The flowchart of multiplex autoantibody by the Luminex xMap system.

<p>(1) The HaloTag Amine Ligands were conjugated to Luminex magnetic beads via the activated carboxylic acid of COOH radical group. Different color represents various types of Luminex beads. (2) The recombinant Halo-tagged proteins were covalently immobilized onto the Luminex beads conjugated with HaloTag ligand via reactive chloroalkane linkers on ligands. Each bead was immobilized with different protein separately. (3)–(4) Due to the covalent binding, each bead-protein complex individually went through vigorously washing and then combined to make the microsphere pool. (5) The protein-coupled microsphere pool was then equally aliquotted into a 96-well plate. Each well contains multiple autoantigen biomarkers and can be used for a single serum sample. (6) The autoantibodies in sera bound to the antigen conjugated microspheres, and were detected by R-phycoerythrin conjugated anti-human antibody. The signal was read on Luminex 200 bioanalyzer.