Microsphere Arrays

Robust and flexible microsphere-based array systems have been developed over the last decade [ 5]. For the purposes of this review, there is only one microsphere-based array developed and commercialized in a manner parallel to the planar array discussed previously that has succeeded in garnering the most significant portion of the multiplexed immunoassay market: xMAP from Luminex Corporation (http://www.luminexcorp.com). Luminex technology can perform up to 100 multiplexed, microsphere-based assays in a single reaction vessel by combining optical classification schemes, biochemical assays, flow cytometry, and advanced digital signal-processing hardware and software.

Multiplexing is accomplished by assigning each analyte-specific assay a microsphere set labeled with a unique fluorescence signature. To attain 100 distinct microsphere signatures, two fluorescent dyes, red and infrared, are mixed in various combinations using 10 intensity levels of each dye (i.e., 10 x 10). Each batch or set of microspheres is encoded with a fluorescent signature by impregnating the microspheres with one of these dye combinations. After the encoding process, assay-specific capture antibodies are conjugated covalently to each unique set of microspheres. Coupling is performed on large numbers of individual microspheres (107 to 109 microspheres) simultaneously within each unique set, resulting in low microsphere-to-microsphere variability.

After optimizing the parameters of each assay separately, multianalyte profiles (MAP) are performed by mixing up to 100 different sets of the micro-spheres in a single well of a 96- well microtiter plate. A few microliters of sample is added to the well and allowed to react with the microspheres. The assay - specific capture reagent on each microsphere binds the analyte of interest. A cocktail of assay- specific, biotinylated antibodies is reacted with the microsphere mixture, followed by a streptavidin-labeled fluorescent "reporter" molecule (typically, phycoerythrin). Finally, the multiplex is washed to remove unbound detecting reagents.

After washing, the mixture of microspheres is analyzed using a Luminex instrument which uses hydrodynamic focusing to pass the microspheres in single file through two lasers. As each individual microsphere passes through the excitation beams, it is analyzed for size, encoded fluorescence signature, and the amount of fluorescence generated in proportion to the analyte. Microsphere size, determined by measuring the 90 ° light scatter as the microspheres pass through a red diode laser (633nm), is used to eliminate microsphere aggregates from the analysis. While in the red excitation beam, the encoded red and far-red dyes are excited and the resulting fluorescence signature (ratio 660nm/720nm) is filtered, measured using avalanche photodiodes, and classified to a microsphere set. Since each microsphere is encoded with a unique signature, the classification identifies the analyte being measured on that individual microsphere. As the microsphere passes through a green diode-pumped solid-state laser (532 nm), the fluorescence "reporter" signal (580 nm) is generated in proportion to the analyte concentration, filtered, and measured using a photomultiplier tube. Data acquisition, analysis, and reporting are performed in real time on all microsphere sets included in the MAP. A minimum of 50 individual microspheres from each unique set are analyzed and the median value of the analyte -specific, or reporter, fluorescence is logged. Using calibrators and controls of known analyte quantity, sensitive and quantitative results are achieved with precision enhanced by the redundant oversampling at each data point.

xMAP provided several key advantages over the two-dimensional planar arrays. First, consistent coating of the polystyrene microsphere surface with antibodies has been performed in the diagnostic industry for decades, so it is a reproducible technique. Microsphere coating does not suffer from the irre-producibility that plagues planar arrays. Second, the lot size for manufacturing of a microsphere array is generally in the thousands to millions of tests per lot. Lower lot-to-l ot variability coupled with extremely large manufacturing lots means that the problem of lot variation is greatly diminished. The primary disadvantage of microsphere arrays is throughput, as each sample must be read in turn in the flow system. So far, no group has solved this problem be developing multiple-channel flow systems, but this is the next logical step of the technology.

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