Fig. 4. Overview of workflow and study setup for MS detection in CSF.

(A) The suspension bead array technology enables the analysis of directly labeled samples for multiplex protein profiling. Crude samples are distributed in a randomized fashion in 96-well plates before they are diluted and labeled with biotin. In parallel, antibodies are immobilized on magnetic, color-coded beads and all bead identities are subsequently pooled to create an antibody array in suspension. The labeled samples are heat treated before their incubation with the bead array. After this, unbound proteins are washed away and a streptavidin-conjugated fluorophore added for target detection. In the used instrumentation, one laser is for the identity of beads and the other laser for the fluorophore used for detection of captured, biotinylated target proteins.

(B) In this study, a small set of samples (n ¼ 65) was utilized for assay development and an initial screening with 71 antibodies targeting 69 unique proteins. At a later stage, a subset of 43 targets selected based on results from the screening were additionally analyzed in the set of 339 CSF samples. At this point, multiple antibodies per target were included, resulting in a 101-plex bead array. Adapted from Ref. [52]. With permission. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)