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| | Human FD Proteolysis Assay (ELISA Ba) |
| Description: | Recombinant human FD (10 nM concentration) was incubated with compound at various concentrations for 1 h at room temperature in 0.1 M PBS (pH 7.4) containing 7.5 mM MgCl2 and 0.075% (w/v) CHAPS. The preformed CVF-human-FB-substrate complex was added to a final concentration of 200 nM. After 1 h incubation time at room temperature, the enzyme reaction was stopped by addition of a 0.1 M Na2CO3 buffer solution (pH 9.0) containing 0.15 M NaCl and 40 mM EDTA. Generation of Ba as the enzymatic cleavage product was quantified by an enzyme-linked-immunosorbent assay (ELISA). Aliquots of reaction samples were pipetted into 384-well high-capacity protein-binding plates (NUNC MaxiSorp) pre-filled with 96 μL/well of coating buffer. After an overnight incubation at 4 °C, assay plates were washed with PBS-Tween 20. Remaining free binding capacity was saturated by the addition of Starting Block T20 for 5 min at room temperature, and assay plates were then washed with PBS-Tween 20. Anti Ba neoepitope antibody was added to each well, followed by incubation for 60 min at room temperature and removal of excess antibody by washing with PBS-Tween 20. Then, goat anti-rabbit antibody labeled with HRP (172-1019; Bio-Rad) (1 μg/well in PBS-Tween 20) was incubated for 60 min at room temperature as in the former step, and excess antibodies was removed by extensive washing with PBS-Tween 20. HRP activity was measured after 20 min incubation time with QuantaBlu fluorogenic peroxidase substrate (100 μL) at room temperature. |
| Affinity data for this assay | |
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