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 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.

Protocol

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 (see formula below).

    3. Prepare 10 nM solutions for each protein by diluting stock solution of recombinant protein with assay buffer.

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

Table 1: Exemplary concentration calculations 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) 0.161.21.63
Recombinant SARS-CoV-2 Spike-Prot (HEK)- Biotin 0.1136.00.74
Recombinant Human ACE2 (HEK)-Biotin 0.187.21.15
Recombinant Human ACE2 (insect cells)0.172.81.37

Sample preparation

Dilute serum or plasma samples with assay buffer (recommended is 1:10 to 1:500 dilution).

Materials

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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