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

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Title: Microfluidic technologies for rapid, high-throughput screening and selection of monoclonal antibodies from single cells
Author: Singhal, Anupam
Degree Doctor of Philosophy - PhD
Program Chemical and Biological Engineering
Copyright Date: 2012
Publicly Available in cIRcle 2013-04-30
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.
URI: http://hdl.handle.net/2429/43575
Scholarly Level: Graduate

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