• Human Fetuin A ELISA kit (R & D Systems, Cat. No. DY1184e) !CAUTION Store reconstituted Ab and antigen at 4°C if they are to be used within a month. Otherwise make the aliquots and store them at -80°C if they are to be used within 6 months. The kit contains
• Mouse anti-HFA capture Ab (720 μg mL-1)
• HFA (20 ng mL-1)
• Biotin-labeled goat anti-HFA detection Ab
• HRP-conjugated streptavidin !CAUTION Store in dark as streptavidin is light-sensitive.
• Blocker bovine serum albumin (BSA) in PBS (10X), pH 7.4, 10% (w/v) (Thermo Scientific, Cat. No. 37525) CRITICAL Filter prior to use to avoid clumping and any microbial contamination.
• KOH pellets (99.99%), semiconductor grade (Sigma Aldrich, Cat. No. 306568) !CAUTION Avoid contact with skin and eyes as it can cause severe burns. Use personal protective equipment (PPE) and handle it only in a safety cabinet. CRITICAL The concentration of KOH used must be 1% (w/v) in autoclaved deionized water (DIW, 18 Ω) as it may affect the surface properties of the Au surface, which can significantly affect the antibody immobilization.
• 3-aminopropyltriethoxysilane (3-APTES) (Sigma Aldrich, Cat. No. A3684) !CAUTION Avoid contact with skin and eyes as it is an irritant. Handle it only in a safety cabinet. The higher concentrations are potentially toxic. CRITICAL Prepare it in autoclaved DIW (18Ω), see REAGENT SETUP.
• BupH Phosphate Buffered Saline Packs (0.1 M sodium phosphate, 0.15 M sodium chloride, pH 7.2) (Thermo Scientific, Cat. No. 18372) !CAUTION Avoid direct inhalation. CRITICAL Prepare it in autoclaved DIW (18Ω), see REAGENT SETUP.
• HBS-EP (0.01 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v surfactant P20) (GE Healthcare, UK, Cat No. BR-1001-88).
• Glycine-HCl (10 mM, pH 2.0) (GE Healthcare, UK, Cat. No. BR-1003-55).
• Deionized water (18 Ω DIW). (Direct-Q® 3 Water Purification System, Millipore, USA)
• SigmaPlot software bundle version 11.2 from Systat
• Eppendorf® microtubes (1.5 mL; Sigma Aldrich, Cat. No. Z606340)
PBS Add a BupH PBS pack containing 0.1M phosphate and 0.15M NaCl to 100 mL of autoclaved DIW. Dissolve well and make the volume up to 500 mL with autoclaved DIW. Each pack makes 500 mL of PBS at pH 7.2 that can be stored at RT for a week and at 4ºC for up to four weeks.
APTES The commercially-available APTES solution has a purity of 98%. Reconstitute in autoclaved DIW to make an effective 1% (v/v) solution just prior to mixing with capture anti-HFA Ab.