Goat anti human igg pe

Optimization experiments determined the optimal concentration of the sera for studying the humoral vaccination response to be 100.000-fold dilution. As previously described [34 (link)], 50 μL of a bead mixture containing all different spike proteins in a concentration of 20 beads per μL were added to 50 μL of diluted serum and incubated overnight on a rotator at 4°C. The next day, plates were washed with TBS containing 0.05% Tween-20 (TBST) and resuspended in 50 μL of Goat-anti-human IgG-PE (Southern Biotech). After 2 hours of incubation on a rotator at room temperature, the beads were washed with TBST and resuspended in 70 μL Magpix drive fluid (Luminex). Read-out of the plates was performed on a Magpix (Luminex). The binding of antibodies was determined as the Median Fluorescence Intensity (MFI) of approximately 50 to 100 beads per well, corrected for background signals by subtracting the MFI of wells containing only buffer and beads and converted into binding antibody units per ml (BAU/ml) using the WHO International Standard for anti-SARS-CoV-2 immunoglobulin (NIBSC 20/136). To confirm assay performance, a titration of serum of one convalescent COVID-19 patient as well as positive and negative controls were included on each plate. In addition, 15 to 20% of samples of each run were replicated to confirm the results.

SU-DHL-1(CRL-2955, ATCC) or HEK293T-CD25 cells were harvested and washed by FACS buffer (0.2% BSA in PBS) two times. Serially diluted antibodies were mixed with 1 × 10⁶ cells at 4 °C for 1 h. After washing two times by FACS buffer, cells were incubated in dark with Goat Anti-Human IgG-PE (2040-09, Southern Biotech) at 4 °C for 30 min and then analyzed by NovoCyte 2060R flow cytometry. Experiments were performed in triplicate, value = mean ± SEM.

Antibody levels in serum and milk were assessed using a custom Luminex assay similar to previously described (29 ). In short, viral antigens were covalently coupled to Luminex Magplex beads with a two-step carbodiimide reaction at a ratio of 37 μg protein to 12.5 million beads for RSV-F glycoprotein and equimolar amounts for other proteins. Following the outcome of optimization experiments, serum was diluted 1:10,000, and milk was diluted 1:100. Beads and diluted samples were incubated overnight, followed by detection with goat-anti-human IgG-PE or goat-anti-human IgA-PE (Southern Biotech). Read-out was performed on a Magpix (Luminex). The resulting median fluorescence intensity (MFI) values are the median of at least 50 beads per well and were corrected by subtraction of MFI values from the buffer and beads-only wells.