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.
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
- Reconstitute lyophilized recombinant proteins to a final concentration of 0.1 mg/mL with deionized, sterile-filtered water.
- Calculate the molar concentration (molarity) of the protein solution (figure 1).
- Prepare 10 nM solutions for each protein by diluting the stock solution of the recombinant protein with assay buffer.
Table 1: Exemplary calculation of the concentrations 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. It is recommended to use a 1:10−1:500 dilution.
- 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 2× with 1 mL assay buffer. For this, centrifuge at 1000×g for 1 min at room temperature, aspirate supernatant, and resuspend beads in 1 mL of assay buffer.
- Resuspend Streptavidin-coated beads in 500 µL of 10 nM solution of recombinant proteins.
- Incubate for at least 15 min at room temperature using the MACSmix™ Tube Rotator. Set on maximum permanent run speed.
- Centrifuge beads coupled to recombinant proteins at 1000×g for 1 min at room temperature.
- Wash beads coupled to recombinant proteins 1× with with 1 mL assay buffer. For this, centrifuge at 1000×g for 1 min at room temperature, aspirate supernatant, and resuspend beads in 1 mL of assay buffer.
- Resuspend beads coupled to recombinant proteins in 1 mL of assay buffer.
- Per sample, transfer 50 µL of beads coupled to recombinant protein to suitable polypropylene or polystyrene reagent tubes or appropriate wells of a MACSPlex Filter Plate.
- To remove the assay buffer from the sample, centrifuge at 1000×g for 1 min at room temperature and aspirate the supernatant.
- Resuspend beads in 100 µL of diluted plasma or serum.
- Incubate for 1 hour at room temperature while shaking.
▲Note: When working with tubes, use the MACSmix™ Tube Rotator set on maximum permanent run speed. When working with MACSPlex Filter Plate, use an orbital shaker for microtiter plates with a shaking speed set at 500 rpm. - To remove the plasma/serum after incubation, centrifuge at 1000×g for 1 min at room temperature and aspirate the supernatant.
- Wash 4× with assay buffer, e.g., when using standard reagent tubes, centrifuge at 1000×g for 1 min at room temperature, aspirate supernatant, and resuspend in 1 mL of assay buffer.
- After the last washing step, aspirate supernatant and resuspend in 100 µL of diluted Detection Reagent.
- Incubate in the dark for 10 min at room temperature while shaking.
▲Note: When working with tubes, use the MACSmix Tube Rotator set on maximum permanent run speed. When working with MACSPlex Filter Plate, use an orbital shaker for microtiter plates with a shaking speed set at 500 rpm. - Wash to remove unbound Detection Reagent, e.g., when using standard reagent tubes, centrifuge at 1000×g for 1 min at room temperature and aspirate supernatant.
- Wash 1× with assay buffer as described above.
- Resuspend in 100 µL of assay buffer and proceed to flow cytometric analysis.
- Prior acquisition, make sure lasers of the flow cytometer, e.g., the MACSQuant® Analyzer 10, are properly calibrated by using, e.g., MACSQuant Calibration Beads. In case more than one fluorochrome is used, compensation of the spillover of fluorochrome emissions has to be established in advance using cells or the MACS Comp Bead Kit, anti-REA.
- Adjust forward scatter (FSC) and side scatter (SSC) parameters.
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.
- 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
Ig λ Light Chain Antibody, anti-human, PE
IgG Antibody, anti-human, PE
IgM Antibody, anti-human, PE
IgA Antibody, anti-human, PE
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 as used in this protocol. If other beads are used, volumes used for conjugation of recombinant proteins and amount of beads per test have to be titrated in advance to achieve optimal performance.
- Plasma samples of COVID-19 convalescents and healthy donors
- Detection Reagent
Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use.
If not otherwise specified, all marks displayed on the Miltenyi Biotec Internet site and documents are subject to the trademark rights of Miltenyi Biotec, including each of Miltenyi Biotec's primary brands, its model name plates, and its corporate logos and emblems.
For copyright note and a complete list of trademarks visit www.miltenyibiotec.com/legalnotes.
