Prominence rf 20axs

To determine equilibrium constants, IgG2, G3*01, IgG4, and IgG1-K409R and DyLight-488 labeled equivalents were reduced with 10 mm DTT (60 min/37 °C) and alkylated with 22 mm iodoacetamide. Serial 4-fold dilutions of reduced/alkylated IgG (0.001–300 nm half-molecules) were incubated with 0.05 ng/ml reduced/alkylated IgG2–488, 0.2 ng/ml IgG3, or 1 ng/ml IgG4/IgG1-K409R in PBS containing as a carrier protein 0.1 mg/ml certolizumab pegol, and 0.02% Tween 20 and incubated at 37 °C for up to 3 days before analysis. Between 50 and 1000 μl of a sample was applied using an autosampler to a Superdex 200 HR 10/300 column that was connected to an ÄKTAexplorer HPLC and eluted at 0.5 ml/min. Elution profiles were monitored by measuring the fluorescence (excitation/emission 488/525 nm) with a Prominence RF-20Axs in-line fluorescence detector (Shimadzu, Kyoto, Japan). Dissociation constants were calculated as described before (34 (link)).

Stability of TNF was assessed by incubating TNF-488 at 0.3–3 ng/mL in PBS-I for various times between 0 and 5 days, alone or in the presence of unlabeled TNF (30 or 300 ng/mL), adalimumab, certolizumab pegol, or etanercept (1 μg/mL) in the dark at 37 °C. Samples were subsequently analyzed applying 500 μl to a Superdex 200 HR 10/300 column (GE Healthcare, Uppsala Sweden) at 0.5 ml/min using a thermostatted autosampler (4 °C), which was connected to an ÄKTAexplorer HPLC system (GE Healthcare, Uppsala Sweden). Elution profiles of TNF-488 were monitored by measuring the fluorescence (excitation/emission 488/525 nm) with a Prominence RF-20Axs on-line fluorescence detector (Shimadzu, Kyoto, Japan).

High Performance Liquid Chromatography with fluorimetric detection (HPLC-FLD) was used for the determination of each of the PAHs. The Nexera X2 (Shimadzu Corporation, Kyoto, Japan) chromatographic system was used, which consisted of SIL-30AC auto-sampler, two LC-30AD pumps, Prominence RF-20Axs fluorescence detector, connected to Lab Solution software ver. 5.57 (Shimadzu Corporation, Kyoto, Japan). The Hypersil Green PAH (Thermo Scientific, Waltham, MA, USA) column (250 × 4.6 mm, I.D., 5 μm) and guard column (10 × 4.0 mm, I.D., 5 μm) were used.