Establishment and application of a competitive enzyme-linked immunosorbent assay differentiating PCV2 antibodies from mixture of PCV1/PCV2 antibodies in pig sera

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 10^5.0 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.

Sera from experimentally infected piglets

Standard PCV2-negative and positive serum samples were house-made by infecting pig with PCV2. Sera from these pigs sampled at 28, 35 and 42 day post-infection (dpi) were reactive with PCV2-infected cells by IPMA and were used as PCV2-postive serum samples. Sera at 0, 7, and 14 dpi were not reactive with PCV2 and were used as negative serum samples in this study. The positive sera had OD₄₅₀ values < 0.719 when tested by cELISA established in this study with the inhibition in excess of 77.8 %. By contrast, the negative sera had OD₄₅₀ values larger than 1.270 with inhibition less than 60.8 %.

Determination of the cut-off of cELISA by using clinical sera

A total of 160 field serum samples tested by IPMA were used to determine the cut-off value of established cELISA (Table 1). Among them, 75 samples were confirmed to be serological PCV2-negative by IPMA. These serum samples were tested by the established cELISA in this study. Distributions of the cELISA PI values showing the frequency of IPMA-positive and -negative samples are shown in Fig. 1. The average PI values (x-axis) of the 75 IPMA negative sera detected by cELISA were 34.4 % ± 12.0 % (mean ± SD). When 58.4 % (mean + 2SD) was used as the cutoff value for percentage inhibition, the sensitivity was 96.5 % and specificity was 96.0 %, giving a 95 % confidence. When the cutoff increased to 70.4 % (mean + 3SD), the sensitivity and specificity of the cELISA was 91.8 and 100 %, giving a 99 % confidence.

cELISA*	IPMA*
	Positive	Negative	Total
Positive	82a	3a	85a
	78b	0b	78b
Negative	3a	72a	75a
	7b	75b	80b
Total	85	75	160

Note:*IPMA immunoperoxidase monolayer assay, cELISA competitive enzyme-linked immunosorbent assay

^aComepetitive ELISA positive 58.4 % inhibition or greater

^bComepetitive ELISA positive 70.4 % inhibition or greater

Reproducibility, thermal stability and specificity of the cELISA

The thermal stability assay was performed by comparing the PI values of 5 PCV2 positive serum samples on different ELISA plates which have been stored at 4 and 37 °C for six days. The results showed there was no significant difference (p = 0.107) existed between these two temperature conditions.

In addition, the cross-reactivity of the cELISA with several positive sera against CSFV, HP-PRRSV, PPV, PRV and PCV1 was also tested according to the cELISA procedures. The OD₄₅₀ values of all these sera were in excess of 1.930 (PI ≤ 38.5 %) and were negative.

Serological survey of PCV2-vaccinated pigs using the established cELISA