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