Gu Min Jeong

Site-specific immobilization of proteins on non-conventional substrates via solvent-free initiated chemical vapour deposition (iCVD) process

Currently, the development of protein chips faces new requirements of improved environmental stability, disposability, and cost-effectiveness for their wide applicability in our daily life. Here, we demonstrate a new protein detection platform on low-cost plastic and paper substrates. To achieve this platform, a highly selective thiol–ene photochemical reaction between vinyl functionality on the surface and cysteine-linked protein was utilized to immobilize the relevant proteins on the disposable substrates. First, the surface of the flexible substrates was modified with a vinyl-containing polymer, poly(2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane) (pV4D4), via a solvent-free initiated chemical vapour deposition (iCVD) process. Next, on the vinyl functionalized surface, cysteine-linked proteins were covalently immobilized by a UV-assisted thiol–ene click reaction. The solvent-free characteristic of iCVD enabled us to directly pattern the functionality on the substrate via shadow mask patterning. On the patterned substrate, selective immobilization of the proteins was possible. With four model proteins, including two fluorescent proteins, single-chain variable fragment (scFv), and non-immunoglobulin protein scaffold (human kringle domain), we successfully demonstrated the immobilization of proteins on various substrates including polyethylene (PE) film and chromatography paper.

Rapid Isolation of Antibody from a Synthetic Human Antibody Library by Repeated Fluorescence-Activated Cell Sorting (FACS)

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.

Development of high-affinity single chain Fv against foot-and-mouth disease virus

Foot-and-mouth disease (FMD) is caused by the FMD virus (FMDV) and results in severe economic losses in livestock farming. For rapid FMD diagnostic and therapeutic purposes, an effective antibody against FMDV is needed. Here, we developed a high-affinity antibody against FMDV by FACS-based high throughput screening of a random library. With the FITC-conjugated VP1 epitope of FMDV and high-speed FACS sorting, we screened the synthetic antibody (scFv) library in which antibody variants are displayed in the periplasm of Escherichia coli. After three rounds of sorting, we isolated one antibody fragment (#138-scFv) against the VP1 epitope of FMDV. Next, to improve its affinity, a mutation library of #138-scFV was constructed by error-prone PCR and screened by FACS. After three rounds of sorting, we isolated one antibody (AM-32 scFv), which has a higher binding affinity (KD=42.7nM) than that of the original #138-scFv. We also confirmed that it specifically binds to whole inactivated FMDV.

A human kringle domain-based fluorescence-linked immunosorbent assay system.

As a non-immunoglobulin protein scaffold, human kringle domain (KD) has attractive properties such as high specificity, stability, and production in bacterial hosts. Here, we developed a rapid and sensitive fluorescence-linked immunosorbent assay (FLISA) system using a fluorescent kringle domain (fluoKD), a fusion protein of a green fluorescent protein (GFP), and a kringle domain variant (KD548). Two kinds of fluoKDs in which KD was fused to the N terminus of GFP (N-fluoKD) or the C terminus of GFP (C-fluoKD) were constructed and characterized. In Escherichia coli host, both fluoKDs were produced in high yield and solubility and were successfully purified by a simple procedure. The purified fluoKDs exhibited strong fluorescent activities and high affinities to the target antigen. Furthermore, it was successfully demonstrated that the FLISA with purified fluoKDs allowed for more rapid detection of target antigens with higher sensitivity compared with conventional enzyme-linked immunosorbent assay (ELISA), indicating that a simple, rapid, and sensitive immunoassay system could be developed by using KD instead of antibody or antibody fragments.