Construction of the recombinant plasmid encoding SBP-Cap∆41 fusion protein
The SBP gene with a GS linker (coding amino acid sequence: GGGGSGGGGSGGGGS) and a 6× His-Tag was obtained by polymerase chain reaction (PCR) with the pBEn-SBPa vector (Stratagene, USA) as a template. The primer sequences for the PCR were 5′-TACCATGGGCTGCAAGCTGGGCCTG-3′ (upstream) and 5′-TGAATTCCGAGCCGCCACCACCTGAACCGCCACCACCCGAGCCACCACCGCCGTGATGATGATGATGGTGCAGGCCCAGCTTGCAGC-3′ (downstream). The PCR products were double digested with NcoI and EcoRI and cloned into pET-28a(+) vector (Novagen, USA), producing pET-28S. The PCV2 Cap∆41 protein (capsid protein without 41-amino acid [aa] N-termina nuclear localization signal peptide) gene was amplified using the genome of PCV2 strain YZH (GenBank Accession Number AY943819) as template according to previously study (Ge et al. 2012). The PCR products were double digested with EcoRI and SalI and cloned into pET-28S, producing pET28S-Cap∆41. The construct was verified by DNA sequencing (GenScript, Nanjing, China).
SBP-Cap∆41 fusion protein expression and purification
For expression of SBP-Cap∆41 fusion protein, Rosetta (DE3) pLysS E. coli cells (Novagen, USA) were transfected with plasmid pET28S-Cap∆41. A single colony of transformants was cultivated in Luria–Bertani medium in an incubator shaker at 37 °C to an optical density of 0.6 at 600 nm. Isopropylthio-β-d-galactopyranoside (IPTG) was added to a final concentration of 1 mM. After induction at 30 °C for 6 h, cells were harvested by centrifugation. Purification of the expressed SBP-Cap∆41 fusion protein was by immobilized metal affinity chromatography using the His-Bind Purification kit (Novagen, USA) according to the manufacturer’s instructions.
Preparation of HRP-streptavidin bound Cap∆41 (Hsb-Cap∆41)
The Hsb-Cap∆41 was constructed by simply mixing HRP-SA (Pierce, USA; ~7.0 × 10−8 M) and SBP-Cap∆41 (2.8 × 10−7 M) in equal volumes, and incubating for 48 h at 4 °C.
Reactivity between the Hsb-Cap∆41 and PCV2 serum antibodies
An immune assay was performed to determine whether the Hsb-Cap∆41 had specific reactivity to PCV2 antibody. The recombinant Cap∆41 protein was prepared according to the method described in the previous study (Ge et al. 2012) and diluted in 0.05 M NaHCO3/Na2CO3 buffer (pH 9.6). The wells of high binding 96-well microtitration plates (Costar, Corning, NY, USA) were coated with 100 µl Cap∆41 protein (100 ng/well) at 4 °C for 24 h. After incubation, the wells were washed 3 times with PBST and blocked with 250 µl 5% dried skim milk in 0.01 mM PBST (pH 7.4) at 37 °C for 2 h. Following three washes with PBST, the plates were dried at room temperature. Eight serum samples (four positive and four negative) were diluted 1:9 with PBST, and 100 µl of each dilution was added to the microtitration plate wells. After incubation for 60 min at 37 °C, followed by five rounds of washing with PBST, 100 µl aliquots of Hsb-Cap∆41 diluted 1/10, 1/100 or 1/1000 were then added. After a further incubation of 60 min at 37 °C, followed by five rounds of washing with PBST, 50 µl/well of 3,3′,5,5′-tetramethylethylenediamine solution (SureBlue Reserve TMB Microwell Peroxidase Substrate, KPL) was added and the plates were incubated for 15 min at 37 °C. The chromogenic reaction was stopped by the addition of 50 µl 2 M sulfuric acid, and the optical density at 450 nm (OD450) was recorded using a microplate reader (MK3; Thermo Lab system, Helsinki, Finland).
Double-antigen sandwich ELISA based on Hsb-Cap∆41
The Hsb-Cap∆41 based double-antigen sandwich ELISA (HBDS-ELISA) was developed according to the method described in the previous study (Ge et al. 2012). All the conditions of the two ELISAs were kept the same except for the HRP-conjugated antigen. The detailed process was as follows: Cap∆41 protein coated plates were prepared as described above. Hsb-Cap∆41 was serially diluted twofold from 1:25 to 1:400 in PBST. Each dilution was mixed with positive and negative control serum in ratios of 1:9, and then 100 µl aliquots of the mixtures were added to the microtitration plate wells. After incubation for 60 min at 37 °C, followed by five rounds of washing with PBST, the chromogenic reaction and the following steps were as described above. The dilution of Hsb-Cap∆41 with the highest P/N ratios (positive control OD450/negative control OD450) and the OD450 value of the positive serum closest to 1.0 were considered optimal.
To confirm the negative–positive cutoff value for the HBDS-ELISA, 60 serum samples collected sequentially from 12 PCV-free pigs testing negative for PCV2 antibody by DS-ELISA and a commercial indicated ELISA Kit (JENO Biotech Inc, Korea) were tested using the HBDS-ELISA. Antibody titers of the samples were calculated according to the formula:
$${\text{S}}/{\text{P}} = \left( {{\text{sample OD}}_{ 450} - {\text{negative control OD}}_{ 450} } \right)/\left( {{\text{positive control OD}}_{ 450} - {\text{negative control OD}}_{ 450} } \right).$$
Mean S/P (X) and standard deviations (SD) of the 60 negative sera were calculated, and the negative–positive cutoff value was determined as X + 3SD.
Reproducibility of the HBDS-ELISA
Twelve HBDS-ELISA positive and 12 HBDS-ELISA negative field serum samples were selected to evaluate the reproducibility of the assay and the procedure was performed as proposed by Jacobson (1998). For intra-assay (within plate) reproducibility, three replicates of each serum sample were assigned in the same plate. For interassay (between run) reproducibility, three replicates of each sample were run in different plates. Mean S/P ratio, standard deviation (SD) and coefficient of variation (CV) were calculated.
Performance evaluation of the HBDS-ELISA
Both DS-ELISA and the commercial indirect ELISA kit were used as the reference methods to evaluate the HBDS-ELISA. A total of 269 serum samples were used to compare the three methods. Of these, 60 were the negative sera as described above. Ninety-two sera were selected at random from field samples testing positive by DS-ELISA. Sixty-three samples were collected every 4 weeks from nine piglets ranging from 4 to 28 weeks old in a PCV2 infected farm. The remaining 54 samples came from nine PCV-free pigs injected with PCV2 and collected at 0, 3, 7, 10, 13 and 16 days p.i. The diagnostic sensitivity (DSN) and specificity (DSP) were calculated as follows: \({\text{DSN}} = {\text{TP}}/\left( {{\text{TP}} + {\text{FN}}} \right) \times 100,\)
\({\text{DSP}} = {\text{TN}}/\left( {{\text{TN}} + {\text{FP}}} \right) \times 100,\) where TP, FN, TN and FP represent true-positive, false-negative, true-negative and false-positive, respectively (Jacobson 1998). The kappa statistic was used to measure the strength of agreement between the results of the HBDS-ELISA and the reference methods. A kappa statistic of >0.75 represents excellent agreement, 0.40–0.75, good to fair agreement, and <0.40, poor agreement (Pottumarthy et al. 1999).
For further comparison of the three methods, the overall dynamics of serum PCV2 antibody production in the nine naturally infected and nine experimentally infected pigs were analyzed. Additionally, to check for possible cross-reactivity of the HBDS-ELISA with a view to confirming its specificity, positive sera of classic swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine pseudorabies virus (PRV), and porcine parvovirus (PPV) from PCV-free pigs were tested according to the HBDS-ELISA procedure.
