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Uppsala Team Adapts Proximity Ligation Tech to More Convenient Clinical Format

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NEW YORK (360Dx) – Uppsala University researchers have developed a version of the proximity ligation assay (PLA) aimed at clinical use.

Described in a paper published last week in Clinical Chemistry, the assay is designed to work in a standard microtiter plate format and can be read out using conventional ELISA plate readers, which are commonly available in hospitals and other clinical laboratories.

The goal of the work is to move the PLA technology, which offers potential sensitivity and specificity benefits compared to standard immunoassays, into the clinic, said Masood Kamali-Moghaddam, an Uppsala researcher and senior author on the study.

He added that while the researchers have not yet selected a specific marker for which they hope to develop a clinical assay, they are collaborating with researchers at Uppsala University Hospital, as well as outside groups on applications of the technology.

The PLA technology was developed in the lab of Uppsala researcher Ulf Landegren, a coauthor on the Clinical Chemistry study. The assay uses pairs of antibodies attached to unique DNA sequences to detect proteins of interest. When the antibodies bind to their targets, 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.

Another form of the PLA assay, which is intended for in situ detection of proteins, uses rolling circle amplification (RCA) instead of PCR to amplify the nucleic acid strand produced upon protein detection. Because RCA can be read out using fluorophore- or enzyme-labeled probes, they are compatible with plate readers commonly used for conventional ELISA assays.

"So we have combined these two PLA formats to establish a new form that can be used for detection of proteins and also, for instance, interactions between proteins, in a standard ELISA plate," said Kamali-Moghaddam.

Called PLARCA (proximity ligation assays with rolling circle amplification), the approach uses a capture antibody that is immobilized in a microtiter well much like in a standard ELISA. The sample is added and the capture antibody binds to the target molecule. The pair of DNA-linked detection antibodies are then added, forming a new piece of DNA upon binding to the target, which is then amplified using RCA and read out using a standard microtiter plate reader.

Because two antibodies must bind for a signal to be generated, PLA reduces background binding, compared to conventional immunoassays, which boosts the sensitivity and specificity of detection. In the Clinical Chemistry study, the researchers looked at the proteins IL-4, GDF-15, IL-6, IL-7, and p53, measuring them in serum using PLARCA and comparing the results to those from other techniques (ELISA for IL-4, GDF-15, IL-6; immune-rolling circle amplification for IL-7 and p53.)

For each molecule, PLARCA provided an improvement in terms of limit of detection, limit of quantitation, and dynamic range. For several of the molecules measured, the authors noted, PLARCA offered more than a hundredfold greater analytical sensitivity than the comparison methods.

The PLARCA assay time was somewhat longer than ELISA assay time (6.25 hours compared to 4.5 hours), while reproducibility was higher for ELISA — coefficients of variation roughly 5 percent for ELSIA versus 10 percent for PLARCA — though both figures are within the desired range for a typical clinical assay.

The researchers also looked at the performance of the assay in actual clinical samples, measuring levels of IL-6 in tissue lysate from tumor tissue and surrounding healthy tissue in 22 patients with colon cancer. They found that PLARCA was able to measure IL-6 in these samples at a limit of detection more than an order of magnitude more sensitive than ELISA.

Looking at IL-4 and IL-6 in plasma samples from 25 prostate cancer patients and 24 healthy controls, PLARCA detected IL-4 and IL-6 in 80 percent and 84 percent of patients, respectively, while ELISA detected these proteins in 16 percent and 32 percent of patients.

Kamali-Moghaddam said he and his colleagues are currently working with collaborators to explore use of the test for a particular clinically relevant marker, though he declined to name it. He added, though, that they are still early in the process of identifying proteins that might be good candidates for clinical PLARCA assays.

"The study just came out," he noted. "We would be happy if in one or two years we have a molecule that [PLARCA] can be used for in the clinic."

In the past, some researchers have noted that PLA, while a powerful research tool, is a somewhat complicated assay with a high number of steps and different buffer conditions. This could be an issue from a clinical standpoint, as streamlined and reproducible workflows are highly desirable in clinical assays.

The PLARCA assay time and reproducibility data presented in the Clinical Chemistry study suggest, though, that the technique can be done on essentially the same time scale as a standard ELISA and with suitable reproducibility.

Kamali-Moghaddam added that PLA has been offered as a commercial product for years and is "used quite frequently" in labs around the world. "I don't think it is that complicated anymore."

Sigma-Aldrich owns the rights to the original PLA technology, which it purchased in 2015 from Olink Bioscience, the Uppsala spinout that was formed in 2004 to commercialize technologies developed by Landegren's lab.

Last year, Olink reorganized into two separate companies, Olink Proteomics and Olink Biosciences with the former focused on developing and selling any technologies for measuring proteins in solution, and the latter focused on developing other aspects of the company's IP portfolio, including technologies for genomic and in situ protein analysis.