Superblock reagent

Pembrolizumab ADA levels were measured according to manufacturers instructions (Bio-Rad). Briefly, Pembrolizumab (Meck & Co) was utilised as a capture antibody was diluted to 1 μg/mL in phosphate-buffered saline (PBS) and coated on Nunc Maxisorp™ 96-well plates (Thermo Fisher Scientific) overnight at 4 °C. Following washing with PBS/0.05% Tween-20 (Sigma) plates were blocked using SuperBlock™ reagent(Thermo Fisher Scientific). 100 μL of assay standard (Bio-Rad; 0–10,000 ng/μL) or plasma/serum was incubated for 1 h at RT.
For this assay HRP was conjugated to Pembrolizumab for use as a detection antibody, using the LYNX Rapid conjugation kit (Bio-Rad). Following washing, HRP-Pembrolizumab was added for 1 h at RT. Following washing, assay was developed using TMB substrate (Biolegend) for 30 min prior to stopping. Colourmetric change was measured at 450 nm using a FLUOStar microplate reader (BMG Labtech). Pembrolizumab ADA assay overview and a representative standard curve are shown in Fig. 1.

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).

Biotinylated peptides were synthesized with N-terminal Biotin-Ahx groups by GenScript (Piscataway, NJ, USA). The peptides were diluted to 10 µg/mL in PBS and captured on streptavidin plates prepared by layering Nunc Maxisorp 96-well plates with 1 µg/mL streptavidin (Thermo Fisher Scientific) in carbonate buffer (pH 9.6) overnight at 4 °C. The coated plates were washed three times with PBST and blocked with Superblock reagent (Thermo Fisher Scientific) for 1 h at room temperature. The plates were then sealed using a liquid plate sealer (Candor BioScience, Wangen, Germany), air-dried, shrink-wrapped, and stored at room temperature. Following incubation with the biotinylated peptide overnight, the plates were washed three times with PBST and blocked with Superblock reagent as above. After removing the blocking reagent by tapping the plates, we added hybridoma culture supernatants and incubated the plates for 30 min. The plates were then washed three times as above and incubated with the HRP-conjugated anti-mouse IgG secondary antibody for 20 min at room temperature. The plates were washed again and incubated with the HRP substrate TMB-E to quantify the signal as described above.