Magattract suspension g

Streptavidin-coated Dynabeads (M-280 Streptavidin, Thermo Fisher Scientific) and MagAttract Suspension G (1026883, QIAGEN) were transferred to a microcentrifuge tube and washed twice with washing solution. To immobilize the SARS-CoV-2 spike protein, 10 μL of reconstituted biotinylated protein and 90 μL of carbonate buffer solution (CBS) were added to the microcentrifuge tube and incubated for two hours at room temperature. CBS was prepared by dissolving 3.03 g of sodium carbonate (S7795, Sigma‒Aldrich) and 6.0 g of sodium bicarbonate (S5761, Sigma‒Aldrich) in 1 L of water. The conjugated Dynabeads were washed twice with the washing solution after incubation, and the buffer was removed after the second wash. To immobilize human CRP and TnC, 0.625 μL of biotin-conjugated CRP capture antibody or 0.625 μL of TnC capture antibody was added to 100 μL of Dynabeads and incubated for two hours at room temperature. The Dynabeads were washed twice after incubation, and 100 μL of PBS was added after the second wash to resuspend the processed Dynabeads. MagAttract Suspension G (Qiagen) was used to enhance the magnetic response. All antibody-conjugated Dynabeads and MagAttract Suspension G were blocked with 100 μL of blocking buffer (37539, Thermo Fisher Scientific) for 1 h. They were washed twice with 100 μL of washing solution and resuspended in 100 μL of PBS.

DNA was extracted from 20–50 mg of culture material (Ectocarpus) or silica dried specimens (Scytosiphon). Samples were ground in liquid nitrogen and extracted using CTAB buffer (100 mM Tris–HCl, pH 8.0, 1.4 M NaCl, 20 mM EDTA, 1 % w/v CTAB, 1 % w/v PVP, 0,2 mg/ml proteinase K), followed by two extractions with chloroform:isoamylalcohol (24:1). DNA was precipitated with 80 % isopropanol and cleaned with Qiagen MagAttract Suspension G according to the manufacturer’s instructions.

DNA was purified using the Biosprint^® 96 One-for-all Vet kit (Qiagen). Briefly, 400 µL of the supernatant obtained above was transferred to a 96-well lysate plate containing 40 µL proteinase K (Qiagen) to which 300 µL of magnetic bead suspension comprising 25 µL MagAttract suspension G (Qiagen) in 300 µL isopropanol (Sigma-Aldrich) was added. Magnetic bead-based purification of DNA was performed using an automated platform (MagMAX Express-96; Life Technologies), which included transfer of the bead-bound DNA through three wash steps using two different wash buffers (Buffer AW1 and RPE, Qiagen) contained in separate 96-well plates (Qiagen), air drying of any wash buffer residue and DNA elution in Buffer AVE (Qiagen). The eluted DNA was stored at −80°C before use. The DNA extract was tested neat and also as a fivefold dilution in Buffer AVE (Qiagen).