Immulon clear 4hbx 96 well plates

CRF from Bachem was coated to Immulon clear 4HBX 96-well plates (#14245153; Thermo Fisher) at 10 nM and 1,000 nM in 100 mM NaCO₃ overnight at 4°C. Plates were then washed with PBS twice and blocked with Block Ace solution for 3 h at room temperature. Plates were washed with PBS twice, and solutions containing final concentrations of 20 nM CTRND05 and varying concentrations of UCN1, UCN2, or UCN3 were placed onto the plate and incubated for 2 h at room temperature. Plates were washed again with PBS twice, and goat anti-mouse secondary antibody conjugated to HRP (#115-035-003; Jackson ImmunoResearch Labs) secondary antibody was diluted (1:5,000) in buffer and incubated on the plate for 1 h. Plates were washed with PBS with Tween twice followed by PBS twice, and TMB substrates (#5067422; Pierce) were applied for 10 min. Reactions were then quenched with 1 M HCl or 85% O-phosphoric acid, and absorbance was measured at 450 nm using the Clariostar spectrophotometer from BMG Labtech.

For detection of anti-CRF antibodies, CRF-KLH or CRF-OVA from above was coated to Immulon clear 4HBX 96-well plates (#14245153; Thermo Fisher) at 1 µg/ml in 100 mM NaCO₃ overnight at 4°C. Plates were washed with PBS twice and blocked with Block Ace solution for 3 h at room temperature. Plates were washed with PBS twice, and sera were diluted in phosphate-based assay buffer and incubated on the plate for 2 h at room temperature. Plates were washed again with PBS twice, and goat anti-mouse secondary antibody conjugated to HRP (#115-035-003; Jackson ImmunoResearch Labs) secondary antibody was diluted (1:5,000) in buffer and incubated on the plate for 1 h. Plates were then washed with PBST twice followed by PBS twice, and TMB substrates (#5067422; Pierce) were applied for 10 min. Reactions were quenched with 1 M HCl or 85% O-phosphoric acid, and absorbance was measured at 450 nm using a Clariostar spectrophotometer from BMG Labtech.