1 step ultra 3 3 5 5 tetramethylbenzidine tmb elisa substrate solution

In preenriched, enriched, and depleted B cell culture, supernatant IgG concentration and SLO specificity were quantified by enzyme-linked immunosorbent assay (ELISA). Briefly, to quantify IgG, high-binding 96-well plates were coated with 0.5 μg/ml unconjugated goat anti-human IgG Fc capture antibody (Invitrogen, Thermo Fisher Scientific). Human IgG1-lambda (Sigma-Aldrich, St. Louis, MO) was used as a standard. To quantify antibodies binding SLO, streptavidin-coated 96-well plates were incubated with biotinylated SLOwt at 37°C for 1 h. Cell culture supernatants were diluted in sample buffer (PBS plus 5% nonfat dry milk) and incubated on plates for 1 h at 37°C. Horseradish peroxidase-conjugated goat anti-human IgG antibody (Thermo Scientific) was used for detection, and 1-step Ultra 3,3′,5,5′-tetramethylbenzidine (TMB)-ELISA substrate solution (Thermo Scientific) was used to detect horseradish peroxidase activity, according to the manufacturer’s protocol. The reaction was stopped with the addition of 2 M sulfuric acid, and the absorbance at 450 nm was read. ELISAs were performed on 2 or more biological replicates.

The phage-displayed SARS-CoV-2 epitopes were used in phage ELISAs with patient plasma samples diluted 100-fold in coating buffer (50 mM Na₂CO₃, pH 9.6). After incubation in a 96-well Nunc MaxiSorp flat-bottom microtiter plate with shaking at 150 rpm at 4°C for 12 to 18 h, plasma was aspirated by a plate washer (BioTek). Next, the plate was treated with 100 μl per well of ChonBlock blocking/sample dilution buffer (Chondrex, Inc.) for 1 h with shaking at 150 rpm at room temperature and washed three times with wash buffer (0.05% [vol/vol] Tween 20 in PBS). The epitope displaying phage and controls were diluted to 1 nM in ChonBlock blocking/sample dilution buffer, and 100 μl was added to each well before incubating for 2 h with shaking (150 rpm) at room temperature. The plate was then washed three times with wash buffer. The primary antibody, anti-M13-horseradish peroxidase (HRP) (Creative Diagnostics), was diluted 1:5,000 in ChonBlock secondary antibody buffer, and 100 μl was added per well; the plate was incubated for 1 h at 150 rpm and room temperature. Following three washes with wash buffer, 1-Step Ultra 3,3′,5,5′-tetramethylbenzidine (TMB)-ELISA substrate solution (100 μl per well; Thermo Scientific) was added. Absorbance of TMB substrate was measured twice at 652 nm by a UV-visible (UV-Vis) plate reader (BioTek) after 5 and 15 min of incubation.

MDTCS and MDTCS-Fc were produced in Chinese hamster ovary (CHO) cells by a serum-free cell culture method (GC Biopharma, Republic of Korea) and their respective activity was measured with TECHNOZYM^® ADAMTS13 activity enzyme-linked immunosorbent assay (ELISA) kit (Technoclone Herstellung von Diagnostika und Arzneimitteln GmbH, Vienna, Austria) following the recommended protocol. A human ADAMTS13 DuoSet ELISA kit (R&D systems, Minneapolis, United States) was used to measure the concentrations of MDTCS or MDTCS-Fc in the plasma samples collected from the mice receiving MDTCS or MDTCS-Fc. The 96-well plates [Enzyme Immunoassay/Radioimmunoassay (EIA/RIA) plate or Greiner 96 well plate] were from Merck KGaA (Darmstadt, Germany) and 1-Step™ Ultra 3,3′,5,5′-Tetramethylbenzidine (TMB)-ELISA Substrate Solution was from Thermo Scientific (Massachusetts, United States). The biotinylated mouse FcRn protein was from Acro Biosystems (Delaware, United States). The streptavidin biosensor was from Sartorius AG (Göttingen, Germany).