Begin the human sera sample dilution by mixing five microliters of the serum sample with 120 microliters of assay buffer in a 96-well plate. Dilute to an additional eightfold by mixing 10 microliters of the initial dilution with 70 microliters of assay buffer. Now, prepare 100 microliters of the bead mix containing target antigen-coupled beads.
Next, dilute the prepared bead mix in 2.4 milliliters of the assay buffer in a Falcon tube. Start serum incubation by pipetting 25 microliters of the bead suspension into designated wells. Add 25 microliters of 200-fold diluted serum into the wells with the bead suspension.
Cover the 96-well reaction plate with a microplate seal. Then incubate the plate with beads on a plate shaker. Following incubation, remove the 96-well reaction plate from the plate shaker.
Place the plate into a magnetic plate washer, and take off the adhesive plate seal. Next, wash the beads three times with 100 microliters of wash buffer, then aspirate the final wash volume from the beads after the last washing step. For the first antibody incubation, start by preparing a fresh IgG and IgM dual detection reagent mixture in assay buffer.
Pipette 30 microliters of the detection reagent into each designated well, Then cover the 96-well reaction plate with a microplate seal. Incubate the plate on a plate shaker for 45 minutes at 20 degrees Celsius at 750 revolutions per minute. After the incubation, place the plate into a magnetic plate washer, and remove the adhesive plate seal pipette 30 microliters of diluted BV421-labeled striptavidin into each reaction well after washing the plates as before.
Then place the sealed plate on a plate shaker for subsequent incubation. Once incubation is complete, wash the plate, and resuspend the beads within the wells to a final volume of 100 microliters using the wash buffer. To analyze the results, cover the 96-well reaction plate with a microplate seal.
Next, incubate the covered plate for three minutes at 20 degrees Celsius at 1000 revolutions per minute on a plate shaker. Transfer the plate from the shaker to the dual reporter flow analyzer instrument. Finally, assess the sample median fluorescent intensity, or MFI, following the manufacturer's instructions.
Spearman correlation analysis for three representative Borrelia antigens demonstrated uniform reproducibility of MFI values when detecting anti-Borrelia antibodies present in human sera. The dual reporter assay showed good assay-to-assay reproducibility with high inter-assay precision demonstrated by Levey-Jennings charts. At sample dilutions of 100-fold to 12, 800-fold, the dilution curves for both IgM detection and IgG detection were similar for both instruments.
The MFI values were slightly higher in the single reporter instrument.
