Goat anti bat igg heavy and light chain antibody a140 118b

Bat-specific IgG was analysed via dot blot. Bat-mice were cheek bled post NP-KLH immunizations, sera samples were dotted onto nitrocellulose membrane (Pore size 0.45 µm; BIO-RAD, USA) and left to dry for 30 minutes. Blocking buffer (Tris-buffered saline with polysorbate 20 (TBS-T) and 5% low-fat milk powder) was used to block non-specific sites for 30 minutes at RT with agitation. After washing, membrane was incubated for 1 hour at RT with goat anti-bat IgG heavy and light chain antibody (A140-118B; Bethyl Laboratories, USA) in PBS-T containing 2.5% milk and for 1 hour with agitation, followed by 3 times 5 minutes wash with TBS-T. Secondary antibody incubation was done with donkey anti-goat IgG-HRP (SC-2056; Santa Cruz Biotechnology, USA) in PBS-T containing 2.5% milk for 1 hour at RT with agitation and subsequently washed with TBS-T for 3 times 5 minutes before reaction development using enhanced ECL reagent (Thermo Fisher Scientific, USA). Results were developed in the dark room for 10 seconds with CL-XPosure^TM Film (Thermo Fisher Scientific, USA).

Specificity of bat immunoglobulin G (IgG) was determined using western blot. Pooled sera from two individual bats (E. spelaea) and sera from C57BL/6 mice, both diluted 1:50 in PBS, were ran on a 12% SDS-PAGE gel, followed by transfer onto Polyvinylidene difluoride (PVDF) transfer membranes (Millipore, USA). Membranes were blocked in Tris-buffered saline with 0.1% Tween (TBS-T 0.1%) and 2.5% skim milk for 1 hour at room temperature (RT), followed by labelling with goat anti-bat IgG heavy and light chain antibody (A140-118B; Bethyl Laboratories, USA) diluted 1:1,000 in TBS-T 0.1% overnight at 4 °C. Membrane was washed 3 times for 5 minutes with TBS-T 0.1%, then labelled with donkey anti-goat IgG-HRP (SC-2056; Santa Cruz Biotechnology, USA) diluted 1:10,000 in TBS-T 0.1% at RT for 1 hour, and subsequently undergo a final 3 times 5 minutes wash. Membrane was visualized using enhanced chemiluminescence (ECL) prime chemi-luminescence reagent (GE Healthcare, USA) and a myECL Imager (Thermo Fisher Scientific, USA).