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 labelled 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

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

Once circularized, this DNA molecule undergoes rolling-circle amplification (RCA), a highly sensitive polymerase-driven process that generates long, repetitive DNA sequences anchored to the site of the 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 is used to extend one probe along the other, instead of ligating the oligonucleotide probes. PEA is preferred for quantitative multiplex applications and large-scale biomarker detection technologies.

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 site of protein interaction, 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

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

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 the detection of viral protein-host protein interactions during the infection cycles of HIV, influenza and the 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

References

1. Li H, Ma X, Shi D, Wang P. Proximity Ligation Assay: From a Foundational Principle to a Versatile Platform for Molecular and Translational Research. Biomolecules 2025;15(10):1468.
2. Wang P, Yang Y, Hong T, Zhu G. Proximity ligation assay: an ultrasensitive method for protein quantification and its applications in pathogen detection. Appl Microbiol Biotechnol 2021;105(3):923-935.
3. Kageler L, Perr J, Flynn RA. Tools to investigate the cell surface: Proximity as a central concept in glycoRNA biology. Cell Chem Biol 2024;31(6):1132-1144.
4. Narváez M, Crespo-Ramírez M, Fores-Pons R, Pita-Rodríguez M, Ciruela F, Filip M, et al. Study of GPCR homo-and heteroreceptor complexes in specific neuronal cell populations using the in situ proximity ligation assay. Receptor and ion channel detection in the brain: Springer; 2021:117-134.
5. Arioz BI, Cotuk A, Yaka EC, Genc S. Proximity extension assay‐based proteomics studies in neurodegenerative disorders and multiple sclerosis. Eur J Neurosci 2024;59(6):1348-1358.
6. Alam MS. Proximity ligation assay (PLA). Immunohistochemistry and immunocytochemistry: methods and protocols: Springer; 2021:191-201.
7. Liu R, Ota K, Iwama E, Yoneshima Y, Tanaka K, Inoue H, et al. Quantification of HER family dimers by proximity ligation assay and its clinical evaluation in non–small cell lung cancer patients treated with osimertinib. Lung Cancer 2021;158:156-161.
8. Sharanek A, Raco L, Soleimani VD, Jahani-Asl A. In situ detection of protein-protein interaction by proximity ligation assay in patient derived brain tumor stem cells. STAR protocols 2022;3(3):101554.
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.