Dylight405 conjugated anti human igm

The anti‐SARS‐CoV‐2 RBD antibody TRES224 (human IgGκ) [34 (link), 35 (link)] has been described previously and was produced by Celltheon (San Francisco, CA). TRES328 (human IgG1κ, NTD binder), TRES480, and TRES567 (human IgG1κ, nonbinders) were generated during the same immunization and purified from transfected HEK293T cells according to standard procedures [35 (link)]. For flow cytometric analyses, anti‐human IgG FITC antibody and DyLight405‐conjugated anti‐human IgM were purchased from Jackson (Dianova, Hamburg), PE‐conjugated anti‐human IgA, and AF647‐ and Cy5‐conjugated anti‐human IgG were obtained from Southern Biotech (Birmingham, AL). Anti‐SARS‐CoV‐2 RBD antibody R10933 (Regeneron) [19 (link)] was produced by Sino Biological (Eschborn, Germany). Flow cytometric analysis of surface SARS‐CoV‐2 spike protein on Ramos‐null spike cells (RSp and mutants) and transfected HEK293T cells (HEK‐Dox‐Spike) was performed by staining cells with anti‐RBD‐binding antibody TRES224 in FACS buffer (PBS/2% FCS/0.05 % sodium azide) for 15 min on ice. Next, cells were washed with FACS buffer and then stained with AF647‐ or Cy5‐conjugated anti‐human IgG antibodies in FACS buffer for 15 min on ice. After washing with FACS buffer, cells were analyzed by using a Gallios or Cytoflex flow cytometer (Beckman Coulter) and Kaluza flow cytometry software (Beckman Coulter).

HEK293T cells were transiently transfected using the PEI method with the SARS‐CoV‐2‐spike‐encoding plasmid pCG1_CoV_2019S [41 (link)] in combination with a GFP encoding plasmid. HEK293T cells transfected only with a GFP‐encoding plasmid served as a negative control. Two days after transfection, SARS‐CoV‐2 spike/GFP‐co‐transfected and GFP‐only transfected HEK293T cells were stained with serum samples (1:100 dilution) from SARS‐CoV‐2‐infected patients and noninfected controls followed by staining with a secondary antibody mixture (2° antibody mix) consisting of PE‐conjugated anti‐human IgA (Southern Biotech, Birmingham, AL), AF647‐conjugated anti‐human IgG (Southern Biotech), and DyLight405‐conjugated anti‐human IgM (Jackson Immuno Research, Dianova, Hamburg) antibodies. Cells were analyzed using a Gallios flow cytometer (Beckman Coulter) or a Cytoflex flow cytometer (Beckman Coulter) and Kaluza flow cytometry software (Beckman Coulter).