Multianalyte Immunobead Assay: A Flow-Based Immunoassay Technique for Concurrent Detection of Multiple Antibody Subtypes in Human Serum Sample

Multianalyte immunobead assay uses multiple antigen-coated microspheres to recognize their corresponding target antibodies simultaneously.

To begin this assay, take a mixture of microbeads in a tube containing the appropriate buffer. Each bead is coated with a specific antigen on its surface, allowing easy differentiation of beads from one another.

Pipette the coated immunobead mixture into the wells of a microtiter assay plate. Supplement the wells with the serum sample containing different isotypes of target antibodies. These variants have subtle differences in their structures, allowing each well to have multiple primary antibodies.

Incubate, to allow sample antibody isotypes to attach to specific antigens captured on respective immunobeads. Wash with an assay buffer to remove unbound primary antibodies. Add fluorescent dye tagged-secondary antibodies, corresponding to each variant of primary antibody, into the well.

Each labeled secondary antibody binds to the Fc site of the respective primary antibody. Multiple bead-captured antigen-primary antibody complexes are fluorescently labeled. Wash the plate to remove unbound secondary antibodies.

Read the plate using a fluorescent analyzer capable of concurrently detecting different fluorophores at their respective wavelengths. Depending upon the signal detection, various isotypes of serum antibodies and their binding to different antigens can be assessed.