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

Take a mixture of different sets of microspheres — or micron-sized polystyrene beads.

Each set is coupled to a unique antigen from an environmental pathogen and is uniquely fluorescently labeled.

Add the mixture to a filter plate containing a membrane at the bottom.

Introduce human saliva containing antibodies against environmental pathogens, which bind to cognate antigens on the microspheres.

Remove unbound antibodies by vacuum. Microsphere-bound antibodies are retained due to the small pore size of the membrane.

Add biotinylated secondary antibodies to bind to the microsphere-bound antibodies. Remove unbound antibodies.

Add streptavidin conjugated to a fluorescent reporter dye, that binds to biotin on the secondary antibodies.

Remove the unbound dye, resuspend the microspheres, and pass them through a multiplex immunoassay analyzer.

A laser beam detects the microsphere fluorescence, identifying the antigen, while another beam detects the reporter signal, confirming a bound antibody.

Antigen-specific antibodies in the saliva indicate prior exposure to the corresponding pathogens.

After resuspending the antigen-coupled bead stocks by vortexing and sonication for 20 seconds, dilute the coupled bead stocks to a final concentration of 100 beads per microliter of each unique bead set in PBS plus BSA. Then, prepare a 1 to 4 dilution of room temperature saliva in PBS plus BSA in a 96-well deep well plate. Pre-wet a filter plate with 100 microliters of wash buffer, and aspirate the supernatant.

Next, add 50 microliters of antigen-coupled beads and an equal volume of the diluted saliva to 95 wells of the 96-well filter plate for a one-to-eight final dilution. At the same time, add 50 microliters of antigen-coupled beads plus 50 microliters of PBS plus BSA alone to the control well.

Incubate the beads in the dark at room temperature for one hour on the microplate shaker at 500 RPM. Then, aspirate the supernatant and wash the wells two times with 100 microliters of wash buffer. After the second wash, resuspend the beads in 50 microliters of PBS plus BSA, dilute the secondary antibody, and add 50 microliters of it to each well.

After mixing the well contents with a multichannel pipette, incubate the plate on the shaker in the dark for 30 minutes at room temperature. Then, wash the wells two times with 100 microliters of wash buffer, and resuspend the beads in 50 microliters of fresh PBS plus BSA.

Now, dilute the reporter and add 50 microliters to each well. Mix thoroughly with the multi-channel pipette. After 30 minutes at room temperature on the shaker in the dark, wash the wells two times as demonstrated. Then, resuspend the beads in 100 microliters of PBS plus BSA, and evaluate the samples on the analyzer.