Realizing the promise of reverse phase protein arrays for clinical, translational, and basic research: a workshop report: the RPPA (Reverse Phase Protein Array) society

Abstract

Reverse phase protein array (RPPA) technology introduced a miniaturized "antigen-down" or "dot-blot" immunoassay suitable for quantifying the relative, semi-quantitative or quantitative (if a well-accepted reference standard exists) abundance of total protein levels and post-translational modifications across a variety of biological samples including cultured cells, tissues, and body fluids. The recent evolution of RPPA combined with more sophisticated sample handling, optical detection, quality control, and better quality affinity reagents provides exquisite sensitivity and high sample throughput at a reasonable cost per sample. This facilitates large-scale multiplex analysis of multiple post-translational markers across samples from in vitro, preclinical, or clinical samples. The technical power of RPPA is stimulating the application and widespread adoption of RPPA methods within academic, clinical, and industrial research laboratories. Advances in RPPA technology now offer scientists the opportunity to quantify protein analytes with high precision, sensitivity, throughput, and robustness. As a result, adopters of RPPA technology have recognized critical success factors for useful and maximum exploitation of RPPA technologies, including the following: preservation and optimization of pre-analytical sample quality, application of validated high-affinity and specific antibody (or other protein affinity) detection reagents, dedicated informatics solutions to ensure accurate and robust quantification of protein analytes, and quality-assured procedures and data analysis workflows compatible with application within regulated clinical environments. In 2011, 2012, and 2013, the first three Global RPPA workshops were held in the United States, Europe, and Japan, respectively. These workshops provided an opportunity for RPPA laboratories, vendors, and users to share and discuss results, the latest technology platforms, best practices, and future challenges and opportunities. The outcomes of the workshops included a number of key opportunities to advance the RPPA field and provide added benefit to existing and future participants in the RPPA research community. The purpose of this report is to share and disseminate, as a community, current knowledge and future directions of the RPPA technology.

Fig. 1.

Schematic representation of the RPPA workflow. Reprinted from Mueller, C. M., Liotta, L. A., and Espina, V. (2010) Reverse phase protein microarrays advance to use in clinical trials. Mol. Oncol. 4, 461–481. Copyright (2010), with permission from Elsevier.

Fig. 2.

A global database providing a quality index for antibodies and comprehensive information about the validation process will be the first stop for researchers performing the RPPA technology.

Fig. 3.

How RPPA can be used as a calibrated immunoassay able to classify a given patient value above or below a predetermined cutoff point using low and high controls and a calibration curve.