Skip to main content
Premium Trial:

Request an Annual Quote

Expanded Proximity Ligation Assay Could Prove Valuable for Biomarker Studies, Top-Down Proteomics


By Adam Bonislawski

Uppsala University scientists have developed an expanded proximity ligation assay that employs up to five antibodies to confirm the simultaneous presence of multiple proteins on a given target.

The researchers used the technique to isolate prostasomes — microvesicles secreted by prostate acinar cells that could be useful as prostate cancer biomarkers — but it could have wider applicability as a tool for top-down proteomics investigations into protein complexes and isoforms.

Conventional PLA technology, which is sold by Swedish biotech firm Olink Bioscience under the DuoLink name and by Life Technologies as part of Applied Biosystems' TaqMan product line, uses pairs of antibodies attached to unique DNA sequences to detect a protein of interest. When the antibodies bind their target, the attached DNA strands are brought into proximity and ligate, forming a new DNA amplicon that can then be quantified using real-time PCR. The quantity of the DNA corresponds to the quantity of the target protein.

The expanded PLA system, or 4PLA, which was detailed in a paper published this week in the online edition of PNAS, uses five antibodies — one immobilized capture antibody and four oligonucleotide-linked antibody probes — to detect the presence of four different target proteins at once.

The capture antibody, which is attached to the surface of a reaction vessel, captures the molecule of interest. Then, the four probes attach to four additional binding sites on this molecule. Only when all four antibodies are bound to their epitopes do the linked oligonucleotides hybridize to one another, guiding the hybridization of another nucleotide, which can then be quantified to determine the amount of target molecule present as in a conventional PLA.

Ulf Landegren, an Uppsala professor and co-author on the paper, is a founder of Olink Bioscience, which has rights to the new technique.

The Uppsala team developed the technique to isolate prostasomes in blood in hopes that they would be useful biomarkers for prostate cancer. These microvesicles are normally released into seminal fluid, but it has been hypothesized that invasive growth of malignant prostate cells might cause them to appear in interstitial space, from which they could make their way into the blood.

Historically, however, researchers have been unable to isolate prostasomes in blood, noted Masood Kamali-Moghaddama, an Uppsala researcher and one of the authors on the PNAS paper. For one thing, they are typically present in blood only in very low concentrations. Additionally, he told ProteoMonitor, many of the proteins expressed on their surface are present in other configurations or as free molecules in blood, making it difficult to be certain that affinity assays for the vesicles are specifically detecting prostasomes, as opposed to a mix of prostasomes and various other versions of their surface proteins.

The 4PLA system addresses this issue by requiring that all five antibodies bind to specific prostasome surface proteins in order to generate a signal. "In this way, once we detect the prostasome we know that it's the whole prostasome, not just, for instance, [the protein] CD13," Kamali-Moghaddama said.

Using the technique, the researchers detected significantly elevated levels of prostasomes in patients with prostate cancer before radical prostatectomy compared to controls and patients with benign biopsy results. In two studies involving roughly 79 subjects, prostasome levels in the blood of prostate cancer patients were 2.5-fold to sevenfold higher than in controls.

The assay also distinguished between patients with high, medium, and low Gleason scores, a scoring system used to guide prognosis of the disease by distinguishing between more aggressive — high scoring — and less aggressive — low scoring — cases, offering clearer divisions between these groups than tests relying on prostate-specific antigen, currently the standard biomarker for prostate cancer.

The team now plans to test the assay in a study involving roughly 1,000 subjects that it hopes to start some time this summer, Kamali-Moghaddama said, adding that the researchers hope to commercialize the test as a diagnostic if the results hold up in the larger study.

He noted, however, that the variability of the assay would need to be improved for diagnostic use. Although the researchers haven't calculated the coefficients of variation rigorously, they are currently "above the acceptable level for routine diagnostics," he said.

The approach potentially "could be generalized to other epithelial tumors" as well, said Landegren. "Cancer is a disease of epithelial organs so we are hoping to demonstrate that similar exosomes released from other areas like the breast, the colon, and so forth" could be used as biomarkers.

Beyond that, it could provide a broadly useful approach to isolating particular protein isoforms and protein complexes, Kamali-Moghaddama said.

Ben Garcia, assistant professor of molecular biology at Princeton University, who was not involved in the research, noted that it could be particularly useful for chromatin biology, which focuses significantly on the post-translational modification patterns of histone proteins. 4PLA using antibodies targeting modification sites of interest could allow researchers to identify proteins with specific modification patterns.

"I think this could have immediate impact in the chromatin biology field," Garcia told ProteoMonitor.

Currently, "mass spec is really the only way to detect which combinations of modifications on histones — methylations, acetylations — are present," he said. "This could be a great potential affinity-capture type of system that could be used to look at combinatorial modification patterns, either just to detect them or to affinity-purify proteins with certain combinatorial patterns."

One potential problem with such an approach would be attaching five antibodies to a single protein, said Randall Nelson, a researcher at Arizona State University's Biodesign Institute. "Sterically, it's pretty hard to conceive five antibodies — each at 150 kDa — bound to a single molecule of, say, 30 kDa," he told ProteoMonitor in an e-mail.

Garcia also noted this as a possible difficulty, but suggested it could be worked around, saying that "obviously you could change the [oligonucleotide] linker distances and things like that."

Simon Fredriksson, Olink's chief scientific officer and one of the original inventors of the PLA technique, told ProteoMonitor that the company is looking at 4PLA "very seriously to see how we could move it forward."

One possibility, he said, would be using it as an alternative to conventional fluorescence-activated cell sorting assays in order to sort cells based on what combinations of epitopes they presented.

"It's a rather new situation. This is the first time anyone has been able to, on one reporter system, assess on one molecule so many determinants," Fredriksson said. "So this opens up a whole new opportunity for doing research and diagnostics. We're of course looking into how this can be commercialized and made available."

Have topics you'd like to see covered in ProteoMonitor? Contact the editor at abonislawski [at] genomeweb [.] com.