Denosumab bridging ELISA for anti-drug antibody (ADA) assay developmentEnlarge
A microtiter plate was coated overnight with denosumab at a concentration of 1 μg/ml. After washing and blocking with PBST+5% BSA, 10% human serum was added spiked with increasing concentrations of Human Anti-Denosumab Antibodies, clone AbD26295_hIgG1 (HCA280, black line; KD = 1.8 nM), clone AbD26296_hIgG1 (HCA281, red line; KD = 7.1 nM) or clone AbD26862_hIgG1, HCA282 (blue line; KD = 0.8 nM). Detection was performed using HRP conjugated denosumab followed by QuantaBlu Fluorogenic Peroxidase Substrate. HRP conjugation of the denosumab used in this assay was performed using a LYNX Rapid HRP Conjugation Kit®. Data is shown as the mean of three measurements.