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

Abstract

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. 1-3 Protein microarray analysis is particularly well suited for such methodology. In this work we demonstrate a high-throughput, quantitative, label-free 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 and the binding specifity of each antibody toward antigens confirmed that the activity of the antibodies remained after microarray fabrication. Goldcoated glass slides presenting amine reactive moieties were prepared to facilitate protein microarray printing. Antigen microarrays were printed similarly with various allergens including human serum albumin as well as bovine serum albumin. As a demonstration of quantitative, low molecular weight drug-molecule screening in real-time with SPR microscopy, the binding kinetics of 1,400 Da biotinylated peptides were monitored on streptavidin (SA) spots attached on biotinylated BSA arrays on functionalized gold-coated glass slides. 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 antigen-antibody binding kinetics in “real-time” without labels with a sensitivity of 1.2 ng/cm 2