Proximity ligation assay
Proximity ligation assay (in situ PLA) is a technology that extends the capabilities of traditional immunoassays to include direct detection of proteins, protein interactions and 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
Two primary antibodies raised in different species recognize the target antigen or antigens of interest. Species-specific secondary antibodies, called PLA probes, each with a unique short DNA strand attached to it, bind to the primary antibodies. When the PLA probes are in close proximity, the DNA strands can interact through a subsequent addition of two other circle-forming DNA oligonucleotides.
After joining of the two added oligonucleotides by enzymatic ligation, they are amplified via rolling circle amplification using a polymerase. After the amplification reaction, several-hundredfold replication of the DNA circle has occurred, and labeled complementary oligonucleotide probes highlight the product. The resulting high concentration of fluorescence in each single-molecule amplification product is easily visible as a distinct bright spot when viewed with a fluorescence microscope.[4]
References
- ^ Fredriksson S, Gullberg M, Jarvius J, Olsson C, Pietras K, Gústafsdóttir SM, Ostman A, Landegren U. Nat Biotechnol. 2002 May;20(5):473-7. Protein detection using proximity-dependent DNA ligation assays.
- ^ Gullberg M, Gústafsdóttir SM, Schallmeiner E, Jarvius J, Bjarnegård M, Betsholtz C, Landegren U, Fredriksson S. Proc Natl Acad Sci U S A. 2004 Jun 1;101(22):8420-4. Cytokine detection by antibody-based proximity ligation.
- ^ Söderberg O, Gullberg M, Jarvius M, Ridderstråle K, Leuchowius KJ, Jarvius J, Wester K, Hydbring P, Bahram F, Larsson LG, Landegren U. Nat Methods. 2006 Dec;3(12):995-1000. Direct observation of individual endogenous protein complexes in situ by proximity ligation.
- ^ Gustafsdottir SM, Schallmeiner E, Fredriksson S, Gullberg M, Söderberg O, Jarvius M, Jarvius J, Howell M, Landegren U. Anal Biochem. 2005 Oct 1;345(1):2-9. Proximity ligation assays for sensitive and specific protein analyses.