for at least 30?min and then subjected to centrifugation before collection of serum. of an FcRn-binding affibody molecule (ZFcRn)20. Affibody molecules are affinity protein domains, 58 amino acids long, that have a folded anti-parallel three-helix bundle structure. They have been generated to bind to a variety of target proteins with high affinity and specificity21. We investigated if one of the previously generated affibody molecules was able to interfere with the IgG/FcRn interaction and purified to homogeneity. The proteins were analyzed by SDS-PAGE (Fig. ?(Fig.1b,1b, Supplementary Figure 1) followed by LC/MS analysis (Fig. ?(Fig.1c),1c), which showed proteins of 98% purity with correct molecular masses. The level of potential contaminating endotoxins was measured and was found to be below the limit of detection. The tendency to precipitate was also investigated, where the proteins were frozen at ?80?C. Upon thawing no precipitation could be detected. Blocking the IgG/FcRn interaction blocking of the IgG/FcRn interaction. HeLa cells expressing the mouse or Amitriptyline HCl human ortholog of FcRn as a fusion to eGFP, hFcRn-eGFP-HeLa hB2m and mFcRn-eGFP-HeLa mB2m respectively, were stained with Alexa647-labeled human or mouse Amitriptyline HCl IgG. During staining ZFcRn or ZFcRn-ABD were added at different concentrations. After staining, the cells were analyzed by flow cytometry where mean fluorescence intensity (MFI) values corresponding to Alexa647-IgG fluorescence were determined. The Y-axis corresponds to the measured values as percentage of the MFI measured without addition of affibody. The X-axis corresponds to the added concentration of ZFcRn or ZFcRn-ABD. (a) Cells expressing human FcRn-eGFP were stained with human IgG in the presence of ZFcRn; (b) Cells expressing mouse FcRn-eGFP were stained with mouse IgG in the presence of ZFcRn; (c) Cells expressing human FcRn-eGFP were stained with human IgG in the presence of ZFcRn-ABD; (d) Cells expressing mouse FcRn-eGFP were stained with mouse IgG in the presence of ZFcRn-ABD. Detailed characterization of affinities to FcRn and serum albumin A detailed characterization of the interactions of ZFcRn and ZFcRn-ABD with both FcRn Amitriptyline HCl and serum albumin were conducted by biosensor analysis. First, ZFcRn and ZFcRn-ABD were injected over a surface with immobilized human FcRn at pH 6.0 and 7.4 in the presence or absence of mouse serum albumin (Fig. ?(Fig.3).3). The equilibrium response when injecting ZFcRn was appreciably higher at pH 6.0 than at pH 7.4 suggesting a higher affinity at pH 6.0 (Fig. ?(Fig.3a).3a). The equilibrium Amitriptyline HCl response was largely unaffected by the presence of MSA, which was expected since MSA should not interact with ZFcRn and its interaction with human FcRn at the concentration used is below the limit of detection in the assay. A control experiment where only MSA at the same concentration was injected over the surface gave no detectable response (Supplementary Figure 2). The equilibrium response when injecting ZFcRn-ABD was similarly higher at pH 6.0 than at 7.4 also suggesting a higher affinity at 6.0 (Fig. ?(Fig.3b).3b). Here the presence of MSA resulted in an increase in the equilibrium response and a decrease in the on-rate, which is indicative of a larger complex interacting with the surface, suggesting that the complex ZFcRn-ABD/MSA is able to interact with FcRn. Open in a separate window Figure 3 Interaction of ZFcRn constructs with FcRn. The interaction of ZFcRn and ZFcRn-ABD with human FcRn at different pH and in the presence or absence of SA was investigated by biosensor analysis. The panels NNT1 show overlays of representative sensorgrams recorded after injection of ZFcRn (a) and ZFcRn-ABD (b). The affinities to FcRn were also determined by injecting dilution series of ZFcRn Amitriptyline HCl and ZFcRn-ABD at pH 6.0 and 7.4 (Fig. ?(Fig.4,4, Table ?Table1).1). The affinity of ZFcRn was found to be approx. 40 times stronger at pH 6.0 compared to pH 7.4 (KD: 9?nM versus 400?nM; Figs 4a,b). Similarly, the affinity of ZFcRn-ABD was approximately 10 times stronger at pH 6.0 compared to pH 7.4 (KD: 3?nM versus 40?nM; Figs 4c,d). The difference in affinity between ZFcRn and ZFcRn-ABD at pH 6.0 is within the margin of error, with a tendency for a higher affinity for the ABD-tagged construct. At pH 7.4 the difference in affinity between ZFcRn and ZFcRn-ABD is ten-fold..