UBC Theses and Dissertations
Microfluidic technologies for rapid, high-throughput screening and selection of monoclonal antibodies from single cells Singhal, Anupam
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.
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