PCV2 antigen and monoclonal antibody preparation
VLPs formed by recombinant Cap protein were produced in E.coli BL21 (DE3) strain as previously described [16] and used as the coating antigen for cELISA. Briefly, the supernatant of cell lysates containing recombinant Cap (rCap) protein was precipitated by 60 % saturated ammonium sulfate and resuspended, followed by anion ion-exchange chromatographic purification. The purified recombinant PCV2 Cap proteins have been completely re-assembled into VLPs in a buffer of 50 mM Tris–HCl and 500 mM NaCl. 200 μl (0.4 μg/μl) recombinant PCV2 Cap protein plus equal volume of Freund’s complete adjuvant was used as an immunogen to inject each of five female Balb/c mice (purchased from Vital Rivea Experimental Animal Technology Ltd., Beijing) via intraperitoneal injection for Mab production. Three booster immunizations with same dose of antigen plus Freund’s incomplete adjuvant were conducted at two-week intervals. Three days after the final booster injection, the mice were euthanized and spleen cells were fused with SP2/0 cells using standard procedure [17]. The hybridoma cells were maintained in RPMI1640 medium (Gibco, USA) with 17 % fetal bovine serum (Hyclone, USA). The supernatant of the hybridoma cells were harvested and tested for antibodies to PCV2 and PCV1 by IPMA. The colony of 3H11 MAb reactive to PCV2 but not to PCV1 tested by IPMA was subcloned two times and selected for use in the cELISA. The MAbs were labeled with horseradish peroxidase (HRP) according to the conventional methods [18].
Serum samples
Five colostrum-deprived specific-pathogen-free piglets (Purchased from SPF Swine Breeding and Management Centre, Beijing) were intranasally inoculated with 105.0 TCID50 infective doses of PCV2 SH strain. Serum samples were collected 0, 7, 14, 21, 28, 35, and 42 days post-vaccination (dpi) and separated for serological testing by IPMA and cELISA. Serum samples collected at 0 dpi worked as negative controls. One hundred and sixty clinical serum samples stored at National Research Center for Veterinary Medicine were tested by IPMA for cELISA development.
In the retrospective serologic study, a total of 1297 field pig serum samples were collected by Veterinary Diagnostic Laboratory from Beijing (377 samples), Hunan (432 samples) and Henan (488 samples) provinces in China. The experiments were carried out under the consent of animal owners. The serum samples were tested by the cELISA established in this study.
Immunoperoxidase monolayer assay (IPMA)
IPMA was used to detect the presence of antibodies to PCV2. Briefly, the confluent monolayer of PKK cells (a PK-15 deprived cell line) infected with PCV2 SH (MOI=0.01) or free of PCV, were fixed in 80 % acetone for 30 min at 4 °C. The plates were stored at −20 °C after three times washing with PBS (0.01 mol/L, pH7.2). The MAb or serum samples were diluted 1:50 with PBS (0.01 mol/L, pH7.2), and added into the PCV2- and mock-infected PKK cells, respectively, 50 μl/well, and then incubated at 37 °C for 40 min. The anti-PCV polyclonal antiserum (VMRD, USA) and negative serum gained from colostrums-deprived piglets were respectively prepared as the positive and negative control. After three times washing, 50 μl of 1:200 dilution of HRP-conjugated goat anti-mouse or goat-pig IgG (Sigma, USA) was added and incubated at 37 °C for 30 min. After three times washing, 50 μl of the substrates 3-amino-9-ethylcarbazole was added and incubated for 30 min at room temperature. After three times washing, the 1:10 dilution hematoxylin was added. Twenty seconds later, the plates were washed with water and examined under an inverted light microscope.
Development of cELISA
Optimized dilution of PCV2 VLP antigen and horseradish peroxidase-conjugated PCV2-specific MAb were established by systematic checkerboard titrations. The polystyrene microliter ELISA plates were coated with 1.6 μg PCV2 VLP in phosphate buffer (0.02 mol/L, pH7.4) at 4 °C for 16 ~ 24 h. After three times washing, the plates were blocked with 200 μl of 20 % calf bovine serum in phosphate buffer (0.02 mol/L, pH7.4) for 2 h at 37 °C. After three times washing, 50 μl of the serum samples were added to each well, then 50 μl of 1:2000 dilution MAb 3H11 conjugated with HRP (Sigma, USA) were added to the wells except the blank well. The plates were incubated at 37 °C for 30 min. After five times washing, 100 μl of the substrate solution (0.2 mg/ml of TMB and 0.2 % H2O2 in 0.05 mol/L citrate buffer, pH4.6) was added and the colorimetric reaction was developed at 37 °C for 15 min. The reaction was stopped by adding 50 μl of 2 mol/L sulphuric acid. The optical density (OD) was measured at 450 nm. The controls included positive control (in triplicate), negative control (in triple) and one blank control. The OD450 of the samples were converted to a percent inhibition (PI) value using the following formulation: PI (%) = (OD450 value of negative value − OD450 value of sample)/OD450 value of negative value × 100 %.
Reproducibility, thermal stability and specificity of the cELISA
Inter-assay and intra-assay repeatability for the established cELISA was evaluated by testing the sixty filed sera. For the inter-assay repeatability, three replicates of each serum samples were detected by the same batch of pre-coated ELISA plates. For the intra-assay repeatability, each serum samples were detected by three batches of pre-coated ELISA plates. Mean PI value and coefficient of variation (CV) of three replications of each test were calculated.
Thermal stability for the established cELISA was evaluated by testing five filed serums using the plates stored at 4 and 37 °C for six days, respectively. The PI value of each serum detected with the plates stored at 37 °C for six days was compared with those had been stored at 4 °C. Statistical analysis of the PI value was carried out by Student’s test using the SPSS 19.0 software. The significance level was set at 0.05 (p < 0.05).
To explore the specificity, positive sera for PCV1, classical swine fever virus (CSFV), high pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), porcine parvovirus (PPV), and porcine pseudorabies virus (PRV) were tested with the established cELISA.
