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Utveckling av analys för detektion av muskel dystrophy specifika autoantikroppar
KTH, School of Biotechnology (BIO).
2015 (Swedish)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesisAlternative title
Development of a Bead-based Immunoassay for detecting circulating autoantibodies in duchenne muscular dystrophy (English)
Abstract [en]

Duchenne muscular dystrophy is caused primarily by mutations in the dystrophin gene with possible involvement of the autoantibodies in the aggravating or initiating the disease and there is a great need for sensitive biomarkers for the disease. Suspension bed arrays enables high throughput of autoantibody reactivities toward multiple targets proteins simultaneously and the aim of this project is to identify, autoantibodies associated with Duchenne Muscular Dystrophy. 54 target proteins were selected through a hypothesis-driven selection for which 91 protein fragments fused to a His6ABP-tag were received. These were transformed then expressed in Escherichia Coli Rosetta (DE3) with ZYP-5052 auto-induction medium, purified with immobilised metal affinity chromatography and then immobilised on carboxylated magnetic beads. Serological assays were done with labelled plasma samples for binding assessment and non-labelled for accurate of autoantibodies detection. Out of 91 protein fragments, 85% (77) were successfully transformed, expressed, purified and coupled to magnetic beads with the developed pipeline. In the final assay, autoantibody reactivities toward 67 analytes were analysed in 83 plasma samples. Data from the serological assay were mostly inconclusive with mild indications of reactivities associated with DMD. In brief, we have developed an efficient pipeline from antigen production to immobilisation of magnetic beads and used a bead-based serological assay for the purpose of analysing autoantibody reactivities in plasma samples. A total of 83 samples were analysed and although primary results are inconclusive there are some indications suggesting that conslusive results could be achieved in a further improved assay. 

Place, publisher, year, edition, pages
Keyword [en]
protein arrays, autoantibodies antibody-based proteomics, duchenne muscle dystrophy, immune-response
National Category
Engineering and Technology
URN: urn:nbn:se:kth:diva-179373OAI: diva2:882778
Available from: 2015-12-15 Created: 2015-12-15 Last updated: 2015-12-15Bibliographically approved

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