Figure 4. Conjugation to Nb218-H4 and detection of hFcRn.
Binding of hFcRn to titrated amounts of (A) human IgG1 and (B) albumin coated in wells. Binding of hFcRn to the ligands at pH 7.4 and pH 6.0 was visualized using HRP-conjugated Nb218-H4. The numbers given represent the mean of triplicates. HeLa cells transfected to over-express hFcRn (hFcRn-RFP and human β2-microclobulin) were stained with (C) Alexa-conjugated (Nb218-H4)2 or (D) a control bivalent Nb. (C–D) Cells were also allowed to take up human albumin conjugated to Alexa647. (E) HeLa cells transfected with a plasmid encoding only the RFP-fused hFcRn HC and then incubated with Alexa-conjugated (Nb218-H4)2 or a control Nb. All cells were inspected by confocal microscopy as described in Material and Methods. n = 3. All data are presented as mean±s.d.