N ethyl n 3 dimethylaminopropyl carbodiimide hydrochloride

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).

Equilibrium binding-affinity measurements were made using a Biacore 3000 system. Cyclonal IgG or scFv antibodies were covalently linked to the surface of a CM5 sensor chip through amine coupling chemistry. First, the surface of the CM5 sensor chip was activated using N-hydroxysuccinimide and N-ethyl-N-(3-dimethylaminopropyl)carbodiimide hydrochloride (GE Healthcare). Purified anti-Gcn4 cyclonals or scFvs diluted to 50 μg ml⁻¹ in 10 mM sodium acetate pH 4.0 were immobilized to the surface of the CM5 chip with a target level of 900 response units (RUs). Unreacted sites on the chip surface were subsequently blocked with 1 M ethanolamine hydrochloride. Serial dilutions of the antigen, MBP–TEV–Gcn4, prepared in 10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% polysorbate-20 (HBS-EP buffer, GE Healthcare) at concentrations ranging from 0.5 to 32 nM were injected over the chip using the same buffer at a flow rate of 30 μl min⁻¹ (2 min injection time, 5 min dissociation). The surface of the chip was regenerated between the injections of each serial dilution with 10 mM glycine, pH 2.0 (2 min injection time, 2 min stabilization time). Kinetic parameters (k_a, k_d and K_D) were determined by fitting the response curves with a Langmuir 1:1 binding model within the BIAevaluation software.