Skip to main content
. Author manuscript; available in PMC: 2020 Jan 13.
Published in final edited form as: Nat Protoc. 2016 Jan 7;11(2):193–213. doi: 10.1038/nprot.2016.009

Figure 5.

Figure 5.

Membrane presented antibody as a means to pre-screen candidate immunogens for the capacity to engage germline BCR sequences of interest. Candidate immunogens are fluorescently labeled and their binding to mIgM antibody expressed on the surface of 239F cells is evaluated by flow cytometry. Presented is CDRH2-dependent recognition of the influenza HA stem region by germline CR6261 (colored lines; grey is binding to 239F expressing VRC01 mIgM, an irrelevant antibody sequence). Surface trafficking of both wildtype and CR6261 germline mIgM containing a I53A/F54A mutation in CDRH26 is confirmed using a fluorescent anti-light chain antibody. Specificity for the HA stem is defined by reactivity to wildtype HA but not HA containing the mutation I45R/T49R in the stem epitope6.