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.