Biomarker Analysis with Grating Coupled Surface Plasmon Coupled Fluorescence

One of the greatest hurdles in protein microarray technology has been the limited sensitivity. However, the strategy of amplifying the SPR signal with fluorescence improves sensitivity. Herein, we report a simple and efficient method, Grating Coupled Surface Plasmon Coupled Fluorescence (GCSPCF) with a peptide microarray to screen sera of human subjects and a mouse strain with autistic-like behavior for quantification of the epitope specificities of their circulating antibodies. The GCSPCF peptide microarray is a novel assay that facilitates high-throughput screening of antibody epitope specificities or binding of interacting analytes with a small quantity (<50 µl) of sample. This study utilized over 600 peptides with the eventual goal of being able to identify antibodies that could be used as biomarkers of particular disorders. The GCSPCF technology was able to detect antibodies to the tripeptide glutathione; it was also capable of sequential screening antibodies of different immunoglobulin isotypes (IgG1, IgG4 and IgE) to the same peptide. The GCSPCF broad-based screening approach was able to begin the identification of epitope-specific antibody binding patterns in an effort to develop a predictive signature that could be further developed for clinical trials. In addition to evaluation of antibody specificities with hundreds of spotted peptides at multiple regions of interest (ROI), the addition of spotted antibodies could provide an even more complete biomarker profile with capture of analytes, such as stress proteins and cytokines. Differences between a mouse strain with normal behaviors (C57BL/6J) and a strain with autistic-like behaviors (BTBR T+tf/J) as well as healthy and autistic participants are used for comparative analyses.