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.
