• KOH pellets (99.99%), semiconductor grade (Sigma Aldrich, cat. no. 306568) !CAUTION Use PPE and handle in a safety cabinet. Avoid contact with skin and eyes as it can cause severe burns. CRITICAL The concentration of KOH must be 1% (w/v) in autoclaved DIW. Higher concentrations may affect the surface properties, thereby leading to decreased binding of capture antibodies.
• 3-aminopropyltriethoxysilane (3-APTES) (Sigma Aldrich, cat. no. A3684) !CAUTION Use PPE and handle in a safety cabinet. Avoid contact with skin and eyes as it is a skin and eye irritant, and highly toxic to kidney. CRITICAL Prepare in autoclaved DIW, see REAGENT SETUP.
• 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) (Thermo Scientific, cat. no. 22981) !CAUTION Use PPE and handle inside a fume cupboard as it is an irritant. Being hygroscopic, it absorbs moisture that leads to loss of its activity. Equilibrate to room temperature (RT) before opening the container. CRITICAL Store at recommended temperature (-20 °C). Reconstitute in 0.1 M MES, pH 4.7, see REAGENT SETUP.
• Human HFA Duoset kit (R & D Systems, cat. no. DY1184e) !CAUTION Store reconstituted Ab and antigen at 2-8 °C, if they are to be used within a month. Otherwise, make aliquots and store at -80 °C for up to 6 months. The kit comprises of
• Mouse anti-HFA capture Ab
• Recombinant HFA
• Biotinylated goat anti-HFA Ab
• Streptavidin-conjugated horseradish peroxidase (SA-HRP) !CAUTION Do not freeze. Store in the dark as streptavidin is light-sensitive.
The HFA Duoset kit’s components can also be purchased separately.
• Blocker BSA in PBS (10X), pH 7.4, 10% (w/v) (Thermo Scientific, cat. no. 37525) CRITICAL Filter with 0.2 µm pore size filter paper prior to use to avoid contamination.
• 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.
• BupH MES Buffered Saline Packs (0.1 M MES [2-(N- morpholino)ethane sulfonic acid], 0.9 % (w/v) sodium chloride, pH 4.7) (Thermo Scientific, cat no. 28390) !CAUTION Avoid inhalation. CRITICAL Prepare in autoclaved DIW, see REAGENT SETUP.
• TMB substrate kit (Thermo Scientific, cat. no. 34021)
• TMB solution (0.4 g/L) !CAUTION Skin, eye and lung irritant. In case of skin contact, wash with plenty of water. CRITICAL Maintain the TMB to peroxide ratio as 1:1 as it is critical for color development.
• Hydrogen peroxide solution (containing 0.02 % v/v H2O2 in citric acid buffer) (Thermo Scientific). !CAUTION Use PPE and work in a safety cabinet or fume cupboard. It is a strong oxidizing agent, harmful if swallowed, and carries severe risk of damage to eyes. In case of contact, rinse immediately with plenty of water and seek medical attention.
• Sulfuric acid (Aldrich, cat. no. 339741) !CAUTION Use personal protective equipment (PPE), such as chemical safety glasses, chemical-resistant shoes and lab coats, for handling. Handle only in a fume cabinet. Avoid skin contact as it is a strong corrosive agent and an irritant. In case of skin contact, wash immediately with acid neutralizers and seek medical advice as soon as possible.
• Human whole blood (HQ-Chex level 2) (Streck, cat. no. 232754) 180 day closed-vial stability and 30 day open-vial stability.
• Human serum (CRP free) (HyTest Ltd., cat. no. 8CFS)
• Deionized water (18 Ω, DIW). (Millipore, Direct-Q®3 Water Purification System)
• 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 (Systat)
PBS. Add a BupH PBS 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 RT for a week and at 4ºC for up to four weeks.
MES. Add a BupH MES 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 MES at pH 4.7, which can be stored at RT for up to two weeks.
APTES. Reconstitute the commercially supplied APTES solution (99% purity) in autoclaved DIW to make a 1% (v/v) solution. Prepare a fresh solution for each DIA run.
EDC. Each pack contains 25 g EDC. Reconstitute in 0.1M MES buffer, pH 4.7, at a concentration of 4 mg mL-1. Aliquots can be stored effectively for six months at -20 ºC.
EDC-activated anti-HFA Ab. Dissolve 0.4 mg EDC in 100 µL of 0.1M MES, pH 4.7. Incubate 990 μL of the anti-HFA Ab (8 μg mL-1) with 10 μL of EDC (4 mg mL-1) solution for 15 min at RT. CRITICAL STEP The concentration of EDC is important for optimal cross-linking. Use the recommended concentration of EDC. ? TROUBLESHOOTING
Biotinylated anti-HFA detection Ab conjugated to SA-HRP. Biotinylated anti-HFA detection Ab conjugated to SA-HRP was prepared by adding 1 µL of biotinylated anti-HFA detection Ab (0.5 mg mL-1) to 1 µL of SA-HRP to 2998 µL of the binding buffer followed by 20 min of incubation at room temperature (RT). As a result, the concentration of biotinylated anti-HFA detection Ab used was 0.17 µg mL-1, while SA-HRP dilution employed was 1:3000.
HFA spiked diluted human whole blood or serum. The HFA spiked samples, containing the final concentration in the range of 0.1-243 ng mL-1, were prepared by mixing the desired HFA concentrations in 1:100 diluted human whole blood/serum.
EDTA plasma samples from anonymized patients. The EDTA plasma samples from anonymized patients were diluted 1:1000 and 1:3000 in the binding buffer. The resulting HFA concentration in these samples falls within the linear range of the DIA, thereby enabling the detection of entire pathophysiological concentration range of HFA.