Proximity ligation assay

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Figure 1: PLA starts with the binding of antibodies from different species to 2 proteins of interest, in this case protein * (star) and protein# ProximityAssay14TC.jpg
Figure 1: PLA starts with the binding of antibodies from different species to 2 proteins of interest, in this case protein * (star) and protein#

Proximity ligation assay (in situ PLA) is a technology that extends the capabilities of traditional immunoassays to include direct detection of proteins, protein interactions, extracellular vesicles and post translational modifications with high specificity and sensitivity. [1] [2] Protein targets can be readily detected and localized with single molecule resolution and objectively quantified in unmodified cells and tissues. Utilizing only a few cells, sub-cellular events, even transient or weak interactions, are revealed in situ and sub-populations of cells can be differentiated. Within hours, results from conventional co-immunoprecipitation and co-localization techniques can be confirmed. [3]

Contents

The PLA principle

Figure 2: Binding of PLA probes. ProximityAssay24TC.jpg
Figure 2: Binding of PLA probes.

Two primary antibodies raised in different species recognize the target antigen on the proteins of interest (Figure 1). Secondary antibodies (2o Ab) directed against the constant regions of the different primary antibodies, called PLA probes, bind to the primary antibodies (Figure 2).

Figure 3: Rolling circle DNA synthesis starts. ProximityAssay34TC.jpg
Figure 3: Rolling circle DNA synthesis starts.

Each of the PLA probes has a short sequence specific DNA strand attached to it. If the PLA probes are in proximity (that is, if the two original proteins of interest are in proximity, or part of a protein complex, as shown in the figures), the DNA strands can participate in rolling circle DNA synthesis upon addition of two other sequence-specific DNA oligonucleotides together with appropriate substrates and enzymes (Figure 3).

Figure 4: Fluorescent probes bind to the amplified DNA. ProximityAssay44TC.jpg
Figure 4: Fluorescent probes bind to the amplified DNA.

The DNA synthesis reaction results in several-hundredfold amplification of the DNA circle. Next, fluorescent-labeled complementary oligonucleotide probes are added, and they bind to the amplified DNA (Figure 4). The resulting high concentration of fluorescence is easily visible as a distinct bright spot when viewed with a fluorescence microscope. [4] In the specific case shown (Figure 5), the nucleus is enlarged because this is a B-cell lymphoma cell. The two proteins of interest are a B cell receptor and MYD88. The finding of interaction in the cytoplasm was interesting because B cell receptors are thought of as being located in the cell membrane. [5]

Figure 5: Fluorescence microscopy image showing interaction of the proteins in the cytoplasm. Nucleus in blue, PLA product in red. ProximityAssay5Cell4TC.jpg
Figure 5: Fluorescence microscopy image showing interaction of the proteins in the cytoplasm. Nucleus in blue, PLA product in red.

Applications

PLA as described above has been used to study aspects of animal development [6] [7] and breast cancer [8] among many other topics. In situ proximity ligation assays (isPLA) has been applied to antibody validation in human tissues with various advantages over IHC, including increased detection specificity, decreased unspecific staining, and better localization. [9] A variation of the technique (rISH-PLA) has been used to study the association of protein and RNA. [10] Another variation of in situ PLA includes a multiplex PLA assay that makes it possible to visualize multiple protein complexes in parallel. [11] PLA can also be combined with other read out forms such as ELISA, [12] flow cytometry. [13] [14] and Western blotting [15]

Related Research Articles

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The proximity extension assay (PEA) is a method for detecting and quantifying the amount of many specific proteins present in a biological sample such a serum or plasma. The method is used in the research field of proteomics, specifically affinity proteomics, where in one searches for differences in the abundance of many specific proteins in blood for use as a biomarker. Biomarkers and biomarker signature combinations, are useful for determining disease states and drug efficacy. Most methods for detecting proteins involve the use of a solid phase for first capturing and immobilizing the protein analyte, where in one or a few proteins are quantified, such as ELISA. In contrast, PEA is performed without a solid phase in a homogeneous one tube reaction solution where in sets of antibodies coupled to unique DNA sequence tags, so called proximity probes, work in pairs specific for each target protein. PEA is often performed using antibodies and is a type of immunoassay. Target binding by the proximity probes increases their local relative effective concentration of the DNA-tags enabling hybridization of weak complementarity to each other which then enables a DNA polymerase mediated extension forming a united DNA sequence specific for each target protein detected. The use of 3'exonuclease proficient polymerases lowers background noise and hyper thermostable polymerases mediate a simple assay with a natural hot-start reaction. This created pool of extension products of DNA sequence forms amplicons amplified by PCR where each amplicon sequence corresponds to a target proteins identity and the amount reflects its quantity. Subsequently, these amplicons are detected and quantified by either real-time PCR or next generation DNA sequencing by DNA-tag counting. PEA enables the detection of many proteins simultaneously due to the readout requiring the combination of two correctly bound antibodies per protein to generate a detectable DNA sequence from the extension reaction. Only cognate pairs of sequence are detected as true signal, enabling multiplexing beyond solid phase capture methods limited at around 30 proteins at a time. The DNA amplification power also enable minute sample volumes even below one microliter. PEA has been used in over 1000 research publications.

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<span class="mw-page-title-main">PLAC-Seq</span> Proximity ligation assisted chip-seq technology

Proximity ligation-assisted chromatin immunoprecipitation sequencing (PLAC-seq) is a chromatin conformation capture(3C)-based technique to detect and quantify genomic chromatin structure from a protein-centric approach. PLAC-seq combines in situ Hi-C and chromatin immunoprecipitation (ChIP), which allows for the identification of long-range chromatin interactions at a high resolution with low sequencing costs. Mapping long-range 3-dimensional(3D) chromatin interactions is important in identifying transcription enhancers and non-coding variants that can be linked to human diseases.

References

  1. ↑ Löf L, Arngården L, Ebai T, Landegren U, Söderberg O, Kamali-Moghaddam M (2017). "Detection of Extracellular Vesicles Using Proximity Ligation Assay with Flow Cytometry Readout—ExoPLA" . Current Protocols in Cytometry. 81 (1): 4.8.1–4.8.10. doi:10.1002/cpcy.22. ISSN   1934-9300. PMID   28678418. S2CID   4379414.
  2. ↑ Gullberg M, Gústafsdóttir SM, Schallmeiner E, Jarvius J, Bjarnegård M, Betsholtz C, Landegren U, Fredriksson S (June 2004). "Cytokine detection by antibody-based proximity ligation". Proceedings of the National Academy of Sciences of the United States of America. 101 (22): 8420–4. Bibcode:2004PNAS..101.8420G. doi: 10.1073/pnas.0400552101 . PMC   420409 . PMID   15155907.
  3. ↑ Söderberg O, Gullberg M, Jarvius M, Ridderstråle K, Leuchowius KJ, Jarvius J, Wester K, Hydbring P, Bahram F, Larsson LG, Landegren U (December 2006). "Direct observation of individual endogenous protein complexes in situ by proximity ligation". Nature Methods. 3 (12): 995–1000. doi:10.1038/nmeth947. PMID   17072308. S2CID   21819907.
  4. ↑ Gustafsdottir SM, Schallmeiner E, Fredriksson S, Gullberg M, Söderberg O, Jarvius M, Jarvius J, Howell M, Landegren U (October 2005). "Proximity ligation assays for sensitive and specific protein analyses". Analytical Biochemistry. 345 (1): 2–9. doi:10.1016/j.ab.2005.01.018. PMID   15950911.
  5. ↑ Staudt L (February 2017). "Therapy of lymphoma inspired by functional and structural genomics". videocast.nih.gov. National Institutes of Health. Retrieved 3 February 2017.
  6. ↑ Wang S, Yoo S, Kim HY, Wang M, Zheng C, Parkhouse W, Krieger C, Harden N (January 2015). "Detection of in situ protein-protein complexes at the Drosophila larval neuromuscular junction using proximity ligation assay". Journal of Visualized Experiments (95): 52139. doi:10.3791/52139. PMC   4354543 . PMID   25650626.
  7. ↑ Kwon J, Jeong SM, Choi I, Kim NH (2016). "ADAM10 Is Involved in Cell Junction Assembly in Early Porcine Embryo Development". PLOS ONE. 11 (4): e0152921. Bibcode:2016PLoSO..1152921K. doi: 10.1371/journal.pone.0152921 . PMC   4820119 . PMID   27043020.
  8. ↑ Chetan C. Oturkar, Spencer R. Rosario, Gokul M. Das (2024).ESR1 and p53 interactome alteration defines mechanisms of tamoxifen response in luminal breast cancer. iScience. | vauthors = Vincent A, Berthel E, Dacheux E, Magnard C, Venezia NL | title = BRCA1 affects protein phosphatase 6 signalling through its interaction with ANKRD28 | journal = The Biochemical Journal | volume = 473 | issue = 7 | pages = 949–60 | date = April 2016 | pmid = 27026398 | doi = 10.1042/BJ20150797 }}
  9. ↑ Lindskog C, Backman M, Zieba A, Asplund A, Uhlén M, Landegren U, Pontén F (July 2020). "Proximity Ligation Assay as a Tool for Antibody Validation in Human Tissues". Journal of Histochemistry & Cytochemistry. 68 (7): 515–529. doi:10.1369/0022155420936384. ISSN   0022-1554. PMC   7350078 . PMID   32602410.
  10. ↑ Roussis IM, Guille M, Myers FA, Scarlett GP (2016). "RNA Whole-Mount In situ Hybridisation Proximity Ligation Assay (rISH-PLA), an Assay for Detecting RNA-Protein Complexes in Intact Cells". PLOS ONE. 11 (1): e0147967. Bibcode:2016PLoSO..1147967R. doi: 10.1371/journal.pone.0147967 . PMC   4732756 . PMID   26824753.
  11. ↑ Leuchowius KJ, Clausson CM, Grannas K, Erbilgin Y, Botling J, Zieba A, et al. (June 2013). "Parallel visualization of multiple protein complexes in individual cells in tumor tissue". Molecular & Cellular Proteomics. 12 (6): 1563–71. doi: 10.1074/mcp.O112.023374 . PMC   3675814 . PMID   23436906.
  12. ↑ Ebai T, Souza de Oliveira FM, Löf L, Wik L, Schweiger C, Larsson A, et al. (September 2017). "Analytically Sensitive Protein Detection in Microtiter Plates by Proximity Ligation with Rolling Circle Amplification". Clinical Chemistry. 63 (9): 1497–1505. doi: 10.1373/clinchem.2017.271833 . PMID   28667186.
  13. ↑ Leuchowius KJ, Weibrecht I, Landegren U, Gedda L, Söderberg O (October 2009). "Flow cytometric in situ proximity ligation analyses of protein interactions and post-translational modification of the epidermal growth factor receptor family". Cytometry. Part A. 75 (10): 833–9. doi: 10.1002/cyto.a.20771 . PMID   19650109. S2CID   2550136.
  14. ↑ Löf L, Arngården L, Olsson-Strömberg U, Siart B, Jansson M, Dahlin JS, et al. (April 2017). "Flow Cytometric Measurement of Blood Cells with BCR-ABL1 Fusion Protein in Chronic Myeloid Leukemia". Scientific Reports. 7 (1): 623. Bibcode:2017NatSR...7..623L. doi:10.1038/s41598-017-00755-y. PMC   5429594 . PMID   28377570.
  15. ↑ Liu Y, Gu J, Hagner-McWhirter Å, Sathiyanarayanan P, Gullberg M, Söderberg O, et al. (November 2011). "Western blotting via proximity ligation for high performance protein analysis". Molecular & Cellular Proteomics. 10 (11): O111.011031. doi: 10.1074/mcp.O111.011031 . PMC   3226413 . PMID   21813417.
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