Br 1004 07

For the coupling of TNC to the NTA chip (BR-1004-07, GE) using its His-Tag, the NTA Reagent Kit (GE, 28-9950-43) and a Biacore X100 (GE Healthcare, Uppsala, Sweden) were used. The chip was conditioned with regeneration buffer (1350 mM EDTA, 60 s, 10 µL/min) and washed with 3 mM EDTA (60 s, 10 µL/min). NiCl₂ solution was flushed over the active flow cell only (60 s, 10 µL/min) followed by a 0.17 µM TNC solution (60 s, 10 µL/min).
The coupling was verified by an increase in RU units. The binding of CCL2 was tested using a concentration range of 0–20 µM in PBS + 0.005% Tween20. The Kd values were calculated by blotting the RU against the concentration and applying a steady state fit using the Biacore evaluation software version 2.0.

Biosensor analysis was performed on a BIAcore 2000 biosensor (GE Healthcare) using an NTA sensor chip as described in detail [33 (link)]. Briefly, a NTA sensor chip (GE Healthcare, BR1004-07) was loaded with Ni²⁺ and used to immobilize histidine tagged PDGF-CC ligand. The PDGF-CC antibodies were passed over the chip at varying concentrations in order to determine apparent affinity (K_D). Chips were regenerated with ethylene glycol tetraacetic acid (EDTA, Sigma, E3889). The chip was re-equilibrated by washing with HBS (containing no EDTA) before further analysis. Using BIAevaluation software, a 1:1 Langmuir binding analysis model was used to estimate the apparent association (ka) and disassociation (kd) rate constants for each antibody generated (49).