The results

were examined under an inverted light microscope. The IPMA was performed in triplicate. Serum neutralization assays To detect the neutralizing activity of mAb 8E4, a serum neutralization assay was adapted from the method of Lefebvre et al. [14]. Briefly, 104.3 × TCID50 (50% tissue culture infective dose) of PCV2 in a volume of 200 μl was incubated for 1 h at 37°C, with an equal volume of undiluted hybridoma supernatant containing mAb against the PCV2 capsid protein. After incubation, this mixture was Ibrutinib in vitro added to semi-confluent monolayers of PCV-negative PK-15 cells in four wells of a 96-well plate. After 1 h at 37°C, the cell cultures were washed twice with RPMI 1640 and fresh medium was added. The cell cultures were incubated for a further

36 h at 37°C, then detected using the IPMA as described by Liu et al. [17] with PCV2-positive serum. Assays were performed with six different strains of PCV2 (PCV2a/LG, PCV2a/CL, PCV2a/JF2, PCV2b/SH, PCV2b/YJ and PCV2b/JF) and recPCV1/G. PCV2-positive sera and mAb 6F10 (with no neutralization to PCV2) were used as positive and negative controls, respectively. The number of infected cells per well was determined by light microscopy. The neutralizing activity of the hybridoma supernatant was expressed as the percentage reduction in the number of infected cells in comparison with negative control. A mAb was considered to have neutralizing ability when its mean neutralizing activity was > 50%. Capture buy SCH772984 ELISA To develop a PCV2 antigen

capture ELISA, the PCV2-positive serum and the supernatant of mAb 8E4 were purified using protein A Sepharose™CL-4B (GE Healthcare, Uppsala, Sweden), respectively. The purified mAb 8E4 FER was labeled using a peroxidase labeling kit (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s instructions. ELISA plates (Nunc, Glostrup, Denmark) were coated with purified PCV2-positive serum (5 μg/ml) in 0.05 M carbonate buffer (pH 9.6) overnight at 4°C. The plates were washed three times with PBS-T and blocked with 100 μl of PBS-T with 10% horse serum for 1 h at 37°C. One hundred microliters of the PCV2 strain cultures diluted in PBS-T to a final 105 TCID50/ml were distributed in each well and incubated at 37°C for 1 h. After washing with PBS-T, 100 μl mAb (8E4) conjugated with horseradish peroxidase (HRP) diluted (1:500) in PBS-T was added, and the plates were incubated at 37°C for 45 min. After the plates had been washed three times, the colorimetric reaction was developed for 20 min by adding 0.21 mg/ml 2,2-azino-di [3-ethylbenzthiazoline sulfonic acid] in 0.1 M citrate (pH 4.2) containing 0.003% hydrogen peroxide (substrate ABTS). The reaction was stopped by adding 50 μl 1% NaF. The optical density (OD) was measured at 405 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).