Luminex Multiplex Assays

The Luminex is a flexible analyzer based on the principles of flow cytometry, which combines advanced optics, lasers, fluid handling, digital signal processing, and software to give rapid results.  The system enables you to multiplex (simultaneously measure) up to 100 analytes in a single microtitre plate well, using very small sample volumes.  With xMAP technology, biological tests or bioassays are performed on the surface of color-coded microspheres — rather than on the surface of a plate or the bottom of a well. Analytes to be measured within a sample are captured to the surface of the microspheres.

The Technology

The microspheres are dyed to create 100 distinct colours and have a distinct ‘spectral address’ based on red/infrared content.  Colour-coding allows each microsphere to be read individually and to be multiplexed with other microspheres.  The surface of the 5.6µm polystyrene beads are carboxylated to allow easy coupling of biomolecules such as antibodies, oligonucleotides, peptides and receptors.

Each microsphere set is coated with a capture reagent (e.g. antibody or oligonucleotide) that binds the sample analytes, and a fluorescent phyco-erythrin (PE) reporter tag then detects the bound analytes.  The beads are interrogated by a red laser (to identify the bead type) and by a green laser which determines the amount of bound analyte.

The Luminex instrument contains 2 laser lines.  A 635nm red laser excites the hard dyed beads for bead classification and a second 532nm green laser excites the PE reporter tag which determines the amount of bound analyte. 

The concentration of bound analyte in unknown samples is automatically calculated in reference to a standard curve of known cytokine standards using the instrument software.

Applications

Luminex technology has a number of applications including:

1. Immunoassays
Antibody-tagged beads specific for a wide range of soluble proteins including cytokines, chemokines, markers of the endocrine system, cardiovascular system, hormones, bone metabolism, isotyping, growth factors and cell signalling.  Multiplexing can remove the need for multiple ELIZA plates as multiple cytokines can be measured in the sample sample, hence reducing time and cost.  Please contact Tracey Lee-Pullen for a list of commercial kit suppliers.

2. Nucleic acid assays
High throughput gene expression profiling and genotyping of multiple markers.  Quantitation of miRNA expression.

3. Enzyme assays.
E.g. Kinase, phosphatase and protease assays.

4. Receptor ligand assays.

Multiplex Kits

Multiplex kits are available from a number of suppliers.  Kits are either pre-mixed into set panels or can be combined to your particular requirements.  When purchasing a kit take care to make sure all the reagents you require are included as some kit suppliers separate the different components e.g. buffers and reagents and capture beads so you will need to buy a base kit plus the beads. 

There are a wide range of analytes commercially available, however if the protein you are interested in is not currently available you do have the option of labelling your own beads as many companies sell 'blank beads' and labelling kits.  Once you have labelled your beads you can then integrate these with other analytes provided the bead IDs are different.

Setup of most kits requires a plate shaker and a vaccum manifold for a the 96-well filter plates.  We have a small lab setup area with these 2 pieces of equipment available for those users who do not have access to these instriments.  We provide full training on the instrument and software, after which users are free to book and run the instrument unassisted.

If you would like more information about the Luminex Instrument and multiplexing applications please contact Tracey Lee-Pullen. 

We have prepared an information pack with details about Luminex technology and things to consider when setting up your experiments which can be downloaded in pdf format.  Current Luminex users can also access a copy of the users manual from this page.