Anti human igg fc hrp

SDS-PAGE was performed with rAAV particles (1 × 10¹⁰ VG) under reducing conditions and blottet to a membrane using the iBlot Dry Blotting System (Invitrogen™, Carlsbad, CA, USA) and iBlot™ Trans-fer Stack (Invitrogen™, Carlsbad, CA, USA). The membrane was blocked with 5.0% milk in PBS-T (0.1% Tween in PBS) for 1 h at RT following first antibody incubation with primary antibody B1 monoclonal mouse anti VP1, VP2, VP3 (Progen, Heidelberg, Germany) 1:250 and anti-2E3 IgG1 human (own production) 1:250 in 1.0% milk PBS-T overnight at 4 °C. Secondary antibodies anti-human IgG Fc HRP or goat anti-mouse IgG1 (H+L) HRP (Thermo Fisher Scientific Waltham, MA, USA) were used 1:1000 in 1.0% milk PBS-T for 1 h at RT. The SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific Waltham, MA, USA) was used to detect chemiluminescent signals by the Image Quant LAS 4000 (Cytiva, Marlborough, MA, USA).

ELISA plate wells were coated with 2 µg/mL Scl-70 at 4 °C overnight. After blocking with PBS containing 5% milk at room temperature for 2 h, serially diluted phages and antibodies were incubated at room temperature for 1 h. The anti-human-IgG-Fc-HRP (Invitrogen A18817) was diluted to 1:5,000 as secondary antibody. The enzyme reaction was then performed using tetramethylbenzidine as substrate, and color development was stopped by adding 2 M H₂SO₄. The absorbance at 450 nm was measured using a microplate reader (18 (link)). For antibody affinity, ipilimumab was used as negative control.