Amplification and sequencing cloning of genes encodingfrom known monoclonal recombinant antibodies targeting amyloid beta in Alzheimer’s disease. - A new solution to an old problem
Author
Stehr, Andreas Møller
Term
4. term
Publication year
2012
Submitted on
2012-06-30
Pages
37
Abstract
Alzheimers sygdom er den hyppigste årsag til demens, og i Europa er 1,9% af personer over 65 år diagnosticeret. En lovende behandlingsmulighed er monoklonale antistoffer – laboratoriefremstillede proteiner, der er designet til at ramme sygdomsrelaterede molekyler. En stor udfordring er blodhjernebarrieren (BBB), et beskyttende cellelag, som forhindrer mange lægemidler i at nå hjernen. En mulig løsning er at indføre gener, der koder for disse antistoffer, i celler i BBB, så barrieren selv kan producere dem. For at gøre dette skal man kende de præcise antistofsekvenser, så de kan kopieres og indsættes, men antistoffers stærkt variable områder er svære at amplificere. Den sædvanlige metode – polymerasekædereaktion (PCR) med degenererede primere – kan have begrænset nøjagtighed og specificitet. I dette studie præsenteres en ny måde at bestemme antistofsekvenser på ved at kombinere mRNA-sekventering med tandem-massespektrometri, hvilket gav meget valide og specifikke resultater, som ikke tidligere er beskrevet i litteraturen.
Alzheimer’s disease is the leading cause of dementia, and in Europe 1.9% of people over 65 are diagnosed. A promising treatment approach is monoclonal antibodies—lab-made proteins designed to target disease-related molecules. A major hurdle is the blood–brain barrier (BBB), a protective layer of cells that prevents many therapies from reaching the brain. One idea is to introduce genes that encode these antibodies into BBB cells so the barrier itself can produce them. To do this, scientists need the exact antibody sequences to copy and insert, but antibodies have highly variable regions that are difficult to amplify. The usual method—polymerase chain reaction (PCR) with degenerate primers—can lack accuracy and specificity. This study presents a new way to determine antibody sequences by combining mRNA sequencing with tandem mass spectrometry, yielding highly valid and specific results not previously reported in the literature.
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