The wells were then blocked for 2 h with 150 μL of binding buffer (coating buffer with 0.5% bovine serum albumin).
After washing twice with binding buffer (coating buffer with 0.1% (w/v) BSA) the cells were incubated in the presence of the competing ligand (90 μL, 0.5 pM to 500 nM echistatin; 0.05 nM to 50 μM peptide in binding buffer) with [I]echistatin (0.37 kBq/well, 10 μL, 0.05 nM; PerkinElmer, Germany) for 3 h.
Briefly, recombinant antigen at a concentration of 15 μg/mL was diluted in carbonate-bicarbonate buffer, coated in a microtiter plate, and kept overnight at 4°C after which, the wells were postcoated with NS1 monoclonal antibody diluted in 1% gelatin in PBS for 1.5 h at 37°C.
NVP-NAC-BSA, BSA, or KLH (100 μL, 10 μg/mL in carbonate bicarbonate coating buffer) were coated into the wells of a flat-bottom 96-well plate (Costar, Cambridge, MA), and the plate was incubated overnight at 4 °C.
50 μl of 8 μg/ml Pfs Ag, or 50 μl of 10 μg/ml SWA, in bicarbonate coating buffer were coated onto Immulon 2 plates (Thermo Labsystems, Franklin, MA, USA) and incubated overnight at 4°C.
Dilutions of serum (1 200) from patients and blood donors with BSA in PBS-T were then added to the plates in triplicate, two to LHR and one to Carbonate buffer-coated wells, and incubated for 2 h at RT.
Dilutions of serum (1 200) from patients and blood donors with BSA in PBS-T were then added to the plates in triplicate, two to LH-R and one to Carbonate buffer-coated wells, and incubated for 2 h at RT.
Based on these results, the optimum coating conditions for striatal B are summarized as follows: PBS/DMSO (1/1, v/v) buffer, and coating overnight at 24°C.
Then, 100 μL of purified rUbi (10 μg) in PBS buffer was coated onto 8 microwells overnight at 4 °C for both αUbi and M13KO7 sample sets.
A total of 0.2 mg of crude rMERS-NP in 100 µL of PBS buffer was coated to the surface of the microplate wells at 4 °C for 15 h.
