Force differentiation in recognition of cross-reactive antigens by magnetic beads

Functionalized magnetic beads have been suggested recently as active labels for extremely rapid and highly sensitive immunoassay. Here we addressed the problem of specificity and cross-reactivity in such detection, which (unlike conventional immunoassay methods) cannot rely on a difference in the equilibrium binding constants to distinguish between closely related antigens. Microarrays containing spots of nine albumins from sera of different mammals (human, bovine, sheep, goat, pig, dog, rabbit, rat, and mouse) were tested for their interaction with magnetic beads functionalized with monoclonal antibodies against bovine or human serum albumin. It was demonstrated that the magnetic beads bound only those albumin spots to which antibody was reactive or cross-reactive in enzyme-linked immunosorbent assay (ELISA). The effect of cross-reactivity in the assay with magnetic beads detection could be decreased substantially by placing the array into a flow cell and subjecting the tethered beads to increasing shear flow, which removed beads first from the weakest cross-reactive antigens and then from more strong ones. Partial blocking of the antibody molecules on the bead surface was shown to reduce critical shear stress necessary to remove beads from the specific antigens, indicating that multiple antigen–antibody bonds held the beads on the array surface.