High bind microplate

Plates (High Bind Microplate, Corning^®, Corning, NY) were coated with 50 μl of a 1:100,000 dilution of the purified TTSuV1 ORF2 the antigen. Coated plates were blocked with 2% BSA and 2% normal sheep serum in a commercial block (General block, ImmunoChemistry Technologies, Bloomington, MN) for 2 hrs at 37 °C, incubated with 1:50 of the human or swine sera for 2 hours at 37 °C, in duplicate, followed by the respective anti-species HRPO-conjugate (KPL, Gaithersburg, MD), at a 1:5000 dilution for 45 mins at 37 °C, and incubation with the substrate (TMB, KPL, Gaithersburg, MD). The reaction was stopped with a 1 M HCl solution after 2 mins. Plates were read at 450 nm on an ELISA plate reader (Elx800 reader, BioTek Instruments, Inc., Winooski, VT).

The chimeric EG2-hFc monoclonal antibody (mAb) titers were determined by “sandwich” enzyme-linked immunosorbent assay (ELISA). A 96-well high-bind microplate (Corning, ref. 3369) was incubated overnight with goat anti-human Fc-specific antibodies (Sigma, ref. I2136) diluted 1:30,000 in PBS. Wells were washed three times with PBS containing 0.05% Tween 20 (Sigma, ref. P1379) and blocked for 1 h with a solution of 1% bovine serum albumin (BSA, Sigma, ref. A7030) in PBS. Extracellular medium samples were diluted 1:500 to 1:10,000 in the PBS-BSA solution, depending on the day of culture. The plate was washed and loaded with the diluted samples or human IgG whole molecule standards (Cederlane, ref. 009-000-003). After 1 h, the plate was washed and incubated for 1 h with peroxidase-conjugated goat anti-human Fc-specific antibodies (Sigma, ref. A0170). After washing, the plate was revealed with TMB substrate (Sigma, ref. T0440) and incubated for 20 min in the dark. Absorbance was read at 630 nm four times in the span of ten minutes and the slopes of optical density variations were compared to the standards by linear regression to determine the concentration in each well.

Mer and Axl were measured using Mouse Mer and Mouse Axl DuoSet ELISA (R&D Systems). To measure Protein S in plasma, polystyrene High Bind Microplates (Corning) were coated with a polyclonal anti-human Pros1 antibody (Dako) in 75 mM Na-carbonate pH 9.6 o/n at +4 °C. In between each assay step, plates were washed 3× with PBS containing 0.1% Tween 20. Wells were blocked with 2 % BSA in PBS, which was also used as sample and antibody dilution buffer. A standard curve was generated with a pooled plasma sample from 11 mice, and individual samples diluted at 1:400 were analyzed. Samples and standards were incubated on the plate for 2 h at RT, followed by incubation with a rat anti-mouse Pros1 antibody (R&D Systems) for 1 h. After an additional 1 h incubation with a goat anti-rat HRP conjugate (Jackson ImmunoResearch), the plates were developed with a TMB substrate (R&D Systems).

Wells of 96-well High Bind microplates (Corning) were coated with fractionated heparan sulfate polymer (5 μg) or with VEGF capture Ab (100 ng) at RT for 16 h. Wells were then washed three times with wash buffer (R&D Systems), incubated with recombinant VEGF₁₈₉ (2 ng) for 2 h, and washed again. Thereafter, bevacizumab or VEGFR1/R2-Fc (100 ng in a reaction volume of 100 μL) were added to wells that contained VEGF₁₈₉ bound to heparan sulfate polymer or to VEGF capture Ab, and incubated for 2 h at RT. Wells were then washed three times. Binding of bevacizumab and VEGFR1/R2-Fc was detected by incubation with HRP-conjugated goat anti-human IgG (Fc-specific) (1:5000 dilution) for 1 h at RT, followed by washing and addition of HRP substrate solution (R&D Systems). Absorbance was read at 450 nm. The reading of wells that contained heparin-bound VEGF₁₈₉ was normalized to the reading of wells that contained VEGF bound to VEGF capture Ab.