To our knowledge, this is the first example of a bead-based multiplexing protocol that uses two reporter signals to simultaneously measure two results per analyte. This technique allows the user to measure antigen-specific IgM and IgG at the same time, with less sample and a shorter time to results. While this dual reporter method is specific for antibody isotyping, it could be adapted to measure other analyte pairs, such as post-translational modifications or free versus bound drug forms.
Demonstrating our procedure today we have Dr.Steve Angeloni, a senior field application scientist for Luminex Corporation. To begin the dual reporter IgG and IgM serological assay, prepare the required multiplex bead mixes from the individual couple bead and control bead stocks at a concentration of one times 10 to the six beads per milliliter. Next, dilute the serum samples 100-fold by adding 10 microliters of serum to 990 microliters of PBS-TBN buffer, then dilute the samples another tenfold by adding 20 microliters of the 1:100 dilution to 180 microliters of PBS-TBN.
When the bead mixes and serum samples are ready, add 50 microliters of the multiplex bead mixes to the assigned wells of a 96-well non-binding microtiter plate, then add 50 microliters of the diluted serum samples to the appropriate wells. Once all samples have been added, cover the plate with a microplate foil seal and incubate on a heated plate shaker at 37 degrees Celsius for 15 minutes. Then, separate the beads from the reaction mixture by placing the plate onto a magnetic plate separator for two minutes.
Keeping the plate on the magnet, remove the foil seal. Then carefully invert the plate over a waste container and gently flick the supernatant out of the wells. While still holding the plate on the magnet, blot the plate on absorbent paper.
To wash the reaction wells, remove the plate from the plate magnet and add 150 microliters of PBS-TBN to each well. Cover the plate with a fresh foil seal and incubate on the heated shaker for two minutes before placing it back on the plate magnet for another two minutes. While keeping the plate on the magnet, invert the plate to discard the supernatant, and blot the plate on absorbent paper.
After the second PBS-TBN wash, remove the plate from the magnet and add 100 microliters of fresh Detection Reagent mix to each well. After covering the plate with a microplate foil seal, place it on a heated plate shaker for 15 minutes, and then on a magnetic plate separator for two minutes. While the plate is on the magnet, discard the Detection Reagent mix by inverting the plate over a waste container and blotting it on absorbent paper.
Wash the reaction wells with PBS-TBN twice as previously demonstrated, then remove the plate from the magnet. Add 100 microliters of PBS-TBN to each well, then cover the plate with foil seal and shake for two minutes at 37 degrees Celsius. Remove the foil seal and proceed to reading 50 microliters of the sample from each well in the flow analyzer.
To read the plate, select the drop-down menu in the upper-left corner, navigate to Plate Configuration, load the previously configured plate, and select Run Plate. Eject the plate carrier by selecting the Eject icon, then load the plate onto the plate carrier and select the Retract icon to retract the plate carrier. Once the plate carrier has protracted into the analyzer, select the Run icon to begin reading the plate.
For the dual reporter neutralization assay, add 50 microliters of the multiplex bead mixes to assigned wells of a 96-well nonbinding microtiter plate, then add 25 microliters of two micrograms per milliliter ACE2 to each well, and covered the plate with a foil seal. Following a two-minute incubation on a heated plate shaker, add 25 microliters of 1:500 serum dilutions to the assigned wells. Once all samples have been added, perform the incubation, wash, detection, and analysis steps as previously demonstrated for the serological assay.
A test of anti-IgM conjugated to the DyLight 405 reporter dye using serum samples within five to 60 days from symptom onset and an IgG strip serum sample did not produce a high signal for the spike antigen. For samples with a high IgM medium fluorescence intensity, the highest signals were seen for the receptor binding domain and nucleocapsid antigens. While the IgM titer should have been elevated in some samples, the observed medium fluorescence intensity levels did not exceed 140 MFI units.
Furthermore, the control bead for IgM lacked a significant dynamic range for median fluorescence intensity when using DyLight 405 conjugated to anti-IgM compared to a phycoerythrin labeled anti-IgM at the same concentration. For IgG detection, the brilliant violet conjugate had higher median fluorescence intensity signals than the streptavidin conjugate of fluorphore Super Bright 436. However, the signal intensity for the brilliant violet conjugate varied across the ACE2 titrations.
This signal fluctuation by the brilliant violet conjugate across ACE2 concentrations also interfered with determining the percent inhibition by ACE2 across the range of IgG titers. For IgM detection, the phycoerythrin anti-IgM displayed higher signals than those generated by DyLight 549 anti-human IgM. While determining ACE2 percent inhibition of IgM binding, there was a slight but insignificant difference between the two IgM detection reagents.
So one of the most important steps in the procedure is to prepare your multiplex bead mix from your individual bead stocks and make sure you do that correctly. So this procedure can also be used to measure the efficacy of vaccines and monitoring the immune responses to other pathogens as well.
