NEW YORK (360Dx) – A technique with sensitivity levels 5,000 times that of commercial ELISAs may enable detecting miniscule amounts of interferon alpha proteins, offering potentially a new diagnostic method for several autoimmune diseases.
A multidisciplinary team led by researchers at the Pasteur Institute and the Institut Imagine in Paris said that they have validated the ultrasensitive method for detecting interferon in human blood or cerebrospinal fluid to aid in diagnosing systemic lupus erythematosus (SLE) — a collection of diseases in which the human immune system becomes hyperactive and attacks normal, healthy tissues — and dermatomyositis, a rare inflammatory disease marked by muscle weakness and a distinctive skin rash.
Describing their work published online last week in The Journal of Experimental Medicine, Technical Advances, the researchers noted that they began by isolating high-affinity anti-interferon antibodies from patients with APECED syndrome, which is a recessively inherited genetic disease. Then they combined the anti-interferon antibodies with a Simoa single-molecule array digital ELISA from Quanterix.
The digital ELISA identified individual antibody-labeled proteins at attomolar concentrations, equivalent to quadrillionths of a gram per milliliter of sample, and thousands of times more sensitive than existing methods for detecting these proteins, the researchers said.
Digital in nature, the single-molecule system generates a signal that can be counted. The approach employs arrays of femtoliter-sized reaction chambers that can isolate and detect single enzyme molecules, according to Quanterix, and array volumes are about 2 billion times smaller than in a conventional ELISA. During testing, the presence of a labeled protein results in a buildup of fluorescent product.
The Simoa digital ELISA uses thousands of beads, each of which is isolated in an individual nanowell, said Darragh Duffy, a researcher at the Pasteur Institute's department of immunology, who led the development of the interferon alpha detection technique along with Yanick Crow from the Institut Imagine in Paris. "The machine can read the fluorescence of a single bead and therefore provide detection to the level of a single fluorescent molecule. Right away, that helps with achieving sensitivity."
The technique can be applied to almost any protein using the Quanterix Simoa kit, Duffy said, adding, "What's unique and most interesting about our study is that we took the digital ELISA approach and antibodies isolated from a unique cohort of auto immune patients who have autoreactive antibodies against key immune proteins. The antibodies have a really high affinity, so they have strong recognition for this particular protein, and that gives you a really great tool to develop a very sensitive assay."
Interferon proteins are a family of cell signaling molecules that play a critical role in the immune system's antiviral defenses. Inappropriate activation of interferon signaling can cause the immune system to attack healthy tissues in the body, leading to a variety of autoimmune diseases.
Interferon alpha proteins, a subset of interferon type 1, detected by the research team "are present in tiny quantities and they are extremely potent," Duffy said, "so small changes to these quantities can have dramatic effects on the immune system."
Despite decades of research, the direct measurement in biological samples of type I interferon protein that are essential mediators of antiviral responses and that have been implicated in the pathogenesis of autoimmunity, remain elusive, the researchers said.
Traditional, analog ELISA systems require millions of enzyme labels to generate signals that are detectable utilizing conventional plate readers, the researchers said, adding that its sensitivity is limited to the picomolar range and above.
With the Simoa-based technique, the collaborating researchers measured interferon levels in the blood of patients who were healthy, and in others that had type I interferonopathy, SLE, and dermatomyositis. Type I interferonopathies are a class of autoimmune diseases characterized by inappropriate activation of type I interferon.
The researchers noted that the interferon alpha protein levels that they detected correlated well with the results of tests for functional activity and interferon-stimulated gene expression.
They noticed elevated interferon levels in all the autoimmune samples, as well as increased interferon levels in cerebrospinal fluid infected with viral meningitis.
In patients with SLE, higher interferon levels correlated with an increased severity of disease, Duffy said, and added that interferon protein levels were particularly high in patients with type I interferonopathies.
Duffy noted that measurement of interferon alpha attomolar concentrations by digital ELISA will enhance the understanding of interferon biology and potentially improve the diagnosis and stratification of pathologies associated with interferon dysregulation.
He said that the ultrasensitive detection of interferon protein in human material can provide novel insights into disease-causing pathways. It enables the direct measurement of interferon protein as a disease biomarker that clinicians can use for patient stratification and for monitoring the efficacy of treatments, he added.
"There's a lot of interest in targeting this molecule for new therapies," Duffy said. "In our research, we saw three groups, or levels, of this protein for lupus patients. About a third is undetectable, a third has low-to-intermediate levels, and a third has high levels," that indicate high levels of disease activity. The information is particularly useful for researchers who are seeking to stratify patient populations in clinical studies aimed at developing therapies for lupus, he said.
The research team is also looking to use the technique to distinguish viral from bacterial infection, and because of the advanced sensitivity of the test, they may be able to advance the science of detecting tuberculosis infection by analyzing its interferon signature in greater detail than was previously possible.
Duffy said that the group is exploring avenues to commercialize its technique with several companies. They purchased antibodies for their study from ImmunoQure, a biotech company doing R&D into human-derived antibodies for treating human diseases, based in Düsseldorf, Germany.
The firm, which participated in the interferon detection study, aims to develop the interferon antibodies as therapeutic compounds. "We're discussing with them how they can best do that and how we can work with them to provide a companion diagnostic test," that stratifies lupus patients for clinical studies, Duffy said. As a next step, "we're also very interested in exploring interferon-related diseases, and we'd like to better define the role of interferon in TB, so that we could understand whether it could be a therapeutic target or not," he added.
The researchers are interested in better understanding the cellular source of the interferon protein, because it could "give you an opportunity to apply an effective therapeutic approach," Duffy said.
An important success factor for the project was that it was executed by a multidisciplinary team consisting of researchers and clinicians. "It was a strong collaborative project, and that's often what you need when you want to advance something technologically and show that it has clinical relevance, he said, adding that, "You need that mix of expertise."