• Human CRP Duoset kit (R & D Systems, UK, cat. no. DY1707E) !CAUTION Store reconstituted Ab and antigen at 2-8 °C, if they are used within a month. Otherwise, make aliquots and store at -20 °C to -70 °C for up to 6 months. The kit comprises of
• Mouse anti-human CRP capture Ab (360 μg mL-1)
• Recombinant human CRP (90 ng mL-1)
• Biotinylated mouse anti-human CRP detection Ab (22.5 µg mL-1)
• Streptavidin-conjugated horseradish peroxidase (SA-HRP) !CAUTION Do not freeze. Store in the dark as streptavidin is light-sensitive.
The Human CRP Duoset kit’s components can also be procured separately, i.e. human CRP antibody (cat. no. MAB17071), human CRP biotinylated antibody (cat. no. BAM17072) and recombinant human CRP (cat. no. 1707-CR).
• Blocker BSA in PBS (10X), pH 7.4, 10% (w/v) (Thermo Scientific, Ireland, cat. no. 37525) CRITICAL Filter with 0.2 µm pore size filter paper prior to use to avoid contamination.
• Dynabeads® M-280 Tosylactivated (Thermo Fisher Scientific, Ireland)
• 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.
• BupH Phosphate Buffered Saline Packs (0.1 M sodium phosphate, 0.15 M sodium chloride, pH 7.2) (Thermo Scientific, Ireland, cat. no. 18372) !CAUTION Avoid inhalation. CRITICAL Prepare in autoclaved DIW (18Ω), see REAGENT SETUP.
• TMB substrate kit (Thermo Scientific, Ireland, 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 The TMB to peroxide ratio is critical for color development. Maintain the ratio 1:1.
• Hydrogen peroxide solution (containing 0.02 % v/v H2O2 in citric acid buffer) (Thermo Scientific, Ireland). !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 a safety cabinet or fume cupboard.
• Human whole blood (HQ-Chex level 2, cat. no. 232754, Streck) 180 day closed-vial stability and 30 day open-vial stability.
• Human serum (CRP free serum, cat. no. 8CFS, HyTest Ltd., Finland)
• Deionized water (18 Ω, DIW). (Direct-Q®3 Water Purification System, Millipore, USA)
• 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. Z606340)
• Sigmaplot software version 11.2 from Systat
REAGENT SETUP
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 RT for a week and at 4 ºC for up to four weeks.
Binding buffer. 0.1% BSA in PBS, pH 7.2.
Washing buffer. 0.05% Tween® 20 in PBS, pH 7.2.
Anti-CRP capture Ab-bound Dynabeads®. The anti-CRP capture Ab was bound to the tosylated Dynabeads® using the standard immobilization procedure provided by the manufacturer (Invitrogen). The prepared stock solution of anti-CRP capture Ab-bound Dynabeads® was then stored at 4 °C.
Biotinylated anti-CRP detection Ab conjugated to SA-HRP. Biotinylated anti-CRP detection Ab conjugated to SA-HRP was prepared by adding 1 µL of biotinylated anti-CRP detection antibody (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-CRP detection Ab used was 0.17 µg mL-1, while SA-HRP dilution employed was 1:3000.