Horseradish peroxidase hrp conjugated mouse anti human igg

Anti-SARS-CoV-2 antibody reactivities were measured using a protocol based on published ELISA methods [21 (link)]. In brief, ELISA plates were coated at 2 μg/mL with SARS-CoV-2 Spike (Sino Biological, Wayne, PA, USA). Recombinant products, positive control mAb (CR3022; Absolute Antibody, San Diego, CA, USA), plasma-derived polyclonal anti-SARS-CoV-2 research reagent (20/130; NIBSC, Potters Bar, Hertfordshire, UK), anti-SARS-CoV-2 plasma hyperimmune globulin (Grifols, S.A., Sant Cugat del Vallès, Spain), and negative control IVIG (Gamunex; Grifols, S.A., Sant Cugat del Vallès, Spain) were serially diluted in sample buffer (1 × PBS + 0.05% Tween + 0.3% dry milk). After one-hour incubation, bound antibodies were detected using horseradish peroxidase (HRP)-conjugated mouse anti-human IgG (Jackson Immunoresearch, West Grove, PA, USA). Quantitative measurements were performed on a plate reader (Molecular Devices, Fremont, CA, USA) and analyzed using Prism (GraphPad, San Diego, CA, USA) to calculate the 50% effective concentration (EC50) of samples. The concentration of total IgG was calculated by UV280 measurement using an extinction coefficient of 1.5 (mg/mL)⁻¹ cm⁻¹. Triplicate measurements were made for each dilution. EC50 95% confidence interval calculations were performed using GraphPad Prism and the asymmetric profile-likelihood method.

Autoantibody detection was performed via an ELISA using a 96-well microplate coated overnight at 4 °C with SF3B4 recombinant antigens. The remaining binding sites on the microplate were blocked with 20% casein buffer in distilled water. Ten microliters of freshly thawed patient or normal serum samples (diluted 1/250 in 20% casein buffer in PBS) were added to appropriate wells and incubated for 1 h at RT. Each well was washed thrice with PBS with 0.2% Triton X-100 (PBST; Sigma-Aldrich, St.Louis, MO, USA). Diluted horseradish peroxidase (HRP)-conjugated mouse anti-human IgG (Jackson Immuno Research, West Grove, PA, USA) was added to the target wells, and goat anti-mouse IgG was added to the calibrator wells for 1 h at RT. The well was washed with 0.2% PBS-T, and 100 µL of 3,39,5,59-tetramethylbenzidine (TMB; Thermo Fisher, MA, USA) substrate solution was added to each well, followed by incubation for 5 min at RT, and the addition of an equal volume of stopping solution (1 M HCl). The optical density was measured at 450 nm.