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Microfluidic technologies for rapid, high-throughput screening and selection of monoclonal antibodies from single cells

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dc.contributor.author Singhal, Anupam
dc.date.accessioned 2012-11-09T19:17:36Z
dc.date.available 2013-04-30
dc.date.copyright 2012 en
dc.date.issued 2012-11-09
dc.identifier.uri http://hdl.handle.net/2429/43575
dc.description.abstract This thesis describes the development of novel microfluidic technologies for rapid, high-­‐throughput screening and selection of monoclonal antibodies (mAbs) from single cells. Microfluidic devices were used to compartmentalize single antibody-­‐ secreting cells (ASCs) in small-­‐volume chambers (i.e. hundreds of picoliters to nanoliters) in order to concentrate secreted mAbs for measurement of antigen binding kinetics and affinities using a novel microfluidic fluorescence bead assay. Microfluidic single-­‐cell antibody screening was performed on ASCs harvested from antigen-­‐ immunized mice and purified by fluorescence-­‐activated cell sorting (FACS). Following microfluidic selection of ASCs producing antigen-­‐specific mAbs, ASCs were sequentially recovered from the microfluidic device and subjected to single-­‐cell RT-­‐PCR to amplify the antibody-­‐encoding heavy and light chain genes. Antibody genes for selected high-­‐ affinity mAbs are sequenced and cloned into expression vectors for recombinant production in mammalian cell lines. Nearly 200 high-­‐affinity mouse mAbs to the model antigen hen egg lysozyme (HEL) were selected as a validation of this technology, representing a ten-­‐fold increase in the number of high affinity anti-­‐HEL mAbs previously selected using single-­‐cell micro-­‐technologies and the traditional hybridoma approach. Microfluidic single-­‐cell mAb screening also yielded important insights into affinity maturation, immuno-­‐dominance, and antibody stereotypy in the adaptive immune system. By circumventing time-­‐consuming limiting dilution and clonal expansion in the hybridoma approach, microfluidic single-­‐cell screening will enable selection of mAbs from other animal species (e.g. rabbits, humans) for both therapeutic and research applications. en
dc.language.iso eng en
dc.publisher University of British Columbia en
dc.relation.ispartof Electronic Theses and Dissertations (ETDs) 2008+ en
dc.title Microfluidic technologies for rapid, high-throughput screening and selection of monoclonal antibodies from single cells en
dc.type Text en
dc.degree.name Doctor of Philosophy - PhD en
dc.degree.discipline Chemical and Biological Engineering en
dc.degree.grantor University of British Columbia en
dc.date.graduation 2012-11 en
dc.type.text Thesis/Dissertation en
dc.description.affiliation Applied Science, Faculty of
dc.degree.campus UBCV en
dc.description.scholarlevel Graduate en

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