Anti his tag antibody

The antigen-binding activities of the purified nanobodies were assayed by ELISA. Aβ was incubated in 96-well plate for 1 h at 37 °C. After incubation, the plate was washed five times with PBS containing 0.05% Tween 20. BSA was used as the negative control. After coating with Aβ42 monomer at different concentrations, the plate was blocked with 2% BSA, followed by the addition of nanobody, anti-His-tag antibody and HRP-conjugated IgG antibody (Sangon Biotech, Shanghai, China). At last, tetramethylbenzidine at 100 mg/L was added to each well and incubated for 20 min before quenching with 2 M H₂SO₄. The absorbance was measured at 450 nm using a SUNRISE XFLUOR4 reader (TECAN, Männedorf, Swiss).

The supernatant was filtered through a 0.22-μm membrane (Millipore, Burlington, MA, USA) and applied to a Ni-IDA resin that was washed and equilibrated with 20 column volumes in a binding buffer at a flow rate of 1.5 ml/min. Ni-IDA elution buffer (20 mM Tris, 0.15 M NaCl, 500 mM imidazole) was then passed through the column with linear imidazole concentrations (0–50%) for 30 min. rAlt a 1 protein was eluted at a 200 mM imidazole concentration.
Purity was assessed by 12% SDS-PAGE (3 μg/slot) under reducing and non-reducing conditions. The concentration of rAlt a 1 was 1.65 mg/ml, as detected using the BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). For immunoblotting studies, the separated proteins were transferred to polyvinylidene fluoride (PVDF, Millipore, Burlington, MA, USA) membranes and blocked with 5% non-fat milk in TBST (Solarbio, Beijing, China) for 2 h at room temperature, followed by incubation at 4°C overnight with human sera (1:20) and anti-His tag antibody (Sangon Biotech, Shanghai, China). After washing with TBST for four times, the blots were incubated with horseradish peroxidase (HRP)-conjugated anti-human IgE (Abcam, Cambridge, MA, USA) and goat anti-mouse IgG antibody (Sangon Biotech, Shanghai, China) for 1 h. Finally, the blots were detected by enzyme-linked chemiluminescence.