These raw polystyrene (xMAP®) and magnetic (MagPlex®) microspheres have a diameter of 5.4-5.6 microns and are coloured by two fluorochromes. They are previously coupled to specific reagents (probes, antigens, etc...); each ligand-coupled bead is -after an hybridization or immunological reaction and a detection step - analyzed twice. Firstly the beads are individually recognized by a red laser (excitation 633 nm, emission 675 nm -red- and > 712 nm -infrared-) for the Luminex® 200. Secondly, the coupled ligands (antibodies, DNA...) are quantified individually by a reporter molecule (in general Streptavidin-Phycoerythrin) and use of the green laser simultaneously to the red laser analysis. The intensities of red and green fluorescences are both recorded. Differents optical bead reagions, (currently up to 100 on a Luminex® 200 and up to 500 on a Luminex FlexMAP 3D®) can be mixed in a single tube.
The raw beads via their carboxylate groups, can be covalently bound with molecules of interest through their amino groups : e.g. nucleic acid probes corresponding to particular genetic alleles. This is the work of Beamedex® to produce these coupled-beads that are used as research reagents on Luminex® systems.
The beads are excited by a set of two lasers, each having a particular role :
• red diode laser (633 nm), which excites the fluorophores incorporated in polystyrene/magnetic beads emitting in the red and infrared , corresponding to the color code of each bead, and
enabling the precise identification of each ;
• the green laser, which excites the fluorochrome coupled to a reporter molecule. The reporter molecule can detect the ligand/receptor or probe/amplicon reaction, e.g. by hybridization at the surface of the bead, after the addition of a labeled conjugate with a green-emitting fluorochrome. Generally, this is streptavidin-phycoerythrin which, excited at 532 nm, retransmits the light at 575 nm.
The reflected fluorescence emitted determines the positivity of the reaction according to a threshold fluorescence for each bead. The fluorescence emitted from the fluorochrome reporter is detected by a photomultiplier tube at the outlet of the liquid stream.
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