The method we'll be demonstrating today provides a high-throughput solution for evaluating the sero-conversion of antibodies associated with either COVID-19 infections or vaccination for routine analyses. Interestingly, this approach allows us to evaluate multiple epitopes on the same analyte. This has numerous applications in the biological sciences, including cell signaling studies and monitoring immune responses.
Demonstrating this technique for you today will be Dr.Imad Tarhoni, a post-doctoral fellow in my laboratory. To begin, select three different vials of the magnetic microspheres with unique bead regions and record the bead ID and lot information for each vial used. Then, vortex the selected microsphere stock for 60 seconds and sonicate for five minutes.
Transfer 1.0 times 10 to the six beads to a 1.5-milliliter low protein binding microcentrifuge tube and insert the tube into a magnetic separator. to allow the separation for 60 seconds. With the tube still in the magnetic separator, carefully remove the supernatant without disturbing the bead pellet.
Remove the tube from the magnetic separator, then resuspend the beads with 100 microliters of HPLC-grade water and vortex for 30 seconds. Again, place the tube back into the magnetic separator for 60 seconds and subsequently remove the supernatant. Repeat the wash protocol twice.
After the last wash, remove the tube from the magnetic separator and resuspend the washed microspheres in 90 microliters of activation buffer, pH 6.2, by vortex for 30 seconds. Next, sequentially treat the microspheres with 10 microliters of 50 milligrams per milliliter anhydroxy-sulfosuccinamide and 10 microliters of 50 milligrams per milliliter EDC solution by vortex for 10 seconds. Later, incubate the microspheres for 20 minutes at room temperature with a gentle vortex every 10 minutes.
After incubation, wash the microspheres twice with 50-millimolar MES or coupling buffer, pH 5.0, as described earlier. Repeat the washing twice. Once done, remove the tube from the magnetic separator and resuspend the beads with 100 microliters of coupling buffer by vortex for 30 seconds, followed immediately by adding the desired quantity of protein to the tube.
Bring the total volume to 150 microliters with coupling buffer and mix the reaction by vortex for 30 seconds. Then, incubate the tube for two hours by rotation at room temperature. After incubation, wash the microspheres twice with PBS quench buffer and resuspend the washed microspheres in 100 microliters of quench buffer containing 0.05%sodium azide by vortex for 30 seconds.
Count the number of recovered microspheres using an automated cell counter and record the observed bead concentration. For assay performance, resuspend the coupled microspheres by vortex for 30 seconds and sonicate for 60 to 90 seconds. Then, remove the required quantity of each bead colloid from the respective tube and combine the bead colloids in a new 1.5-milliliter microcentrifuge tube.
Next, insert the tube into a magnetic separator and allow separation for 60 seconds. With the tube still in the magnetic separator, remove the supernatant without disturbing the bead pellet. After removing the beads from the separator, resuspend the beads in 100 microliters of assay buffer.
Vortex for 30 seconds before placing the tube into a magnetic separator for 60 seconds to separate the beads. Repeat the washing twice. Next, adjust the concentration of the three-plex working microsphere mixture by adding an appropriate volume of assay buffer to generate a final concentration of 100 microsphere per one microliter for each target.
Aliquot 25 microliters of the microsphere mixture into each well of a 96-well plate. Dilute plasma or serum specimens 500-fold in assay buffer and prepare standard specimens according to titration desired. Add 25 microliters of assay buffer as the blank sample and add each of the diluted specimens or standard into each designated well of a 96-well specimen plate.
When done, cover the plate with an aluminum seal or foil to incubate for one hour at room temperature on a plate shaker set to 700 rotations per minute. Prepare a solution of anti-human detection antibodies or secondary antibody solutions at four micrograms per microliter with assay buffer as described in the manuscript. Place the plate on a magnetic separator to wash rapidly and then forcefully invert the plate over a biohazard container to remove liquid from the wells.
With the plate still inverted, forcefully tap the plate against a thick wad of paper. Then, wash each well with 100 microliters of assay buffer and remove the liquid by forceful inversion over a biohazard container. Repeat the wash twice.
After the last wash, discard all used wads of paper into a biohazard container. Next, add 25 microliters of secondary antibody working solution to each well of a 96-well plate and cover the plate with aluminum foil to incubate on a plate shaker at 700 rotations per minute. After 30 minutes of incubation, wash the wells of the plate twice with assay buffer as demonstrated earlier.
Then, add 100 microliters of assay buffer into each well of the 96-well plate. Cover the plate with aluminum foil and incubate for five minutes on a plate shaker at room temperature. Analyze a 60-microliter sample via the instrument analyzer according to the system manual.
For optimization of the specimen capture incubation time, incubate the plates for a duration of 30, 60, and 120 minutes. After incubation, analyze 60 microliters of the assay mixture on the analyzer. A seven-point standard curve based on a 1:5 serial dilution series was evaluated for the spike S1, the membrane antibodies, and the nucleocapsid.
The intra-assay precision test was performed on the plate to evaluate assay precision calculated as percent coefficient of variation, standard deviation, and average. For inter-assay precision, three distinct batches of the bead sets were prepared for each assay. Precision variables of the assay with the standard and the human plasma samples were calculated.
The average median fluorescent intensity values at 120 minutes of the incubation were optimal for IgG titres for the spike S1, the membrane, and the nucleocapsid antibodies, indicating a fast primary antibody binding kinetics. Secondary antibody concentration optimizations in dual channel performance demonstrated a wide range of signals in linear measurement for all concentrations. The observed signals from different time incubations of the secondary antibodies and the details on the signal changes for all the incubation times are shown here.
In the specificity assessments for dual-channel assays, negligible cross signal contamination was observed between the blank and the single reporter channel. In the illustration of sero-conversion events following the COVID 19 vaccination, the observed spike S1 IgA and IgM values were approximately 40-fold lower than the IgG isotype titres. This method has important implications for monitoring response to vaccines in immunocompromised individuals.
It will permit us to determine if these patients are truly getting protected by those vaccinations.
