Biology: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#

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]

The PLA principle

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.

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.

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.

Applications

PLA as described above has been used to study aspects of animal development[6][7] and breast cancer[8][9] 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.[10] A variation of the technique (rISH-PLA) has been used to study the association of protein and RNA.[11] Another variation of in situ PLA includes a multiplex PLA assay that makes it possible to visualize multiple protein complexes in parallel.[12] PLA can also be combined with other read out forms such as ELISA,[13] flow cytometry.[14][15] and Western blotting[16]

References

  1. ↑ Löf, Liza; Arngården, Linda; Ebai, Tonge; Landegren, Ulf; Söderberg, Ola; Kamali-Moghaddam, Masood (2017). "Detection of Extracellular Vesicles Using Proximity Ligation Assay with Flow Cytometry Readout—ExoPLA" (in en). Current Protocols in Cytometry 81 (1): 4.8.1–4.8.10. doi:10.1002/cpcy.22. ISSN 1934-9300. PMID 28678418. https://currentprotocols.onlinelibrary.wiley.com/doi/abs/10.1002/cpcy.22. 
  2. ↑ "Cytokine detection by antibody-based proximity ligation". Proceedings of the National Academy of Sciences of the United States of America 101 (22): 8420–4. June 2004. doi:10.1073/pnas.0400552101. PMID 15155907. Bibcode: 2004PNAS..101.8420G. 
  3. ↑ "Direct observation of individual endogenous protein complexes in situ by proximity ligation". Nature Methods 3 (12): 995–1000. December 2006. doi:10.1038/nmeth947. PMID 17072308. 
  4. ↑ "Proximity ligation assays for sensitive and specific protein analyses". Analytical Biochemistry 345 (1): 2–9. October 2005. doi:10.1016/j.ab.2005.01.018. PMID 15950911. 
  5. ↑ Staudt, Louis (February 2017). "Therapy of lymphoma inspired by functional and structural genomics". National Institutes of Health. https://videocast.nih.gov/Summary.asp?Live=21795&bhcp=1. Retrieved 3 February 2017. 
  6. ↑ "Detection of in situ protein-protein complexes at the Drosophila larval neuromuscular junction using proximity ligation assay". Journal of Visualized Experiments (95): 52139. January 2015. doi:10.3791/52139. PMID 25650626. 
  7. ↑ "ADAM10 Is Involved in Cell Junction Assembly in Early Porcine Embryo Development". PLOS ONE 11 (4): e0152921. 2016. doi:10.1371/journal.pone.0152921. PMID 27043020. Bibcode: 2016PLoSO..1152921K. 
  8. ↑ "HER-3 targeting alters the dimerization pattern of ErbB protein family members in breast carcinomas". Oncotarget 7 (5): 5576–97. February 2016. doi:10.18632/oncotarget.6762. PMID 26716646. 
  9. ↑ "BRCA1 affects protein phosphatase 6 signalling through its interaction with ANKRD28". The Biochemical Journal 473 (7): 949–60. April 2016. doi:10.1042/BJ20150797. PMID 27026398. 
  10. ↑ Lindskog, Cecilia; Backman, Max; Zieba, Agata; Asplund, Anna; Uhlén, Mathias; Landegren, Ulf; Pontén, Fredrik (July 2020). "Proximity Ligation Assay as a Tool for Antibody Validation in Human Tissues" (in en). Journal of Histochemistry & Cytochemistry 68 (7): 515–529. doi:10.1369/0022155420936384. ISSN 0022-1554. PMID 32602410. 
  11. ↑ "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. 2016. doi:10.1371/journal.pone.0147967. PMID 26824753. Bibcode: 2016PLoSO..1147967R. 
  12. ↑ "Parallel visualization of multiple protein complexes in individual cells in tumor tissue". Molecular & Cellular Proteomics 12 (6): 1563–71. June 2013. doi:10.1074/mcp.O112.023374. PMID 23436906. 
  13. ↑ "Analytically Sensitive Protein Detection in Microtiter Plates by Proximity Ligation with Rolling Circle Amplification". Clinical Chemistry 63 (9): 1497–1505. September 2017. doi:10.1373/clinchem.2017.271833. PMID 28667186. 
  14. ↑ "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. October 2009. doi:10.1002/cyto.a.20771. PMID 19650109. 
  15. ↑ "Flow Cytometric Measurement of Blood Cells with BCR-ABL1 Fusion Protein in Chronic Myeloid Leukemia". Scientific Reports 7 (1): 623. April 2017. doi:10.1038/s41598-017-00755-y. PMID 28377570. Bibcode: 2017NatSR...7..623L. 
  16. ↑ "Western blotting via proximity ligation for high performance protein analysis". Molecular & Cellular Proteomics 10 (11): O111.011031. November 2011. doi:10.1074/mcp.O111.011031. PMID 21813417. 




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