Variance in multiplex suspension array assays: intraplex method improves reliability

An "intraplex" method was developed in order to minimize differences in sample readings between instruments. A full intraplex assay consists of a set m of microparticle set classifications assaying for the same analyte, with each of the m classifier sets having different sensitivity to analyte, and n classifier sets replicating each of the m levels of sensitivity, where m > 1 (generally m > 4 would be used).The intraplex method can compensate adequately for the sources of variance that have been identified in suspended microarray assays. It requires no changes to current equipment in use, and is a superior method of constructing precision assays. Additionally, Luminex？ users may want to consider the evidence that shows that despite calibration to the same standard, two instruments may not give similar results for all concentrations of analytes.A suspended microarray assay system uses small particles, such as microspheres or microrods that contain some method for identifying a set of particles composing one assay. An chemical compound used to bind to a biological (or chemical) target molecule (analyte) is bound to the surface of a set of identical particles, which are generally in the size range of 3–15 microns. Differently labeled particles have different target molecules that they assay for. These particles are added to a liquid (such as serum or cell lysate) containing the potential analytes. (In systems such as "smart dust", the assay may be distributed in the field to detect analytes. A system such as "smart dust" may also use an alternative method of analyte signaling and readout.) The final step in the assay activates a reporter fluorophore that provides a signal. (Essentially, this is an ELISA assay on the surface of a small particle.) The particles are run through a flow cytometer, which may be optimized for the specific assay system. For each particle in the mixture, the cytometer identifies the classifier for the set the particle belongs to together with t