Ez link nhs lc biotin

Antigens were biotinylated with EZ-Link NHS-LC-biotin (ThermoFisher, MA, USA) according to manufacturer's instructions. Excess unbound biotin was removed using a Zeba Spin desalting column (ThermoFisher), and proteins were re-suspended in PBS at 1 mg/mL. Ten micrograms of biotinylated proteins were coupled individually to 10 μl of 1 μm yellow-green fluorescent, neutravidin-coated microspheres (FluoSpheres, Life Technologies, CA, USA) by incubating at 37°C for 2 h. Beads were washed twice with PBS-5% BSA to block. Beads were resuspended in a final volume of 1 mL PBS-0.1% BSA and stored at 4°C in the dark for up to 1 week.

The equilibrium affinity constants (K_a) of scFvs were determined by the Scatchard analysis²¹ . Mixtures of [1, 2, 6, 7-³H]-cortisol (3.53 TBq/mmol; PerkinElmer) (ca. 250 Bq), varying amounts of standard cortisol, and a constant amount of each scFv (adjusted to capture ca. 50% of the tritium-labeled cortisol in the absence of standard cortisol) were incubated in GPB (700 µL) at 4 ºC overnight (~ 16 h). The bound (B) and free (F) fractions were separated by a dextran-coated charcoal method, and radioactivity of the B fraction was measured. Dissociation rate constants (k_d) of the soluble scFvs obtained by the ORD selection were determined by the BLI^{24 (link)} at 25 °C using BLItz (ForteBio), a BLI sensor. Streptavidin-coated biosensor tips, which were saturated with a biotin-labeled CS-BSA, prepared by reaction with EZ-Link NHS-LC-Biotin (Thermo Fisher Scientific)^{44 (link),45 (link)}, were dipped into 4.0-µL scFv solutions in G-PBS (50–1,600 nmol/L). Association of the scFvs with the cortisol residues was monitored for 120 s, and then dissociation was measured for 600 s in G-PBS.

The EZ-Link NHS-LC-Biotin (Thermo Fisher Scientific) was used for biotinylation of indicated antibodies, the reaction was carried out by incubating 20-fold molar excess of biotin reagent with the antibody at room temperature for 30 min. For the Fc N-glycan cleavage, GST-fused endoglycosidase S (EndoS) expressed by E.coli was used to incubate with E4 antibody at a ratio of 1:1000 (w/w) and 37 °C for 1 hour. All antibodies were purified by using Protein G GraviTrap Columns (VWR) according to the manufacturer’s instructions.

Polyclonal antibodies (pAbs) were raised in female New Zealand White rabbits immunized either with an extract of horse dander (Allergon) or with recombinant 6xHis-tagged Equ c 1 (expressed in yeast, affinity purified using IMAC) purchased from Flarebio, College Park, Maryland, USA (Cat. No. CSB-YP839158HO). Immunization was conducted by Charles River (Kisslegg, Germany) using four subcutaneous antigen injections of 0.2 mg each, according to the standardized protocol of the company. The final bleeding and serum collection was carried out 70 days after the first antigen injection.
For the antibody purification, the IgG fractions of both sera were isolated by affinity chromatography with HiTrap protein G columns (GE Healthcare, Uppsala, Sweden) following the manufacturer’s instructions. One part of each of the purified pAbs was used as capture antibodies in sandwich ELISAs. Another part was labeled with biotin and used as detection antibodies. Biotinylation was carried out by incubating the antibodies with a 33-fold molar excess of EZ-Link NHS-LC-Biotin (Thermo Scientific, Rockford, IL, USA) under continuous agitation for 2 h at room temperature. The biotinylated antibodies were dialyzed extensively with PBS to remove free biotin molecules.

A 20-fold molar excess of freshly prepared 10 mM solution of biotin (EZ-Link™ NHS-LC-Biotin, Thermo Scientific, Gdańsk, Poland) in dimethylsulfoxide was added to the solution of anti-cADA affinity-purified IgY antibodies in PBS. The reaction was performed for 2 h at room temperature, followed by dialysis against PBS.

To produce biotin-labeled antibodies specific to the H1 HA head, mAb 5J8 (Krause et al., 2011 (link)) in PBS was mixed with a 20 × molar excess (relative to complete antibodies) of EZ-Link NHS-LC-Biotin (Thermo Fisher Scientific) and allowed to sit for 2 hr at 4°C, followed by two rounds of overnight dialysis against PBS at 4°C to remove excess biotinylation reagent. All biosensors were hydrated in assay buffer (25 mM Tris, 150 mM NaCl, 0.5% bovine serum albumin, 0.01% TWEEN 20, pH 8.0) before use. Biotinylated 5J8 (20 μg/mL in assay buffer) was immobilized on SA biosensors, then briefly dipped in assay buffer prior to exposure to designed H1 HA fusions (500 nM per asymmetric unit, in assay buffer). The biosensor was again dipped in assay buffer and then exposed to the stem-specific mAb CR6261 (20 μg/mL in assay buffer) (Throsby et al., 2008 (link)).