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A Demonstration of High-throughput Immunoassay and Small Molecule Binding on Protein Microarrays with SPR Microscopy

G. Kim, L. Jiang, P.K. Rathod, C.T. Campbell, A. Nishimoto and V. Casasanta
University of Washington, US

immunoassay, spr, protein microarray, biosensor

The use of Surface Plasmon Resonance (SPR) for the detection of biomolecular binding events at the nanometer scale is of great utility when expanded into microarray density formats. Protein microarray analysis is particularly well suited for such methodology. In this work we demonstrate a high-throughput, quantitative immunoassay by employing microarray techniques in combination with SPR microscopy. Antibody microarrays with various immunoglobulin proteins, including human immunoglobulin subclasses were printed on functionalized gold-coated glass slides. Gold-coated glass slides presenting amine reactive moieties were prepared by traditional silation reactions to facilitate protein microarray printing. Antigen microarrays were also printed with various allergens including human serum albumin as well as bovine serum albumin in similar fashion. A spot density of up to 240 120-µm spots was generated using conventional robotic microspotting. Application of antibody/antigen microarrays with SPR microscopy allows for the simultaneous monitoring of immunoglobulin protein binding kinetics over the whole array (allergens or antibodies), at a time resolution of one second. The ability of microarray based SPR to quantitatively monitor (in “real-time”) antigen-antibody binding kinetics allows it to become a complementary tool in support of the multi-step and labor-intensive ELISA method (with a sensitivity of 1.2 ng/cm2 for most proteins).

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