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.

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Protocol

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

1. Things to prepare in advance

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 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

Sample preparation

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

2. Overview

Protocol overview

Figure 2: Bead-based immunoassay for detection of patient-derived SARS-CoV-2 antibodies. Schematic overview illustrating how our biotinylated antigens can be applied to detect patient-derived SARS-CoV-2 antibodies.

3. Coupling of beads and recombinant proteins

Transfer 1x10⁶ Streptavidin-coated beads to suitable polypropylene or polystyrene reagent tubes. Prepare one tube for each recombinant protein to be used in the assay.
Wash Streptavidin-coated beads 2x with 1 mL assay buffer.
▲Note: For washing, centrifuge at room temperature (RT) at 1000xg for 1 minute, aspirate supernatant, and resuspend beads in 1 mL assay buffer.
Resuspend Streptavidin-coated beads in 500 µL of 10 nM solution of recombinant proteins.
Incubate for at least 15 minutes at RT using the MACSmix™ Tube Rotator. Set on maximum permanent run speed.
Centrifuge beads coupled to recombinant proteins at 1000xg for 1 minute at RT.
Wash beads coupled to recombinant proteins 1x with assay buffer as described above.
Resuspend beads coupled to recombinant proteins in 1 mL of assay buffer.

4. Detection of SARS-CoV-2–specific antibodies

Per each sample transfer 50 µL of beads coupled to recombinant protein to suitable polypropylene or polystyrene reagent tube or appropriate well of MACSPlex Filter Plate.
To remove the assay buffer from the sample: centrifuge at 1000xg for 1minute at RT and aspirate afterwards the assay buffer.
Resuspend beads in 100 µL of diluted plasma or serum.
Incubate for 1 hour at RT while shaking.
▲Note: When working with tubes: use MACSmix™ Tube Rotator which is set on maximum permanent run speed. When working with MACSPlex Filter Plate: use orbital shaker for microtiter plates with a shaking speed set at 500 rpm.
To remove the plasma / serum after incubation: centrifuge at 1000xg for 1minute at RT and aspirate the supernatant.
Wash beads 4x with assay buffer.
▲Note: For example, when using standard reagent tubes wash as described above: centrifuge at 1000xg for 1 minute at RT, aspirate supernatant, and resuspend beads in 1 mL assay buffer.
Resuspend beads in 100 µL of diluted Detection Reagent.
Incubate in the dark for 10 minutes at RT while shaking.
▲Note: When working with tubes: use MACSmix™ Tube Rotator which is set on maximum permanent run speed. When working with MACSPlex Filter Plate: use orbital shaker for microtiter plates with a shaking speed set at 500 rpm.
Remove unbound Detection Reagent.
▲Note: For example, when using standard reagent tubes centrifuge at 1000xg for 1 minute at RT and aspirate supernatant.
Wash beads 1x with assay buffer as described above.
Resuspend beads in 100 µL of assay buffer and proceed to flow cytometry analysis.

5. Flow cytometry analysis

Prior acquisition
_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.

Figure 3: Flow cytometric analysis of SARS-CoV-2–specific antibodies in serum samples.
Biotinylated recombinant SARS-CoV-2 proteins were used as above described to detect SARS-CoV-2–specific antibodies in serum samples. Samples were aquired using MACSQuant® Analyzer 10.

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.

Reagent and instrument requirements

Biotinylated SARS-CoV-2 Antigens and control antigens, e.g.:
Recombinant SARS-CoV-2 RBD (HEK)-Biotin (50 µg: # 130-127-457/ 10 µg: # 130-127-458)
Recombinant SARS-CoV-2 RBD (insect cells)-Biotin (50 µg; 130-127-469/ 10 µg, 130-127-445)
Recombinant SARS-CoV-2 Nucleoprotein-Biotin (50 µg; 130-127-467/ 10 µg, 130-127-454)
Recombinant SARS-CoV-2 Spike-Trimer (HEK)-Biotin (25 µg; 130-127-685)
Recombinant SARS-CoV-2 Spike-S1 (HEK)-Biotin (25 µg: 130-127-583)
Recombinant SARS-CoV-2 Spike-S2 (insect cells)-Biotin (25 µg; 130-127-688)
Recombinant SARS-CoV-2 Spike-Prot (HEK)- Biotin (25 µg; 130-127-682)
Recombinant Human ACE2 (HEK)-Biotin (50 µg; 130-127-465/ 10 µg 130-127-442) or Recombinant Human ACE2 (insect cells) (50 µg: # 130-127-464 / 10 µg: # 130-127-468) as control antigens to determine background binding of immunoglobulins
Material for detection of immunoglobulins specific to the antigen, e.g.:
Ig κ Light Chain Antibody, anti-human, PE (# 130-123-246)
Ig λ Light Chain Antibody, anti-human, PE (# 130-119-778)
IgG Antibody, anti-human, PE (# 130-119-878)
IgM Antibody, anti-human, PE (# 130-122-930)
IgA Antibody, anti-human, PE (# 130-113-476)
MACSQuant® Analyzer 10 (# 130-096-343) or other flow cytometers that can excite or detect PE fluorescence
MACS® BSA Stock Solution (# 130-091-376)
autoMACS® Rinsing Solution (# 130-091-222)
MACSPlex Filter Plate with 0.2 µm filters or suitable polypropylene or polystyrene reagent tubes
MACSmix™ Tube Rotator (# 130-090-753)
Streptavidin-coated beads suitable for flow cytometric analysis, e.g., 5 µm transparent PMMA-streptavidin beads from PolyAn were used in this protocol. If other beads are used, volumes used for conjugation of recombinant proteins and amount of beads per test should be titrated to achieve optimal performance.
Plasma samples of COVID-19 convalescents and healthy donors

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