O phenylenediamine tablets

A semi-quantitative in vitro determination of human IgG and IgA antibodies against the SARS-CoV-2 was performed on serum samples by using the Anti-SARS-CoV-2 Spike ELISA (EUROIMMUN), according to the manufacturer’s instructions. Values were then normalized for comparison with a calibrator. Results were evaluated by calculating the ratio between the extinction of samples and the extinction of the calibrator. Results are reported as the ratio between OD sample and OD calibrator. The ratio interpretation was as follows: < 0.8 = negative, ≥ 0.8 to < 1.1 = borderline, ≥ 1.1 = positive. To detect IgM anti-RBD we developed an in-house ELISA [17 (link)]. 96-well plates (Corning) were coated for 1 h at 37 °C with 1 μg/mL of purified SARS-CoV-2 RBD protein (Sino Biological). After washing with PBS 1 × /0.05% Tween and blocking with PBS 1 × /1% BSA, plates were incubated for 1 h at 37 °C with diluted sera (1:100). After washing again, plates were incubated for 1 h at 37 °C with peroxidase-conjugated goat anti-human IgM antibody (Jacksons ImmunoResearch Laboratories). The assay was developed with o-phenylenediamine tablets (Sigma-Aldrich) as a chromogen substrate. Absorbance at 450 nm was measured, and IgM concentrations were calculated by interpolation from the standard curve based on serial dilutions of monoclonal human IgM antibody against SARS-CoV-2 Spike-RBD (Invivogen).

A total of 30 sera were selected to be tested with each peptide. Of these, 23 were from pre-exposed people with high IgM levels for Pap31 or SCS-α (accordingly to the peptide to be tested), 2 from patients with Carrión’s disease, and 5 that had no reactivity to Pap31 or SCS-α. To determine the immunogenicity of each peptide, ELISA tests were carried out using the synthetic biotinylated peptides as an antigen. Briefly, 96-well Nunc-Immuno Maxisorp microtiter plates (Nalgene Nunc International, Roskilde, Denmark) were coated with 100 μL (0.67 μg/well) of NeutrAvidin biotin-binding protein (Invitrogen, Carlsbad, CA, USA) overnight at 4 °C. After washing with phosphate-buffered saline (PBS) 0.1% Tween-20, 100 μL of a single biotinylated peptide (28.6 μg/mL in each well) was added and incubated for 1 h. The plates were washed 3 times, and sera diluted 1:100 with TBS-Tween with 1% BSA were incubated for 2 h. The same negative control and secondary antibody used in the Western blottings were used here. The optical densities were measured as absorbance at 450 nm after using o-phenylenediamine tablets as substrate (Sigma, St. Louis, MO, USA). Each sample was analyzed in triplicate intra-plate.

Three types of clay were supplied from the Clay Mineral Society Source Clays Repository: kaolinite, Warren County, Georgia, USA (KGa-2); Na-rich montmorillonite, Crook County, Wyoming, USA (SWy-2); and Ca-rich montmorillonite, Gonzales County, Texas, USA (STx-1b). NX-illite was obtained from Arginotec, NX Nanopowder, B + M Notenkämper, Munich, Germany. NX-Illite is composed of illite, illite-smectite mixed layer, feldspar, kaolinite, quartz and carbonate^{52 (link)}.
PEG 8 kDa, dextran 10 kDa, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), HEPES sodium salt, sodium bicarbonate, o-phenylenediamine tablets, hydrogen peroxide and 2,3-diaminophenazine were purchased from Sigma-Aldrich (St. Louis, MO). Labeled polymer mPEG-NH₂ MW 5000 was purchased from Shearwater Polymers. Alexa Fluor 647-dextran 10 kDa, Alexa Fluor 488 labeling kits and 20 mm diameter by 0.5 mm deep press-to-seal silicone isolators were obtained from Life Technologies (Carlsbad, CA). Water was deionized to a resistance of 18.2 MΩ with Barnstead NANOpure Diamond water purification system (Van Nuys, CA) and used for all experiments.