Pierce maleic anhydride activated plates

Pig gastric mucin (PGM) and saliva blocking assays were performed as previously described [69 (link)]. Briefly, ELISA plates were coated with 10 μg/ml PGM (Sigma, Germany) or with saliva type A or B diluted in PBS 1:2000. Nanobodies were two-fold serially diluted in PBS containing 2.5 μg/ml GII.10 VLPs (for PGM assay), 0.5 μg/ml GII.10 VLPs (for saliva assay) or 0.5 μg/ml GII.4 2006 VLPs (both PGM and saliva assay) and incubated for 1 h at RT. The VLPs-Nanobodies mixture was added to the plates and bound VLPs were detected with a α-GII.10 or α-GII.4 VLPs rabbit polyclonal antibody. For synthetic HBGA blocking assay, 10 μg/ml synthetic blood type B trisaccharide amine derivative (Dextra, UK) was coated on Pierce maleic anhydride activated plates (Thermo Fisher Scientific) overnight at 4C. Serially diluted Nanobodies were pre-incubated with 5 μg/ml GII.4 VLPs for 1h at RT. Following steps were performed as above. The binding of VLPs-only was set as a reference value corresponding to a 100% binding. The half maximal inhibitory concentrations (IC₅₀) values for Nanobody inhibition were calculated using GraphPad Prism (6.0a).

The FLAA test for SARS-CoV-2 S-protein detection was set in 96-well microplates as previously described [14 (link)]. Briefly, 100 pmole of 5′-amino-C6-modified C7 aptamer was immobilized on clear or black opaque Pierce™ maleic anhydride activated plates (Thermo Fisher Scientific, Waltham, MA, USA) as a capture agent. Plates were blocked using the Super-Block™ reagent (Thermo Fisher Scientific) following the manufacturer’s protocol. Supernatant from Sal, Sw or Sw-D samples was added in TNa7 for the final 10% and 20% concentrations; a volume of 100 μL of each dilution was then poured into C7-coated plates and incubated in an orbital shaker for 1 h at room temperature. Wells were washed five times with TNa7 and then 50 pmole of the 5′-FAM-C9 aptamer were added as a detection agent. After a second incubation for 1 h at RT, plates were washed five times with TNa7, and fluorescence was either acquired immediately (direct protocol) from black opaque plates or incubated in clear plates with 150 μL of 7 M urea for 30 min at RT for C9 aptamer retrieval and posterior transference into black plates (Corning Inc., Corning, NY, USA) for further reading (indirect protocol). Finally, fluorescence in the 96-well black plates was measured at 491ex/516em nm using a BioTek^® Synergy™ H4 Hybrid Multi-mode microplate reader (Thermo Fisher Scientific).