There are two light sources used to collect data from samples in xMAP® Technology based assays. The red light source (laser for Luminex® 100/200™ and LED for MAGPIX®) excites the internal dyes in the microspheres so that they can be classified on the bead map. The green light source excites any reporter bound to the surface of the beads. In today’s Tech Tuesday, we will examine this reporter more closely.
The fluorescent reporter molecule used in the detection step of xMAP and xTAG® Technology is Streptavidin Phycoerythrin (SA-PE). Phycoerythrin is a fluorescent macromolecule isolated from red algae and cyanobacteria. Phycoerythrin is one of the brightest fluorescent molecules isolated. In Luminex applications, SA-PE is excited by the 532 nm green laser or 520 nm green LED. Its emission peak exists at 575 ± 10 nm (orange/yellow).
Graphic courtesy of Life Technologies
In Luminex applications, phycoerythrin is often conjugated to streptavidin. Luminex assays take advantage of the high affinity of biotin to streptavidin by incorporating biotin into PCR products and nucleic acid anti-TAGs in order to detect reporter molecules:
There are a few different formats of this bright molecule used for Luminex applications. Always refer to the package insert or instructions for use provided with the assay. The table below lists the correct SA-PE formulation for each Luminex xTAG IVD assay as well as the proper storage conditions.
It is important to note that different Luminex assays use different formulations of SA-PE. Applications using magnetic beads employ an SA-PE with higher concentrations of BSA. It is extremely important to pay attention to the proper storage conditions listed for each formulation. Improper storage will destroy your SA-PE. Always keep any formulation of SA-PE protected from light as it is photo reactive. Store in an opaque box or wrap in aluminum foil before putting it in the fridge. If you are using multiple xMAP or xTAG assays in your laboratory, do not mix and match these reagents. As always, keep all reagents from a particular kit and lot together (do not carryover leftover reagents from one kit to the next).
When developing your own assay using Luminex Technology, there are many formulations of SA-PE conjugates commercially available. We have tested many of these in both DNA direct hybridization and capture sandwich assays.
The Luminex 100/200 is a class 1(I) laser product.
Products are region specific and may not be approved in some countries/regions. Please contact support@luminexcorp.com to obtain the appropriate product information for your country of residence.