Multiplex immunoassays
Capture antibodies coupled to polystyrene microspheres that are internally dyed in distinguishable ratios, so a flow-based reader classifies each bead and measures its reporter fluorescence separately. Up to 50 or 500 regions are available, the reported value is the median across the beads of a region, and bead count therefore sets the precision.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Luminex beads
Capture antibodies coupled to polystyrene microspheres that are internally dyed in distinguishable ratios, so a flow-based reader classifies each bead and measures its reporter fluorescence separately. Up to 50 or 500 regions are available, the reported value is the median across the beads of a region, and bead count therefore sets the precision.
How it differs from the standard method
- Precision is counting statistics: the usual acceptance criterion is at least 25–35 beads per analyte per well.
- Adjacent bead regions in the classification space can be mis-called, and the reporter channel bleeds between them.
- Magnetic beads allow plate washers and far better reproducibility than filter-plate vacuum washing.
- Beads can be coupled in-house, which makes custom panels possible, and makes re-validating the panel the user's job.
1Many analytes, one well
A multiplex immunoassay is a sandwich immunoassay run many times over in the same volume of sample. Each analyte has its own capture antibody, held on a physically or spectrally distinguishable support (a dyed microsphere, a printed spot on the floor of the well, a feature on a slide) and each has its own biotinylated detection antibody.
Luminex / xMAP
Capture antibodies on polystyrene microspheres coded by internal dyes; a flow reader classifies each bead and measures its reporter fluorescence, reporting the median per bead region.
Planar arrays
Capture antibodies printed as a spot array in each well. Electrochemiluminescent or fluorescent imaging reads all the spots at once.
Proximity extension
Antibody pairs carry DNA oligos that hybridise only when both bind the same molecule; the joined oligos are amplified and counted, so cross-reactive pairs give no product.
Sample
One small aliquot serves the whole panel, which is also why it has to be one dilution.
2Cross-reactivity is a property of the mixture
In a singleplex sandwich assay there are two antibodies and one antigen. In a 25-plex there are twenty-five detection antibodies meeting twenty-five capture reagents, which is six hundred ordered pairs that have to not interact.
3One dilution, many dynamic ranges
The analytes of a serum cytokine panel are not present at comparable concentrations. IL-2 may sit below a picogram per millilitre while CRP is in the milligrams per millilitre, nine orders of magnitude, and each individual immunoassay has a working range of about three.
4The matrix effect has a different sign for each analyte
Serum and plasma contain heterophilic antibodies, soluble receptors, complement, lipids and rheumatoid factor, and each of them interferes with some antibody pairs and not others. The consequence is that spike recovery in a panel is not one number: the same serum may suppress one analyte to 60% and enhance another to 140%.
- Spillover
- Signal reaching the wrong channel or the wrong region: reporter fluorescence bleeding between adjacent bead classes, or light from one printed spot reaching its neighbour. It scales with the brightness of the neighbour, so it matters most for a scarce analyte next to an abundant one.
- Bead count
- How many microspheres of a given region the reader classified. The reported value is the median over those beads, so precision is set by counting statistics; fewer than about 25–35 beads is the usual rejection criterion.
- Spike recovery
- A known amount of analyte added to the real sample matrix and measured. 80–120% is the usual acceptance window, and in a panel it has to be checked analyte by analyte.
- Dilution linearity
- Whether the back-calculated concentration stays the same when the sample is diluted further. A failure means the matrix, not the analyte, is being measured.
- Hook effect
- At very high analyte concentration, capture and detection antibodies bind separate molecules and the signal falls instead of rising, so an enormous concentration can read as a modest one. The reason a sample above range is diluted and re-run rather than extrapolated.
- LLOQ / ULOQ
- Lower and upper limits of quantification: the concentrations between which that analyte's values are accurate. In a panel they are different for every analyte.
5What the software will hand you
Analysis software will print a concentration for a signal below the lowest standard, for a signal above the highest, and for a median taken over eight beads. Those numbers are indistinguishable downstream from real measurements, and in a panel of fifty analytes there will always be several of them.
Key terms
Practise these as flashcards →The number of analytes measured simultaneously. Cross-reactivity risk grows with its square, because every detection antibody meets every capture reagent.
Median fluorescence intensity: the raw readout of a bead-based assay, the median over the beads of one region in that well.
A microsphere population identified by its internal dye ratio, carrying one capture antibody. Adjacent regions in the classification space are the ones that spill into each other.
A standard diluent formulated to behave like the sample matrix, so the standard curve carries the same interference as the samples.
A format in which antibody-linked DNA oligos must both bind the same molecule before they can be extended and amplified, so cross-reactive antibody pairs produce no signal.
Demonstrating specificity, recovery, linearity and precision for every analyte in the combination actually being run, as opposed to in each assay separately.
When to use it
- Measuring a cytokine or biomarker panel in a sample too small to split between separate assays, such as paediatric serum, CSF, tears or a small cell-culture supernatant.
- Screening many candidate analytes across a cohort to decide which few deserve a validated singleplex assay.
- Profiling a signalling or inflammatory response where the pattern across analytes is the result, rather than any single concentration.
- Any study where running the equivalent number of ELISAs would exhaust the sample, the budget or the available time.
Compared with alternatives
Better validated, cheaper per analyte and free of cross-reactivity between pairs; prefer it once you know which one or two analytes matter.
Gives a size as well as an intensity, so it can show that a signal is the protein rather than something the antibody also binds: useful for validating a panel's analytes.
No antibodies, so no cross-reactivity, and it measures hundreds of proteins; but it is far less sensitive for the low-picogram cytokines a panel is usually built for.
Bead-based multiplexing can be read on a flow cytometer, and the same instrument measures intracellular cytokines per cell rather than secreted totals.
Measures what a protein does rather than how much of it is there; an immunoassay cannot distinguish active from inactive.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Capture antibodies coupled to polystyrene microspheres that are internally dyed in distinguishable ratios, so a flow-based reader classifies each bead and measures its reporter fluorescence separately. Up to 50 or 500 regions are available, the reported value is the median across the beads of a region, and bead count therefore sets the precision.
- Precision is counting statistics: the usual acceptance criterion is at least 25–35 beads per analyte per well.
- Adjacent bead regions in the classification space can be mis-called, and the reporter channel bleeds between them.
- Magnetic beads allow plate washers and far better reproducibility than filter-plate vacuum washing.
- Beads can be coupled in-house, which makes custom panels possible, and makes re-validating the panel the user's job.