A Bead-Based Multiplex Immunoassay to Identify Pathogen-Specific Antibodies in Saliva

1. Bead Coupling

1. Couple the antigens to the bead sets using the concentrations shown in Table 1.
2. Add each antigen to the activated beads and bring the total volume to 500 µl in 50 mM MES, pH 5.0. Mix the antigens and beads by vortex.
3. Incubate the antigens and beads for 2 hr with mixing by rotation (~15 rpm) at room temperature in the dark. Pellet the coupled beads by microcentrifugation at 10,000 x g for 2 min.
4. Remove the supernatant and resuspend the beads in 500 µl of phosphate-buffered saline (PBS)-bovine serum albumin (BSA)-polyoxyethylene sorbitan monolaurate (Tween-20)-sodium azide (PBS-TBN) pH 7.4 by vortex and sonication. Pellet the beads by microcentrifugation at 10,000 x g for 2 min and remove the supernatant.
   Caution: Sodium azide is an acutely toxic chemical. It is fatal if swallowed or gets in contact with the skin. Do not breathe dust/fumes/gas/mist/vapors or spray. Wear appropriate personal protective equipment (PPEs) when handling and disposing of it in accordance with appropriate laws.
5. Resuspend the beads in 1 ml of PBS-TBN by vortex and sonication for 20 sec.
6. Pellet the beads by microcentrifugation at 10,000 x g for 2 min.
7. Repeat steps 1.5 and 1.6.
   Note: This provides a total of two washes with PBS-TBN.
8. Resuspend the coupled and washed beads in 1 ml of PBS, 1% BSA, 0.05% Azide, pH 7.4. Store the coupled beads in a 2-8 °C refrigerator in the dark.

2. Salivary Multiplex Immunoassay

1. Remove saliva from the -80 °C freezer and allow it to thaw at room temperature.
2. Resuspend antigen-coupled bead stocks by vortex and sonication for 20 sec.
3. Prepare a working bead mixture by diluting the coupled bead stocks to a final concentration of 100 beads/µl of each unique bead set in PBS-1% BSA buffer.
4. Prepare a 1:4 dilution of saliva with PBS-1% BSA buffer in a 96-well, deep well plate.
5. Pre-wet the filter plate with 100 µl of wash buffer and remove the supernatant by vacuum.
6. Add 50 µl of a working bead mixture and an equal volume of diluted saliva to 95 wells of the 96-well filter plates for a 1:8 final dilution. Mix reactions with a multi-channel pipettor. To the one control well, add 50 µl antigen-coupled beads plus 50 µl of PBS-1% BSA buffer (as a replacement for saliva).
7. Cover and allow to incubate in the dark at room temperature for 1 hr on a microplate shaker at 500 rpm. Remove supernatant by vacuum. Wash wells with 100 µl of wash buffer and remove supernatant by vacuum. Repeat wash 1x.
8. Resuspend beads in 50 µl of PBS-1% BSA with a multi-channel pipettor.
9. Dilute biotinylated goat anti-human IgG secondary detection antibody to 16 µg/ml in PBS-1% BSA.
10. Add 50 µl diluted secondary antibody to each well and mix contents with a multi-channel pipettor.
11. Cover the filter plate and allow it to incubate in the dark at room temperature for 30 min on a plate shaker. Remove supernatant by vacuum. Wash wells with 100 µl of wash buffer and remove supernatant by vacuum. Repeat wash 1x.
12. Resuspend beads in 50 µl of PBS-1% BSA with a multi-channel pipettor.
13. Dilute streptavidin-R-phycoerythrin reporter (SAPE) to 24 µg/ml in PBS-1% BSA.
14. Add 50 µl reporter to each well and mix with a multi-channel pipettor.
15. Cover the filter plate and allow it to incubate in the dark at room temperature for 30 min on a plate shaker. Remove supernatant by vacuum. Wash wells with 100 µl of wash buffer and remove supernatant by vacuum. Repeat wash 1x.
16. Resuspend beads in 100 µl of PBS-1% BSA and analyze 50 µl using the analyzer.
    Note: Results of the multiplex immunoassay are measured in Median Fluorescence Intensity (MFI) units. Always refer to the latest version of the software manual, if available to avoid errors.

Table 1: Working bead mix for the multiplex immunoassay. After coupling and confirmation were completed, a master mix consisting of each bead set was prepared as shown. The master mix was distributed among all of the wells in the 96-well plate. All of the wells were incubated with saliva with the exception of one background control well which contained only beads and PBS-1% BSA buffer.