Begin by mixing casein, polyvinyl alcohol, polyvinylpyrrolidone and BSA with pH seven 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 dilution 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 931g 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 for 21 conjugated anti-FLAG antibody. Add 50 microliters per well of fluorescent solution mix 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 dilution 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 dilution as low as one to 18.
For the remaining two single-chain variable fragments, the virus is detectable in dilutions as low as one to six.
