Bat-mouse bone marrow chimera: a novel animal model for dissecting the uniqueness of the bat immune system

Yong, Kylie Su Mei; Ng, Justin Han Jia; Her, Zhisheng; Hey, Ying Ying; Tan, Sue Yee; Tan, Wilson Wei Sheng; Irac, Sergio Erdal; Liu, Min; Chan, Xue Ying; Gunawan, Merry; Foo, Randy Jee Hiang; Low, Dolyce Hong Wen; Mendenhall, Ian Hewitt; Chionh, Yok Teng; Dutertre, Charles-Antoine; Chen, Qingfeng; Wang, Lin-Fa

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).