The most common format in immunoassay design relies on antigen capture by surface immobilized antibodies.  Because ensuring exposure of antibody binding sites to the sample matrix is pivotal for maximizing antigen binding efficiency and assay performance, considerable effort has been spent to enrich favorable antibody orientations when immobilized on a substrate surface.  However, directly determining antibody orientation has proven difficult, therefore binding capacity assays are commonly utilized to assess immobilized antibody performance.  We employ defocused polarized total internal reflection fluorescence microscopy measurements of single antibodies with rigidly bound fluorescent markers to directly probe the three-dimensional orientation of immobilized antibodies.  Anti-fluorescein mouse monoclonal IgG 4-4-20 antibody is utilized as it has been shown to bind fluorophores in a known orientation based on X-ray crystallography measurements.  The orientation of the captured fluorophore can be directly correlated to overall antibody orientation, thus provides a test system to explore various immobilization protocols.

Imaging immobilized single antibody 3D orientations with polarized total internal reflection fluorescence microscopy

244th National Meeting (2012)

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