UBC Theses and Dissertations
Using a biosensor platform for the detection and characterization of autoantibodies in myasthenia gravis patients Pakzad, Zahra
Myasthenia gravis (MG) is an antibody-mediated autoimmune disease of the neuromuscular junction, characterized by skeletal muscle weakness and fatiguability. 85-90% of MG patients have antibodies targeting the nicotinic acetylcholine receptor (AChR), an ion channel on the postsynaptic muscle membrane involved in neuromuscular transmission. A smaller proportion of MG patients have autoantibodies targeting muscle specific tyrosine kinase (MuSK), involved in AChR clustering. AChR and MuSK antibodies are highly specific for MG and serve as excellent diagnostic biomarkers. However, there is still a need for biomarkers to help predict response to treatment and disease course. Reasonable targets are antibody titer, isotype and IgG subclass, and binding affinity as these parameters dictate autoantibody pathogenicity. Our first objective was to develop and optimize a sensitive assay using a biosensor platform (Biacore™) for the measurement of these parameters in MG patients with MuSK antibodies. MuSK antigen was immobilized onto Biacore™ sensor chips and diluted sera injected over the surface to measure binding levels. Antibody isotype and subclass were determined with sequential injections of anti-human Ig antibodies, and dissociation rate, as a measure of binding affinity, was determined by fitting the dissociation phase to a 1:1 model. This assay is specific, sensitive, and reproducible, and can be performed more rapidly and using low volumes of serum. We demonstrated the use of this assay in providing a comprehensive long-term profiling of MuSK antibodies that has the potential to be useful in a clinical setting for monitoring treatment response. Developing a biosensor assay for the measurement and characterization of AChR antibodies involves the more complex process of immobilizing AChR in its native membrane-bound conformation. Our secondary objective was to determine a method for immobilizing AChR onto a sensor chip in its membrane-bound form. We demonstrate a method using immobilized cell membrane vesicles which, with further study, may be viable for this purpose. Collectively, these studies demonstrate the usefulness of biosensor methods in measuring and characterizing autoantibodies over the course of a patient’s disease. Measuring and characterizing antibodies for use as clinical biomarkers in MG to help with assessing disease progression and response to treatment merits further study.
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Attribution-NonCommercial-NoDerivs 2.5 Canada