Ryan Hamnett, PhD | 17th March 2025
Luminescent proximity assays are bead-based, homogeneous (no wash) assays for detecting and quantifying single analytes or binding partners with high sensitivity. Typically, the set up of the assay requires two beads, donor beads and acceptor beads, which produce light when brought within 200 nm of each other. Introducing a third bead, which can emit light at a different wavelength, enables multiplexed luminescent proximity assays that can detect more than one protein or interaction.
For more information on how luminescent proximity assays work, their advantages and their applications, see our other pages in this guide:
Acceptor beads are activated by singlet oxygen produced by donor beads, and emit light at a defined wavelength. To enable multiplexed assays, we currently offer two distinct acceptor beads: Acceptor Beads 615 use Eu3+ chelates to generate bright chemiluminescence at 615 nm, while Acceptor Beads 545 emit at 545 nm by using Tb3+ chelates (Figure 1). Critically, both Acceptor Beads are activated by singlet oxygen, meaning the same donor beads can be used with both.
Figure 1: Emission spectrum of Acceptor Beads 545 (Tb3+) compared to Acceptor Beads 615 (Eu3+).
Multiplexing allows the detection of multiple analytes, such as detecting the phosphorylated and non-phosphorylated forms of a protein simultaneously for normalization. A common antibody is bound to donor beads, while different antibodies are bound to each acceptor bead (Figure 2).
Figure 2: Multiplexed luminescent proximity assay for normalization of phosphorylated analytes. Total levels of an analyte can be detected by one assay, in this case using Acceptor Beads 545, while the phosphorylated form can be detected by conjugating phospho-specific antibodies to the Acceptor Beds 615.
Multiplexing allows ADA assays (see Anti-Drug Antibody (ADA) Assays) to be performed concomitant with isotyping assays (Figure 3). This combination can determine not only that a patient’s immune system is generating antibodies against a drug, but also provide more nuanced information on the nature of the immune response.
Figure 3: Multiplexed luminescent proximity assay simultaneous ADA assays and isotyping assays.
Isotyping assays distinguish between different antibody classes (e.g. IgM, IgG1, IgG2, IgG3, IgG4, IgA, IgE), which can indicate different types of immune response. For example, an early, acute response is often marked by IgM, while IgE can indicate an allergic response. Other classes can be associated with different clinical outcomes. Immune responses can also be tracked over time. For instance, a shift from IgM classes to IgG may indicate that the immunogenic response is becoming more established.
All detection technologies are susceptible to interference from certain compounds. If those compounds are present in a screening library, they may be mistakenly interpreted as affecting the interaction under investigation, rather than simply affecting the technology itself (i.e. giving a false positive signal). Multiplexed luminescent proximity assay beads can be used to detect interfering compounds and to distinguish between different types of compound (Figure 4).
The most common classes of compounds known to interfere with luminescent proximity assay beads are 1) Singlet oxygen (1O2) quenchers such as NaN3 and some transition metals (Cu2+, Zn2+, Fe2+), and 2) biotin mimetics (when using streptavidin-coated beads). These will decrease the signal generated proportional to their concentration in the assay.
To detect singlet oxygen quenchers, streptavidin donor beads are prebound with biotinylated acceptor beads 545, producing emission at 545 nm. Adding compounds to this complex will reduce the emitted signal only if they affect the transfer of singlet oxygen, or if they are compound aggregates that cause diffraction. Biotin mimetics will not be able to dislodge the prebound acceptor bead because of the extremely high affinity between biotin and streptavidin.
Subsequent addition of acceptor beads 615 can then detect biotin mimetics or pan-assay interference compounds (PAINS), the presence of which will result in less binding and reduced signal at 615 nm. Like with the 545 beads, singlet oxygen quenchers and aggregates can also affect this interaction.
Figure 4: Multiplexed luminescent proximity assays to detect interfering compounds. A, Biotinylated Acceptor Beads 545 are prebound to streptavidin donor beads to emit light at 545 nm. Subsequent addition of biotinylated Acceptor Beads 615 also produces light at 615 nm, unless in the presence of interfering compounds, which will inhibit the generation of light by both beads (singlet oxygen quenchers) or just 615 beads (biotin mimetics). B + C, Graphs demonstrating the effects of adding increasing concentrations of a singlet oxygen quencher (azide, B) or biotin (C) on 545 and 615 bead signal. Note that azide affects both signals equally, whereas biotin addition does not affect the prebound 545 signal.
