(620bc) Site-Specific Immobilization of Nanobodies By Introducing Material-Binding Peptides

Antibodies having a strong affinity and specificity against a certain antigen, have been widely used in immunoassay and immuno-affinity chromatography. Since the conventional monoclonal antibodies (whole Ab) are often produced by mammalian cells or animal, their production cost is considerably high. Therefore, these operation costs using the whole antibody become high, while there is still space to reduce the cost. Recombinant antibody fragments such as single-chain Fvs and nanobodies (VHH) can be successfully produced by microorganisms such as E. coli cells. Furthermore, site-specific immobilization of these fragments can be attained by introducing our original material-binding peptides such as PS-tag, PMMA-tag and so on by this research group. Therefore, economical and high-performance antibody-immobilized supports may be prepared by combination these technologies. Here, we investigated production and immobilization of tagged recombinant antibody fragments for detection of target antigens with high sensitivity. In this study, anti-C-reactive protein (CRP) scFv from mouse and anti-green fluorescent protein (GFP) VHH derived from camel are used as models. We investigated availability of both PS-tag and PMMA-tag for site-specific immobilization of recombinant antibody fragments. When both of them with or without peptide-tags were overexpressed by E. coli cells, they were produced in insoluble fractions. After purification of antibody fragments followed by reconstitution of disulfide bonds, they were refolded by dialysis under the optimum conditions. Antigen-binding activities of antibody fragments in adsorption state were accessed by sandwich ELISA and SPR sensor equipped with polymer-coated Au sensor chip. Consequently, these peptide tags increased antigen-binding capacity of the antibody-immobilized solid supports and lower concentration of antigen was detectable, compared with antibody fragments without peptide-tags. Thus, recombinant antibody fragments fused with material-binding peptides will be useful for detection and/or separation of target substance with high efficiency.