Coating of the standards and samples was performed in a 96-well plate with 100 μl of phosphate-buffered saline coating buffer at 4°C overnight.
50 µL of each antibody mix was prepared in ELISA coating buffer (eBioscience, San Jose, CA) and coated on to ELISA plates (Maxisorp, Nunc).
Assay procedures: Maxisorp plates are coated with 100 μL of coating buffer overnight at 4°C without shaking.
After Ab deposition, the microarray was blocked with PBSC for 1 h, treated with Post Coating Buffer (ALerCHEK, Portland, ME), spin coated, and stored at 4°C.
Briefly, 96-well ELISA plates (Sangon, Shanghai, China) were coated with 0.05% (w/v) poly-L-lysine in coating buffer (0.159% Na2CO3, 0.293% NaHCO3, pH 9.6) for 1 h, followed by washing the plates 3× with coating buffer.
ELISA plates were coated overnight at 37°C with 100 μl, 5 μg MVP per ml coating buffer (0.05 M sodium carbonate, pH 9.6).
rOmpF was dissolved in coating buffer (pH 9.6, 0.015 M sodium carbonate, 0.035 M sodium bicarbonate).
Each well was coated with PVX-specific antibodies (DSMZ, PV-0027) diluted in coating buffer.
Each inactivated influenza virus (5 μg/mL) was coated onto microtiter plates overnight at 4°C in coating buffer.
ELISA conditions such as coating buffer and blocking solution were optimized using sera obtained from mice immunized with amphotropic MLV particles.
Serum samples were analyzed in duplicate with the antigen and once without antigen (coating buffer only).
