Anti mouse igg hrp conjugated antibody

Capsid thermal stability assays were performed as described before [37 (link)]. Briefly, per reaction 1x10¹⁰ viral capsids of a vector preparation were diluted in PBS, incubated at indicated temperatures for 15 min and then further diluted in PBS. The temperature gradient (65 to 85°C) was generated by a LightCycler^® 96 System (Roche Life Science) with the following program: 2 cycles (10 sec at 37°C; 900 sec at temperature gradient; 10 sec at 37°C), 1 cycle (30 sec at 37°C). Samples were transferred to a nitrocellulose membrane using a vacuum blotter, blocked (5% milk in TBS-T) and incubated with hybridoma cell supernatant containing anti-AAV2 specific antibody A20 (recognizing intact capsids) (PROGEN, 1:5 dilution). Anti-mouse IgG HRP-conjugated antibody was used as secondary antibody (Cayman Chemical—Biomol, Hamburg, Germany; 1:2,000 dilution) and signals were detected using SuperSignal^™ West Pico Chemiluminescent Substrate or SuperSignal^™ West Femto Maximum Sensitivity (ThermoFisher Scientific, Waltham, MA) in combination with a Fusion FX Vilber Lourmat system (Peqlab).

Capsid composition was analysed by Western Blot. Iodixanol-purified viral vector particles were concentrated using the StrataClean Resin (Agilent Technologies, Ratingen, Germany) according to manufacturer’s instructions, separated on a 8% SDS-PAGE gel (5x10¹⁰ capsids per lane) and subjected to standard Western blotting procedure using the following primary antibodies: hybridoma cell supernatant containing AAV2 specific antibody A69 (recognizing VP1/VP2 proteins [36 (link)], 1:10 dilution) (PROGEN, Heidelberg, Germany). Anti-mouse IgG HRP-conjugated antibody was used as secondary antibody (Cayman Chemical—Biomol, Hamburg, Germany; 1:2,000 dilution) and signals were detected using the SuperSignal^™ West Pico Chemiluminescent Substrate or SuperSignal^™ West Femto Maximum Sensitivity (ThermoFisher Scientific, Waltham, MA) in combination with a Fusion FX Vilber Lourmat system (Peqlab).