NEW YORK (GenomeWeb) – Researchers at the Mayo Clinic have developed an assay that can distinguish a spectrum of diseases frequently confused with multiple sclerosis. The assay uses cells expressing a particular neurological protein rather than a standard immunoassay, and is innovative in its use of fluorescence-activated cell sorting (FACS) to increase throughput.
The new test detects antibodies in patient serum against a cell surface protein present on oligodendrocytes in the brain called myelin oligodendrocyte glycoprotein, or MOG, and uses a cell-based assay, rather than a more typical type of immunoassay, like an ELISA.
"The problem with ELISAs and immunoprecipitation assays is that they are often using linear proteins, which is not the conformation that would be present in a living cell," said Sean Pittock, a developer of the test who is also director of Mayo Clinic's Center for Multiple Sclerosis and Autoimmune Neurology as well as director of Mayo's Neuroimmunology Research Laboratory.
Cell-based immunoassays involve transfecting HEK 293 human kidney cells and then measuring patient IgG binding, typically with a fluorescence-based readout. Comparing fluorescence on transfected cells to signal from un-transfected cells yields a ratio, and that ratio is set to best distinguish true positives.
In particular, if a patient's antibody is against a protein that is expressed on the outside of a living cell, it is likely to be pathogenic. "For those types of antibodies … you want to use live cells that are expressing the target on the surface to optimize detection," Pittock said, whereas for antibodies against proteins expressed inside of cells, which would more likely be biomarkers of a T cell-mediated disease, linear protein detection using ELISAs is still a good option.
The method of the MOG IgG test is based on previous Mayo Clinic work on a different cell-surface protein called aquaporin-4. Cell-based assays are used by microbiologists to detect antibodies to viral antigens, Pittock said, but the methodology developed at Mayo was the first cell-based assay used to detect an autoantibody in neurological disease, although cell-based assays are now taking over the field of testing for neural antibodies, he said.
Patients with MOG antibodies are often misdiagnosed with MS when they actually have what are called neuromyelitis optica spectrum disorders (NMOSDs), including optic neuritis and transverse myelitis, or a disease called acute disseminated encephalomyelitis (ADEM).
Multiple sclerosis, meanwhile, affects about 1 in 500 people in North America, said Pittock, and it is a disease for which there is no biomarker. Diagnosis is based on a patient history of relapsing changes in vision or balance, MRI abnormalities, and non-specific markers in spinal fluid called oligoclonal bands, he said.
In 2004, the Mayo Neuroimmunology lab published an article in The Lancet describing antibodies against aquaporin-4 — a channel which pumps water across the membrane of astrocytes in the brain — as the first biomarker for an inflammatory demyelinating central nervous system disease that could be used to distinguish MS from another neurodegenerative disease.
Patients with aquaporin-4 antibodies have an illness that resembles MS, but about 80 percent of them have been shown to actually have neuromyelitis optica, or Devic's disease. This disease, which consists of inflammation and demyelination of the optic nerve and spinal cord, can be relapsing and remitting, thus mimicking a signature component of MS.
Mayo began offering the aquaporin-4 IgG test, but in the atypical cell-based format. The lab uses a human kidney cell line, HEK 293, for this purpose. "We found that ELISA had problems with specificity, but when you transfect cells with [aquaporin-4] and then look under the microscope to see if the patient has antibodies that bind to the outside of that cell you dramatically increase specificity," Pittock explained.
This method allowed the lab to distinguish neuromyelitis optica from multiple sclerosis, when previously the two could be confused, Pittock said. "If you are Aquaporin-4 positive, you don't have multiple sclerosis, you have neuromyelitis optica or some spectrum of that disease," he added, noting that among Asian populations the test picks up a distinct disease referred to as optic spinal MS.
Subsequent research at Mayo published in the Journal of Experimental Medicine showed that the cell-based detection method for aquaporin-4 antibodies was effective. The lab now tests around 150 patients each day for aquaporin-4 IgGs, making it a significant proportion of the 1.4 million tests for neural antibodies it runs each year, Pittock said. "You can imagine that looking down a microscope at a cell would be pretty time consuming, which is why we then developed the flow-cytometry method," he said.
Pittock further noted that retrospective studies at Mayo have suggested patients treated with MS drugs meant to modify the course of illness who actually have Devic's disease seem to have more frequent episodes. These patients would be more appropriately treated with immunosuppressants. Now, aquaporin-4 testing has become a standard of care in the evaluation of patients for multiple sclerosis, Pittock said, noting that it can also predict recurrence.
For MOG, the lab is duplicating its aquaporin-4 work, and has also further developed a flow-cytometry method using FACS for the diagnostic test.
Distinguishing demyelinating disease
The MOG protein is present on oligodendrocytes, the cells in the brain that make myelin. MOG has been studied extensively, in part because MS is a demyelinating disease.
For years people used ELISA tests and claimed MOG antibodies were a biomarker for MS, Pittock said. "MOG is a really sticky protein — researchers were screening patient serum on the ELISA and there was a lot of binding," he said. In fact, Mayo's own data showed around 25 percent of normal serum samples will bind on an ELISA test for MOG antibodies.
Further confusing the issue, models for MS that mimic the human disease are created by immunizing animals with MOG protein and having the animals generate antibodies.
But, it turns out "MOG antibodies are a method of telling you that you do not have multiple sclerosis," Pittock said.
In addition, Mayo's research on a small number of patients so far is showing that those whose demyelinating disease is related to MOG also have an increased frequency of episodes, or higher annualized relapse rate, while taking the drugs designed to treat MS, Pittock said.
And, the new MOG test also rules out the spectrum of disease related to aquaporin-4, Pittock said, because there is no overlap between the two in an individual patient.
Mayo now offers a comprehensive central nervous system demyelinating disease evaluation which includes both tests. "It is very important for clinicians to know, because if a patient is positive [for aquaporin-4 or MOG] it tells them something about the immunopathology of the disease and directs them toward a different type of treatment," Pittock said.
In terms of specificity for the MOG test, the lab has tested around 300 patients with classical MS and found only one low positive, and it is also continuously evaluating the cut-off to improve the test. In terms of sensitivity, about 40 percent of patients with ADEM demyelination are positive for MOG antibodies, but longitudinal studies show persistence or decrease of MOG antibodies in serum can also predict relapse in these patients.
In unpublished research, about 15 percent of neuromyelitis optica patients do not express aquaporin-4 antibodies, but 30 percent of them are positive for MOG antibodies, Mayo has found. Also, about 15 percent of patients with recurrent episodes of optic neuritis are MOG IgG positive.
The method of using the aquaporin-4 antibody as a test was licensed by the Mayo Clinic to a company in the UK that now sells an ELISA kit, Pittock said, and that company also gave rights to a company called Euroimmun, which then created a cell-based aquaporin-4 assay sold in the US and Europe. However, a 2012 Neurology study co-authored by Pittock demonstrated that assays detecting IgG by binding to cells expressing aquaporin-4 and using quantitative flow cytometry had superior sensitivity to other tests.
At the moment there is no other lab in the US offering MOG antibody testing, although there are some groups in Europe, particularly a team Mayo is collaborating with at Oxford University. There are also kits in development by other companies, Pittock said, but he suspects that they won't have as high of specificity and sensitivity as the Mayo FACS-based test.
Pittock's team has waited to perfect the assay before reporting data, but has now submitted a number of poster abstracts for an upcoming American Academy of Neurology conference as well as a paper to JAMA Ophthalmology.
The Mayo Clinic Lab has a sales force that will now promote the MOG test with neurologists nationwide. Pittock said that patients whose aquaporin-4 IgG tests are negative are particularly good candidates for MOG testing, since the diseases these antibodies are related to have a very similar phenotype.
In the future, autoimmune antibodies that can distinguish other demyelinating disease from MS could also be FACS- and cell-based tests, Pittock said. The lab is discovering about four of five antibodies per year at the moment that are clinically relevant to other autoimmune-based disease like autoimmune epilepsies and dementia.