One-Step Immunoassay for the Detection of Influenza Virus Based on Screened Nucleoprotein (NP)-like Mimotopes from Fv-Antibody Library

The nucleoprotein (NP)-like mimotopes of influenza-A (Inf-A) virus were screened from autodisplayed Fv-antibody library and these NP-like mimotopes were applied to one-step immunoassay of influenza virus. The NP-like mimotopes were screened from an Fv-antibody library. The Fv-antibody represented the V_H region of heavy chain IgG, and the Fv-antibody library was prepared by randomizing CDR3 region. Therefore, prepared Fv-antibody library was expressed on the outer membrane of Escherichia coli using autodisplay technology. The target Fv-antibodies with the binding affinity to anti-NP monoclonal antibody (mAb) for Inf-A were screened as the NP-like mimotopes, and four clones were screened to have with the binding affinity to anti-NP mAb. The screened Fv-antibodies (NP-like mimotopes) were expressed as fusion proteins with a super folder green fluorescent protein (sfGFP), and the binding affinity (K_D) of NP-like mimotopes was estimated using SPR biosensors. The one-step immunoassay was configured for detecting Inf-A NP by binding NP-like mimotopes to immobilized anti-NP mAbs. The NP-like mimotopes (labeled with sfGFP) were quantitatively dissociated when the Inf-A NP (target analyte) were bound to immobilized anti-NP mAbs. The one-step immunoassay based on NP-like mimotopes was estimated to have a far higher sensitivity than the conventional lateral-flow immunoassay. Additionally, the one-step immunoassay for Inf-A was determined to be used for the detection of influenza-B (Inf-B) because of a high similarity (> 70%) in the amino acid sequence of NPs of Inf-A and Inf-B. Using the real samples of Inf-A and Inf-B (both were heat deactivated), the one-step immunoassay was demonstrated to be feasible for the medical diagnosis of Inf-A as well as Inf-B.