Establishment and Characterisation of New Immunoreagents for Diagnosis of Neurodegenerative Disease
Examensarbete för masterexamen
Biotechnology (MPBIO), MSc
Neurodegenerative diseases are a collection of disorders which can develop any time in adulthood, but with the majority occurring in the elderly population. A common denominator for these diseases is the gradual degradation of neurons in the central nervous system, often taking place long before the manifestation of clinical symptoms. One of the proteins taking part in this process is called neurofilament light, which upon degradation can be found both in the cerebrospinal fluid as well as the blood. In vivo diagnostic tools utilising antibodies have been developed for the detection of NF-L in the CSF, allowing for earlier diagnosis. However, for screening to be able to take place at local healthcare centers, a diagnostic test for NF-L in blood would be more beneficial. Since the concentration of the antigen is much lower in the blood compared to the CSF, antibodies with high specificity and sensitivity are necessary. The aim of this project was to establish high affinity monoclonal antibodies against the protein neurofilament light, specifically when present in plasma, using classic hybridoma technology. Mice were immunised with full length bovine NF-L as well as two shorter custom made peptides, originating from the Coil-2 region of NF-L, having shown to be a valuable epitope sight for clinically effective antibodies. Over 20 hybridomas producing relevant antibodies were established, detected through ELISA screens. All succesfull hybridomas originated from the mice immunised with peptides. The majority of the established antibodies had the desired isotypes and a few had a strong response towards full length bovine NF-L, indicating clinical relevance. To conclude, a number of antibodies with the potential of being used in a sandwich ELISA diagnostic test for the detection of NF-L in blood have been established. These antibodies were proven to recognise the native form of bovine NF-L, epitope mapped to the clinically relevant Coil 2 region and were not reactive to other proteins present in clinical samples of CSF or serum. This project was performed at Fujirebio AB located in Gothenburg, Sweden.
Neurofilament light , biomarker , monoclonal antibodies , hybridoma technology , ELISA