Proximity ligation assays (PLA) are highly sensitive immunoassay technologies designed to detect protein–protein interactions, post-translational modifications or low-abundance proteins directly within cells or tissue samples. In PLA, detection specificity is achieved by using two independent primary antibodies, each recognizing a distinct epitope on the target proteins. The primary antibodies are conjugated to secondary antibodies labeled with short DNA oligonucleotides. When the two targets are in proximity, the attached oligonucleotides are ligated to form a circular DNA template. This circular DNA is then amplified through rolling-circle amplification, producing a localized fluorescent signal that can be quantified at single-molecule resolution.¹

Because PLA combines dual-recognition antibody specificity with DNA-based signal amplification, it allows researchers to visualize molecular interactions with high precision. Therefore, it has become an essential detection tool in drug discovery for validating target engagement, confirming the mechanism of action and monitoring signaling changes in response to candidate compounds. Furthermore, in biomarker research, PLA is used to detect rare or transient protein interactions that traditional immunoassays may miss.¹

What Are Proximity Ligation Assays?

Proximity ligation assays are advanced antibody-based detection methods that translate protein proximity, resulting from interactions, binding events or post-translational modifications, into a quantifiable DNA signal. The core principle involves two antibodies binding to their respective epitopes on the same protein or interacting with their respective protein partners. Each antibody is conjugated to a short DNA oligonucleotide. When the targets lie within a defined nanoscale distance, typically within 30-40 nm, the attached DNA strands are brought close enough to be ligated into a circular DNA molecule.¹

This circular DNA then undergoes rolling-circle amplification (RCA), generating a long, repeated DNA product that can be visualized as a bright fluorescent spot or measured quantitatively. Because the assay requires dual antibody recognition and successful DNA ligation, PLA inherently provides high specificity for proximity events. From this perspective, the ability to detect low-abundance proteins or transient interactions makes PLA a powerful alternative to traditional immunoassays, such as ELISA or immunofluorescence, as well as other methods for measuring protein-protein interactions, including immunoprecipitation and fluorescent resonant energy transfer (FRET).¹

PLA vs. Other Protein Detection and Interaction Analysis Methods

Principle of a Proximity Ligation Assay

The principle of a proximity ligation assay is to convert molecular proximity, typically the close spatial association of two proteins or two epitopes on the same protein, into a detectable and quantifiable DNA signal. PLA relies on a pair of primary antibodies, raised in different species, with each detecting a unique epitope. The primary antibodies are conjugated to short DNA oligonucleotide sequences. When the resulting PLA probes are within 40 nm proximity of each other, their attached DNA strands can be joined by enzymatic ligation, forming a unique circular DNA template.²

In indirect PLA formats that use species-specific secondary probes, the two primary antibodies should generally be raised in different host species, for example, mouse and rabbit, so each PLA probe selectively recognizes one primary antibody. With same-species primary antibodies, both secondary probes may bind either antibody, preventing unambiguous probe assignment and potentially generating artifactual or difficult-to-interpret proximity signals. This restriction does not apply to direct PLA formats in which PLUS and MINUS oligonucleotides are conjugated directly to validated primary antibodies.

Once circularized, this DNA molecule undergoes rolling-circle amplification (RCA), a highly sensitive, polymerase-driven process that generates long, repetitive DNA sequences anchored at the site of protein interaction. Fluorescently labeled complementary probes are subsequently hybridized to the amplified DNA, producing bright, punctate signals that can be detected under fluorescence microscopy.²

Two key enzymes drive the conversion from proximity to an amplified signal:²

• DNA ligase joins adjacent oligonucleotides when they are brought together by antibody-bound proteins, ensuring that only true proximity events generate a DNA circle
• DNA polymerase, through RCA, amplifies the circular DNA thousands-fold, generating a high concentration of fluorescence at the interaction site

Types of Proximity Ligation Assays

Proximity ligation assays are available in several formats, varying in sample type, target complexity and detection method.

Depending on the need for a secondary antibody, a PLA can be direct or indirect.

Direct PLA³

• Direct PLA uses primary antibodies directly conjugated to oligonucleotides
• It is suitable for targeting proteins that have validated antibody conjugates
• Provides high specificity and minimizes background noise due to the straightforward protein-antibody-probe conjugation

Indirect PLA³

• Indirect PLA introduces secondary antibodies to carry the oligonucleotide probes
• Ideal when primary antibodies cannot be readily conjugated to the oligonucleotides
• It can produce a slightly higher background, but it also allows flexibility for researchers to use primary antibodies across a wide range of targets

PLA can also be  in situ  or solution-based depending on the type of sample.

In situ PLA⁴

• In situ PLA detects molecular interactions directly within intact cells or tissue sections
• Preserves spatial context, enabling visualization of where interactions occur within cellular architecture
• Produces discrete fluorescent dots that each represent an interaction event
• Ideal for studying signaling networks, receptor activation or subcellular protein localization

Solution-Based PLA (or Homogeneous PLA⁾¹

• Solution-based PLA is performed in liquid samples, such as cell lysates, plasma or serum
• Can be used to quantify protein–protein interactions or low-abundance biomarkers without microscopy
• DNA amplification products are quantified using qPCR, digital PCR or sequencing
• It is suitable for high-throughput and clinical applications

Proximity Extension Assay (PEA)⁵

In this advanced type of PLA assay, a DNA polymerase extends one probe along the other, rather than ligating the oligonucleotide probes. PEA is preferred for quantitative multiplex applications and large-scale biomarker detection technologies.

PLA Signal Detection Formats

• Fluorescence microscopy — the standard readout for in situ PLA. Fluorescent detection oligonucleotides hybridize to amplified DNA products, which appear as discrete signals that can be counted and localized within fixed cells or tissue sections.
• Brightfield microscopy — an immunohistochemistry-style readout suitable for cells and tissue sections, including formalin-fixed, paraffin-embedded samples. An enzyme-mediated reaction produces a visible precipitate that can be evaluated using a standard light microscope.
• Flow cytometry — enables quantitative, higher-throughput measurement of PLA signals in fixed, permeabilized suspension-cell populations. Conventional flow cytometry measures signal intensity at the individual-cell level but does not preserve subcellular localization.

How Proximity Ligation Assays Work

Sample Preparation

Biological samples, such as cultured cells, tissue sections or protein lysates, are prepared and fixed or lysed depending on the assay format. Proper preparation is essential for preserving protein epitopes and the structural context necessary for accurate detection.⁶

Binding of Primary Antibodies

Samples are incubated with primary antibodies that recognize either two distinct proteins or separate epitopes on the same protein. These antibodies anchor the detection system to the molecules of interest with high specificity.⁶

Addition of PLA Probes

PLA probes, secondary antibodies conjugated to unique DNA oligonucleotides, bind to the primary antibodies. These DNA-tagged probes act as molecular barcodes, reporting whether the recognized proteins are within nanoscale proximity.⁶

Hybridization and Ligation:

Short connector oligonucleotides are added to the sample such that they hybridize only with PLA probes that are within 40 nm of each other. DNA ligase seals the hybridized ends, forming a circular DNA that represents the unique proximity or binding event.⁶

Rolling Circle Amplification (RCA)

The circular DNA serves as a template for rolling-circle amplification (RCA) driven by DNA polymerase. RCA produces a long, single-stranded DNA concatemer anchored at the protein-interaction site, thereby amplifying the signal thousands of times.⁶

Detection

Fluorescently-labeled complementary oligonucleotide probes hybridize to the amplified DNA. Each RCA product appears as a discrete fluorescent spot under a fluorescence microscope. The number and localization of signals reflect the frequency and spatial distribution of protein interactions.⁶

Experimental Design Considerations for PLA

Beyond the core mechanism, several experimental design choices can improve the reliability and interpretability of PLA results. These considerations complement, rather than replace, a validated, format-specific protocol:

• Use appropriate controls — include no-primary and single-primary controls to assess signal arising from nonspecific antibody or probe binding. When possible, add a known positive control to confirm that sample preparation, assay chemistry and detection are functioning as expected.
• Optimize antibody, amplification and imaging conditions — titrate each primary antibody under sample-processing conditions compatible with both targets. Excess antibody can increase background, while high target density, extended amplification or limited image resolution can cause neighboring signals to overlap, reducing counting accuracy.
• Validate the antibody pair before a full experiment — confirm target specificity, epitope accessibility and compatible fixation, permeabilization or antigen-retrieval conditions. For indirect PLA using species-specific secondary probes, the primary antibodies should generally be raised in different host species. Direct PLA formats using independently conjugated PLUS and MINUS primary antibodies can accommodate same-species antibody pairs.

Advantages of Proximity Ligation Assays

Proximity ligation assays offer several advantages over traditional immunoassays, making them powerful tools for studying protein interactions and low-abundance targets.¹

• High sensitivity and specificity: PLA combines dual antibody recognition with DNA ligation and RCA, allowing detection of low-abundance proteins and rare interactions with minimal background noise
• Spatial context:  In situ  PLA preserves cellular and tissue architecture, allowing researchers to visualize exactly where protein interactions occur within the biological sample
• Quantitative analysis: Each proximity event generates a discrete amplified DNA signal, making PLA suitable for single-molecule detection and quantification
• Versatility: PLA can be applied to diverse sample types, including cultured cells, tissue sections, lysates and various other clinical specimens, making it suitable for both basic research and translational applications

Limitations of Proximity Ligation Assays

PLA’s sensitivity and specificity come with tradeoffs that are important to weigh when designing an experiment or interpreting results:

• Proximity is not proof of direct interaction: A positive PLA signal indicates that two antibody-bound epitopes were within the assay’s effective proximity range, commonly described as approximately 40 nm. Signals may therefore reflect proteins within the same larger complex or densely organized compartment rather than direct binding. Orthogonal validation may be needed when the biological claim concerns direct physical interaction.
• Dependent on antibody or affinity-reagent quality: PLA performance depends on the specificity, affinity and validated performance of the antibodies or other affinity reagents used. Poorly performing reagent pairs can reduce sensitivity or increase background, and these limitations cannot always be overcome by increasing probe concentration.
• Background and cross-reactivity: Nonspecific antibody or probe binding, antibody cross-reactivity, high local target density or suboptimal assay conditions can generate background or artifactual signals. Appropriate technical and biological controls are essential for interpretation.
• Semi-quantitative in most formats: in situ PLA signal counts can support relative comparisons of target proximity across samples, but they do not inherently provide absolute molecular quantities. Absolute quantification requires an appropriately calibrated assay and readout designed for that purpose.

Applications of Proximity Ligation Assays

Proximity ligation assays have broad applications across biomedical research, translational science and drug development.

Drug discovery and mechanistic studies

PLA is widely used to validate target engagement, assess drug–protein interactions and monitor signaling modulation in response to candidate compounds. In cancer research, PLA is frequently employed to quantify receptor dimerization (e.g., EGFR and HER2), which promotes overactive signaling. Furthermore, it is instrumental in evaluating dimerization in tumor samples in response to receptor blockers or monoclonal antibodies.^7,8

Protein-protein interaction studies in situ

Because  in situ  PLA detects interactions directly within intact cells or tissue sections, it provides insights into where and when protein complexes form. Thus, it enables researchers to study transient interactions, receptor activation and pathway dynamics in both physiological and pathological settings. In particular, PLA has been applied to detect viral protein-host protein interactions during the infection cycles of HIV, influenza and coronavirus^.9,10

Detection of Post-Translational Modifications and Biomarkers

PLA can identify modifications, such as phosphorylation, ubiquitination or cleavage events, by using antibody pairs that recognize modified epitopes. In clinical and translational research, solution-based PLA formats are used to measure low-abundance biomarkers in liquid samples with high sensitivity. It can help quantify phosphorylation and activation states of key signaling proteins, such as ERK and AKT, as well as the effect of kinase inhibitors on activation and pathway dynamics.^1,11

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References

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9. Chakraborty S, Suresh S, Buch H, Panchapakesan A, Ranga U. Proximity Ligation Assay to Detect the Proximity Between Host Proteins and Viral Proteins of HIV-1. HIV Protocols: Springer; 2024:245-258.
10. Hmila I, Marnissi B, Kamali-Moghaddam M, Ghram A. Aptamer-assisted proximity ligation assay for sensitive detection of infectious bronchitis coronavirus. Microbiol Spectr 2023;11(1):e02081-22.
11. Keeney MT, Hoffman EK, Greenamyre JT, Di Maio R. Measurement of LRRK2 kinase activity by proximity ligation assay. Bio-protocol 2021;11(17):e4140-e4140.