Serum
ELISAs were performed as previously
described.14 (link) Briefly, plates were coated
with 100 μL of protein at 5 μg/mL overnight at 4 °C,
then blocked with 150 μL of 1% BSA in PBS-Tween for 1 h at room
temperature. Sera was added at a starting dilution of 1:40 with a
serial 5-fold dilution and incubated for 90 min at room temperature.
Plates were washed, and 150 μL of HRP-conjugated antimouse IgG
(Abcam) or HRP-conjugated antihuman IgG (Abcam) was added at 1:20,000.
After a 1 h secondary incubation at room temperature, the plates were
washed and 150 μL of 1× ABTS development solution (Thermo
Fisher) was added. Plates were developed for 30 min at room temperature
before stopping with 100 μL of 1% SDS to read on a SpectraMaxiD3
plate reader (Molecular Devices) for absorbance at 405 nm.
ELISAs were performed as previously
described.14 (link) Briefly, plates were coated
with 100 μL of protein at 5 μg/mL overnight at 4 °C,
then blocked with 150 μL of 1% BSA in PBS-Tween for 1 h at room
temperature. Sera was added at a starting dilution of 1:40 with a
serial 5-fold dilution and incubated for 90 min at room temperature.
Plates were washed, and 150 μL of HRP-conjugated antimouse IgG
(Abcam) or HRP-conjugated antihuman IgG (Abcam) was added at 1:20,000.
After a 1 h secondary incubation at room temperature, the plates were
washed and 150 μL of 1× ABTS development solution (Thermo
Fisher) was added. Plates were developed for 30 min at room temperature
before stopping with 100 μL of 1% SDS to read on a SpectraMaxiD3
plate reader (Molecular Devices) for absorbance at 405 nm.
