Cells and viruses
Heterologous goose parvoviruses EP22, G3, GD, HE, and Muscovy duck parvovirus (MDPV) J3D6 were used in this study. The GPV Ep22, G3, GD, and HE strains were isolated from the livers dead geese with hepatitis in China in 2001, 1995, 1989, and 2007, respectively, as described previously . MDPV J3D6 and KL strains were isolated from dead Muscovy ducks in 1999 and 2008. VP3 gene of EP22, G3, GD, and HE diverge 4.1 to 4.7% at amino acids level. VP3 of J3D6 and KL showed 85.9%-88.7% identities to those of GPV. GPV were propagated in goose CGBQ cells (AATC CCL-169) or goose embryo fibroblast cells (GEF) or in the allantoic sacs of 14-day-old embryonated goose eggs. MDPV J3D6 and KL was propagated in 13-day-old Muscovy duck embryonated eggs.
The allantoic fluids containing EP22 and J3D6 were centrifuged at 20 000 × g for 15 min. The supernatants were layered onto 30% (w/w) sucrose solution and concentrated by ultracentrifugation (109 000 × g, 10 h, 4°C). The pellets were suspended in PBS, clarified at 5000 × g for 20 min and ultracentrifuged at 109 000 × g for 2.5 h. The purified viruses were stored at −20°C until used.
The VP3 protein used for the production and characterization of MAbs was synthesized in Escherichia coli BL21 (DE3) as described previously . The expressed His-VP3 and 6.7-kDa His tag proteins were purified by using a Ni-NTA kit (Qiagen, Valencia, CA). The 6.7-kDa His tag protein was used as a negative control during screening for specific antibodies to VP3 in an ELISA.
Monoclonal antibodies production
BALB/C mice (Harbin Veterinary Experimental Central) were immunized intraperitoneally with 30 μg of antigen containing the VP3 fusion protein in complete Freund’s adjuvant and boosted twice with the same amount of antigen in incomplete Freund’s adjuvant at 2-week intervals. Six weeks after the initial immunization and 4 days before the mice were sacrificed for the preparation of hybridomas, a final boost was given via the same route with 30 μg of the same antigen. MAbs were produced by using techniques similar to those described previously . Briefly, spleens were removed from the immunized mice, and their splenocytes were fused with NS1 myeloma cells. Hybridoma cell lines secreting antibodies against the VP3 protein were screened and subcloned at least three times by use of a limiting dilution method and ascitic fluids were prepared with the cloned hybridomas in BALB/C mice. All mice were maintained in the animal facility at Harbin Veterinary Research Institute under standard conditions prescribed by the Institutional Guidelines. The study protocol was approved by the Institutional Animal Care and Use Committee.
Hybridoma culture supernatants or mouse ascetic fluids were screened for antibodies in an indirect ELISA as described for the antibody binding assay. Antibodies that bound to the VP3 protein but failed to bind the 6.7-kDa protein were selected for sub-cloning.
Isotypes of the produced MAbs were determined by using a Mouse Immunoglobulin isotyping kit (Zymed Laboratories, Inc.) according to the manufacturer’s instructions.
Western blotting and dot blotting assays
The samples of expressed His-VP3/His proteins and purified GPV EP22 and MDPV J3D6 were denatured by boiling in SDS and 2-mercaptoethanol. The boiled samples were subjected to 10% SDS-PAGE and transferred to nitrocellulose membranes for Western blotting analysis. The membranes were probed with different MAbs followed by a secondary HRP-conjugated goat anti-mouse antibody (KPL, MD, USA). The purified GPV and MDPV antigen or blank allantoic fluids (as a negative control) were used for Western blotting assays. The native antigens containing His-VP3 and the 6.7 His protein (as a negative control) were used for dot blotting assays. The membranes were then probed with the same MAbs as for Western blotting assays.
Detection of native VP3 protein by immunofluorescence assay
GEF and DEF infected with Ep22 and J3D6 strain (at 10 M.O.I. TCID50/cell), respectively, incubated at 37°C for 48 h. The cells were fixed with cold methanol for 10 min and then probed with different anit-VP3 MAbs and negative normal mouse serum for 1 h at 37°C. Bound antibodies were visualized using fluorescent conjugated antibodies against mouse IgG (1:500 dilutions) under a fluorescence microscope.
Effect of native structure of VP3 on MAbs recognition
To determine if a native GPV or MDPV particles could be recognized by three MAbs, the purified GPV EP22 and MDPV J3D6 particles (about 1 μg) and blank allantoic fluids as negative control were spotted onto nitrocellulose membranes for dot blotting assays.
Coupling of horseradish peroxidase to MAbs
Immunoglobulin fractions were isolated from ascetic fluids by precipitation at 4°C with an equal volume of saturated ammonium sulfate (pH 7.0), and then purified by using an affinity column of protein G-agarose (Boehringer Mannheim). Antibodies were coupled to HRP by means of the periodate method  and stored at -20°C.
Determination of MAbs titres
The titers of the MAbs were determined by using an ELISA. The purified His-VP3 protein (0.1 μg) was coated onto plate wells at 37°C for 2 h. The plates (Nunc MaxiSorp® flat-bottom 96 well plate) were then washed three times with washing buffer (0.01 M phosphate-buffered saline, pH 7.2, 0.05% Tween 20) and blocked with 100 μl of TNE buffer containing 2.5% bovine serum albumin. After washing, two-fold serial dilutions of 1 μg/ml uncoupled or HRP-coupled MAbs were added and incubated for 1 h. For uncoupled MAbs, an additional 50 μl of HRP-coupled goat anti-mouse antibodies (KPL, MD, USA) was added. Absorbance was read at 405 nm with a Microplate Reader (BIO-RAD). The level of binding for the relative activity assessment was measured from the resulting dose–response curve.
Antibody binding assay
For the competitive binding assay, the amount of MAb binding in the ELISA was determined for MAbs uncoupled or coupled with HRP . Briefly, for HRP-unconjugated MAb determination, ELISA plates were coated with 0.1 μg of purified VP3 per well at 37°C for 2 h. After washing, 100 μl of TNE buffer containing 2.5% bovine serum albumin was added to each well to saturate all unbound sites. After washing, 100 μl of purified MAb serially diluted with TNE buffer containing 1% bovine serum albumin was added and incubated for 2 h at 30°C. After washing, 50 μl of a 1:500 dilution of HRP-conjugated goat anti-mouse IgG serum was added and incubated for another 1 h. The enzymatic activity was determined after 20 min of incubation by the addition of 30 ml of 1% sodium azide. Absorbance was measured at 405 nm. For HRP-conjugated MAb determination, the same procedures were carried out except that HRP-conjugated MAbs were directly added to the antigen-coated plates without using the HRP-conjugated goat anti-mouse antiserum. The level of maximum binding for the relative activity assessment and the MAb concentration at which 50% binding occurred were obtained from the resulting dose–response curve.
Competitive binding assay
The competitive binding assay was performed similarly to the procedures described above, except that a mixture of the HRP-conjugated MAbs was used at twice the concentration, giving half-maximal binding. Unconjugated, competing antibodies at different concentrations were also added simultaneously. The competition between two MAbs for the same site was correlated to their relative avidities and concentrations. A spectrum of dose-related interference was tested. Non-specific binding without antigens was used to represent the background. The degree of competitive binding was measured from the absorbance at 405 nm in the presence or absence of unconjugated competing antibodies. Competition was rated as strong (++) if it was more than 60%, significant (+) if it was more than 30%, and negative (--) if it was less than 30%.
Cross-reactivity of MAbs to heterologous GPV strains
To study the cross-reactivity of the MAbs for various GPV and MDPV strains in an antigen-captured ELISA, we tested three GPV (G3, GD, and HE) and MDPV (J3D6 and KL) field isolates. MAbs (4A8 and 2D5) were used to prepare an antigen-capture ELISA and compared with a polyclonal antibody against GPV EP22. Briefly, 100 μl of mouse anti-VP3 polyclonal antibodies (1:200) was coated onto ELISA plates. After washing and blocking, 100 μl of cell extracts of GEF or DEF infected with GPV or MDPV isolates or from mock-infected cells was added and incubated for 1 h at 37°C. For the MAb reactions, 50 μl of HRP-conjugated MAbs (1:1000) was added as the primary antibody. To determine whether the VP3 present in each cell extract from cells infected with each GPV and MDPV isolate was captured by the anti-VP3 antiserum, goose antiserum against GPV EP22 and HRP-coupled goat anti-goose antiserum were used as a primary and secondary antibodies, respectively. Absorbance was measured at 405 nm. Binding to the heterologous virus was expressed as a percentage of the absorbance obtained with GPV EP22, which was set at 100. Binding was rated as strong if it was more than 50%, significant if it was 25%–50%, and negative if it was less than 25%.