Proximity Ligation Assay

Introduction to Proximity Ligation Assay

Proximity Ligation Assay (PLA) is a homogeneous, highly specific and sensitive immunohistochemical tool that couples the specificity of ELISA with the sensitivity of PCR. The technique was developed by Fredriksson and colleagues in 2002 and has been shown to overcome the difficulties that arise when attempting to visualize and study single proteins, protein–protein interactions and post translational modifications (PTMs), such as phosphorylation, acetylation and glycosylation. PLA can be used in advanced protein applications and diverse samples such as biological samples, cell lines and fresh, frozen or formalin fixed, paraffin embedded (FFPE) tissues. It is also a particularly useful technique to elucidate how cells function in health and disease.

Principles of the Proximity Ligation Assay

Enhanced selectivity for this technique comes from the utilization of a pair of DNA proximity probes, each composed of a specific antibody linked to a high affinity oligonucleotide (Ab–Oligo), which simultaneously binds to different epitopes of the same protein, or to two proteins in a complex, thereby converting them to DNA molecules for downstream quantification.

Two different methods exist for Proximity Ligation Assay – direct and indirect, but either method uses antibodies raised in different species (see Figure 1.). The direct primary method uses Ab–Oligo conjugates and the indirect method uses unmodified primary antibodies that are detected with secondary Ab–Oligo conjugates. The technique can use either monoclonal or polyclonal antibodies, or a combination of both.   

Figure 1. Direct and indirect PLA techniques. The direct method uses antibody pairs with primary conjugation and the indirect method uses secondary antibody conjugates.

For either method, additional ‘connector’ oligonucleotides are introduced, which recognise and bind to the free ends of the two Ab–Oligo conjugates and are subsequently brought into close proximity (30–40nm apart), where they become ligated by DNA ligase. This enzymatic ligation joins the 3’ end of the first probe with the 5’ end of the second probe, resulting in the formation of a unique target DNA reporter strand (molecule) that is a surrogate marker for the specific protein to be detected, and which contains specific molecular barcodes. These strands participate in rolling circle amplification (RCA) to amplify the product for quantification by real time PCR.

Types of Proximity Ligation Assay

Homogenous Proximity Ligation Assay

Homogenous Proximity Ligation Assay is suitable for the quantitation of low concentrations of protein molecules in a small homogenous solution, reaching femtomolar detection sensitivity. This method of PLA is  performed in three steps, with no wash step required. Briefly, the target antigen is incubated with two proximity probes (3’ and 5’) that bind to adjacent epitopes on the target antigen, and a connector oligonucleotide (~20 bps), which hybridizes to both probes. The DNA molecule generated is amplified, detected and quantitated by real time PCR. As the connector oligo is added in molar excess, free proximity probes are blocked from further hybridization, resulting in low background noise. The added advantage of homogenous PLA is that a solid support is not required to immobilize the target protein.

Solid Phase Proximity Ligation Assay

Solid Phase Proximity Ligation Assay is an alternative methodology to homogenous PLA and utilizes a capture antibody to immobilize a target protein onto a solid phase, providing some advantages such as investigation of larger sample sets. The sample is combined with a capture antibody and incubated with the proximity probes. Thus, the target antigen is sandwiched between the proximity probes and the capture antibody. Ligation and quantitative PCR (qPCR) are conducted following wash steps. One advantage of solid phase PLA is the elimination of extensive, carefully controlled wash steps, as required for other solid phase assays such as ELISA. Immobilization of the protein on a solid support can serve to purify and concentrate the samples, especially those that contain constituents that may inhibit ligation or amplification, or where the concentration of analyte is extremely low. The wash step may also remove unbound probes prior to ligation, which would otherwise result in nonspecific probe ligation.

In situ Proximity Ligation Assay

In situ PLA is a third proximity-mediated detection method that can detect and visualize target proteins / protein–protein complexes expressed by fixed cells and on tissue slide sections. As well as using a fixative that is appropriate for the antibodies used in the protocol to fix the cells or tissue, they may sometimes need to be permeabilized. If this step is required, then antigen retrieval and antibody specific blocking must also be performed.

In situ PLA uses RCA to amplify ligation products, generated through the hybridization of two connector oligonucleotides to the two Ab–oligo conjugates. The addition of DNA ligase to this complex anneals the two ends to close the gap, resulting in the formation of a circular, single stranded DNA molecule, with one of the Ab-conjugated DNA molecules serving as a primer for RCA. Further addition of a DNA polymerase leads to the formation of a long DNA product that remains covalently attached to one of the PLA probes, which can be detected via the conjugation moiety.

Advantages of Proximity Ligation Assay:

  • Fast, ultra-sensitive and highly efficient assay
  • Usually no need for sample purification prior to PLA
  • Utilizes the specificity of ELISA and the sensitivity of PCR to product reliable results
  • Can be developed for diagnosis of pathogens and proteins
  • Excellent method for the validation of potential biomarkers for clinical diagnostic testing
  • Good resource in identifying several PTMs on the same target
  • Different formats available – Homogeneous, solid phase and in situ
  • Suitable for a wide range of sample types
  • May assist in the development of mutation specific targeted therapies
  • Potential to multiplex for increased throughput.

Limitations of Proximity Ligation Assay:

  • Highly dependent upon the quality of the antibody used in the probe
  • Potential variation in results due to batch-specific antibody performance
  • Background signal due to nonspecific ligation of oligonucleotides
  • Covalent conjugation of oligonucleotides to antibodies can be difficult and time consuming*.

Thunder-Link® PLUS Conjugation Kit

To overcome the complexity of conjugating antibodies to oligonucleotides*, Expedeon offers Thunder-Link® PLUS, an easy to use kit that enables rapid conjugation of antibodies to oligonucleotides, with high recovery of materials and a superior clean-up procedure. The kit is quick and simple to use, overcoming time consuming and lengthy protocols associated with standard conjugation methods.

Features and Benefits:

  • Fast, only 30 minutes antibody and oligo activation and one hour oligo conjugation.
  • Easy to use – step by step optimised procedure – no specialist scientific knowledge required
  • Robust and flexible clean up procedure (See Figure 2.) – No interference from unbound oligo
  • Works with a wide range of antibody fragments and other proteins
  • Flexible procedure allows the use of your own antibody and oligos
  • High levels of antibody and oligo recovery – Saving you precious reagents
  • Unidirectional chemistry so there’s no risk of cross linking
  • Covalent bonds form highly stable conjugates
  • Suitable for single stranded oligos of 10–120 bases and double stranded oligos up to 80 base pairs – Covers the majority of sequences
  • Linking chemistry works at both the 5’ and 3’ end providing the ability to combine with other modifications

Figure 2. The Thunder-Link® PLUS conjugation process. The Ab–Oligo conjugation protocol consists of three main steps: activation of both the antibody and the oligonucleotide followed by desalting, after which the two are mixed and left to incubate overnight. If an unbound oligonucleotide removal step is necessary for the end application, this can be performed the next morning. Following this, the conjugate is ready to use.

Main applications for Thunder-Link® PLUS:

ImmunoPCR (iPCR), Proximity Ligation Assay (PLA), Electrochemical Proximity Assay (ECPA), Lateral Flow.

To find out more about this product please click here. We have an expert technical support team that are happy to help, contact us here.

Custom Services
Optimizing your antibody/oligonucleotide conjugates can be time consuming and expensive, especially when using traditional multi-step conjugation techniques. Our antibody/oligonucleotide micro-optimization is a unique service performed by our experienced scientists. We use unique conjugation that can enhance the performance of even the most temperamental of antibodies. To find out more,click here.

References:

Blokzijl, A., Nong, R., Darmanis, S., Hertz, E., Landegren, U., & Kamali-Moghaddam, M. Protein biomarker validation via proximity ligation assays. Biochimica et Biophysica Acta (BBA) – Proteins and Proteomics. 2014 May;1844(5), 933–939. doi:10.1016/j.bbapap.2013.07.016

Fredriksson, S., Gullberg, M., Jarvius, J., Olsson, C., Pietras, K., Gústafsdóttir, SM., Östman, A., & Landegren, U. Protein detection using proximity dependent DNA ligation assays. Nature Biotechnology. 2002;20(5):473–477. doi:10.1038/nbt0502-473.

Fredriksson, S., Dixon, W., Ji, H., Koong, A. C., Mindrinos, M., & Davis, R. W. Multiplexed protein detection by proximity ligation for cancer biomarker validation. Nature Methods. 2007;4(4), 327–329. doi:10.1038/nmeth1020.

Greenwood, C., Ruff, D., Kirvell, S., Johnson, G., Dhillon, H. S., & Bustin, S. A. (2015). Proximity assays for sensitive quantification of proteins. Biomolecular Detection and Quantification. 2015;4:10–16. doi:10.1016/j.bdq.2015.04.002.

Gustafsdottir, S. M., Schallmeiner, E., Fredriksson, S., Gullberg, M., Söderberg, O., Jarvius, M., & Landegren, U. (2005). Proximity ligation assays for sensitive and specific protein analyses. Analytical Biochemistry. 2005;345(1), 2–9. doi:10.1016/j.ab.2005.01.018.

Leuchowius, K.-J., Weibrecht, I., & Söderberg, O. (2011). In Situ Proximity Ligation Assay for Microscopy and Flow Cytometry. Current Protocols in Cytometry, 56(1), 9.36.1–9.36.15. doi:10.1002/0471142956.cy0936s56.

Proteinatlas.org. (2018). Learn: method proximity ligation assay – The Human Protein Atlas. [online] Available at: https://www.proteinatlas.org/learn/method/proximity+ligation+assay [Accessed September 2018].

Söderberg, O., Gullberg, M., Jarvius, M., Ridderstråle, K., Leuchowius, KJ., Jarvius, J., & Landegren, U. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nature Methods. 2006;3(12):995–1000.

 

 

 

 

 

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