Sia kit

Mouse anti-cortisol monoclonal antibody (association constant K_A = 1.7 × 10⁹ M⁻¹, cortisol antibody) was purchased from Abcam PLC (UK). As the cortisol analog, hydrocortisone 3-(O-carboxymethyl) oxime (hydrocortisone 3-CMO) was purchased from Sigma Aldrich (USA). PEG6-COOH aromatic dialkanethiol (PEG6-COOH) was purchased from SensoPath Technologies, Inc. (USA), and was used as a reagent to form SAMs on the chip surface. The Au sensor chip included in the SIA Kit, the N-ethyl-N-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) included in an amine coupling kit, the N-hydroxysuccinimide (NHS) for sensor surface fabrication, a borate buffer (pH 8.5, 10 mM disodium tetraborate, 1 M NaCl), and 50 mM NaOH were purchased from GE Healthcare Bio-Science AB (Sweden). Ethylenediamine was purchased from Wako Pure Chemical Industries, Ltd. (Japan). An ELISA kit was used as a conventional salivary cortisol detection method, and Salimetrics oral swabs, used as a salivary collection material were purchased from Salimetrics LLC (USA). Water purified using a Milli-Q integral water purification system was used as the solvent. All aqueous solutions were prepared with Milli-Q water obtained from a Milli-Q system (Millipore Corporation, USA).

SPR measurements were performed using a Biocore T200 (GE Healthcare, Life Sciences, Piscataway, NJ, USA). The thiol-modified Nup98-FG-SH protein and the inert control polymer PUT3 (C₁₇H₃₆O₄S, hydroxyl-terminated tri-ethylene glycol undecane thiol, HS-(CH₂)-(OCH₂CH₂)₃-OH) were immobilized to the gold surface of the sensor chip (SIA kit; GE Healthcare, Life Science, Piscataway, NJ, USA). Tau proteins were then injected in increasing concentrations between 7.6 nM to 4 μM, and differences of the Nup98-FG layer height were determined. BSA was injected after each Tau injection as described previously [36 (link)]. The equilibrium binding constants were calculated using one- or two-component Langmuir fits [53 (link)]. Sensograms presented in one graph were obtained from measurements using the same gold sensor chip and the same immobilization of Nups. A detailed protocol is provided by Diez et al. [54 ].