The full name of PLA technology is Proximity Ligation Assay, also known as adjacent connection or adjacent connection technology. PLA technology is a special immunoassay method that can be used to detect target proteins, protein interactions, and more. This method recognizes the target protein through a pair of monoclonal or polyclonal antibody probes labeled with a segment of oligodeoxynucleotides (single-stranded DNA), namely PLA probes. When the two probes recognize the same When a protein is present, the distance between the two probes is close, resulting in the so-called proximity effect.
At this time, by adding a segment of connector oligonucleotides that are complementary to the DNA attached to the antibody, the DNA on the PLA probe will be complementary to this segment of DNA through pairing complementation, and then ligase Under the action of the PLA probe, the fragmented DNAs on the PLA probe are linked together to form a new DNA fragment. The new DNA fragment can be amplified and quantified by fluorescent PCR, so that the corresponding target protein can be quantified.
The first reported PLA technology is based on two DNA aptamers that bind to different epitopes of thrombin. Aptamers are single-stranded nucleic acids with recognition performance comparable to antibodies, and they can fold into complex 3D structures to specifically bind to a large number of target proteins. They were obtained by screening a combinatorial library of 1012-1016 oligonucleotides that could bind to the protein motif of interest. Despite their early appearance, aptamers have not been widely used due to their weak affinity (and thus low sensitivity).
At present, the most widely used is the introduction of a capture antibody on the basis of liquid-phase PLA. The capture antibody is immobilized on a solid-phase carrier (magnetic beads, microplates, etc.). Identification and quantification.
In 2011, researchers at The Ohio State University developed a new technique called fluorescent in situ gene protein assay, which combines traditional fluorescence in situ hybridization (FISH) with in situ proximity ligation analysis ( In situ proximity ligation assay, referred to as PLA), can simultaneously observe the number of genes and protein expression in cells. This technology enables detailed analysis of the association between gene states and corresponding protein expression, and provides a better understanding of cancer and other complex diseases. The results were published in Neuro-Oncology.
The approach of proximity ligation assays differs from other in situ hybridization applications based on antigen-antibody reactions in that it increases protein specificity by using two antibodies (rather than a single antibody) that recognize different epitopes of the same protein. In addition, using two antibodies to recognize different proteins can both reveal the location and quantify protein interactions. Signal enhancement at the detection step provides a unique ability to study stable and transient interactions at the endogenous expression level of the protein of interest. Combined with various image processing and analysis systems, PLA is highly suitable for high-throughput cell-based screening experiments.