A Novel Method for Saliva Proteomic Analysis

DESCRIPTION (provided by applicant): Changes of salivary protein profile often reflect the pathological processes of many oral diseases such as oral cancer, desquamative gingivitis, and periodontitis, as well as systemic diseases such as Sjogren's syndrome, systemic lupus erythematosus, cancers and HIV/AIDS. Whereas a large number of abundant proteins in saliva have been identified, the technologies required for multiplex analysis of less abundant salivary proteins that may play vital roles in innate defense, immune regulation, and disease pathogenesis remain to be developed. This proposal presents a novel design of protein array (chip) that aims at detecting less abundant proteins in saliva in large-scale. It overcomes the shortcomings of current protein arrays including the detection of limited number of proteins due to low sensitivity and low specificity. We propose a two-step binding approach to replace the conventional ELISA-based protein array. In the proposed procedure, (1) the targeted proteins bind to antibodies randomly coated on a solid surface and are then biotinylated (step-one); (2) the biotin- labeled proteins, after release in a low pH glycine buffer and subsequent neutralization with HEPES buffer, bind to antibodies coated in array format on a slide (step-two). The goal is to detect multiple less abundant proteins using a small quantity of sample protein while maintaining the high specificity and sensitivity. In phase I period, this approach will be used to design a prototype salivary protein array for 30 low abundant proteins (pg-ng/ml) with emphasis towards proteins involved in oral inflammation and oral cancers. These 30 proteins include (1) inflammatory and anti-inflammatory cytokines and (2) growth factors and tumor markers. By using conventional chemiluminescent (ECL) detection, this design may be used by most basic, clinical, and forensic research laboratories that have interest in saliva proteomic analysis. The proposed work in the phase I period will establish the groundwork necessary for eventual creation of commercial saliva protein arrays capable of detecting a large number of saliva proteins during the phase II period.