEV ORF2 protein and was produced following the method described previously (Zhao et al., 2013). The recombinant protein was expressed with isopropyl␤-d-thiogalactopyranoside (IPTG) in Rosetta (DE3) pLysS cells, purified using the Bug Buster Ni-NTA His Bind Purification Kit (JinSiTe Company, Nanjing, China) and renatured in the renaturation buffer (1 mM EDTA, pH 7.2 PBS containing 6, 4, 2 or 0 mM urea). Six mAbs designated 7H12, 3E8, 1B5, 1H5, 3A1 and 6G8 produced previously in our laboratory (Dong et al., 2011) were purified using a goat-anti-mouse IgG affinity column following the manufacturer’s instruction (Jinsite Company, Nanjing, China) and stored in 0.01 M PBS (pH 7.2). 2.2. Serum samples A total of 265 chicken serum samples were collected from six adult chicken flocks without a history of avian HEV infection. Those flocks were confirmed to be non-infected by testing serum antiavian HEV antibodies with iELISA and avian HEV RNA in fecal samples with RT-PCR. A total of 116 serum samples positive for anti-avian HEV antibodies as confirmed by iELISA were collected from SPF chickens inoculated intravenously with 200 ␮l of CaHEV infectious stock containing 104 genomic equivalents (GE)/ml at 28

day post challenged (DPC). In addition, a panel of 160 sequential chicken serum samples collected from 20 challenged chickens at 0, 3, 7, 10, 14, 21, 28 and 35 DPC and was tested by both bELISA and iELISA. The animal experiments were reviewed and approved by Animal Care Committee of Northwest A&F University. To evaluate the bELISA compared with iELISA and Western blot, 300 clinical chicken serum samples were collected from healthy chickens with different ages in the field. These serum samples were used to determine the cut-off value, sensitivity and specificity of bELISA. 2.3. Analysis of affinity between six mAbs and truncated ORF2 protein Each of six mAbs (1 mg/ml) diluted 1:10 to 1:104 were tested in the iELISA as described previously (Dong et al., 2011). One mAb that had the highest affinity with the truncated ORF2 protein of CaHEV was selected to be a blocking antibody in the bELISA and the optimal concentration of mAb reaction with antigen in iELISA was determined when the OD450 nm value was approximately 1.0. 2.4. The bELISA The optimal coating antigen concentration and testing serum dilution for the bELISA were determined using a checkerboard titration. The concentrations of the coating antigen were 2 ␮g/ml, 4 ␮g/ml and 8 ␮g/ml and the dilution of chicken serum samples were from 1:10, 1:20 and 1:40. The final conditions were determined from the reaction that produced the highest percent inhibition (PI) of the positive and negative serum samples. The bELISA procedure was as follows: the MaxiSorp ELISA plates (Nunc-immunoplate, Roskilde, Denmark) were coated with the recombinant truncated ORF2 protein of CaHEV (100 ␮l/well) diluted in 0.01 M PBS (pH 7.2) and incubated at 4 ◦ C for 16 h. Plates were washed three times with PBST [PBS containing 0.5% (v/v) Tween-20] and then blocked with blocking buffer (containing 2.5% non-fat dry milk in PBST) at room temperature (RT) for 1 h. Following three further washes, 100 ␮l of testing serum samples, positive or negative control samples diluted in the blocking buffer was added to the wells. After the plates were washed three times again, the blocking mAb (100 ␮l/well) was added to each well and the plates were incubated at RT for 1 h. Plates were washed three times, an HRP-goat anti-mouse IgG (BoAoSeng Company, Beijing, China) at 1:4000 was added to the wells (100 ␮l/well) and then the plates were incubated at RT for 1 h again. After final three washes, tetramethylbenzidine [A: 205 mM potassium citrate (pH 4.0); B: 41 mM tetramethylbenzidine; A:B (v/v) = 39:1, 100 ␮l/well] was added to each well and the plates were incubated in the dark for 15 min at RT. The colorimetric reaction was stopped by adding 3 M H2 SO4 (50 ␮l/well) and the OD450 nm values were read using an automated ELISA plate reader (Bio-Rad, USA). The OD450 nm value was used to calculate the PI using the following formula: PI (%) = [1 − (OD value of testing serum samples/OD value of negative serum samples)] × 100%. The 265 negative serum samples from six avian HEV-free adult chicken flocks were used to determine the cut-off value between the positive and negative sera samples. The cut-off value for the bELISA was set at the mean PI of 265 negative serum samples plus 2 or 3 standard deviations (SD), which could give either 95% or 99% confidence for the negative sera sample fell within the defined range. 2.5. Validation of the bELISA The 116 positive serum samples collected from SPF chickens inoculated intravenously with CaHEV infectious stock and confirmed by iELISA were used to assess the sensitivity of the bELISA. To analyze the cross-reaction with other anti-avian virus

B. Liu et al. / Journal of Virological Methods 204 (2014) 1–5

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Fig. 1. Analysis of the affinities of six mAbs 7H12, 3E8, 1B5, 1H5, 3A1 and 6G8 with the truncated ORF2 protein containing C-terminal 268 amino acids from CaHEV using iELISA. The production and characterization of six mAbs were documented previously by Dong et al. (2011). The concentrations of six mAbs (1 mg/ml) were at a range of dilutions 10−1 to 10−4 for reaction with the truncated ORF2 protein in the iELISA.

antibodies of the bELISA, a cross-blocking assay was performed by testing the known reference positive sera against Newcastle disease virus (NDV), avian influenza H5N1 and H9N2, Marek’s disease virus (MDV), J subtype of avian leucosis virus (ALV-J) and egg drop syndrome virus (EDSV) obtained from the China Institute of Veterinary Drug Control (Beijing, China). The precision of the bELISA was evaluated by testing six selected positive serum samples from the challenged chickens with CaHEV. The coefficient of variation (CV) was used to evaluate the interassay variation (between plates) and the intra-assay variation (within a plate). Each sample was tested in three different plates on different occasions to determine the inter-assay CV and three replicates within each plate were used to calculate the intra-assay CV. 2.6. Comparisons of the bELISA with iELISA and with Western blot To test the coincidence of the bELISA with iELISA and with Western blot, 300 clinical chicken serum samples were detected separately by iELISA, Western blot and bELISA methods. The methods of iELISA and Western blot were performed according to the procedure described previously (Zhao et al., 2013). Coincidence rates of the bELISA with iELISA and with Western blot methods were calculated using Microsoft Excel’s CORREL function. 2.7. Statistical analysis The Kappa values were calculated to estimate respectively the agreements between bELISA and iELISA and between bELISA and Western blot using the Kappa analysis with a web-based program (http://faculty.vassar.edu/lowry/kappa.html) (Rosner, 2006). 3. Results 3.1. Selection of a blocking mAb for the bELISA The affinities of six mAbs, designated 7H12, 3E8, 1B5, 1H5, 3A1 and 6G8, with the truncated ORF2 protein of CaHEV were tested using iELISA. As indicated in Fig. 1, the mAb 1H5 which showed the strongest positive interaction with the truncated ORF2 protein was used as the blocking mAb in the bELISA. In addition, when mAb 1H5 was diluted 104 times the OD450 nm value was approximately 1.0 (Fig. 1). Thus, the optimal concentration of mAb 1H5 for the bELISA was 1 mg/ml × 10−4 = 0.1 ␮g/ml.

Fig. 2. Frequency distribution of PI values based on the results from the 116 positive anti-avian HEV antibody serum samples tested using bELISA. The serum samples were positive for avian HEV when PI was ≥20.7% and were negative when PI was ≤15.7%. The serum samples were tested at optimized dilution of 1:10 in the bELISA.

3.2. Development of bELISA with mAb1H5 A bELISA was developed by performing checkerboard titrations with coated antigen, and positive and negative chicken sera. The results showed that the optimal concentration of the coated antigen was 4 ␮g/ml and the optimal dilution for the serum under test was 1:10. The 265 negative chicken serum samples were tested using the bELISA to determine the cut-off value for the assay. The results showed that the average PI (X) of the 265 negative samples was 5.7%, with an SD of 5.0% and then the cut-off values for the bELISA were 15.7% (X + 2SD) and 20.7% (X + 3SD). Therefore, testing samples with PI ≥ 20.7% and ≤15.7% were designated positive and negative, respectively. Samples with PI between 15.7% and 20.7% were considered inconclusive and were re-tested. If the PI was still 0.4).

4. Discussion At present, serological tests are the primarily diagnostic methods of avian HEV infection including iELISA and Western blot methods (Huang et al., 2002; Zhao et al., 2013). Nevertheless, the iELISA often produces non-specific background signals and false positive and the Western blot assay is not convenient for large-scale serological investigations. However, there have been no reports about the development of bELISA for detection of antibodies against avian HEV, which can overcome these limitations. In the present study, a bELISA for detecting anti-avian HEV antibodies using the truncated ORF2 protein from CaHEV as coated antigen and a mAb 1H5 as blocking antibody was developed and showed a higher specificity than iELISA. In a previous study, it was found that the C-terminal 268-amino acid region of avian HEV capsid protein contained the primary epitopes of the virus and induced strong specific antibodies against virus in chickens (Haqshenas et al., 2002; Guo et al., 2006; Dong et al., 2011). Therefore, the region of capsid protein from CaHEV expressed by bacterial system was used as the coated antigen in the developed bELISA, which was the same as the coated antigen of iELISA developed previously (Zhao et al., 2013). Through detecting anti-avian HEV antibodies in positive and clinical chicken serum samples with the bELISA, the results further confirmed that this region of avian HEV capsid protein was an optimal target antigen for detecting anti-avian HEV antibodies. The mAb 1H5 used in the development of bELISA was selected from six mAbs produced by traditional hybridoma technique using the truncated ORF2 protein as antigen (Dong et al., 2011). In a previous study, the epitope recognized by mAb 1H5 located in the amino acid 384–414 region of CaHEV (genotype 3) capsid protein which shares 100% amino acid sequence identities with other genotype avian HEV isolate in GenBank (Dong et al., 2011). The results indicated the bELISA using mAb 1H5 as blocking antibody can detect antibodies against other genotypes of avian HEV strains. Conventionally, when a new assay is developed, it is a common practice to compare the new assay with the “gold standard” assay

to estimate the accuracy of the new assay. However, since no “gold standard” assay for avian HEV and iELISA and Western blot methods are used universally to detect antibodies against avian HEV, the bELISA was compared separately to iELISA and Western blot. The results of high reproducibility and no significant differences between bELISA and iELISA (Kappa = 0.68) and between bELISA and Western blot (Kappa = 0.86) suggested that the bELISA can replace the iELISA and Western blot to screen anti-avian HEV antibodies in poultry. In summary, a mAb 1H5-based bELISA has been developed and can be used to detect IgG antibody against avian HEV. The bELISA has a higher specificity than iELISA and more convenient than Western blot. Therefore, this assay is ideal for large-scale serological studies of avian HEV infection.

Acknowledgements This study was co-supported by Natural Science Foundation of China (31372464) to EMZ, the Open Fund of Key Laboratory of Veterinary Etiologic Biology (SKLVEB2013KFKT008) to QZ and by the Specialized Fund for the Basic Research Operating Expenses Program of Central College (Z109021307) to QZ.

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Development of a blocking ELISA for detection of antibodies against avian hepatitis E virus.

A blocking enzyme-linked immunosorbent assay (bELISA) was developed for the detection of immunoglobulin G antibodies against avian hepatitis E virus (...
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