Bead-based immunoassay for detection of patient-derived SARS-CoV-2 antibodies

This application protocol describes how SARS-CoV-2–specific antibodies can be detected in plasma or serum samples of COVID-19 convalescents using Miltenyi Biotec's biotinylated recombinant SARS-CoV-2 proteins in a bead-based flow cytometric assay. The humoral response of SARS-CoV-2−convalescent patients is an essential background information to analyze cellular responses, such as antigen-specific B cells, more in detail.


The materials and methods described in this protocol are for detection of patient-derived SARS-CoV-2 antibodies.

Reagent preparation

  • Assay buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS® BSA Stock Solution (# 130-091-376) 1:20 with autoMACS® Rinsing Solution (# 130-091-222).
  • Dilute selected Detection Reagent 1:50 with assay buffer.

Recombinant proteins

  1. Reconstitute lyophilized recombinant proteins to a final concentration of 0.1 mg/mL with deionized, sterile-filtered water.
  2. Calculate the molar concentration (molarity) of the protein solution (figure 1).
  3. Prepare 10 nM solutions for each protein by diluting the stock solution of the recombinant protein with assay buffer.

Figure 1: Formula to define the molar concentration (molarity) of the protein solution.

Table 1: Exemplary calculation of the concentrations of reconstituted recombinant proteins

Recombinant protein variantConcentration after reconstitution (mg/mL)MW (kDa)Corresponding molar concentration (µM)
Recombinant SARS-CoV-2 RBD (HEK)-Biotin0.133.82.96
Recombinant SARS-CoV-2 RBD (Insect cells)-Biotin0.134.22.92
Recombinant SARS-CoV-2 Nucleoprotein-Biotin0.149.12.04
Recombinant SARS-CoV-2 Spike-Trimer (HEK)-Biotin  0.1418.00.24
Recombinant SARS-CoV-2 Spike-S2 (insect cells)
Recombinant SARS-CoV-2 Spike-Prot (HEK)- Biotin 0.1136.00.74
Recombinant Human ACE2 (HEK)-Biotin
Recombinant Human ACE2 (insect cells)

Sample preparation

  • Dilute serum or plasma samples with assay buffer. It is recommended to use a 1:10−1:500 dilution.


The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.
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