Hrp conjugated goat anti mouse igg

The hemolymph of the silkworm larvae wascollected 6 days post-infection with recombinant viruses, the supernatant and pellet (polyhedra) were obtained by centrifugation at 6000× g for 5 min. The obtained pellet was washed 3–5 times with 0.1% SDS and 2 times with 1× PBS to obtain the purified polyhedra. To dissolve polyhedra, the purified polyhedra were incubated with a lysis buffer (0.2 mol/L Na₂CO₃-NaHCO₃) for 30 min at 30 °C until the suspension became clear and this was followed by adjusting the pH value to 8.0–9.0 with 1 mol/L HCl. Protein (50 μg) from the hemolymph, supernatant, and polyhedrawassubjected to SDS-PAGE [35 (link)]. Proteins were transferred onto PVDF membranes and Western blotting was carried out with mouse anti-ORF72 (1:500) and HRP-conjugated goat anti-mouse IgG (1:20,000) (Sino Biological Inc., Peking, China).

To determine the antibody titers in the immunized fish, blood (n = 3) harvested from the caudal vein of fish 3 weeks post-vaccination wasstored overnight at 4 °C. After centrifugation for 10 min at 2000× g, the sera were collected and antibody titers were determined according to our previous report [31 (link)]. Briefly, ELISA plates were coated with different concentrations of recombinant ORF72, followed by blocking with 5% bovine serum albumin overnight at 4 °C. Plates were incubated with different dilutions of sera from vaccinated or unvaccinated fish for 2 h. After washing five times with Tris-buffered saline containing Tween-20 (Sangon, Shanghai, China), the ORF72 antibody (1:500) was added and incubated for 2 h. After further washing with TBST, HRP-conjugated goat anti-mouse IgG (Sino Biological Inc., Peking, China) was added and incubated. Finally, the absorbance at 492 nm was measured after coloration.

The interaction between CD177 and PECAM-1 was detected by ELISA with recombinant soluble PECAM-1 (sPECAM-1) at 2 μg/ml as the solid-phase antigen. sPECAM-1 in a coating buffer (0.05 M bicarbonate buffer, pH 9.6) was used to coat the wells of half of a polystyrene microtiter plate (Nunc-Immuno plate; Nunc, Roskilde, Denmark) and was incubated overnight at 4 °C. The other half of the plate was coated with coating buffer alone to establish antigen-free wells. The wells were blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) and incubated with CD177 at various concentrations for 1 h at 37 °C. After washing three times with PBS-tween 20 (PBS-T), the wells were incubated with mouse anti-human CD177 antibody (1:1000; Sino Biological) for 1 h at 37 °C, and HRP-conjugated goat anti-mouse IgG (1:1000) was used as the secondary antibody. Tetramethylbenzidine (TMB; Sigma, St. Louis, MO) was used as the substrate and the reaction was stopped by the addition of sulfuric acid 0.5 mol/L (Carl Roth GmbH, Germany). Optical densities of formed complexes were measured at 450 nm using a microplate reader (Bio-Rad, Tokyo, Japan).