Victortm x5 multilabel plate reader

Cytotoxicities of Mab-5G2 and PRA mutant proteins were evaluated using the Cell Counting Kit-8 (CCK-8) (Beyotime) according to the manufacturer’s instructions. Briefly, cells were seeded into 96-well plates (1 × 105/well) and incubated with indicated treatments at 37 °C for 24 h. Next, CCK-8 reagent (10 µL/well) was added to cells followed by incubation for another 2 h. Viable cells were determined from absorbance measurements at 450 nm using a VictorTM X5 Multilabel Plate Reader (PerkinElmer).

Indirect enzyme-linked immunosorbent assays (IELISAs) were conducted to detect the interaction between recombinant PRA and GP5Δ. Briefly, 96-well polystyrene microtiter plates (Corning, NY, United States) was coated with CP5Δ-His or PRA at the indicated concentration overnight at 4°C, blocked with 5% skim milk in PBS-T buffer [PBS containing 0.5% Tween 20 (Sigma-Aldrich)] and incubated with PRA or CP5Δ-His for 1 h at 37°C. After washed by PBS-T buffer for three times, interaction was detected using anti-His or Mab2-5G2 antibodies, HRP-conjugated goat anti-mouse IgG antibody as secondary antibody and visualized using a 3,3′,5,5′-tetramethylbenzidine (TMB) kit (TianGen, Beijing, China). The values of absorbance at 450 nm were evaluated using a Victor^TM X5 Multilabel Plate Reader (PerkinElmer, Waltham, MA, United States). Protein p239 was used as negative protein control.

A sandwich ELISA-based PRRSV virion capture assay was conducted as previously described [26 (link)]. Briefly, the wells of 96-well polystyrene microplates (Corning, Corning, NY, USA) were coated with 400 ng of mAb-PN9cx3 in 100 μL of PBS (pH 8.0) per well. The plates were incubated overnight at 4 °C, and each well was then blocked with 2.5% gelatine in PBS supplemented with 0.5% Tween 20 (Sigma-Aldrich). Subsequently, HP-PRRSV-SD16 (100 μL, 10^6.5 TCID₅₀/mL) was added to the wells, and the plates were incubated for 1 h at 37 °C. For the isotype control, all the steps were performed using mAb-2G8 at the same amount as mAb-PN9cx3. Anti-PRRSV convalescent pig serum (1:200 dilution in PBS) was used to detect the captured PRRSV virions. The binding of pig antibodies was revealed using HRP-conjugated goat anti-pig IgG (Jackson ImmunoResearch, West Grove, PA, USA) and 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (TianGen Biotech, Beijing, China). The absorbances of the individual wells were measured using a Victor^TMX5 Multilabel Plate Reader (PerkinElmer, Waltham, MA, USA) at a wavelength of 450 nm.