Proximity ligation assay

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 (2^o 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.

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]