Traditionally, methods like ELISA (Enzyme-Linked Immunosorbent Assay) or Western Blot are in use for a single-analyte, or analysis of a single intracellular or extracellular protein at a time. These are also called singleplex protein detection methods. Both these methods are costly, time-consuming, and sample-depleting when one wishes to measure multiple markers in each sample.
An alternative to these laborious techniques is Luminex xMAP (Multi-Analyte Profiling) technology. It enables scientists to measure large numbers of proteins present in the given sample within a single reaction. It is also known as Multiplexing. It is a type of solid-phase Immunoassays, where the result analysis is carried out by the Luminex 100/200™ instrument.
Principle of Luminex multiplex assays
Luminex Assay make use of different colored beads of superparamagnet, which are having a coating of antibodies specific to the analyte under study. These beads are added to and incubated with the sample. If the analyte has the molecules specific for the antibodies on the beads, they will get captured by these beads. The detection of these conjugates needs a unique instrument.
How Luminex Multiplex technology works?
Luminex technology is a combination of advanced fluidics, digital signal processing, and optics. xMAP technology has a flexible design, which helps in performing a variety of bioassays accurately, cost-effectively, and quickly.
Mechanism of Luminex Multiplex assay
- xMAP technology makes use of microspheres or beads made up of superparamagnet having 6.5-micron size.
- The core of the sphere has a magnet, and the surface is of polystyrene.
- Red and infrared fluorophore or dyes are present in each bead in different proportions.
- These differing proportions of the fluorophores result in the generation of 100 unique spectral peaks, each representing an individual microsphere.
- These spectra are then recognized and identified by the detection system of Luminex xMAP.
- It allows the qualitative and quantitative analysis of multiple analytes using a single well.
The procedural guidelines
- Take a filter bottom microplate with 96 or more wells.
- Mix beads having varying proportions of a fluorophore and different analyte-specific antibodies with the analyte in the well.
- There will be a separate well for Standard, sample, and control analyte concentrations.
- Allow the mixture in the wells to incubate.
- Initially, the analytes bind to the antibodies on the beads.
- Wash the wells to removes unbound beads.
- Now is the time for the addition of detector antibodies, which are analyte-specific and having some tagging and incubation.
- The detector antibodies bind to the epitopes of the appropriate analytes, which in turn is present on the beads.
- After washing, add streptavidin and R-Phycoerythrin (Streptavidin-RPE) conjugate to the system and incubate.
- Here, the biotinylated detector antibodies in association with the analytes on the beads form a four-member sandwich.
- After washing, the beads are subject to the Luminex 100/200™ instrument for analysis.
- You can find The concentration of the analytes by monitoring the level of R-Phycoerythrin (RPE) fluorescence and the spectral properties of the beads.
Luminex Multiplex is a solid-phase immunological assay that makes the simultaneous detection of multiple proteins in a sample quick and efficient.