Donkey f ab 2 anti rabbit igg pod

Example 6

The binding analysis was carried out using an enzyme-linked immunosorbent assay (ELISA)-based technology. The antigen murine IL-1beta (Sino Biologics, Cat. No. 50101-MNAE) was immobilized at a concentration of 0.5 μg/mL in 25 μL in PBS on a 384 well microtiter plate (Thermo Scientific, Cat. No. 464718). Every of the following steps was followed by a washing routine of 3 times 90 μL PBS, 0.5% BSA, 0.05% Tween with dispense and aspiration: 1) blocking step: saturating unbound surface (1 hour, 2% BSA); 2) anti-IL-1beta antibody in increasing concentrations for 1 hour; 3) detection antibody, dilution=1:2000 (Donkey F(ab)₂ anti-rabbit IgG POD, Amersham, NA9340V or sheep IgG anti-mouse IgG POD, Amersham RPN4201). 20-30 min. after adding the substrate 3,3′,5,5′-tetramethylbenzidine (TMB, Roche Diagnostics GmbH, Mannheim, Germany, Cat. No 11835033001) the optical density was determined at 370 nm. The EC₅₀ was calculated with a four parameter logistic model using GraphPad Prism 6.0 software.

Example 5

The binding analysis was carried out using an enzyme-linked immunosorbent assay (ELISA)-based technology. The antigen human IL-1beta (Sino Biologics, Cat. No. 90010CNAE) was immobilized at a concentration of 0.5 μg/mL in 25 μL in PBS on a 384 well microtiter plate (Thermo Scientific, Cat. No. 464718). Every of the following steps was followed by a washing routine of 3 times 90 μL PBS, 0.5% BSA, 0.05% Tween with dispense and aspiration: 1) blocking step: saturating unbound surface (1 hour, 2% BSA); 2) anti-IL-1beta antibody in increasing concentrations for 1 hour; 3) detection antibody, dilution=1:2000 (Donkey F(ab)₂ anti-rabbit IgG POD, Amersham, NA9340V or sheep IgG anti-mouse IgG POD, Amersham RPN4201). 20-30 min. after adding the substrate 3,3′,5,5′-tetramethylbenzidine (TMB, Roche Diagnostics GmbH, Mannheim, Germany, Cat. No 11835033001) the optical density was determined at 370 nm. The EC₅₀ was calculated with a four parameter logistic model using GraphPad Prism 6.0 software.