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Affinity-based SARS-CoV-2 Surrogate Virus Neutralization Test

Scientist-inspecting-a-Microfluidic-Diffusional-Sizing-chip

Advent Letters for Phrase - IThis 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 an affinity-based receptor competition assays to detect virus-neutralizing antibodies (NAbs) directly in COVID-19 patient serum samples by use of the Fluidity One-W Serum instrument.

1.1. Background

COVID-19 is a respiratory disease caused by the coronavirus SARS-CoV-2. In order to infect a human cell, SARS-CoV-2 utilizes spike proteins on its surface to bind to the human angiotensin-converting enzyme 2 (ACE2) receptor protein which is typically found on the surface of human lung cells. Upon entry into the cell, the virus replicates causing the immune system to respond by producing different types of antibodies. These include virus-neutralizing antibodies (NAbs) which are crucial to protect the human body from future infections.

While conventional cell neutralization assays offer a well-established way to determine the presence of neutralizing antibodies, these assays are cumbersome and lengthy and often require live biological materials and strict biosafety regulations.

To overcome these limitations we have developed a rapid surrogate virus-neutralization test that mimics the virus neutralization process by measuring the binding interaction between the ACE2 receptor and the SARS-CoV-2 spike S1 protein, as well as the subsequent displacement of the spike protein in the presence of virus-neutralizing antibodies (NAbs) directly in patient serum.

 

2. Affinity-Based Receptor Competition Assay

This User Guide describes the use of a rapid affinity-based receptor binding competition assay as an alternative to conventional cell-based neutralization assays. The assay can be performed in less than two hours (excluding fluorescence labeling) and requires prior knowledge of antibody concentrations.

For the assay, labeled ACE2 is mixed with unlabeled SARS-CoV-2 spike S1 protein. After incubating at room temperature (RT) for 40 min to allow complex formation, patient serum is added followed by a second incubation of 60 min at RT. In the presence of virus-neutralizing antibodies (NAbs), the SARS-CoV-2 spike S1 protein will dissociate from the ACE2/SARS-CoV-2 spike S1 complex and bind to the neutralizing antibodies. As a result, the hydrodynamic radius (Rh) will decrease compared to the Rh measured for the complex. In the absence of NAbs, ACE2 remains bound to SARS-CoV-2 spike S1 protein resulting in an unchanged Rh.

NeutralizingQUICK_compromise2b

Figure 1: Labeled ACE2 is mixed with unlabeled SARS-CoV-2 spike S1 protein. After complex formation, patient serum is added. In the presence of virus-neutralizing antibodies (NAbs), the SARS‑CoV‑2 spike S1 protein will dissociate from the ACE2/SARS‑CoV‑2 spike S1 complex and bind to the neutralizing antibodies. As a result, Rh will decrease compared to Rh measured for the complex. In the absence of NAbs, ACE2 remains bound to SARS‑CoV‑2 spike S1 protein resulting in an unchanged Rh.

user guide 2 workflow

3. Before you start

Good laboratory practice when working with serum samples

Serum or plasma collected from COVID-19 patients (whether newly infected or recovered) as well as “uninfected” people who could still harbour infectious agents must be handled using a high level of precaution and at the recommended biosafety level (dependent on national legislation, will vary between countries). In addition, all serum samples should be handled under the assumption that they contain infectious agents.

 

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 the increased sample viscosity will negatively impact the data

If samples are 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.

 

4. Required equipment, reagents and consumables

 

4.1. Equipment

  • Fluidity One-W Serum (Fluidic Analytics F1W0001 - SRM)
  • Nanodrop (Thermo Fisher ND-ONE-W or equivalent)
  • Pipettes (1000 µL, 200 µL, 10 µL)
  • pH meter (various suppliers)
  • Centrifuge 5430R (Eppendorf 5428000060, or equivalent)
  • Optional: Table-top centrifuge (various suppliers)
  • Optional: AEKTA protein-chromtography system (Cytiva, AEKTA pure 25)

 

4.2. Reagents

  • Alexa Fluor™ 647 NHS Ester (Thermo Fisher A20006)
  • DMSO (anhydrous) (Invitrogen, D12345-10 x 3 mL)
  • Human Angiotensin converting enzyme 2 (ACE2): 50 µg (1 µg of labeled protein will be sufficient for >50 samples) (ACROBiosystems, AC2-H52H8-50ug)
  • SARS-CoV-2 spike S1: 100 µg (sufficient for testing >5,000 patient samples) (ACROBiosystems, S1N-C52H4-100ug)
  • SARS-CoV-2 negative human serum (Merck H5667) – to be used as negative control
  • 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)
  • NaOH solution at 1 M for pH adjustment (various suppliers)

 

4.3. Consumables

  • Fluidity One-W consumables – Medium; 12 boxes of chips (sufficient for 288 measurements), 3 filled cartridges (F1W0003)
  • Zeba™ Desalting Chromatography Cartridges, 7K MWCO, 1 mL (Thermo Fisher 89934)
  • 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)
  • 5 mL syringe (various suppliers)
  • 2 x 1 mL syringe (various suppliers)
  • Eppendorf rack (various suppliers)
  • Disposable needle (various suppliers)
  • Optional: 2 x Amicon® Ultra 0.5 mL Centrifugal Filters (MWCO 10 kDa) (Merck UFC5010)
  • Optional: Superdex 200 Increase 3.2/300 column (Cytiva 28990946)

 

5. Protocol

 

5.1. ACE2 Labeling

Required equipment, reagents and consumables

Required Equipment:

  • Nanodrop (Thermo Fisher ND-ONE-W or equivalent)
  • Pipettes (1000 µL, 200 µL, 10 µL)
  • pH meter (various suppliers)
  • Optional: Table-top centrifuge (various suppliers)
  • Optional: AEKTA protein-chromtography system (Cytiva, AEKTA pure 25)

 

Required Reagents:

  • Alexa Fluor™ 647 NHS Ester (Thermo Fisher A20006)
  • DMSO (anhydrous) (Invitrogen, D12345-10 x 3 mL)
  • Human Angiotensin converting enzyme 2 (ACE2): 50 µg (1 µg of labeled protein will be sufficient for >50 samples) (ACROBiosystems, AC2-H52H8-50ug)
  • SARS-CoV-2 negative human serum (Merck H5667) – to be used as negative control
  • NaHCO₃ (Merck S6014)
  • Glycerol (Sigma G9012)
  • Ultrapure water (various suppliers)
  • HCl solution at 1 M for pH adjustment (various suppliers)
  • NaOH solution at 1 M for pH adjustment
  • PBS buffer at pH 7.4 (Merck P4417)
  • PBS-T buffer at pH 7.4 (PBS plus 0.05% Tween 20); sterile filtered

 

Required consumables:

  • Zeba™ Desalting Chromatography Cartridges, 7K MWCO, 1 mL (Thermo Fisher 89934)
  • 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)
  • 5 mL syringe (various suppliers)
  • 2 x 1 mL syringe (various suppliers)
  • Disposable needle (various suppliers)
  • Optional: 2 x Amicon® Ultra 0.5 mL Centrifugal Filters (MWCO 10 kDa) (Merck UFC5010)
  • Optional: Superdex 200 Increase 3.2/300 column (Cytiva 28990946)

 

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 to achieve a 1 M solution
    •   Note: This might take 10 – 15 min depending on stirring speed and temperature
  • Adjust pH to 8.3 with 1 M HCl or 1 M NaOH as 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

Human Angiotensin Converting Enzyme2 (ACE2) Labeling

  • Dissolve 50 µg of ACE2 at a concentration of 0.5 mg/mL in 100 µL PBS buffer at pH 7.4 (final concentration of 5.8 µM)

  • Note: If the ACE2 protein is delivered already dissolved in Tris-buffer, buffer exchange into PBS buffer (pH 7.4) prior to labeling

  • Add 20 µL of 6 x labeling buffer and mix carefully by pipetting up and down 5 times. Do not vortex

  • Dilute label stock solution to a final concentration of 1 mM using DMSO

  • Add 1.7 µL of the 1 mM label solution to the ACE2 solution and carefully pipette up and down 5 times. 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 for this equilibration step
  • 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

 

Removal of unbound Alexa Fluor™ 647 NHS Ester using an size-exclusion chromatography

If an protein-chromatography system (i.e., AEKTA protein-chromatography system) is available in the lab, we would recommend using size‑exclusion chromatography for clean-up of ACE2.

  • Concentrate the labeling mixture to a volume of 60 µL using Amicon® Ultra 0.5 mL Centrifugal Filters with a molecular weight cut-off of 10 kDa
  • Mount a Superdex 200 Increase 3.2/300 column to an protein-chromatography system and equilibrate with PBS buffer pH 7.4
  • Set the detection wavelength to absorbance at 645 nm to measure labeled ACE2 and unbound Alexa Fluor™ 647 NHS Ester (use both 280 nm and 645 nm for multi-wavelength systems)
  • Inject 60 µL of concentrated labeling mixture using a 50 µL sample loop
  • Purify ACE2 at the recommended flow rate and collect fractions of 100 µL
  • Collect and pool fractions of the main peak which elutes at approximately 1.5 mL
  • Concentrate pooled fractions to a volume of 60 µL using Amicon® Ultra 0.5 mL Centrifugal Filters with a molecular-weight cut off of 10 kDa
  • Discard fractions containing potential aggregates eluting <1.3 mL
  • Discard fractions containing unbound Alexa Fluor™ 647 NHS Ester eluting >2.1 mL

 

Labeling QC

  • Measure protein yield as well as labeling ratio
    •   To determine the protein yield as well as labeling ratio, measure the absorbance of labeled ACE2 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)
         o Use the “Proteins & Labels” function of the nanodrop, select Alexa Fluor 647 as a dye and use 169180 M-1 cm-1 as an extinction coefficient for ACE2
         o The labeling ratio should be close to 1 label per protein
         o Typical yields are ≥1 µg
         o Typical concentrations are >1 µM
    •   If the label-to-protein ratios are considerably higher, unbound label was not completely removed, and the purification should be repeated
  • Determine the Rh of ACE2 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
         o The typical Rh of ACE2 in PBS-T is 6.5 ± 0.2 nm
         Note: The size will decrease to 5.0 ± 0.2 nm when measuring in serum
    •   If an Rh lower than 6.5 nm is measured in PBS-T, unbound label was not completely removed, and the desalting procedure should be repeated
  • Add 10% glycerol to the labeled protein
  • Prepare 5 µL aliquots at a concentration of 200 nM
  • Store at -80 °C for ~4 weeks

 

5.2. 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 NHS Ester–ACE2 probe (see step 5.1)
  • SARS-CoV-2 spike S1: 100 µg (sufficient for testing >5,000 patient samples) (ACROBiosystems, S1N-C52H4-100ug)
  • Serum samples
  • SARS-CoV-2 negative human serum (Merck H5667) – to be used as negative control

 

Required consumables

  • 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)

 

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 Instructions

 

Preparation of required solutions

Reconstitution of SARS-CoV-2 spike S1 protein

  • Reconstitute 100 µg of SARS-CoV-2 spike S1 protein in 167 µL of deionised water to achieve a concentration of 7.8 µM
  • Take 5 µL of this stock solution and mix with 190 µL PBS/10% glycerol to achieve a final concentration of 200 nM (final volume 195 µL)
  • Prepare 5 µL aliquots and store aliquots as well as stock solution at -80 °C for ~4 weeks

 

Protocol

  • Thaw a 5 µL aliquot of labeled ACE2 stock solution (200 nM) on ice
  • Thaw a 5 µL aliquot of SARS-CoV-2 spike S1 stock solution (200 nM) on ice
  • In a tube, prepare a mastermix by combining 1.1 µL ACE2 stock solution and 1.1 µL SARS-CoV-2 spike S1 solution per sample (inclusive negative control serum or plasma) to be analysed
  • Incubate protected from light for 40 min at room temperature
  • For the measurements, pipette 18 µL of patient serum or plasma at an antibody concentration of 28 nM into a tube or well (dilute to the appropriate concentration if needed using 0.2% PBS-T buffer)
  • Add 2 µL of the ACE2/SARS-CoV-2 mastermix to each sample
  • Mix samples by slowly pipetting up and down five times
  • Incubate the samples for 60 min at room temperature, protect from light

 

Serum background measurements

Whilst the ACE2/SARS-CoV-2 spike S1/serum samples are are incubating, measure serum background:

  • Purpose: each serum sample has a different level of autofluorescence. This will be subtracted from the data during the analysis. Serum background has to be measured on the same day as the samples
  • For each measurement, pipette 5 µL of a 50% serum dilution 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 background sample in duplicate

 

Sample Measurements

  • Measure each patient sample as well as the SARS-CoV-2 negative serum (negative control) in triplicate at 1.5 – 8 nm setting
    •   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”: SARS-CoV-2 spike S1 / ACE2 Alexa 647
         o “Protein concentration”: 10 nM
    •   Press “start”

 

6. Data Analysis

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

 

7. Results

After data submission, background subtraction will be performed for each measurement. If virus-neutralizing antibodies (NAbs) are present in the sample, the SARS-CoV-2 spike S1 protein will dissociate from the ACE2/SARS-CoV-2 spike S1 complex and bind to the neutralizing antibodies. As a consequence, the Rh will decrease compared to Rh measured for the complex in the negative control. In the absence of NAbs, ACE2 remains bound to SARS-CoV-2 spike S1 protein resulting in an unchanged Rh compared to the negative control.

This principle has been described in Schneider et al. https://www.medrxiv.org/content/10.1101/2020.09.20.20196907v1


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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