# Design of Antibody-Functionalized Polymeric Membranes for the Immunoisolation of Pancreatic Islets
## 2 Materials and Methods
### Site-Directed Functionalization of the Antibody and Characterization Procedures
Activation of the carboxyl groups in Fc fragment and the subsequently bonding to Acryl-PEG-NHS were performed byadding Acryl-PEG-NHS (2 \(\upmu\)M) and EDC (20 \(\upmu\)M) in PBS solution 4 h at 4 \({}^{\circ}\)C. Finally, the samples were abundantly rinsed with PBS in order to remove physically adsorbed molecules, and stored at 4 \({}^{\circ}\)C.  
_Detachment and quantification of the protein antigen._ To quantify the protein attached to the SAM, the samples were exposed to the eluent buffer (150 mM NaCl, 10 mM HEPES, 500 mM Imidazole) for 1 h at 4 \({}^{\circ}\)C. The amount of the protein detached from the substrate and eluted was determined comparing the GFP fluorescent emission of eluted solution with the feeding solution (400 nM) as well as the recovered solution (that corresponds to the unbound protein fraction, recovered during the washing steps). Photoluminescence spectra were recorded using a Cary Eclipse spectrofluorometer. Then, the relative peak intensities (at 508 nm) of the eluted and recovered samples were compared to that of the feeding GFP solution and plotted in a graph. _Western Blot analysis._ An alternative method to quantify the attached antigen was based on Western Blot assays. The proteins were run on 10% Tris-Glycine SDS/PAGE (Bio-Rad) under denaturing conditions and transferred to a PVDF membrane. After blocking with 5% (_w/v_) nonfat dry milk in 1X PBS/0.1% (_v_/_v_) Tween 20, the membrane was incubated over night at 4 \({}^{\circ}\)C with an antibody against  
Figure 1: Chemical modification of the gold-coated silicon surface: (**a**) derivatization with MUA, (**b**) NTA-lysine binding (**c**) formation of the NTA-lysine-Ni\({}^{2+}\) complex. GFP (1:2500 in 5% (_w/v_) nonfat dry milk in PBS/0.1% Tween 20 (_v/v_)). The membrane was then washed, incubated with anti-chicken peroxidase-conjugated secondary antibody (1:1000 in 1% (_w/v_) nonfat dry milk in PBS/0.1% (_v/v_) Tween 20) at room temperature for 1 h, and developed by ECL plus (Bio-Rad). _Detachment and quantification of the Acryl-PEG-Antibody complex._ To elute the Acryl-PEG-Antibody (either Anti-GFP or CTLA-4-IgG) complex from the functionalized wafer, it was incubated with 0.1 M glycine-HCI, pH 2.8, at 4 \({}^{\circ}\)C for 10 min. This buffer dissociates most protein-protein and Ag-Ab binding complexes without permanently affecting protein structure [29]. Afterwards, the collected solution was concentrated by ultrafiltration with a 50 kDa centrifugal filter device (Amicon, Millipore). The Acryl-PEG-antibody complex was then quantified using Western Blot. _Acryl-PEG-Anti-GFP photo-crosslinking on PEG hydrogel using a selective mask._ The functionality of acrylic groups on Acryl-PEG-Anti-GFP complex was assessed by photo-crosslinking, a hydrogel dish [30]. Briefly, a solution of Acryl-PEG-Anti-GFP 20 nM and Darocur(r) 1173 (0.3% _w/v_) was irradiated to conjugate the antibody to the hydrogel. A TEM grid was used as UV photomask to direct the radiation in limited and defined areas, and consequently to have a selective photopolymerization (Figure 2). Irradiation with a 365 nm UV light at 2 mW cm\({}^{-2}\) for 3 min was performed. During UV irradiation, the grid was in contact with the hydrogel to limit UV light diffraction due to air gap [31]. The presence of Acryl-PEG-Anti-GFP on the hydrogel was then visualized through a secondary FITC-conjugated antibody (Alexa Fluor(r) 488 anti-chicken, Thermo Fisher Scientific), incubated for 1 h at RT (1:250).