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This application protocol describes how SARS-CoV-2–specific antibodies can be detected in plasma or serum samples of COVID-19 Convalescents using our biotinylated recombinant SARS-CoV-2 proteins in a bead-based flow cytometric assay. Knowing the humoral response of SARS-CoV-2 convalescent patients is an essential background information to analyze the cellular responses, such as antigen-specific B cell analysis, more in detail.
Table 1: Exemplary concentration calculations of reconstituted recombinant proteins
|Recombinant protein variant||Concentration after reconstitution (mg/mL)||MW (kDa)||Corresponding molar concentration (µM)|
|Recombinant SARS-CoV-2 RBD (HEK)-Biotin||0.1||33.8||2.96|
|Recombinant SARS-CoV-2 RBD (Insect cells)-Biotin||0.1||34.2||2.92|
|Recombinant SARS-CoV-2 Nucleoprotein-Biotin||0.1||49.1||2.04|
|Recombinant SARS-CoV-2 Spike-Trimer (HEK)-Biotin||0.1||418.0||0.24|
|Recombinant SARS-CoV-2 Spike-S2 (insect cells)||0.1||61.2||1.63|
|Recombinant SARS-CoV-2 Spike-Prot (HEK)- Biotin||0.1||136.0||0.74|
|Recombinant Human ACE2 (HEK)-Biotin||0.1||87.2||1.15|
|Recombinant Human ACE2 (insect cells)||0.1||72.8||1.37|
Dilute serum or plasma samples with assay buffer (recommended is 1:10 to 1:500 dilution).
_Make sure lasers in the flow cytometer, e.g., the MACSQuant® Analyzer 10, are properly calibrated
(by using, e.g., the MACSQuant Calibration Beads), and in case more than one fluorochrome is used, compensation of the spillover of fluorochrome emissions has been established in advance using cells or the MACS® Comp Bead Kit, anti-REA.
_Adjust forward scatter (FSC) and side scatter (SSC) parameters.
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