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High-throughput antibody sequencing for therapeutic applications using droplet-based microfluidics
KTH, Skolan för bioteknologi (BIO).
2014 (engelsk)Independent thesis Advanced level (degree of Master (Two Years)), 20 poäng / 30 hpOppgave
Abstract [en]

The aim of the thesis was to develop an innovative high-throughput approach for charaterization of primary B cells repertoires. This system combining droplet-based microfluidics and next generation Illumina sequencing technology, will allow the heavy chain and light chan gene pairs of targeted antibodies to be identified and sequenced.

 

Droplet-based microfluidics involves generating highly monodisperse aqueuous droplets in the range of picoliter volume that function as independent biocompatible microreactors for chemical or biological assays. In the thesis, droplet-based microfluidics is used to compartmentalize a single cell inside a droplet, capture the mRNA released by the B cells and labelled it with a unique barcode DNA sequence. Cells are encapsulated with reverse transcription reagents and a single bead carrying a unique CNA barcode sequence and a specific sequense targeting the heavy chain and light chain genes of the antibodies o finterest. Up tp 85 millipn pf DNA barcodes are generated by combinatorial indexing method using beads carriers. A split-pool sythesis method allows the construction of a unique 4 indices barcode onto the beads with an additional gene specific sequence tareting the light chain or heavy chain gene sequences of the antibodies. After incubation, droplets are collected, broken and the generated cDNA  is exponentially amplified by PCR prior to sequencing. Bioinformatic analysis will allow the identification of VL and VH genes from the same antibody as they bear the same barcode.

This master thesis was realized in the Laboratory of Biochemistry (LBC) directed by Pr. Andrew Griffiths at EXPCI ParisTech and is part of the antibody –screening project developed in collaboration between the LBC and HiFiBIO, a spin-off company hosted at ESPCI and based on single cells technologies for drug discovery.

 

sted, utgiver, år, opplag, sider
2014.
Emneord [en]
antibody, droplet-based microfluidics, DNA barcoding technology
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-173581OAI: oai:DiVA.org:kth-173581DiVA, id: diva2:853705
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Examiner
Tilgjengelig fra: 2015-09-15 Laget: 2015-09-14 Sist oppdatert: 2015-09-15bibliografisk kontrollert

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