• 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, aliquot out and stored at -80°C for up to 6 months. The kit comprises of
• 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 the dark as streptavidin is light-sensitive.
• Blocker BSA in PBS (10X), pH 7.4, 10% (w/v) (Thermo Scientific, cat. no. 37525) CRITICAL To avoid microbial contamination in BSA, filter prior to use.
• Sulfuric acid (Aldrich, cat. no. 339741) !CAUTION Avoid skin contact as it is a strongly corrosive agent and an irritant. Use personal protective equipment (PPE) such as chemical safety glasses, chemical-resistant shoes and lab coats, for handling. Handle only in a fume cabinet. In case of skin contact, wash immediately with acid neutralizers and seek medical advice as soon as possible.
• 3-Aminopropyltriethoxysilane (APTES) (Sigma Aldrich, cat. no. A3684) !CAUTION Higher concentrations are potentially toxic. It is a skin and eye irritant. Avoid contact with skin and eyes. Handle inside a fume cabinet. CRITICAL Prepare 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 inhalation. CRITICAL Prepare in autoclaved DIW (18Ω), see REAGENT SETUP.
• TMB substrate kit (Thermo Scientific, cat. no. 34021)
• TMB solution (0.4 g L-1) !CAUTION Skin, eye and lung irritant. In case of skin contact, wash with plenty of water. CRITICAL TMB to peroxide ratio is critical for color development. Maintain the ratio 1:1.
• Peroxide solution (containing 0.02 % (v/v) H2O2 in citric acid buffer). !CAUTION Strong oxidizing agent. Harmful if swallowed. Severe risk of damage to eyes. Rinse immediately with plenty of water in case of contact and seek medical attention. Wear suitable protection and work in safety cabinet or fume cupboard.
• Deionized water (18Ω, DIW). (Direct-Q® 3Water Purification System, Millipore)
• Nunc microwell 96-well polystyrene plates, flat bottom (non-treated), sterile (Sigma Aldrich, cat. no. P7491)
• Eppendorf microtubes (1.5 mL; Sigma Aldrich, cat. no. Z 606340)
• Sigmaplot software version 11.2 from Systat
PBS Add a BupH phosphate buffered saline pack to 100 mL of autoclaved DIW, dissolve well and make the volume up to 500 mL using autoclaved DIW. Each pack makes 500 mL of PBS at pH 7.2, which can be stored at room temperature (RT) for a week and at 4ºC for up to four weeks.
APTES The procured APTES solution has a purity of 99%. Reconstitute in autoclaved DIW to make 1% (v/v) APTES just before mixing with capture anti-HFA Ab.