Goat anti human igg alexa fluor 647

Streptavidin-coated beads with different intensities of PE-channel fluorescence (Spherotech SVFA-2558–6K and SVFB-2558–6K) were incubated with the following biotinylated antigens: 10 μg/mL MERS spike, NL63 spike, 229E spike, HKU1 spike, OC43 spike (all from Sino Biological), SARS-CoV-2 spike, SARS-CoV-2 RBD, SARS-CoV-2 NTD, SARS-CoV-1 spike, SARS-CoV-1 RBD or 10 μg/mL CD4 as a control. Excess streptavidin sites were blocked with 10 μg/mL of CD4 and the beads were washed and mixed. The beads were stained with 1/50, 1/250 or 1/1250 plasma for 30 min at room temperature, washed, and stained with 2.5 μg/mL goat anti-human IgG Alexa Fluor 647 (Jackson Immunoresearch 109-606-170). The samples were read with the iQue Screener Plus (Intellicyt) high-throughput flow cytometer and FACS data were analysed with Flowjo. Data from titrations were analysed by calculating area under the curve (AUC) for the titration and subtracting the AUC of the negative control antigen.

Four-fold serial dilutions of recombinant mAbs in 0.5% BSA w/v in PBS, for a final dilution series of 47.7 pg/mL - 200 μg/mL, were incubated with multiplexed CoV antigen beads at room temperature for 30 min. Beads were then washed and stained with 2.5 μg/mL goat anti-human IgG Alexa Fluor 647 (Jackson Immunoresearch, 109-606-170). Samples were acquired on the iQue Screener Plus (Intellicyt) and resulting data were analysed with FlowJo (Version 10.8.1). Titration curves and AUC analyses were performed on GraphPad Prism (Version 9.3.1); values were reported after subtraction of binding values to the negative control antigen CD4. For binding of mAbs to the SARS-CoV-2 spike and stem helix peptide (peptide 154), the L9 negative control curves are the same in Figures S3B and S3E.

Streptavidin-coated beads with different intensities of phycoerythrin (PE)–channel fluorescence (Spherotech, SVFA-2558-6K and SVFB-2558-6K) were incubated with the following biotinylated antigens: 10 μg/ml of Middle East respiratory syndrome (MERS) spike (Sino Biological, 40069-V08B), NL63 spike (Sino Biological, 40604-V08B), 229E spike (Sino Biological, 40605-V08B), HKU1 spike (Sino Biological, 40606-V08B), OC43 spike (Sino Biological, 40607-V08B), SARS-CoV-2 spike, SARS-CoV-2 RBD, SARS-CoV-2 NTD, SARS-CoV-1 spike, and SARS-CoV-1 RBD or CD4 (10 μg/ml) (56 (link)) as a control. SARS-CoV-2 and SARS-CoV-1 antigens were produced in-house as described above. Excess streptavidin sites were blocked with CD4 (10 μg/ml), and the beads were washed and mixed. The beads were stained with 1:50, 1:250, or 1:1250 plasma for 30 min at room temperature, washed, and stained with 2.5 μg/ml of goat anti-human IgG Alexa Fluor 647 (Jackson ImmunoResearch, 109-606-170), anti-human IgA Alexa Fluor 647 (Jackson ImmunoResearch, 109-606-011), or anti-human IgM Alexa Fluor 647 (Jackson ImmunoResearch, 109-606-129). The samples were read with the iQue Screener Plus (IntelliCyt) high-throughput flow cytometer, and FACS data were analyzed with FlowJo. Data from titrations were analyzed by calculating the area under the curve (AUC) for the titration and subtracting the AUC of the negative control antigen.

P. falciparum NF54 sporozoites dissected from salivary glands were stained with 1:2000 SYBR Green and four dilutions of VRC 314 U.S. plasma (1:20, 1:100, 1:500, and 1:2500) for 30 minutes at 4˚C, washed, and then incubated with 2.5 μg/mL goat anti-human IgA-Alexa Fluor 647 (Jackson Immunoresearch 109-606-011) or goat anti-human IgG-Alexa Fluor 647 (Jackson Immunoresearch 109-606-170) for 30 minutes at 4˚C. The binding data with the sporozoites were acquired with the iQue Screener Plus high-throughput flow cytometer and analyzed with FlowJo software (Tree Star).