Next generation ligand binding assays-review of emerging technologies' capabilities to enhance throughput and multiplexing.

The purpose of this manuscript is to provide a summary of the evaluation done by the Throughput and Multiplexing subteam on five emerging technologies: Single molecule array (Simoa™), Optimiser™, CyTOF® (Mass cytometry), SQIDLite™, and iLite™. Most of the information is presented with a minimum amount of published data and much is based on discussions with users and the vendor, to help provide the reader with an unbiased assessment of where the subteam sees each technology fitting best in the bioanalysis of large molecules. The evaluation focuses on technologies with advantages in throughput and multiplexing, but is wide enough to capture their strengths in other areas. While all platforms may be suited to support bioanalysis in the discovery space, because of their emergent nature, only Optimiser and SQIDLite are currently ready to be used in the regulated space. With the exception of Optimiser, each instrument/technology requires an up-front investment from the bioanalytical lab that will need justification during capital budget discussions. Ultimately, the platform choice should be driven by the quality of data, project needs, and the intended use of the data generated. In a time- and resource-constrained environment, it is not possible to evaluate all emergent technologies available in the market; we hope that this review gives the reader some of the information needed to decide which technology he/she may want to consider evaluating to support their drug development program in comparison to the options they already have in their hands.

Multiplexing molecular diagnostics and immunoassays using emerging microarray technologies.

Enzyme-linked immunosorbent assays (ELISA) are frequently used for quantitative measurement of the presence of protein, for single-analyte testing, in a sample. The application of ELISA in a microarray format has the potential to simultaneously measure the presence and/or concentrations of numerous proteins, in multiplex testing, all contained in a small drop of test fluid. Microspot microarray technology, in combination with protein biomarkers and nucleic acid diagnostics, appears to be the future high-performance analytical platform of choice. Validation of a large number of disease markers in both molecular and protein diagnostics has paved the way for the emergence of the multiplex assay. Initially, simple low-throughput multiplex assays were tested using the immunoassay format. These were followed by low-level multiplexing and high-throughput array-based immunoassays. More recently, two types of high-level multiplexing and high-throughput diagnostic methods using microspot arrays and bead arrays have been successfully developed to complement single-analyte assays. The value in rapid diagnostic evaluation for high-throughput multiplex, diagnostic test systems based on sound assay design must take into account data screening, normalization and statistical evaluation of possible concentration measurement, data errors and automated operation. Benefits of using multiplex array platforms include improved-quality patient care, as well as cost effectiveness and time saving. These multiplex methods also set the stage for future protein/nucleic acid codetection. Currently, the one analyte at a time test scheme is still dominant; nonetheless, the multiplex microspot microarray tests evaluated in a single multiassay analyzer are expected to become a significant part of clinical diagnostic testing within the next 5-10 years. This review is focused on microspot array and bead array methods for providing high throughput and a high degree of multiplexing in diagnostic testing.