Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy
January 12th, 2024
Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.
Proteomic analysis of enveloped virus revealed that host proteins are incorporated in new formed virus, affecting replication, effectivity, and pathogenesis. Our research focuses on profiling surface proteins in viral particles to find key elements in virus/host interaction, leading to new targets for antivirals and vaccines. Immune electron microscopy is the only imaging technique allowing the direct visualization of proteins expressing virus.
However, mass spectrometry immunocapture with magnetic beads and emerging flow biometry are now more commonly used to enable broader proteomic profiling and to screen a large number of viral particles. Our method uses color code illuminate speeds and dual list readout for immunocapture. It enables multiplex profiling of host surface proteins on intact variants, and screening of antibodies and antiviral drugs on multiple viruses and antigens simultaneously.
Mass spectrometry and affinity proteomics are powerful tools to identify both viral and host proteins on the variant surface. The strategy we describe will lead to a better understanding of the role of host proteins on virus particles, enabling high throughput profiling of infected samples in various experimental setups. To begin, prepare the working solution of NeutrAvidin in MES buffer in a 1.5 milliliter low protein binding microfuge tube.
Then, prepare the working solution of Goat immunoglobulin G control antibody in MES buffer. Vortex the diluted antibody solution. And centrifuge the tube.
Next, take the microtiter plate containing activated beads and place it on a magnetic plate separator for 30 seconds to immobilize the beads. With the microtiter plate still positioned on the magnetic separator, aspirate the supernatant from magnet-immobilized beads. Then, add 100 microliters of NeutrAvidin solution, antibody solution, and MES buffer to appropriate wells containing beads.
Seal the microtiter plate. And incubate for two hours on an orbital shaker at 650 RPM in the dark at room temperature. Then, wash beads with PBST using an automated washer.
After overnight incubation, prepare the recombinant human biotinylated ACE2 and biotin working solution in 10 millimolar PBS. Immobilize the microspheres on the magnetic plate separator for 30 seconds. And aspirate the supernatant from magnet-immobilized microspheres as shown earlier.
Remove the microtiter plate from the magnetic separator. And add 50 microliters of 10 millimolar PBS to each well. Then, add 100 microliters of the biotinylated ACE2 and biotin working solution to appropriate wells containing NeutrAvidin-conjugated microspheres.
Seal the microtiter plate. And incubate for one hour on an orbital shaker as shown earlier. After washing the microspheres, store the ACE2 and biotin-conjugated microspheres.
Begin by mixing casein, polyvinyl alcohol, polyvinylpyrrolidone, and BSA with pH7 to prepare assay buffer. Next, prepare 10%rabbit immunoglobulin G in assay buffer to make the sample dilution buffer. Prepare the volume of the working bead mixture necessary for testing.
Then, thaw the prepared SARS-CoV-2 and control supernatants at four degrees Celsius for one hour. Label and arrange eight 1.5 milliliter microfuge tubes each for SARS-CoV-2 and control supernatants. To create the highest dilution of each supernatant, combine 600 microliters of the supernatant with the sample dilution buffer in appropriately labeled tubes.
Followed by briefly vortexing the tube to mix. Sequentially transfer 400 microliters of the one is to one diluted supernatant to the next dilution tube. Briefly vortex each diluted supernatant before proceeding with the next dilution.
Then, take the pre-prepared working bead mixture after vortexing for 30 seconds. And add five microliters to each assigned well of a flat bottom 384 well microtiter plate. Add 45 microliters of the prepared supernatant dilutions to assigned wells containing microspheres in the 384 well plate.
Seal the plate and incubate overnight on an orbital shaker at 650 RPM in the dark at room temperature. After removing the microtiter plate from the orbital shaker, centrifuge the plate at 931 G for one minute. Then, remove the plate sealer.
To immobilize the beads, place the microtiter plate on a magnetic plate separator for 30 seconds. With the microtiter plate still positioned on the magnetic separator, aspirate the supernatant from magnet-immobilized beads. After removing the microtiter plate from the magnetic separator, add 60 microliters of PBST to each bead-containing well.
Next, take five 1.5 milliliter tubes to prepare different detection mixes. Add human monoclonal anti-S1 antibody and flag-tagged single chain variable fragments targeting the spike protein on the SARS-CoV-2 particle to each tube. Add 50 microliters per well of the appropriate single chain variable fragment specific spike detection mix to the washed microspheres.
Seal the microtiter plate and incubate as shown earlier. Then, centrifuge the microtiter plate. Wash excess spike detection reagent from beads with 60 microliters of PBST three times.
Next, prepare a fluorescent solution consisting of PE-conjugated anti-human immunoglobulin G together with brilliant violet 421 conjugated anti-FLAG antibody. Add 50 microliters per well of fluorescent solution mixed to the washed microspheres and incubate as shown earlier. Then, spin down the microtiter plate.
Wash the excess fluorescent solution mix from the microspheres with 60 microliters of PBST. Suspend the microspheres in 60 microliters of PBST from the last wash step. Then analyze the plate on a dual reporter flow analysis system with the desired settings.
In the dilutions of SARS-CoV-2 infected cell supernatants, in both reporter channels, a concentration dependent signal was observed. Three out of the five single chain variable fragments can detect the virus in dilutions as low as 1:18. For the remaining two single chain variable fragments, the virus is detectable in dilutions as low as 1:6.
Overview
This study presents a novel multiplex fluorescent immunoassay designed to detect two unique spike protein epitopes on intact SARS-CoV-2 viral particles. Utilizing a dual-reporter flow cytometric system, the method enhances the profiling of viral particles for better understanding of virus-host interactions.
Key Study Components
Area of Science
- Virology
- Immunology
- Proteomics
Background
- Host proteins are incorporated into newly formed viruses, influencing their replication and pathogenesis.
- Profiling surface proteins on viral particles can reveal critical elements in virus-host interactions.
- Traditional imaging techniques are being supplemented by mass spectrometry and flow biometry for broader proteomic analysis.
- Understanding these interactions may lead to new antiviral targets and vaccine development.
Purpose of Study
- To develop a method for multiplex profiling of host surface proteins on intact viral variants.
- To screen antibodies and antiviral drugs against multiple viruses and antigens simultaneously.
- To enhance the understanding of the role of host proteins on virus particles.
Methods Used
- Preparation of NeutrAvidin and antibody solutions in MES buffer.
- Use of magnetic microspheres for immunocapture of viral particles.
- Sequential dilution of SARS-CoV-2 supernatants for testing.
- Flow cytometric analysis to detect signals from viral particles.
Main Results
- Detection of SARS-CoV-2 in dilutions as low as 1:18 using specific single chain variable fragments.
- Successful multiplex profiling of host proteins on viral particles.
- Demonstration of the method's capability to screen multiple antibodies and antiviral drugs.
Conclusions
- The developed immunoassay provides a powerful tool for understanding virus-host interactions.
- This method can facilitate high-throughput profiling of infected samples.
- Future applications may lead to improved antiviral strategies and vaccine design.
Frequently Asked Questions
Executive Industry Relevance
Multiplex dual-reporter profiling of viral surface protein epitopes enables high-throughput, quantitative assessment of intact viral particles, directly supporting early-stage antiviral and vaccine target validation. This approach increases predictive confidence in epitope accessibility and antibody binding, informing portfolio triage and mechanistic de-risking for biologics discovery. The method's specificity and parallel detection capabilities position it as a reusable platform for translational virology and immunotherapeutic R&D.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables direct interrogation of viral surface protein epitopes for functional target validation.
- Supports mechanistic de-risking by confirming epitope presence and accessibility on intact viral particles.
- Facilitates predictive confidence in antibody and antiviral candidate selection.
Screening & Assay Development
- Provides validated, multiplexed detection of viral surface proteins for downstream screening workflows.
- Delivers quantitative, reproducible readouts for antibody and drug affinity assessment.
- Enables high-throughput profiling and assay standardization across multiple viral antigens.
Translational & Preclinical Research
- Aligns with translational biomarker strategies by profiling disease-relevant viral epitopes in physiologically relevant matrices.
- Supports continuity from discovery through preclinical validation by enabling robust, scalable epitope detection.
- Reduces biological risk in candidate advancement by confirming target engagement in complex samples.
Pipeline & Workflow Integration
This multiplex profiling strategy integrates from early discovery through lead identification and preclinical validation, supporting iterative hypothesis testing and candidate prioritization.
- Discovery Biology: Confirms epitope presence and host-virus interaction targets for hypothesis-driven research.
- Screening: Delivers reproducible, quantitative outputs for compound and antibody evaluation.
- Analytics: Provides dose-dependent, multi-channel readouts to compare binding and specificity across candidates.
- Translational Research: Enables profiling in infected supernatants and body fluids, supporting biomarker alignment.
- Enterprise Reuse: Offers a scalable, adaptable platform for profiling diverse viral and extracellular vesicle targets.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in target validation.
- Operational Value: Standardizes multiplex detection and supports high-throughput, reproducible workflows.
- Strategic Value: Improves go/no-go decisions and capital efficiency by enabling robust candidate triage.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of antiviral and immunotherapeutic assets.
Implementation Considerations
- Requires expertise in flow cytometry, immunocapture, and multiplex assay design.
- Needs access to dual-reporter flow cytometric instrumentation and magnetic bead handling infrastructure.
- Demands cross-team standardization of reagents, controls, and analytical thresholds.
- Adaptable to various viral and extracellular vesicle systems with appropriate affinity reagents.
- Assay performance and specificity depend on reagent quality and sample matrix complexity.
