Journal: Nature Communications

Article Title: Tailored collagen binding of albumin-fused hyperactive coagulation factor IX dictates in vivo distribution and functional properties

doi: 10.1038/s41467-025-62955-9

Figure Lengend Snippet: a Illustration of the ELISA-based binding assay used to study binding between hFcRn or mFcRn and FIX-HSA variants at pH 5.5. Soluble truncated FcRn (light gray) was captured on IgG1 MST/HN (blue) coated in the well, before FIX-HSA (red/black) was added to the wells, and the HSA region of the fusion protein was detected by an ALP-conjugated anti-HSA antibody (dark gray). b , c Results from the ELISA-based hFcRn and mFcRn binding assays performed at pH 5.5. Data represent the mean ± SD of technical duplicates from one representative experiment. d Illustration of the SPR experiment used to study the interaction between FcRn (gray) and FIX-HSA (orange/black) performed by immobilizing FIX-HSA fusion proteins on a CM5 sensor chip before injecting a truncated form of soluble FcRn in a concentration gradient. Representative sensorgrams from SPR performed by injecting soluble truncated hFcRn ( e – h ) or mFcRn ( i – l ) over immobilized FIX-HSA variants at pH 5.5, showing one out of three independent experiments performed. The dotted lines show curves fitted by the 1:1 Langmuir binding model. m An illustration of the HERA setup designed to study hFcRn-mediated cellular ( n ), uptake and ( o ), recycling of FIX-HSA fusion proteins. Data are presented as relative to FIX-HSA WT (=1, dotted line) and represent the group mean ± SD of three independent experiments performed in technical triplicates (uptake: n = 9, except n = 8 for FIX KA -HSA WT and FIX KR -HSA WT , n = 6 for Padua KR -HSA QMP ; recycling: n = 9, except n = 7 for FIX KA -HSA WT ). Statistical significance was tested by unpaired two-tailed Student’s t tests with 95% confidence level (precise p -values are given in Supplementary Table ). FIX-HSA WT , black; Padua-HSA QMP , gray; FIX KA -HSA WT , blue; FIX KR -HSA WT , orange ; Padua KA -HSA QMP , pink; Padua KR -HSA QMP , purple. Source data are provided as a Source Data file. a , d , m Created in BioRender. Hovden Aaen, K. (2025) https://BioRender.com/auh41ln .

Article Snippet: After incubation for 90 min at 37 °C, the wells were washed as before, and bound proteins were detected by adding a polyclonal goat anti-HSA HRP-conjugated antibody (Bethyl Laboratories, #A80-129P, lot 32) diluted 1:2000 in sample buffer and incubating the wells as before.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay, Two Tailed Test