Ryan Hamnett, PhD | 17th March 2025
Luminescent proximity assays are highly versatile tools for detecting and quantitating single analytes or interactions between two binding partners. Targeted detection is usually enabled by specific antibodies, which facilitate energy transfer between two beads to generate a chemiluminescent signal. Based on luminescent oxygen-channeling immunoassay (LOCI) chemistry, luminescent proximity assays are homogeneous assays, meaning they require no wash steps and are therefore highly amenable to automation and high-throughput screening.
Luminescent proximity assays rely on the transfer of energy via singlet oxygen (1O2) between acceptor and donor beads to produce a chemiluminescent signal (Figure 1).
Figure 1: Energy transfer via singlet oxygen (1O2) in luminescent proximity assays.
Chemiluminescence is generated via the following steps:
| Donor Beads 680 | Acceptor Beads 615 | |
|---|---|---|
| Chemical Constituents | Contain a porphyrin like molecule, phthalocyanine | Contain a 1O2 trap and a lanthanide (Eu3+) chelate |
| Excitation | 680 nm | Singlet oxygen reaction with the 1O2 trap produces 320-360 nm light, which excites the Eu3+ chelate, causing it to emit light |
| Emission | Conversion of molecular oxygen (O2) to singlet oxygen (1O2) | 615 nm |
| Size | 250 nm diameter – this size prevents most sedimentation in buffers and clogging of pipette tips, but the beads can be still be centrifuged and filtered, making handling more convenient | |
| Bead Coating | Hydrogel coating minimizes non-specific binding and self-aggregation, while allowing conjugation of biomolecules to the beads, such as glutathione, streptavidin, protein A, and antibodies. | |
Table 1: Key features of donor beads and acceptor beads
Mechanism of Action
Luminescence at 615 nm is only produced by the combination of chemistries that underly the donor and acceptor beads (Figure 2). Phthalocyanine in donor beads converts molecular oxygen to singlet oxygen, which is then trapped by acceptor beads. The resulting emission at 320-360 nm excites the lanthanide (e.g. Eu3+) chelate to cause emission at 615 nm.
Figure 2: Donor and acceptor bead activation. The phthalocyanine in the donor beads is used as an energy antenna to convert molecular oxygen to singlet oxygen after excitation at 680 nm. Singlet oxygen travels up to 200 nm to acceptor beads and reacts with the oxygen trap. The resulting emission at 320-360 nm activates the Eu3+ chelate to cause emission at 615 nm, which can be measured.
In order to detect analytes with luminescent proximity assays, those analytes must be bound to the donor or acceptor beads. This can be achieved in a number of ways. The most common format is for donor beads to be conjugated to streptavidin to bind to biotinylated molecules, while acceptor beads are conjugated to antibodies raised against a specific target (Figure 3).
Figure 3: General format to bind target biomolecules to donor and acceptor beads. Donor beads are conjugated to streptavidin, giving them an extremely high binding affinity for biotinylated targets. Meanwhile, acceptor beads are conjugated to specific antibodies raised against their target. Other binding substrates, such as protein A/G, glutathione or nickel, can also be used with the appropriate binding partner (e.g. GST, 6xHis).
A generalized protocol for a luminescent proximity assay would be:
Note that the exact order of addition and which protein is bound to which bead can influence the ultimate sensitivity of the assay. All 4 components (Protein A, Protein B, donor beads and acceptor beads) can be added simultaneously, but this may result in slower reaction times. Conversely, adding the beads and incubating sequentially after step 1 can result in greater sensitivity. Incubation times should be optimized, but 60 minutes is a recommended starting point.
This flexible and versatile set up can measure interactions between any biomolecules, and drugs that alter those interactions, using cell-free systems or cell lysates. For examples of how luminescent proximity assays can be applied, see Measuring Protein-Protein Interactions with Luminescent Proximity Assays and ELISA-like Luminescent Proximity Assays for Single Analytes.
Luminescent proximity assays offer many advantages for detecting single analytes or binding partners over comparable techniques such as ELISA and time-resolved Förster resonance energy transfer (TR-FRET).
Like any assay, luminescent proximity assays should be optimized to ensure optimal signal, including parameters such as bead concentration, analyte concentration, ATP levels and substrate design. Other potential considerations are discussed below.
Signal Interference
Signal interference can occur from singlet oxygen quenchers, such as antioxidants, biotin mimetics, and colored compounds that absorb light near the 615 nm range. Culture medium, particularly containing serum, may also reduce signal, so cell samples should be washed with a buffer like PBS before performing the assay.
To detect compounds that interfere with the light emitted, see Detection of Interfering Compounds to learn how our multiplex beads can provide a solution to identify the potential source of interference.
Selection of Antibody Pairs
As in sandwich ELISAs, selecting the right antibody pair is usually critical to ensuring the specificity of the assay.
Thanks to the flexibility of luminescent proximity assays, many experiments can be performed with more general, highly optimized antibodies, such as those targeting tags. When using target-specific antibodies, each antibody should recognize a distinct epitope, and validations should be performed to check that the antibodies function as a matched pair. Both monoclonal and purified polyclonal antibodies can be used.
One antibody is usually biotinylated and bound to the donor bead, while the other is conjugated to the acceptor bead. Conjugation to the acceptor bead can be direct (e.g. via thiolation), or indirect via Protein A/G or anti-species antibodies. It is recommended that the configuration of antibodies be tested to determine which provides the optimal signal, because sensitivity can differ significantly:
Antibodies should not be in amine-based buffers, such as Tris or glycine buffers, nor should they contain any protein-based stabilizer such as BSA or gelatin. Neutral or slightly alkaline (~pH 8) buffers should be used.
The Hook Effect
At very high concentrations of analyte, the signal can begin to decrease due to a phenomenon called the ‘hook effect’ (or ‘hooking effect’). This effect is common to reagents used to detect analytes that can become saturated, such as beads, antibodies and streptavidin.
Below the hook point (signal peak) and within the dynamic range of the assay, the beads bind to progressively more analyte as the analyte concentration increases, and show a concomitant and proportional increase in signal. However, beyond the hook point, the excess analyte inhibits the beads being brought into proximity, reducing signal (Figure 4).
Figure 4: The hook effect due to target saturation.
