Development and evaluation of a competitive ELISA using a monoclonal antibody for antibody detection after goose parvovirus virus-like particles (VLPs) and vaccine immunization in goose sera

Journal of Virological Methods 209 (2014) 69–75

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Development and evaluation of a competitive ELISA using a monoclonal antibody for antibody detection after goose parvovirus virus-like particles (VLPs) and vaccine immunization in goose sera Qian Wang, Huanyu Ju, Yanwei Li, Zhiqiang Jing, Lu Guo, Yu Zhao, Bo Ma, Mingchun Gao, Wenlong Zhang, Junwei Wang ∗ College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China

a b s t r a c t Article history: Received 15 April 2014 Received in revised form 6 August 2014 Accepted 12 August 2014 Available online 6 September 2014 Keywords: Goose parvovirus Monoclonal antibody Competitive ELISA Antibody detection

An assay protocol based on a monoclonal antibody-based competitive enzyme-linked immunosorbent assay (MAb-based C-ELISA) for detecting antibodies against goose parvovirus (GPV) and its virus-like particles (VLPs) is described. The assay was developed using baculovirus-expressed recombinant VP2 virus-like particles (rVP2-VLPs) as antigens and a monoclonal antibody against GPV as the competitive antibody. Of the four anti-GPV MAbs that were screened, MAb 1G3 was selected as it was blocked by the GPV positive serum. Based on the distribution of percent inhibition (PI) of the known negative sera (n = 225), a cut-off value was set at 36% inhibition. Using this cut-off value, the sensitivity of the assay was 93.3% and the specificity was 95.8%, as compared with the gold standard (virus neutralization assay). The rVP2-VLPs did not react with anti-sera to other goose pathogens, indicating that it is specific for the recognization of goose parvovirus antibodies. The assay was then validated with serum samples from goslings vaccinated with several VLPs (rVP1-VLPs, rVP2-VLPs, rVP3-VLPs, and rCGV-VLPs) and other vaccines (inactivated and attenuated). The C-ELISA described in this study is a sensitive and specific diagnostic test and should have wide applications for the sero-diagnosis and immunologic surveillance of GPV. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Goose parvovirus (GPV) infection, also known as the gosling plague, is an important disease for geese and Musovy ducklings under the age of 4-20 days (Gough, 1991; Kisary, 1993). It was first described in Yangzhou (Jiangsu Province in China) by Fang (1962). Mortality due to the disease ranges from 0 to 100% depending on age. In geese, GPV is the etiological agent of gosling plague, which is also known as Derzsy’s disease, goose hepatitis, goose plague, and viral enteritis (Derzsy, 1967; Nagy and Derzsy, 1968; Schettler, 1971), which has been described as a small virus particle (20–22 nm in diameter) with an icosahedral outer appearance and has been designated as an Autonomously Replicating Parvovirus belonging to the Dependovirus genus of the Parvoviridae family.

∗ Corresponding author at: College of Veterinary Medicine, Northeast Agricultural University, No. 59 Mucai Street, Harbin, Heilongjiang 150030, China. Tel.: +86 0451 55191244; fax: +86 0451 55191672. E-mail addresses: [email protected], [email protected], [email protected] (J. Wang). http://dx.doi.org/10.1016/j.jviromet.2014.08.021 0166-0934/© 2014 Elsevier B.V. All rights reserved.

The GPV genome is 5106 nucleotides long, single-stranded DNA and contains two open reading frames (ORFs) that encode regulatory and structural proteins. The left ORF encodes for the regulatory proteins, while the right ORF encodes for three capsid proteins: VP1, VP2, and VP3. VP1, VP2, and VP3 are derived from the same gene by differential splicing. Additionally, VP2 is contained within the carboxyl terminal portion of VP1, and VP3 is contained within that of VP2 (Zádori et al., 1994; Tatar-Kis et al., 2004). The gosling plague is an acute, contagious, and fatal disease, which has high pathogenicity and mortality in goslings, however adults are not susceptible to the disease. Now available vaccines are used to immunize adults, so parent flocks should be monitored for antibody titers after vaccination against GPV to determine whether antibody titers are high enough for the protection of offspring from infection. Current testing for serum GPV antibodies can be performed by agar gel precipitation (AGP), serum neutralization assay (Gough, 1984), virus antigen-based enzyme-linked immunosorbent assay (ELISA) (Jestin et al., 1991; Kardi and Szegletes, 1996), an indirect fluorescent antibody test (Takehara et al., 1999) and a VP3 indirect ELISA (Zhang et al., 2010). The serum neutralization assay is the standard method for GPV neutralizing antibody detection;

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however, it is a laborious procedure that takes 2–3 days to complete. The assay is inappropriate for mass serological surveillance because of this aspect. The AGP assay is the traditional lower sensitivity test for GPV antibody detection. The procedure of indirect ELISA is difficult to optimize because of sera’s higher nonspecific adsorption (Sun, 2006). To overcome these limitations, a novel competitive ELISA has been developed that utilizes baculovirusexpressed recombinant VP2 virus-like particles (rVP2-VLPs) (Ju et al., 2011) and a newly developed monoclonal antibody against GPV. The VP2 protein is the main GPV immunologic functional area that can induce neutralizing antibodies in GPV-infected geese. Some GPV MAbs can recognize GPV, while these GPV MAbs only recognize the VP2 and VP3 proteins in Western blot assay (Li, 1998), suggesting that the VP2 protein is a viral protective antigen and has favorable antigenicity. Therefore, the VP2 protein may be a suitable candidate for the detection of GPV specific antibodies. In the present study, a competitive ELISA for the detection of antibodies to GPV in goose sera was developed and validated. The principle of the competitive ELISA relies on the use of monoclonal antibodies (MAbs), so that only the serum antibody that corresponds to the same epitope with the selected MAb will specifically block the virus-MAb reaction. This assay may be useful for the detection of antibodies against GPV infection and vaccines based on rVP2-VLPs. 2. Materials and methods 2.1. Ethics statement The animal use in this study was approved by The Laboratory Animal Ethical Committee of Northeast Agricultural University. The animal treatment was performed according to the Chinese Regulations of Laboratory Animals and the Guidelines for the Care of Laboratory Animals (Ministry of Science and Technology of People’s Republic of China), and GB 14925-2010 Laboratory Animal-Requirements of Environment and Housing Facilities (National Laboratory Animal Standardization Technical Committee). 2.2. Cells, viruses and antigens SP2/0 cells, cultured with RPMI 1640 medium (Gibco, USA) containing 20% fetal bovine serum (FBS; Haoyang, Tianjin, China), were used for MAbs preparation. Goose parvovirus (GPV) 98E strain (105.15 ELD50 /0.2 ml) was used as an immunogen for the production of the MAbs that were specific against the goose parvovirus. The viruses were inoculated into 12-day-old embryonated eggs free of GPV (Gough et al., 1981). The infective allantoic fluid was collected from the dead eggs after 96–120 h and was then put through three freeze–thaw cycles and centrifuged at 5000 × g for 30 min at 4 ◦ C. The supernatants containing the viruses were layered onto a NTE buffer (100 mM NaCl, 50 mM Tris–Cl, 1 mM EDTA, pH 7.4) containing 25% (w/v) sucrose in a 9:1 ratio and ultracentrifuged at 30,000 × g for 3 h at 4 ◦ C. The pellet was resuspended in PBS and stored at −20 ◦ C. After determining the GPV protein concentration and purity with a BCA Assay Kit (Beyotime, Jiangsu, China) and Bandscan (Alpha Innotech, CA, USA) respectively, the virus was adjusted with PBS to a final protein concentration and mice were immunized with 50 ␮g of the virus, or rather 107.0 ELD50 . A duck embryo attenuated GPV strain (GPV-DE, 10−5.676 TCID50 /0.1 ml) was used in the IFA. The virus was derived from GPV 98E at the 15th-egg-passage, which resulted in a 100% duck embryo mortality rate (from the Laboratory Animal Center of Harbin Veterinary Research Institute, CAAS) after the 7th-egg-passage.

rVP2-VLPs (Ju et al., 2011) were used as antigens for the MAbs screening by an indirect ELISA and were also the antigens that were used for the competitive ELISA. A total of 2 × 106 Sf9 cells in 4 × 100 cm2 plastic flakes were infected with the recombinant baculoviruses VP2 at a MOI of 4. The cells were incubated at 28 ◦ C for 72 h and were then washed with cold PBS, pelleted by centrifugation, and resuspended in a buffer (1% deoxycholate, 10 mM Tris–HCl, pH 8.0). Then, the cells were incubated on ice for 30 min and centrifuged (12,000 × g, 10 min, 4 ◦ C). The supernatant was stored at −20 ◦ C (Ju et al., 2011). The GPV AGP antigens (40 ␮g/10 ␮l) were gifted by Professor Hongbin Li at the Heilongjiang Institute of Veterinary Science (in China). Ten microliters of AGP antigen was added into the central well, while individual serum samples (10 ␮l) at serial dilutions (1:2–1:128) were added to the peripheral wells in a 1% agar gel in a 4% NaCl solution at 37 ◦ C for 48 h. The results were evaluated in a blinded manner. 2.3. Sera GPV-positive serum: a 60-day-old healthy goose that had not been immunized was selected and inoculated intramuscularly with 1 ml of GPV 98E strain cultures at 105.15 ELD50 /0.2 ml four times at 1- mouth intervals. The blood samples were taken weekly. When the antibody titer detected by AGP was 1:32, the animal was killed, and its serum was isolated and designated as GPV-positive serum. GPV-negative serum: a healthy goose coming from a nonimmunized district was selected, and its GPV serum antibodies were assessed for negativity by AGP. This serum was isolated and designated as GPV-negative serum. Both the positive serum and negative serum were determined to be positive and negative by the neutralization assay. The other 225 GPV negative sera were determined to be negative by the neutralization assay. Positive reference duck enteritis virus (DEV), goose paramyxovirus (GPMV), Escherichia coli and avian influenza virus (AIV)-H5 serum samples were conserved by our lab and provided by Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences (CAAS), respectively. Serum samples (15 geese per group, seven groups, and 0–8th weeks) were collected (Ju et al., 2011). Specifically, 4-day-old geese from Datong Farm were housed in a specific pathogen-free facility at the NEAU Experimental Animal Center with free access to water and food ad libitum. The geese were randomized into seven groups, and individual geese were vaccinated subcutaneously with 20 ␮g of the individual VLPs types (rVP1-VLPs, rVP2-VLPs, rVP3-VLPs, and CGV-VLPs) in 50% mineral oil (Sigma, NY, USA). Additionally, similar protein amounts of inactivated GPV vaccine (Binzhou Huahong Biological Products, Shandong, China) in 50% mineral oil and 105 ELD50 /0.1 ml of attenuated Gosling Plague vaccine (SYG41-50 Strain, Yang Zhou Vacbio Bio-Engineering, China) were also used. An additional negative control group of geese was injected with the same amount of mineral oil. Their blood samples were obtained from their leg veins weekly up to 8 weeks post-immunization, and their sera were prepared and stored at −20 ◦ C. 2.4. Production and identification of monoclonol antibodies (MAbs) Two 6-week-old female BALB/c mice (from the Laboratory Animal Center of Harbin Veterinary Research Institute, CAAS) were immunized subcutaneously three times with partially purified goose parvovirus at 2-week intervals. Additionally, complete Freund’s adjuvant (Sigma, NY, USA) was used for the first immunization and incomplete Freund’s adjuvant was used for the other two, and then booster immunizations were given intraperitoneally with the same antigen in PBS. Three days after the final booster

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injection, spleen cells were fused with SP2/0 cells using standard procedure (Galfre and Milstein, 1981). The resulting hybridoma cells were cultured successively in RPMI 1640 medium containing HAT (Sigma, NY, USA), HT (Sigma, NY, USA), and then in DMEM supplemented only with 20% fetal bovine serum (FBS; Haoyang, Tianjin, China). Hybridomas were screened for secretion of the desired antibodies by indirect ELISAs and only the GPV specific MAbs were further characterized. Indirect ELISAs for MAbs screening were performed to comfirm the reactivity of produced the MAbs. rVP2-VLPs, at a saturating dilution (300 ng/well), were adsorbed onto 96-well micro-plates (Jet, Canada) by overnight at 4 ◦ C incubation in 0.01 M PBS (pH 7.4). The hybridoma supernatants and an HRP-conjugated goat anti-mouse IgG antibody were sequentially reacted for 1 h at 37 ◦ C. The diluting buffer consisted of PBS (pH 7.4) with 0.05% Tween 20 (PBST). The substrate solution (0.1 mg/ml of TMB and 0.1 mg/ml of hydrogen peroxide urea in 0.1 M citrate buffer, pH 4.6) was then added. After 10 min, the colorimetric reaction was stopped by adding 50 ␮l of 1 M sulphuric acid, and absorbance values were read at 450 nm using an ELISA reader (Bio-Tek Instruments Inc., Winooski, VT). One hundred microliters per well of the reagent was used, and three PBST washes were performed after each incubation. The isotypes of the produced MAbs were determined using the Mouse MonoAb-ID Kit (HRP) (Cellway, Luoyang, China) according to the manufacturer’s instructions. The assay identifies the IgG1, IgG2a, IgG2b, IgG3, IgA and IgM isotype classes using monospecific goat polyclonal antibodies (PAbs). The hybridoma culture supernatants were harvested after in vivo culture. The hybridomas were used as sources of MAbs, and their antibody titers of them were measured by the indirect ELISA. 2.5. MAbs characterization and the selection for the C-ELISA 2.5.1. The reactivity to antigen The reactivity of the MAbs to rVP2-VLPs was determined using a Western blot assay. The rVP2-VLPs were mixed with an equal volume of Laemmli buffer (2×) (Laemmli, 1970) and boiled for 5 min, separated by standard SDS-PAGE, and then transferred to a PVDF membrane (Millipore, MA, USA) in transfer buffer using a TransBlot SD Semi-dry Transfer Cell (Liuyi, Beijing, China) at 50 mA for 50 min. The membrane was blocked with PBS containing 5% skim milk overnight at 4 ◦ C and then incubated with the MAbs at 37 ◦ C for 2 h. After three PBST washes, the membranes were incubated with a 1:10,000 dilution of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (H + L) (Zsbio, Beijing, China) at 37 ◦ C for 1 h. After three PBST washes, the recombinant proteins were visualized with an EasySee Western blot Kit (Transgen, Beijing, China) by exposing the membranes to film and processing with medical film processor. 2.5.2. Immunofluorescence assay (IFA) An IFA was used to detect the reactivity of the MAbs to GPV. Briefly, duck embryo fibroblasts (DEF) were infected in triplicate with 1000 TCID50 GPV-DE per well in 12-well tissue-culture plates for 72 h. The cells were washed with PBS (pH 7.4) and fixed with 4% paraformaldehyde for 20 min at RT. GPV probing was conducted with anti-GPV MAbs for 1 h at 37 ◦ C. The bound antibodies were visualized using a 1:200 dilution of FITC-goat anti-mouse IgG (Zsbio, Beijing, China) under a fluorescence microscope (TE2000U, Nikon, Japan). 2.5.3. Blocking ELISA A blocking ELISA was designed to analyze the capability of the GPV-positive serum to inhibit the binding of the anti-GPV MAbs to rVP2-VLPs. One hundred microliters of known positive and negative sera, at 2-fold sequential dilutions starting at 1:2 dilution, were incubated for 1 h at 37 ◦ C in rVP2-VLPs-coated ELISA plate, and then

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100 ␮l of a pre-determined hybridoma supernatant dilution was added that gave a 1–1.5 absorbance value in a preliminary titration. The binding of the MAbs was detected with a HRP-conjugated anti-mouse immunoglobulin and the colorimetric reaction was achieved as described above. Then, one MAb was chosen as a competitive antibody that could be blocked by the GPV positive serum. 2.6. The competitive ELISA First, 96-well polystyrene microtiter ELISA plates (Jet, Canada) were coated with 100 ␮l of rVP2-VLPs diluted in 0.05 M carbonate buffer (pH 9.6) by a 4 ◦ C incubation overnight. The plates were then washed three times with PBST (pH 7.4) and then blocked with 300 ␮l of 1% BSA (Sigma, NY, USA) in PBST for 1 h at 37 ◦ C. After the plates were washed three times, 50 ␮l of serum (the known positive and negative sera and the serum samples in Section 2.3) diluted 1:2 in PBST and 50 ␮l of the 1G3 supernatants were incubated for 0.5 h at 37 ◦ C. After washing with PBST, 100 ␮l of goat anti mouse IgG (H + L) conjugated with HRP (Zsbio, Beijing, China) diluted in PBST was added and the plates were incubated for 0.5 h at 37 ◦ C. After the plates were washed three times, a substrate (described above) was added for 20 min with continuous orbital shaking. The reaction was stopped by adding 50 ␮l of 1 M sulphuric acid. The optical density (OD) was then measured at 450 nm. The blocking ELISA conditions for each step were investigated by varying conditions for at each step while maintaining constant conditions for all other steps constant, except for the colorimetric and stopping reaction steps. The controls (in duplicate) included a positive serum sample from an immunized goose, a serum from a negative animal, and a buffer control (only monoclonal antibody and buffer). The OD value was converted to a percent inhibition (PI) value using the following formula: PI (%) = 100 × [1 − (test serum OD450 nm/negative reference serum OD450 nm)]. The cut-off value between the positive and negative sera was calculated from the mean percentage inhibition of 225 GPV negative sera plus 2 standard deviations (SD) from the mean. This calculation provides 95.4% confidence that all negative values would fall within the defined range. A total of 54 goose serum samples from commercial goose farms in the Heilongjiang province of China were detected with the competitive ELISA. Additionally, the 54 serum samples were validated with the neutralization assay (Ju et al., 2011) to calculate the sensitivity and the specificity of the C-ELISA. The known positive reference sera against AIV-H5, DEV, GPMV, and E. coli were detected with the competitive ELISA to confirming whether the sera competed with the MAb against GPV. Intraassay and interassay variations for the competitive ELISA were evaluated by testing control sera and four serum samples in quadruple, respectively. 2.7. Detection of the goslings antibody dynamics after VLPs and vaccine immunizations The serum samples after the VLPs and vaccine immunizations were tested using the C-ELISA (above procedure optimized) to analyze the antibody dynamics. Then, the C-ELISA performance was compared with the neutralization assay, the gold standard for GPV antibody detection. The goose sera were diluted serially 2-fold in PBST and DMEM for the C-ELISA and the neutralization assay, respectively (starting at 1:2 dilution in both methods). 2.8. Statistical analysis Data are expressed as the means ± standard deviations. The significance of the variability among the animal trials was determined by a one-way analysis of variance using the GraphPad Prism

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Fig. 1. SDS-PAGE analysis of immunogen and antigen. Lane M: protein molecular weight marker; Lane 1: ultracentrifuged GPV protein; Lane 2: rVP2-VLP protein extracted from Sf9 cells that were infected with recombinant baculoviruses VP2. Ultracentrifuged GPV protein exhibited three dark bands with molecular weights of 87, 72, and 60 kDa. The rVP2-VLPs had an approximate molecular mass of 75 kDa, which was consistent with the expected size.

(version 5.0) software. A p-value of <0.05 was considered statistically significant. 3. Results 3.1. SDS-PAGE analysis of the immunogens and antigens After determining the protein concentrations with a BCA Assay Kit (Beyotime, China), GPV capsid proteins were characterized by SDS-PAGE. The protein bands were subsequently visualized with Coomassie brilliant blue staining. The ultracentrifuged GPV exhibited three dark bands with molecular weights of 87, 72, and 60 kDa respectively (Fig. 1A). Sf9 cells were infected with recombinant baculoviruses VP2, and the SDS-PAGE analysis of the VP2 expression in the Sf9 cells revealed a VP2 fusion protein with an approximate molecular mass of 75 kDa (Fig. 1B), which was consistent with the expected size. 3.2. Monoclonal antibodies production and characterization The monoclonal antibodies (MAbs) against goose parvovirus were obtained by fusing SP2/0 myeloma cells and spleen cells after 6-week-old BALB/c mice were immunized with the goose

Fig. 2. Western blot analysis of rVP2-VLPs by anti-GPV MAbs. The purified rVP2VLPs were separated by SDS-PAGE, transferred to PVDF membrane, and then incubated with the four MAbs. Lane M, Western Marker; Lane 1, 1G3 MAb; Lane 2, 2G8 MAb; Lane 3, 3B8 MAb; Lane 4, 3B11 MAb. All of the four MAbs reacted with the rVP2-VLPs.

parvovirus 98E three times. Cloning by limiting dilution resulted in four stable hybridomas at last. These MAbs were designated as 1G3, 2G8, 3B8, and 3B11. The MAbs isotypes were identified using the Mouse MonoAb-ID Kit (HRP). The result showed that the 1G3 and 3B11 antibody isotypes were IgG2b and the 2G8 and 3B8 antibody isotypes were IgM. The antibody titers of the four hybridoma culture supernatants were measured with the indirect ELISA. The antibody titers in the culture supernatants of 1G3, 2G8, 3B8, and 3B11 MAb culture supernatants were 1:1024, 1:4, 1:4, and 1:2048, respectively. The reactivity of the four MAbs to rVP2-VLPs was determined using Western blot analysis. MAbs 1G3, 2G8, 3B8, and 3B11 gave reactions with rVP2-VLPs (Fig. 2). An IFA was performed on the GPV-DE infected DEF cells to assess whether the GPV MAbs recognized the GPV (Fig. 3). The four GPV MAbs reacted with the GPV-DE infected cells, whereas they showed no reaction with noninfected cells. This result indicated that all MAbs were able to detect the GPV in the GPV infected cells. The blocking-specificity of the anti-GPV MAbs was further confirmed by results of the blocking ELISA, which was designed to detect the capability of the positive serum, with a known specificity, to inhibit the binding of each MAb to the rVP2-VLPs (Fig. 4). In these assays, the binding of the 1G3 and 3B11 MAbs was inhibited, but only by the positive sera. That is, the serum containing antibody

Fig. 3. GPV detection with an indirect immunofluorescence assay from GPV-DE infected DEF cells. 1–4: IFA results of GPV-DE inoculated in DEF cells with the 1G3, 2G8, 3B8 and 3B11 MAbs, respectively; 5–8: IFA results of uninfected DEF cells with the same four MAbs. The four GPV MAbs strongly reacted with the GPV-DE infected cells, whereas they showed no reaction with uninfected DEF cells.

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Table 1 Coincidence of the C-ELISA and the serum neutralization assay for 54 serum samples from commercial goose farms in the Heilongjiang province of China. Detection methods

The serum neutralization assay Testing positive numbers

Testing negative numbers

Total

The C-ELISA Testing positive numbers Testing negative numbers

28 2

1 23

29 25

Total

30

24

54

−1.6 ± 19% (mean ± SD), whereby X + 2SD = 36%; therefore, PI ≥ 36% was considered positive. At a PI ≤ 36%, the serum antibody was considered negative. Fig. 4. The blocked result of the four MAbs with the GPV-positive serum. With GPVpositive and -negative serum dilution increasing, the P/N value rose. As the negative value was deemed to be invariable, the positive value rose. That is, more and more MAbs could combined with the rVP2-VLPs. The 1G3 and 3B11 MAbs binding to the rVP2-VLPs could be blocked by GPV positive serum. That was to say, the two MAbs could competed with the GPV-positive serum for the available epitopes.

to GPV could compete with the pre-titrated 1G3 and 3B11 MAbs for the available epitopes. Additionally, the 1G3 MAb had a better cell state than the 3B11 antibody. Finally, the 1G3 MAb were chosen as the C-ELISA competitive antibody. 3.3. Establishment of the competitive ELISA The competitive ELISA was established using the rVP2-VLPs, GPV positive/negative sera and the 1G3 MAb, and it was standardized by checker board titrations. The optimal dilution of the coated rVP2-VLPs was 1:2000 (300 ng/well). The tested goose serum was diluted to 1:2, and the 1G3 supernatant was used, whose titer was 1:1024. Additionally, a the HRP-labeled goat antimouse IgG antibody working concentration was determined to be a 1:10,000 dilution. Coloration was developed by 0.1 mg/ml of TMB and 0.1 mg/ml of hydrogen peroxide urea in 0.1 M citrate buffer pH 4.6, and terminated with 1 M sulphuric acid, and the OD450 nm value for each well was read using a microplate reader. To determine the competitive ELISA PI cut-off level, 225 known negative goose serum samples (as determined by the neutralization assay) were examined with the competitive ELISA. The histogram obtained was similar to a normal distribution (Fig. 5). The average PI (X) of the 225 goose serum samples by the competitive ELISA was

Fig. 5. The percentage inhibition frequency distribution of the negative sera. X + 2SD was used as a cut-off value, which resulted in a 95.4% confidence. The average PI (X) of the 225 goose serum samples by the competitive ELISA was −1.6 ± 19% (mean ± SD), whereby X + 2SD = 36%. PI ≥ 36% was considered positive. At a PI ≤ 36%, the serum antibody value was considered negative.

3.4. Competitive ELISA validation Based on the C-ELISA cut-off level, the results of the comparative experiments using the 54 geese sera from the commercial goose farms in Heilongjiang province are shown in Table 1. Using a serum neutralization assay, 30 samples were positive and 24 were negative, whereas with the C-ELISA, 29 were positive and 25 were negative. Two samples were negative by the C-ELISA but were positive with the serum neutralization assay, and one sample was positive with the C-ELISA but was negative with the serum neutralization assay. Using the serum neutralization assay as a reference standard, the C-ELISA sensitivity was 93.3% (28/30) and the specificity was 95.8% (23/24), indicating that the C-ELISA approach was associated with a high sensitivity and specificity. The cross-reactivity, as demonstrated by the competitive ELISA, with several positive reference goose virus sera (AIV-H5, DEV, GPMV, and E. coli) showed that all of the sera except for the GPVpositive serum were negative. This demonstrates that the competitive ELISA can specifically detect GPV antibodies and that there was no serum cross-reaction with the other viruses. The competitive ELISA reaction plates coated in the same and different batches were used to detect its repeatability. The intra-batch variation coefficients ranged from 6.81% to 8.03% and the inter-batch variation coefficients ranged from 3.42% to 9.08%, indicating that the results obtained by the competitive ELISA were easily reproducible. 3.5. The serum antibody dynamics in the experimental goslings Based on the competitive ELISA cut-off level, the experimental goslings sera test results by this method are shown in Fig. 6. Only the antibodies in the rVP2-VLPs and rVP3-VLPs groups were positive at

Fig. 6. The competitive ELISA serological profile of the anti-GPV antibodies from several VLPs as well as the inactivated and attenuated vaccine immunizations. The immunization strategy can be seen in Section 2.3. The mean PI value of each weekly goslings sera group was used as the y-coordinate. The rVLPs and inactivated vaccine antibodies were generated faster than attenuated vaccine after immunization. The goslings control group sera were negative for anti-GPV antibodies over the entire time course.

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Fig. 7. C-ELISA antibody and neutralizing antibody titers comparisons. The rVP2-VLPs and rVP3-VLPs group neutralizing and competitive antibodies could only be detected at 1 week following immunization in the sera of the six groups. Additionally, the neutralizing antibody titers were higher at all weeks time points in all of the six groups.

2 weeks after immunization; however the antibodies in all groups, except for the attenuated vaccine and control groups, were positive at 3 weeks after immunization. Additionally, the antibody levels in all of the groups were maintained at a high level from week 4, except for attenuated vaccine group, whose antibody level in attenuated vaccine group became higher only after week 8 (data not shown).

all of the groups were higher than that of competitive antibody after immunization. It can therefore be concluded that the antibody titer detected by the competitive ELISA may reflect directly the neutralizing antibody level and that the sensitivity of the neutralization assay was higher than that of the competitive ELISA.

3.6. Comparison of the C-ELISA antibody and neutralizing antibody titers

The aim of this study was to develop a simple and reliable method for GPV infection detection, as well as to analyze the immune status of vaccinated flocks. The conventional serum antibody detection methods against GPV include the neutralization assay, AGP assay, IFA, and indirect ELISA, of which the AGP assay and indirect ELISA are most commonly used. The AGP assay has a lower sensitivity, and it is difficult to achieve high-throughput and automated detection, which limits its application. The sensitivity of the indirect ELISA approach, which is based on recombinant protein, is sometimes too high (or a lower specificity) because of the total antibody detection and the nonspecific reactions with the goose

The sera from the six groups (the four VLPs group, the inactivated vaccine group, and the attenuated vaccine group) were detected by the C-ELISA and the neutralization assay, and their antibody titers were calculated. The results are shown in Fig. 7. The neutralizing and competitive antibodies of the rVP2-VLPs and rVP3-VLPs groups could only be detected at 1 week after immunization in the sera from the six groups. Additionally, their neutralizing antibody titers were also higher. The neutralizing antibody titers of

4. Discussion

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serum (Sun, 2006). Only an indirect ELISA (Zhang et al., 2010) has been developed to test for the presence of anti-GPV antibodies in goose sera, which utilized a recombinant GPV VP3 capsid protein and a secondary antibody directed against goose IgY. Because of goose sera’s nonspecific adsorption, the indirect ELISA established previously was not successful. Consequently, novel diagnostic tests for both the virus and the anti-GPV antibodies in serum are highly desirable. C-ELISAs have been developed for the detection of antibodies to other avian viruses and have the distinct advantage over indirect ELISAs because secondary antibodies specific to the immunoglobulins of the species being tested are not required. For current large-scale screening and sero-surveillance, C-ELISAs have largely replaced indirect ELISAs (Gorham, 2004). In this study, the method used to develop a novel competitive ELISA for the detection of antiGPV antibodies in goose serum was presented. The rVP2-VLPs was selected as the coating antigen based on the following considerations. VLPs, particularly those from DNA viruses, usually have high immunogenicity and antigenicity because they contain important antigen epitopes, which are presented to immune cells in a native conformation. The rVLPs can be produced in large quantities in insect cells using a baculovirus expression system. Additionally, only the GPV rVP2-VLPs formed precipitation line in the AGP assay in rVPs-VLPs of GPV, which presumed that the exposed rVP2-VLPs’ antigen epitopes were better than the other two rVPs-VLPs (Ju et al., 2011). All four MAbs were positive by an indirect ELISA based on the rVP2-VLPs. Western blot and IFA results showed that the four MAbs could combine with rVP2-VLPs and the natural GPV, but only the 1G3 and 3B11 antibodies could be blocked by the GPV-positive serum, suggesting that the epitopes recognized by the other two MAbs were non-immunogenic during GPV infection in geese. A 1:2 serum dilution was chosen to allow for direct dilution into the wells of a 96-well plate, obviating the requirement for a tube for preparing the dilutions. Both incubation periods have been decreased to 30 min each, compared with a single 1-h stage during previous indirect ELISAs. Because the nonspecific reaction in the method is within the allowed range, the related diluents are conventional, which enhances its stability greatly. The gosling antibody levels after GPV rVLPs and vaccine immunization were detected using the C-ELISA. The antibody levels against the rVLPs and inactivated vaccine were more rapidly generated and higher than attenuated vaccine after immunization until the 8th observed week. The reason may be that the former groups were immunized with inactivated proteins while attenuated vaccine group were immunized with live virus in which the amount of protein was less than the former ones. However, the antibody levels against attenuated vaccine were still high after the 8th week (data not shown). The C-ELISA antibody titers in all of the groups were generally lower than 8 log 2. This is the feature of C-ELISA, which used much lower serum dilution. In that case, the binding of the antigens and MAbs was able to be blocked by serum with higher concentration. The assay will have good specificity and better sensibility than AGP in practical applications. A comparison between the C-ELISA antibody titers and neutralizing antibody titers in the goslings was conducted. The C-ELISA antibody could be detected at 2–3 weeks after immunization, and this titer was 2-fold lower than that of the neutralization assay. The C-ELISA has a number of advantages over the neutralization assay. First and most importantly, the C-ELISA is much more easily standardized, as the amounts of both the recombinant protein and monoclonal antibody used in the assay can be more accurately quantified. Although both the recombinant protein and monoclonal

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antibody are initially expensive to develop and produce, they are a much more reliable and consistent source of reagents than SPF goose embryos. The competitive ELISA also has the advantage that serum containing both IgM and IgY can be reliably tested using the one assay, as it does not rely on secondary antibodies directed against either immunoglobulin class. Overall, the C-ELISA described in this paper should be a useful tool for GPV infection. It is more readily standardized, simpler to perform, and more repeatable although it has a lower sensitivity than the neutralization assay. In the future, it should also provide valuable information in subsequent studies regarding the pathophysiology of the virus, such as fluctuations in antibody levels at various disease stages and the transfer of maternal antibodies and their effect on infection and immunity. Acknowledgements This research was supported financially by a grant from the “Twelfth five” Ministry of Science & Technology of Heilongjiang Province (GA09B302) and Specialized Research Fund for the Doctoral Program of Higher Education (20102325110004). References Derzsy, D., 1967. A viral disease of goslings. Acta Vet. Acad. Sci. Hung. 17, 443–448. Fang, D.Y., 1962. Recommendation of GPV. China Anim. Husb. Vet. Med. 8, 19–20 (in chinese). Galfre, G., Milstein, C., 1981. Preparation of monoclonal antibodies, strategies and procedure. Methods Enzymol. 75, 3–53. Gorham, J., 2004. Biotechnology in the diagnosis of infectious diseases and vaccine development. In: OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 5th ed. Office International des Epizooties, Paris. Gough, R.E., 1991. Goose parvovirus infection. In: Calnek, B.W., Barnes, H.J., Beard, C.W., Reid, W.M., Yoder Jr., H.W. (Eds.), Diseases of Poultry. , 9th ed. Iowa State Univ. Press, Ames, IA, pp. 684–690. Gough, R.E., 1984. Application of the agar gel precipitation and virus neutralization tests to the serological study of goose parvovirus. Avian Pathol. 13, 501–509. Gough, R.E., Spackman, D., Collins, M.S., 1981. Isolation and characterisation of a parvovirus from goslings. Vet. Rec. 108, 399–400. Jestin, V., Le Bras, M.O., Cherbonnel, M., Le Gall, G., Bennejean, G., 1991. Isolement de virus de la maladie de Derzsy très pathogens chez le canard de Barbarie. Recueil de Mèdecine Vètèinaaire 167, 849–857. Ju, H.Y., Wei, N., Wang, Q., Wang, C.Y., Jing, Z.Q., Guo, L., Liu, D.P., Gao, M.C., Ma, B., Wang, J.W., 2011. Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose. Biochem. Biophys. Res. Commun. 409, 131–136. Kardi, V., Szegletes, E., 1996. Use of ELISA procedures for the detection of Derzsy’s disease virus of geese and of antibodies produced against it. Avian Pathol. 25, 25–34. Kisary, J., 1993. Derzsy’s disease of geese. In: McFerran, J.B., McNulty, M.S. (Eds.), Virus Infection of Birds. Elsevier Science Publishers, Amsterdam, pp. 157–162. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Li, X.H., 1998. Preparation of neutralizing monoclonal antibodies against goose parvovirus and their experimental control effects. Chin. J. Anim. Poultry Infect. Dis. 20, 247–249 (in Chinese). Nagy, Z., Derzsy, D., 1968. A viral disease of goslings. II. Microscopic lesions. Acta Vet. Hung. 18, 3–18. Schettler, C.H., 1971. Virus hepatitis of geese. II. Host range of goose hepatitis virus. Avian Dis. 15, 809–823. Sun, L.S., 2006. Development of indirect-ELISA for detecting duck and goose antibodies against type A influenza. Northeast Agricultural University master degree theses. Takehara, K., Nakata, T., Takizawa, K., Limn, C.K., Mutoh, K., Nakamura, M., 1999. Expression of goose parvovirus VP1 capsid protein by a baculovirus expression system and establishment of fluorescent antibody test to diagnose goose parvovirus infection. Arch. Virol. 144, 1639–1645. Tatar-Kis, T., Mato, T., Markos, B., Palya, V., 2004. Phylogenetic analysis of Hungarian goose parvovirus isolates and vaccine strains. Avian Pathol. 33, 438–444. Zádori, Z., Erdei, J., Nagy, J., Kisary, J., 1994. Characteristics of the genome of goose parvovirus. Avian Pathol. 23, 359–364. Zhang, Y., Li, Y.F., Liu, M., Zhang, D.B., Guo, D.C., Liu, C.G., Zhi, H.D., Wang, X.M., Li, G., Li, N., Liu, S.G., Xiang, W.H., Tong, G.Z., 2010. Development and evaluation of a VP3-ELISA for the detection of goose and Muscovy duck parvovirus antibodies. J. Virol. Methods 163, 405–409.