Heat shock proteins (Hsps), a class of stress-response chaperone proteins, are an integral part of the maintenance of proteostasis in the body. Hsps prevent aberrant protein misfolding, aggregation, and degradation, which are often disease pathologies. The proteostasis network, built of Hsp and co-chaperone proteins, responds differentially dependent on the type of stress condition or disease pathology, yet a clear understanding of the specific proteins impacted by various conditions remains to be determined. Therefore, developing methodology to evaluate protein expression changes in response to multiple conditions will lead to better understanding of how different components of the proteostasis network are altered in response to stress. We present a novel immunoassay to quantify multiplexed protein expression changes and apply this method to generate baseline Hsp expression patterns in mouse brain samples.<br/><br/>To study the stress response of Hsps under various conditions, our group is developing a high-throughput assay of multiple heat shock proteins. To accomplish this, Hsp-specific fluorescent microbeads are constructed and used in conjunction with xMAP microbead technology to carry out a multiplex immunoassay. Multiple microbeads can be used in a single assay, allowing for the identification and quantification of multiple Hsps in a sample.<br/><br/>Previous work in our group has achieved antibody-microbead conjugation and confirmation of Hsp27 and Hsp70-conjugated microbeads using the Luminex MagPix. Current work confirms that the phycoerythrin-conjugated secondary antibodies are specific to targeted Hsps by western blot. Furthermore, Hsps in processed mouse brain samples are appropriately visualized and quantified. Continued optimization of this process is ongoing along with confirmation of our methodology using a cross-reactivity assay. Successful development of a multiplex immunoassay of Hsps will contribute to the study of proteostasis and our understanding of how the chaperone network responds to stress and prevents protein aggregation diseases.

Development of a novel immunoassay for multiplex detection of heat shock proteins using xMAP technology

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