Multiplex (assay)

A multiplex assay is a type of assay that simultaneously measures multiple analytes (dozens or more) in a single run/cycle of the assay. It is distinguished from procedures that measure one analyte at a time. Multiplex assays within a given application area or class of technology can be further stratified based on how many analytes can be measured per assay, where "multiplex" refers to those with the highest number of analyte measurements per assay (up to millions) and "low-plex" or "mid-plex" refers to procedures that process fewer (10s to 1000s), though there are no formal guidelines for calling a procedure multi-, mid-, or low-plex based on number of analytes measured. Single-analyte assays or low-to-mid-plex procedures typically predate the rise of their multiplex versions, which often require specialized technologies or miniaturization to achieve a higher degree of parallelization.

Multiplex assays are widely used in functional genomics experiments that endeavor to detect or to assay the state of all biomolecules of a given class (e.g., mRNAs, proteins) within a biological sample, to determine the effect of an experimental treatment or the effect of a DNA mutation over all of the biomolecules or pathways in the sample. The ability to perform such multiplex assay experiments measuring large numbers of biomolecular analytes has been facilitated by the completion of the human genome sequence and that of many other model organisms.

"Multiplex" versus "High-throughput"

Multiplex assays are often used in high-throughput screening settings, where many specimens can be analyzed using a multiplex (or other) assay. Strictly speaking, a multiplex assay is not necessarily high-throughput. When the execution of a single multiplex assay generates data for a large number of analytes (e.g., gene expression levels for all genes in the human genome), it is considered high-throughput. However, it is more the ability to rapidly process multiple specimens in an automated fashion that characterizes high-throughput techniques. Massive parallelization of assays is one way to achieve "high-throughput" status. Another way is via automating a manual laboratory procedure.

Example multiplex assay techniques

Nucleic acid-based multiplex techniques

• DNA microarray used for gene expression or SNP detection assays
• SAGE for gene expression
• High-throughput sequencing which can produce millions of short DNA sequences in parallel
• Multiplex PCR for applications requiring the amplification or sequencing of DNA or RNA
• Multiplex Ligation-dependent Probe Amplification (MLPA)
• DNA sequencing by ligation
• Luminex/XMAP is bead based multiplexing, where beads are internally dyed with fluorescent dyes to produce a specific spectral address. Biomolecules (such as an oligo or antibody) can be conjugated to the surface of beads to capture analytes of interest. This technology uses flow cytometric or imaging technologies for characterization of the beads as well as detection of phycoerythrin emission due to analyte presence. The Luminex technology enables up to 500 proteins or genes to be detected in each well of a 96 or 384-well plate, using very small sample volume. Common applications include cytokines, metabolic markers, and phosphorylated proteins.

Protein-based multiplex techniques

• Protein microarray for measuring protein-protein interactions or small molecule binding
• Antibody microarray a type of protein array in which antibodies are arrayed
• Phage display for screening large protein libraries for interacting proteins or other molecules
• Antibody profiling (e.g. multiple HLA antibody identification or reactivity prediction against a panel of organ donor population): Luminex/XMAP principle based muliplexing is done for Anti-HLA antibody identification in serum samples where a mixture of color-coded luminex beads are coated either in such a way that individual beads (PRA beads) carry HLA peptide libraries from individual donors thus in combination simulating a donor population (for Panel reactive antibody or PRA testing), or single purified synthetic HLA antigens captured onto the beads (single antigen beads).
• Luminex/XMAP is bead based multiplexing, where beads are internally dyed with fluorescent dyes to produce a specific spectral address. Biomolecules (such as an oligo or antibody) can be conjugated to the surface of beads to capture analytes of interest. This technology uses flow cytometric or imaging technologies for characterization of the beads as well as detection of phycoerythrin emission due to analyte presence. The Luminex technology enables up to 500 proteins or genes to be detected in each well of a 96 or 384-well plate, using very small sample volume. Common applications include cytokines, metabolic markers, and phosphorylated proteins.
• Binding Antibody Multiplex Assay (BAMA) can be run using multiple ligands to profile multiple antibody isotypes and subclasses.^[1][2][3]

Other multiplex techniques

• Tissue microarray for analyzing multiple tissue samples
• Cellular microarray for observing cellular responses against a panel of materials
• Chemical compound microarray to assay multiple chemical compounds for specific activities
• Multiplex detection enables the simultaneous detection of two or more targets on a western blot.^[4]
• Multiplex biomarker analysis of urine.^[5]
• ELISAs are a type of assay that is not multiplex per se because each assay detects a single analyte, but it is typically parallelized via microtiter plates to achieve high-throughput sample processing.

References