Fig 2: Immunocapture of drug/TNF complexes by Type 3 antibodies. Type 3 anti-golimumab/TNF antibody AbD25705 and Type 3 anti-adalimumab/TNF antibody AbD20350 were coated on a microtiter plate at 1 µg/mL. Golimumab and adalimumab at a fixed concentration (1 µg/mL = 7 nM) were incubated for 1 hour with an increasing amount of TNF and added to the plate. Detection was performed with anti-human IgG:HRP antibody specific for the CH2 domain that detects golimumab or adalimumab but not the Fab antibodies, followed by QuantaBlu fluorogenic peroxidase substrate.

Fig 3: Comparison of assay formats. (a) Schematic view of the assay set-up: bridging assay set-up using a Type 1 reagent for capture and an HRP-conjugated Type 1 antibody for detection of a bivalent drug and (b) antigen capture assay using an HRP-conjugated Type 3 antibody for detecting a monovalent or bivalent drug in complex with its drug target. (c) Detection of adalimumab using a Type 3 antibody in antigen capture assay (black), or Type 1 capture and detection antibodies in a bridging assay (red). For the antigen capture assay, human TNF was coated at 5 µg/mL on a microtiter plate overnight. After washing and blocking, adalimumab spiked into 10% human serum was added. HRP-conjugated anti-adalimumab/TNF antibody AbD18754 (KD = 67 nM) was added at 2 µg/mL in HISPEC assay diluent, followed by QuantaBlu fluorogenic peroxidase substrate. For the adalimumab bridging assay, the anti-adalimumab Type 1 antibody AbD18654 (KD = 0.16 nM) was coated at 1 µg/mL on a microtiter plate overnight. After washing and blocking, adalimumab spiked into 10% human serum was added, followed by HRP-conjugated Type 1 anti-adalimumab antibody AbD18655 (KD = 0.06 nM) at 2 µg/mL in HISPEC assay diluent and QuantaBlu fluorogenic peroxidase substrate.