(582dq) Refolding and Immobilization of Peptide-Tag-Fused VHH Antibodies and Its Application to the Sensitive Immunoassays

Antibodies can specifically recognize a target molecule with strong binding affinity. Therefore, they have been utilized as diagnostic reagents as well as drugs. The conventional antibodies have been considered to have some problems for use in such fields, for example, high production cost and low binding activity after immobilization due to lower density and unfavorable orientation. On the other hand, recombinant antibody fragments such as Fab, scFv, and VHH have been developed to overcome these problems.

   Here, we focus on VH domain of heavy-chain antibodies (VHH antibodies) from camel for use as a ligand antibody in immunoassay. VHH antibodies are functional recombinant antibodies that consist only of the VH domain of heavy-chain. Therefore they are expected to be the smallest size for antigen recognition. Its molecular weight is less than one-tenth of whole antibody. Therefore, immobilization density must be higher than those of whole antibody and scFv. Furthermore, VHH antibodies may easily refold because they have only single intramolecular disulfide bond. For above reasons, VHH antibodies are expected to be useful ligand among the recombinant antibody fragments.

   In this study, we investigated to produce VHH antibodies by E. coli cells and immobilize them onto the various plastic plates for use in immunoassay. We recently developed affinity peptide-tags, which can strongly recognize the surfaces of polystyrene (PS-tag:RIIIRRIRR) and poly(methylmethacrylate) (PMMA-tag:DVEGIGDVDLVNYFEVGATYTFNK).

Consequently, we successfully produced VHH antibodies genetically-fused with both PS-tag and PMMA-tag, and they were efficiently refolded by dialysis method. Both VHH antibodies refolded showed strong binding activities against the model antigen, hCG. Both PS-tag-fused VHH and PMMA-tag-fused VHH were successfully immobilized onto the surface of hydrophilic polystyrene plates and the VHH-immobilized plates detect the antigen with much high sensitivity. Furthermore, PMMA-tag-fused VHH could be used as a ligand for maxisorp and PMMA plates, indicating that PMMA-tag possesses relatively strong affinity against a variety of plastic plates. Thus, peptide-tag-fused VHH as a ligand will be significantly useful for sensitive immunodiagnosis.