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SARS-CoV-2 Antibody Profiling User Guide

This workflow and its products described here are for research use only and is not to be used for any other purposes, including, but not limited to, in vitro diagnostics, clinical diagnostics, or use in humans. The document and its content are proprietary to Fluidic Analytics and is intended for use only in connection with the products described herein and for no other purposes.

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1. Intended use

This user guide describes affinity-based antibody profiling against the SARS‑CoV‑2 receptor binding domain (RBD) protein directly in COVID‑19 patient serum samples by use of the Fuidity One‑W Serum instrument.

 

2. Background

COVID‑19 is a respiratory disease caused by the coronavirus SARS‑CoV‑2 that produces mild symptoms in most individuals. However, dependent on underlying health issues and other factors such as age, gender, or genetic makeup, symptoms can vary dramatically in severity. A complete and accurate immune response assessment of COVID-19 patients or vaccinated individuals based on the two key determining factors, namely antibody concentration and affinity, is key to:

  • Understanding the differences in disease severity
  • Assessing the strength and duration of acquired immunity
  • Evaluating the mechanisms and immunological effects of virus-neutralizing antibodies
  • Characterizing cross-reactivity with other, less harmful, coronaviruses

This user guide describes affinity-based antibody profiling against the SARS‑CoV‑2 RBD for a comprehensive immune response assessment in COVID‑19 patients or vaccinated individuals.

 

3. Affinity-based antibody profiling – assay principle

This user guide describes all steps required to characterize the immune response against the SARS‑CoV‑2 RBD directly in minimally diluted serum samples. After RBD labeling, three independent serum titration curves are measured to enable the simultaneous determination of antibody affinity and concentration.

 

4. Before you start

Good laboratory practice when working with serum samples

For use of this user guide we recommend working with serum rather than plasma. Serum collected from COVID‑19 patients (whether newly infected or recovered), and uninfected people who could still harbour infectious agents must be handled using a high level of precaution and at the required biosafety level for your country’s regulations. In addition, all serum samples should be handled as if capable of transmitting infectious agents.

SARS-CoV-2 Antibody profiling infograph

Serum preparation and storage

For preparation of serum samples, leave whole blood clotting for half an hour at room temperature immediately after collection. Spin down at 2,000 × g for 10 min at 4 °C.

  • Immediately prepare 50 µL aliquots from the supernatant and freeze samples at -80 °C
  • Do not snap freeze
  • Only thaw samples once; additional freeze-thaw cycles will negatively impact the data
  • Additives are not recommended, and addition of surfactants is strongly discouraged as it will negatively impact the data

If serum is received frozen, store immediately at -80 °C. After thawing the sample for the first time, remove the volume required for the assay, and aliquot the remaining volume into 50 µL aliquots before re-freezing at -80 °C.

When using plasma be aware that recurring freeze-thaw cycles can lead to precipitation which might negatively impact the measurements.

 

5. Required equipment, reagents and consumables

5.1. Equipment

  • Fluidity One‑W Serum (Fluidic Analytics F1W0001 - SRM)
  • Nanodrop (Thermo Fisher ND‑ONE‑W or equivalent)
  • Pipettes (1000 µL, 200 µL, 10 µL)
  • Zeba™ Desalting Chromatography Cartridges, 7K MWCO, 1 mL (Thermo Fisher 89934)
  • pH meter (various suppliers)
  • Centrifuge 5430R (Eppendorf 5428000060, or equivalent)

 

5.2. Reagents

  • Alexa Fluor™ 647 NHS Ester (Thermo Fisher A20006)
  • DMSO (anhydrous) (Invitrogen, D12345-10 x 3 mL)
  • SARS‑CoV‑2 RBD: 100 µg (sufficient for affinity measurements of 100 patient samples) (ACROBiosystems, SPD‑C52H3‑100ug)
  • NaHCO₃ (Merck S6014)
  • PBS buffer at pH 7.4 (Merck P4417)
  • Tween® 20 (Merck P7949)
  • Glycerol (Sigma G9012)
  • Ultrapure water (various suppliers)
  • HCl solution at 1 M for pH adjustment (various suppliers)

 

5.3. Consumables

  • Fluidity One‑W consumables, 4 boxes of chips (sufficient for 96 measurements), 1 filled cartridge (F1W0002)
    Alternatively: Fluidity One‑W consumables – Medium, 12 boxes of chips (sufficient for 288 measurements), 3 filled cartridges (F1W0003)
  • 1000 µL, 200 µL, 10 µL low-retention graduated tips (various suppliers)
  • Protein Lo-Bind tubes (Eppendorf 022431081 1.5 mL; Eppendorf 0030108302 5 mL)
  • Sterile filters, 0.22 µm, PVDF (sterile, various suppliers)
  • 50 mL syringes, non-sterile (various suppliers)
  • 10 mL syringes, non-sterile (various suppliers)
  • 5 mL syringe (various suppliers)
  • 2 x 1 mL syringe (various suppliers)
  • 96-well assay plates, flat bottom clear, black polystyrene, non-binding surface (Cat. No. 3881, Corning®)
  • TopSeal™-A PLUS, clear adhesive microplate seal (Cat. No. 6050185, PerkinElmer)
  • 50 mL Falcon tubes (various suppliers)
  • Eppendorf rack (various suppliers)
  • Disposable needle (various suppliers)

 

6. Protocol

  

6.1. RBD Labeling

Required equipment, reagents and consumables

 

Required equipment:

  • Nanodrop (Thermo Fisher ND‑ONE‑W or equivalent)
  • Pipettes (1000 µL, 200 µL, 10 µL)
  • Zeba™ Desalting Chromatography Cartridges, 7K MWCO, 1 mL (Thermo Fisher 89934)
  • pH meter (various suppliers)

 

 Required reagents:

  • Alexa Fluor™ 647 NHS Ester (Thermo Fisher A20006)
  • DMSO (anhydrous) (Invitrogen, D12345-10 x 3 mL)
  • SARS‑CoV‑2 RBD: 100 µg (sufficient for affinity measurements of 100 patient samples) (ACROBiosystems, SPD‑C52H3‑100ug)
  • Sodium bicarbonate NaHCO₃ (Merck S6014)
  • Ultrapure water (various suppliers)
  • HCl solution at 1 M for pH adjustment (various suppliers)
  • PBS buffer at pH 7.4 (Merck P4417); sterile filtered
  • PBS-T buffer at pH 7.4 (Merck P4417, 0.05% Tween® 20 – Merck P7949); sterile filtered
  • Glycerol (Sigma G9012)

 

Required consumables:

  • Protein Lo-Bind tubes 1.5 mL (Eppendorf 022431081)
  • 1000 µL, 200 µL, 10 µL low-retention graduated tips (various suppliers)
  • Sterile syringe filters, 0.22 µm, PVDF (various suppliers)
  • 5 mL syringes (various suppliers)
  • 1 mL syringes (various suppliers)
  • Disposable needle (various suppliers)

  

Preparation of required solutions

Prepare label stock solution

  • Immediately before use, dissolve 1 mg of Alexa Fluor™ 647 NHS Ester in 80 µL of DMSO to prepare a 10 mM solution
  • Aliquot in 5 µL portions and store at -20 °C
  • Label stock solution can be used for at least 4 weeks if stored at -20 °C
  • Do not re-freeze aliquots once thawed

 

Prepare 6-fold concentration labeling buffer

  • Dissolve 8.4 g of sodium bicarbonate (NaHCO₃) in 95 mL of ultrapure water
    •   Note: This might take 10 – 15 min depending on stirring speed and temperature
  • Adjust pH to 8.3 with 1 M HCl if required
  • Add ultrapure water to a final volume of 100 mL
  • Filter using a pore size of 0.22 µm
  • Store at -20 °C in 1 mL aliquots. If stored at -20 °C, 6-fold concentration labeling buffer can be used for up to 4 weeks

 

Protocol

SARS-CoV-2 spike RBD labeling

  • Use 100 µg of RBD at a concentration of 1 mg/mL (i.e., 40 µM) in PBS buffer (pH 7.4)
  • Add 20 µL of 6-fold concentration labeling buffer and mix carefully by pipetting up and down 5 times. Do not vortex.
  • Add 1.5 µL of label stock solution to SARS-CoV-2 spike RBD and carefully pipette up and down 5 times for mixing, using a 100 µL pipette. Do not vortex.
  • Incubate the labeling reaction at 4 °C overnight. Protect from light.

 

Removal of unbound Alexa Fluor™ 647 NHS Ester using a desalting column

  • Connect a 5 mL plastic syringe to the desalting column and equilibrate with 5 mL of PBS (pH 7.4). Note: do not use labeling buffer
  • Place 10 Eppendorf tubes in a rack, leave the lids open
  • Connect a disposable needle to the 1 mL plastic syringe
  • Draw the labeling mixture into the 1 mL plastic syringe
  • Remove the needle from the syringe and dispose accordingly
  • Remove trapped air from the syringe before connecting it to the desalting column
  • Push the sample through the column. Note: It is not necessary to collect the flow- through at this stage
  • Fill a second, unused 1 mL plastic syringe with 1 mL of PBS (pH 7.4) buffer
  • Elute the sample by collecting 100 µL fractions in the prepared Eppendorf tubes. Read the fraction volumes using the scale of the syringe
  • The protein will elute in fractions 2 – 4, and higher number fractions will contain unbound label

 

Labeling QC

  • Measure protein yield as well as labeling ratio
    •   To determine the protein yield as well as labeling ratio, measure the absorbance of SARS‑CoV‑2 spike RBD at a wavelength of 280 nm and the absorbance of the conjugated label at a wavelength of 645 nm on a Nanodrop (select Alexa Fluor 647)
  • The labeling ratio should be close to 1 label per protein
  • Typical yields are ≥75 µg
  • Typical concentrations are >1 µM
    •   If considerably higher label-to-protein ratios are measured, unbound label was not completely removed, and the desalting procedure should be repeated
  • Check Rh of SARS‑CoV‑2 RBD on Fluidity One‑W Serum
    •   Dilute the protein to a concentration of 100 nM in a volume of 10 µL using PBS-T buffer (pH 7.4)
    •   Pipette 5 µL on a microfluidic chip and perform a single measurement at the 1.5 – 8 nm setting
  • The typical Rh of SARS‑CoV‑2 spike RBD is 3.6 ± 0.2 nm
    •   If an Rh lower than 3.3 nm is measured, unbound label was not completely removed, and the desalting procedure needs to be repeated
  • Stock solution of the labeled protein (Alexa Fluor 647–RBD) is stored in PBS, containing 10% glycerol at -80 °C

 

6.2. Serum measurements

Required equipment, reagents and consumables

Required equipment

  • Fluidity One‑W Serum (Fluidic Analytics F1W0001 - SRM)
  • Pipettes (1000 µL, 200 µL, 10 µL)
  • Centrifuge 5430R (Eppendorf 5428000060, or equivalent)

 

Required reagents

  • PBS buffer at pH 7.4 (Merck P4417)
  • Tween® 20 (Merck P7949)
  • Alexa Fluor 647–RBD probe (see above)
  • Serum samples

 

Required consumables

  • Fluidity One‑W consumables, 4 boxes of chips (sufficient for 96 measurements), 1 filled cartridge (F1W0002)
    Alternatively: Fluidity One‑W consumables – Medium, 12 boxes of chips (sufficient for 288 measurements), 3 filled cartridges (F1W0003)
  • 1000 µL, 200 µL, 10 µL low-retention graduated tips (various suppliers)
  • Protein Lo-Bind tubes (Eppendorf 022431081 1.5 mL; Eppendorf 0030108302 5 mL)
  • Sterile syringe filters, 0.22 µm, PVDF (various suppliers)
  • 50 mL syringes, non-sterile (various suppliers)
  • 10 mL syringes, non-sterile (various suppliers)
  • 96 Well Assay Plates, flat bottom clear, black polystyrene, non-binding surface (Cat. No. 3881, Corning®)
  • TopSeal™-A PLUS, clear adhesive microplate seal (Cat. No. 6050185, PerkinElmer)

 

Preparation of required solutions

1% PBS-T (stock solution)

  • Add 0.5 mL of Tween® 20 to 49.5 mL PBS and stir for at least 30 min at room temperature to obtain a stock solution of 1% Tween® 20 in PBS (pH 7.4)(PBS-T)
  • Filter sterilize the solution and store at 4 °C

 

0.2% PBS-T (working solution)

  • Mix 4 mL of filter sterilized PBS (pH 7.4) with 1 mL of 1% PBS-T stock solution
  • Vortex for 10 sec
  • Store at room temperature

 

Alexa Fluor 647–RBD (probe)

  • Thaw one aliquot of stock solution and determine the concentration if not known
  • For the preparations of the binding curves, prepare the following three stock dilutions:
    •   1 µM, 400 nM and 100 nM
    •   Dilute the RBD stock solution with 0.2% PBS-T to obtain 50 µL of each solution
    •   Keep on ice

 

Serum samples

  • Thaw three serum aliquots (150 µL) slowly on ice. Pool the aliquots and spin down
    5 min at 14,000 × g

 

Serum dilutions for background determination

  • For each serum sample, prepare 100%, 50% and 25% serum solutions using 0.2% PBS-T
    •   100% serum: 0 µL 0.2% PBS-T + 16 µL of 100% serum
    •   50% serum: 12 µL 0.2% PBS-T + 12 µL of 100% serum
    •   25% serum: 8 µL 0.2% PBS-T + 8 µL of 50% serum

 

Instrument set-up

  • Switch on the instrument (power button on the back of the instrument)
  • Wait until the instrument has booted up
  • Log into your account or use guest account
  • Check cartridge level. If required change cartridge as per the instrument User Guide

 

Protocol

Preparation of serum titration series

We recommend three titration curves with different concentrations of Alexa Fluor 647–RBD for the simultaneous determination of antibody affinity (KD) and concentration;

  • Prepare the titration series for the three affinity binding curves in a 96-well assay plate as follows:
    •   Cover the plate with a sheet of adhesive microplate seal and protect from light
    •   Incubate for 1 hour at room temperature
SARS-CoV-2 antibody profiling table 1

 

SARS-CoV-2 antibody profiling table 2

 

SARS-CoV-2 antibody profiling table 3

 

Serum background measurements

Whilst the solutions for the affinity binding curves are incubating, measure serum background:

  • Purpose: each serum sample has a different level of autofluorescence. This will be subtracted from the datapoints of the titration curves later. Serum background has to be measured on the same day as the samples
  • For each measurement, pipette 5 µL of the respective serum dilutions (100%, 50%, 25% and 0% serum in 0.2% PBS-T) onto a fresh Fluidity One‑W chip and insert it gently into the instrument
  • Press “single run”
  • Choose medium flow rate setting (1.5 – 8 nm)
  • Add the following labels:
    •   “Ligand”: Insert the name or ID of the patient serum sample
    •   “Ligand concentration”: Serum concentration
  • Press “start”
  • Measure each concentration in duplicate

 

Measurements of affinity binding curves

  • Measure each datapoint of titration series 1 (10 nM Alexa Fluor 647–RBD, constant) in duplicate at medium flow setting; repeat the measurement for any potential outlier
    •   Press “single run”
    •   Choose medium flow rate setting (1.5 – 8 nm)
    •   Add the following labels:
        o   “Ligand”: Insert the name or ID of the patient serum sample
        o   “Ligand concentration”: Serum concentration
        o   “Protein”: Alexa Fluor 647–RBD
        o   “Protein concentration”: 10 nM
    •   Press “start”
  • Measure each datapoint of titration series 2 (40 nM Alexa Fluor 647–RBD, constant) in duplicate at medium flow setting; repeat the measurement for any potential outlier
    •   Press “single run”
    •   Choose medium flow rate setting (1.5 – 8 nm)
    •   Add the following labels:
        o   “Ligand”: Insert the name or ID of the patient serum sample
        o   “Ligand concentration”: Serum concentration
        o   “Protein”: Alexa Fluor 647–RBD
        o   “Protein concentration”: 40 nM
    •   Press “start”
  • Measure each datapoint of titration series 3 (100 nM Alexa Fluor 647–RBD, constant) in duplicate at medium flow setting; repeat the measurement for any potential outlier
    •   Press “single run”
    •   Choose medium flow rate setting (1.5 – 8 nm)
    •   Add the following labels:
        o   “Ligand”: Insert the name or ID of the patient serum sample
        o   “Ligand concentration”: Serum concentration
        o   “Protein”: Alexa Fluor 647–RBD
        o   “Protein concentration”: 100 nM
    •   Press “start”

 

7. Data analysis

  

7.1. Data analysis support

For data analysis support, please contact Fluidic Analytics technical support at techsupport@fluidic.com

 

7.2. Results

After data submission, background subtraction will be performed for each measurement and the resulting data for all three titration series subjected to a global, non-linear fit to determine the following parameters:

Parameter Definition Constraints
Rh_free Hydrodynamic radius of the unbound Alexa Fluor 647–RBD (nm) Shared, must be greater than zero
Rh_complex Hydrodynamic radius of the antibody-bound Alexa Fluor 647–RBD (nm) Shared, must be greater than zero
A Antibody concentration in the sample serum (nM) Shared, must be greater than zero
n Stoichiometry factor Fixed to 1.0 
P Concentration of Alexa Fluor 647–RBD in each experiment (nM) Fixed to 10, 40 and 100 nM respectively
KD Dissociation constant (nM) Shared, must be greater than zero
SARS-CoV-2 antibody profiling graph

Concentration of antibody binding sites: [ab] = 34.5 nM (± 2 nM)
KD = 1 nM (± 0.5 nM)

Nanodrop™, Alexa Fluor™, and Zeba™, are trademarks of Thermo Fisher Scientific
TopSeal™ is a trademark of PerkinElmer
Tween® is a trademark of Merck


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