Phage immunoprecipitation sequencing identifies previously unknown disease biomarkers

Eye on Innovation

In a world of ever-faster technical change, Mayo Clinic Laboratories is uniquely positioned to innovate. Collaboration with clinicians pinpoints unmet patient needs and facilitates the development of diagnostic testing that provides answers.

Phage immunoprecipitation sequencing identifies previously unknown disease biomarkers

In Mayo Clinic’s Advanced Diagnostics Laboratory, there are dozens of projects underway at once to develop new technologies, discover novel findings, validate new tests, and support physicians in providing advanced patient care. Researchers are relentlessly innovating to build the tests of the future to transform diagnostic treatment.

One example of these innovations is the use of phage immunoprecipitation sequencing to discover new serological biomarkers for autoimmune diseases. This approach is a multiplexed, phage-display based methodology for analyzing antibody binding specificities. The method combines barcoded DNA high-throughput sequencing to determine the levels of binding of antibodies to epitopes.

“We use this approach to present all known human proteins in the form of short peptides displayed on the surface of bacteriophages to antibodies in patients’ serum or CSF, in an attempt to discover novel autoantibody biomarkers for various autoimmune disorders,” says Divyanshu Dubey, M.B.B.S., a neurologist in Mayo Clinic’s Department of Laboratory Medicine and Pathology. “This method has the potential to accelerate the rate of biomarker discovery faster than traditional autoantibody discovery methods and allows us to test up to 96 samples at once.”

Divyanshu Dubey, M.B.B.S.,

The traditional approach of autoantibody biomarker discovery involves identifying patient samples, finding a good antigen source, looking for proteins bound by patients’ antibodies through mass spectrometry, and confirmation.

With phage immunoprecipitation sequencing (PhIP-Seq), Mayo Clinic researchers curated human proteins — approximately 60,000 of them — to develop a comprehensive library of DNA sequence fragments. Then they put proteins or peptides in bacteriophages, in a manner that they are displayed on the surface, along with the viral coat protein allowing peptide-antibody interaction.

Technology generates a novel discovery

This three-year project yielded an exciting development. Working with Mayo Clinic’s Neuroimmunology Laboratory and Margherita Milone, M.D., Ph.D., from the Muscle Laboratory, researchers sought to identify a biomarker for immune-mediated rippling muscle disease (iRMD). This disease is a rare myopathy causing wavelike muscle contractions (rippling) and percussion- or stretch-induced muscle mounding.

In a recent study using PhIP-Seq, Mayo Clinic researchers discovered a previously unknown antibody marker for iRMD: caveolae-associated protein 4 (cavin-4).

From left to right: Cavin-4 specific rabbit immunoglobulin G (IgG). IgG of patient with immune-mediated rippling muscle disease. Co-localization with patient IgG.

“In this study, autoantibodies to cavin-4 were identified and orthogonally validated in 8 of 10 patients with iRMD, and results for all healthy and disease-control individuals were seronegative,” says Dr. Dubey. “In addition, the cavin-4 expression was depleted in the muscle biopsies of patients with iRMD.”

This finding, published in JAMA Neurology, will support testing options and accurate diagnosis of iRMD, helping physicians treat patients with iRMD and restore their quality of life.

“With this discovery, we are helping clinicians to bypass all that workup and indecision and providing them with a specific biomarker that can guide diagnosis and management,” says Dr. Dubey.

Team-based collaboration

Phage immunoprecipitation sequencing is an innovative, team-based approach that requires close collaboration with many experts. Researcher John Mills, Ph.D., co-director of Mayo Clinic’s Neuroimmunology Laboratory, describes the teamwork involved in this multidisciplinary project.

John Mills, Ph.D.

“The key to success in bringing this new technology to Mayo Clinic was bringing together a multidisciplinary team” says Dr. Mills. “We couldn’t accomplish this with the resources and expertise we had in our own laboratory. This is a very complex technology that very few medical facilities have the capability to develop. The fact that we can do these types of complex projects at Mayo Clinic speaks volumes about our investment in innovation and our dedication to paving the way for future discoveries to benefit patients.” 

With over 60 specialty-dedicated labs, Mayo Clinic Laboratories is backed by the knowledge and expertise of more than 160 consultants and over 4,000 employees in a wide range of professional roles. This deep experience boosts researchers’ ability to pursue ongoing projects and grant applications built around new technology like PhIP-Seq.

“There is the obvious lab aspect to this, but there is also the clinical side where people are asking the right questions, the pathology side where we work with people to understand what the biomarkers mean, and the patient side where they provide the investment in providing samples and contributing to the studies,” says Dr. Dubey.

“These technologies are great, but they do require a lot of validation. We must use a lot of other methodologies to confirm the finding is accurate. When you get a biomarker hit, you cannot just run with it. There is a lot of time and effort involved to ensure we are confident that the identified biomarker is disease specific,” explains Dr. Dubey.

Once a promising antibody biomarker is discovered and proven to be clinically useful, the biomarker is transferred into the clinical test development arena, where a test is developed, optimized, and after rigorous validation is offered to patients.

“The ability of Mayo Clinic to make discoveries and translate these into solutions for patients is critical to our mission,” says Dr. Mills.

Future application

The iRMD study is just one example how this phage technology can be used. Researchers look forward to applying the technology to future collaborative efforts to discover antibodies for cancer and other neurological and rheumatological disorders.

In Mayo Clinic’s Advanced Diagnostics Laboratory, researchers will continue to optimize the PhIP-Seq platform, work on a more robust, data-driven analysis pipeline, expand machine learning, and immunoprofile more patients over time.

“Because of the high throughput potential, relatively low cost, and scaled-up, semi-automated process, this technology has utility beyond neuroimmunology questions,” says Dr. Dubey. “We discovered this iRMD specific biomarker first, but there are more to come.”

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Luci Gens

Luci Gens is a marketing manager at Mayo Clinic Laboratories. She joined Mayo Clinic in 2022 and has over ten years of experience in hospital-based marketing and communications.