Tmb stop solution

High-binding, 384-well polystyrene plates (Corning, Corning, NY) coated with 30 ng antigen/well were incubated overnight at 4°C or for 2 hours at RT. Plates were then washed with wash buffer (PBS, 0.1% Tween 20) by an automatic microplate washer (BioTek, Winooski, VT) and were blocked with Superblock buffer (PBS, 4% whey protein, 15% goat serum, 0.5% Tween 20) for 1–2 hours at RT. After one wash, standard and sample dilutions were added in duplicates and plates were incubated for 1–2 hours at RT. Plates were washed twice and peroxidase goat anti-human IgG (Jackson Immunoresearch, West Grove, PA) was added to each well. After 1 hour at RT, plates were washed and then incubated with SureBlue Reserve substrate (VWR, Radnor, PA) for 5–10 minutes at RT in the dark. TMB Stop Solution (VWR, Radnor, PA) was added and plates were immediately read on a SpectraMax Plus Plate Reader (Molecular Devices, Sunnyvale,CA) to determine optical density (OD) at 450 nm. SoftMax Pro 6.3 software was used to interpolate concentrations from standard curves.

To measure rhesus gB-specific IgG binding, 384-well ELISA plates were coated with 1.5 μg/ml of rhCMV gB and incubated at 4°C overnight. Plates were washed one time (95% DI water, 4% 25X PBS, 1% Tween-20), blocked (4% whey, 15% goat serum, 0.5% tween 20, 80.5% 1X PBS), and incubated at 4°C overnight. Samples and controls were diluted in blocking buffer (1:30) and serially diluted 1:3. Plates were washed and diluted samples were added to the plate in duplicate. After a 2 hour incubation at RT, plates were washed two times and secondary antibody (Mouse anti-Monkey IgG-HRP, Southern Biotech) was added to each well (1:5000). Plates were incubated for 1 hour at RT and subsequently washed four times. Room temperature SureBlue Reserve TMB Substrate (VWR) was added to each well and plates were incubated for 10 min while shielded from light. TMB Stop Solution (VWR) was added to each well and plates were immediately read at 450 nm on the SpectraMax (Molecular Devices) using the SoftMax software interface. Data are reported as median effective dilution that resulted in a 50% decrease in optical density (ED50).