Site‐oriented immobilization of fusion antigen directed by an affinity ligand,and its validation in an immunoassay

Immobilizing proteins on a solid surface in a site‐specific orientation and maintaining their bioactivity are crucial to the construction of high‐performance immunoassays. In this study, an affinity ligand for polystyrene (PS) surface screened from a phage display peptide library, named Lig1, was genetically fused to the N/C‐terminus of chimeric antigen HCV that could be recognized by specific antibodies against hepatitis C virus (HCV). Immunoassay characteristics of lig1‐fused HCVs immobilized on PS surface were compared to that of original HCV in both direct and indirect enzyme‐linked immunosorbent assay (ELISA). The results indicated that HCV‐Lig1 (Lig1 fused to HCV C‐terminus) was preferentially adsorbed on PS surface in a site‐oriented manner and would expose specific antibody‐binding sites well, which resulted in a substantial enhancement of detection sensitivity. AFM images showed that, compared to the original one, HCV‐Lig1 was arranged on PS surface in an ordered state and its conformational and steric distortions induced during the interfacial binding process were much slighter. As long as the specific epitope of a coating antigen is not located on both its N and C‐terminus, the ligand fusion approach could be an ideal strategy for site‐oriented protein immobilization. Copyright © 2010 John Wiley & Sons, Ltd.