FcRn binding and transport
properties of the Quad molecules. (A,
B) FcRn-ELISA binding assays were obtained for NIP-IgG1-WT, Q187,
Q190, and Q191 at acidic pH (pH 5.5) and neutral pH (pH 7.4). (C)
Schematic overview of the HERA protocol. Quads and anti-NIP-IgG1 were
added to starved HMEC1-hFcRn cells (1–2) and incubated for
3 h to allow for uptake (3), followed by lysis. Samples were removed,
followed by a new 3 h incubation period with fresh medium to allow
recycling and release into the medium, or retention inside the cells
measured after lysis of the cells (4). Proteins present in the lysates
and recycling medium were quantified by two-way anti-Fc ELISA (5).
The figure was created with Biorender.com. (D–F) ELISA quantification
of the amounts taken up, recycled, or accumulated. Data represents
three independent experiments; mean ± SD, unpaired Student’s t-test: *p > 0.05, **p > 0.01, ***p > 0.001, **** p >
0.0001.