Abstract
Antibodies and their derivatives are the most important agents in therapeutics and diagnostics. Even after the significant progress in the technology for antibody screening from huge libraries, it takes a long time to isolate an antibody, which prevents a prompt action against the spread of a disease. Here, we report a new strategy for isolating desired antibodies from a combinatorial library in one day by repeated fluorescence-activated cell sorting (FACS). First, we constructed a library of synthetic human antibody in which single-chain variable fragment (scFv) was expressed in the periplasm of Escherichia coli. After labeling the cells with fluorescent antigen probes, the highly fluorescent cells were sorted by using a high-speed cell sorter, and these cells were reused without regeneration in the next round of sorting. After repeating this sorting, the positive clones were completely enriched in several hours. Thus, we screened the library against three viral antigens, including the H1N1 influenza virus, Hepatitis B virus, and Foot-and-mouth disease virus. Finally, the potential antibody candidates, which show KD values between 10 and 100 nM against the target antigens, could be successfully isolated even though the library was relatively small (∼106). These results show that repeated FACS screening without regeneration of the sorted cells can be a powerful method when a rapid response to a spreading disease is required.
Highlights
For the last two decades, monoclonal antibodies and antibody fragments have been proven to be effective as therapeutic and diagnostic agents, and have long been invaluable tools in various fields of biological research [1,2]
The proof of repeated fluorescence-activated cell sorting (FACS) screening strategy To prove the concept of our strategy (Fig. 1), we first conducted the enrichment of probe-specific cells from mixture with non-specific cells
The quality and diversity are of particular importance in the isolation of the antibody; the six complementarity-determining regions are fully randomized and inserted into a single human antibody framework
Summary
For the last two decades, monoclonal antibodies and antibody fragments have been proven to be effective as therapeutic and diagnostic agents, and have long been invaluable tools in various fields of biological research [1,2]. For the development of antigenspecific antibodies, hybridoma technology that relies on animal immunization has been traditionally employed [3]. The recent progress in combinatorial technologies because of in vitro antibody repertoires and high-throughput screening methodologies has allowed the development of target-specific antibodies without animal immunization [4,5]. In these technologies, various protein display systems including phage display, ribosome display, and cell-surface display, have been widely used for the initial isolation of antibodies specific to antigens from huge libraries, as well as for engineering the antibodies towards desired functions, e.g., enhanced affinity and higher thermostability. The development of new tools to quickly isolate antibodies against rapidly spreading infectious viruses for treatment as well as early diagnosis is urgently required
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