Anti human igg pe

PBMCs were stained with anti-CD3-PerCP (BD, Franklin Lakes, NJ, USA), anti-CD19-Alexa Flour 700 (Beckman Coulter, Pasadena, CA, USA), anti-Human IgG-PE (BD, Franklin Lakes, NJ, USA), and Strep-tag Gn at 2 μg/5 × 10⁵ cells. After they were washed three times with FPBS (2% FBS), the PBMCs were stained with anti-Strep-APC (Biolegend, San Diego, CA, USA). Gn-specific memory B cells were defined as CD19⁺ CD3⁻ hIgG⁺ Strep⁺ and sorted into 96-well plates (single cell/well) using a MoFlo XDP cell sorter (Beckman Coulter, Pasadena, CA, USA).

Functional CBA beads were purchased from BD Biosciences (San Jose, CA). Recombinant SARS COV2 antigens were purchased from commercial vendors: RBD-His was purchased from (Raybiotech, #230–30162) and (Sino Biological #40150-V08B2); S1 ECD-His (40591-V08H), S1D614G (40591-V08H3), S2 ECD-His (40590-V08B), S1+S2 ECD (40589-V08B1) were purchased from Sino Biological; Recombinant SARS-CoV-2 Nucleocapsid Protein (NP) was purchased from (Raybiotech).
Streptavidin-PE, streptavidin-BV421, anti-human IgG-PE, anti-human IgM- V450, and biotinylated anti-human IgA monoclonal antibodies were purchased from BD Biosciences (San Jose, CA). The specificity of these reagents was confirmed by ELISA and/or flow cytometry.

Antigen-coupled microbeads were added to protein LoBind 1.5 mL Eppendorf tubes (Eppendorf Cat#525-0133) in a volume of 50 μL of PBS containing a total of 5000–6000 beads. After centrifugation (2000 r.p.m., 5 min), microbeads were resuspended with 100 µL of pre-diluted (PBS) serum samples or serially diluted commercial antibodies against RBD (GenScript Cat#A02038) or N (Acrobiosystems Cat#NUN-S41) starting from a 1 mg/mL concentration. Negative control samples were prepared with PBS. After a 30 min incubation (RT protected from light), samples were washed three times in PBS. Secondary antibodies were diluted in 100 µL of PBS containing 5% FBS: anti-human IgG-PE (1:50) (Clone G18-145, BD Biosciences Cat#555787) and anti-human IgM-BV421 antibodies (1:1000) (Clone G20-127, BD Biosciences Cat#555783). The mix was incubated with the samples for 15 min at RT protected from light. One final wash was performed, and microbeads were resuspended in 200 µL of PBS supplemented with 5% FBS for acquisition. At least 600 events for each type of microbead were acquired in a FACSymphony flow cytometer (BD Biosciences) and geometric mean fluorescence intensities (gMFI) were obtained. Results were analysed using FlowJo version 10 (BD Biosciences).

Lymphocytes were isolated from cranial bone marrow using a previously reported protocol^{37 (link)}. In brief, the calvaria was cut into small pieces using sterile scissors and dissociated in PBS + 2% FBS with a pestle. Spleens were harvested from mice, washed in PBS and cut into 0.5 mm cubes in ice-cold PBS. Spleen or bone marrow isolates were transferred to a 70 μm cell strainer (Falcon, catalog no. 352350); cells were washed with PBS and resuspended in red blood cell lysis buffer (BioLegend, catalog no. 420301). Cells were stained for 30 min on ice using the following antibodies: anti-CD3 BV711 or anti-CD3-AF647 (clone okt3, BD Biosciences, catalog nos. 750983 and 566686)), anti-MuSK PE (clone 189-1 or 24C10), antihuman Ig light chain λ PE (clone MHL-38, BioLegend, catalog no. 316608), antihuman Ig light chain κ APC (clone MHK-49, BioLegend, catalog no. 316510), antihuman IgG PE (BD Biosciences, catalog no. 555787) and/or antimouse IgG APC (clone A85-1, BD Biosciences).