(530a) Invited Talk: Sensitive Immunological Detection Using Rabbit Scfv Antibodies Fused with Nitrocellulose-Binding Protein in Lateral-Flow Immunoassay

Lateral-flow immunoassay (LFIA) using a nitrocellulose membrane is well-known as a simple and rapid immunological detection system mainly utilized in the fields of pathogen infection tests and food allergy tests. While a variety of test kits are commercially available, there are some demands to increase signal/noise (S/N) ratio, diminish non-specific background signals. LFIA using recombinant antibodies and/or antibody fragments are also highly demanded to avoid ethical problem to use experimental animals in antibody production. Although single-chain Fv antibodies are one of candidates as a ligand molecule to capture the target antigens on the surface of nitrocellulose membrane, direct immobilization of scFvs on NC membrane resulted in serious conformational changes of antigen-binding domain, and consequently, antigen-binding signals were barely detectable in LFIA.

Here, we developed a sensitive lateral-flow immunoassay system using a fusion protein consisting of rabbit scFv antibodies and a nitrocellulose-binding protein (NBP). Among more than 20 kinds of commercially-available proteins, candidates of NBPs has been successfully selected by dot-blot analysis in the presence of 0.1 – 1.0 % CHAPS as a competitor. Two of candidates, LF and ConA could significantly improve both adsorption and antigen-binding activity of scFv by chemical conjugation. The fusion protein, that LF was genetically connected at the C-terminus of rabbit scFv via flexible linker (G₄S)₃ was expressed by ExpiCHO expression system, and successfully purified by use of protein L affinity chromatography. The scFv-LF was preferentially adsorbed by strong nitrocellulose-binding affinity of LF, and consequently, strong antigen-binding signals could be detected in both LFIA as well as dot-blot immunoassay. Furthermore, the original CDR-grafting method for rabbit scFvs were successfully adopted to accept a variety of antigen-binding specificities in a same scFv-LF format. The fusion protein, rabbit scFv-LF developed in this study is a promising ligand molecule to contribute establishment of sensitive and animal-free LFIA.