Ryan Hamnett, PhD | 17th March 2025
Luminescent proximity assays are highly sensitive bead-based, homogeneous (no wash) assays that can be used to detect and quantify single analytes, similar to ELISA. Any sandwich ELISA can be easily converted to a luminescent proximity assay using the same matched antibody pairs. Compared to ELISA, these assays benefit from:
Luminescent proximity assays work on the basis of energy transfer between two beads, donor beads and acceptor beads. Donor beads are excited by 680 nm light to convert oxygen (O2) to singlet oxygen (1O2), which diffuses up to 200 nm to acceptor beads, causing them to emit light at 615 nm.
For more information on how luminescent proximity assays work, their advantages and their applications beyond ELISAs, see our other pages:
The same matched pair of antibodies used in a sandwich ELISA can be applied to luminescent proximity assays. Streptavidin-coated donor beads are bound to the biotinylated detection antibody, while acceptor beads are directly conjugated to capture antibodies (Figure 1). As with a sandwich ELISA, the capture and detection antibodies recognize distinct epitopes on the same antigen. Once both have bound to the analyte, the beads are brought together to generate light at 615 nm.
No wash steps are needed because only beads in close proximity will result in emission from the acceptor beads, while effective amplification of the signal is achieved through the thousands of singlet oxygen molecules released by the donor beads.
Figure 1: Replacing ELISAs with luminescent proximity assays. In luminescent proximity assays, capture and biotinylated detection antibodies recognize different epitopes on the same antigen, bringing together acceptor and donor beads to produce a signal. With short incubation steps and no washes, the overall protocol takes only 1.5 hours. Note that although all components can be added at the same time, adding the streptavidin-coated donor beads later to bind the biotinylated detection antibody tends to result in faster reaction times.
| Luminescent Proximity Assay | ELISA | |
|---|---|---|
| Protocol time | 1 – 2 hours | ~4.5 hours |
| Automation | Easy | Moderate |
| Manual throughput (tests/day) | Thousands | Hundreds |
| Dynamic range | Up to 5 logs | Up to 2 logs |
| Typical sensitivity (lower limit) | 1-10 pg/ml | 10-50 pg/ml |
| Signal stability | Up to 24 hours | Up to 1 hour |
| Typical signal:noise ratio | >100 | ~10 |
| Typical cost per test ($) | 0.5 - 5 | 1 - 10 |
Table 1: Comparison of the main features of luminescent proximity assays and ELISAs
Small molecules are traditionally difficult to measure by sandwich ELISA because their small size means that two antibodies cannot bind to them simultaneously. Competition ELISA solves this issue, and a similar approach can be taken with luminescent proximity assays (Figure 2).
One prominent target for such competition assays are second messengers, such as cAMP and IP3, which are small molecules that are essential to signal transduction cascades, carrying information from activated receptors into the cell to effector proteins. Measuring the changing levels of second messengers can therefore be useful as a proxy for receptor activation, such as G-protein coupled receptor (GPCR) activation.
To measure cAMP in cells, a biotinylated cAMP probe can be sandwiched between streptavidin-donor beads and anti-cAMP antibody-conjugated acceptor beads, producing a high signal. On addition of cell lysate, a decrease in the signal will be observed because cAMP from the cell will compete for the anti-cAMP antibodies, resulting in the beads becoming separated.
Figure 2: Competitive luminescent proximity assay to measure second messengers such as cAMP.
This competitive approach can be used for many other small molecules, such as the thyroid hormone T4, though in some cases the competitive probe will need to be conjugated to something larger, such as BSA, to act as a carrier so that both beads can bind to it. Alternative methods of assessing GPCR activation include looking at phosphorylation events downstream, such as kinase activity at ERK1/2.
Anti-drug antibody (ADA) assays are essential for determining the immunogenicity of biologic drugs by detecting whether a patient’s immune system has generated antibodies against the drug.
High sensitivity ADA assays can be performed using luminescent proximity beads in the standard ‘bridging’ format (Figure 3). Here, both the donor and acceptor beads are bound to the drug. If a sample contains ADAs, the beads will be brought together, bridged by the antibody, and produce a signal that is proportional to the concentration of ADA in the sample.
Figure 3: Anti-drug antibody (ADA) assay with luminescent proximity beads. Both the donor and the acceptor beads are bound to the drug, in this case a therapeutic monoclonal antibody. Anti-drug antibodies (green) will bind to the drug and act as a bridge to bring the donor and acceptor beads into proximity.
Single nucleotide polymorphisms (SNPs) are single base mutations in genomic DNA that occur in at least 1% of the population. SNPs and other allelic variations can be detected using luminescent proximity assays by using oligonucleotide probes that are complementary to a specific SNP sequence of interest, which would be conjugated to acceptor beads. Attached to the donor beads is a biotinylated oligonucleotide probe that will bind to DNA near to the SNP site. If the SNP is present, the acceptor beads will bind to the DNA and be brought into close proximity with the donor beads to generate a signal.
Our Fluorescent Beads 680/615 combine the features of donor and acceptor beads to make a single bead that emits a bright, long-lived signal at 615 nm. They can be excited at either 680 nm like traditional acceptor beads, or at 340 nm (detectable using Fluorescence Intensity or Time Resolved Fluorescence modes) to directly excite the Eu3+ chelate (see Features of Luminescent Proximity Assay Donor and Acceptor Beads).
Fluorescent beads can be coated with streptavidin or anti-species (human, mouse, rabbit) IgG to easily replace any fluorometric (e.g. FITC) or chromogenic (e.g. HRP) conjugates used in existing ELISA protocols (Figure 4).
Figure 4: ELISA comparison performed using traditional chromogenic reagents vs fluorescent beads. Streptavidin-coated fluorescent beads are added to bind to the biotinylated detection antibody. After removal of unbound beads, bound fluorescent beads are excited at either 680 nm or 340 nm to result in 615 nm emission. No stop reaction is needed.
Advantages of Fluorescent Beads in ELISA
