Development of single-chain Fv against the nucleoprotein of type A influenza virus and its use in ELISA

Single chain fragment variable (ScFv) antibodies specific to the nucleoprotein (NP) of avian influenza virus (AIV) were developed using a phage display system. The variable heavy (VH) and the variable light (VL) chain gene fragments were derived from spleen cells of Balb/c mouse immunized with a recombinant NP (rNP) antigen (∼63 kDa) of H5N1 influenza virus. The VH and the VL DNA fragments were assembled through a flexible linker DNA to generate ScFv DNA that was cloned subsequently in a phagemid to express ScFv protein in Escherichia coli cells. The specific reactivity of the ScFv with the rNP antigen and viral antigen (H5N1) was confirmed by Western blot and ELISA. A competitive inhibition ELISA (CI-ELISA) was developed using the rNP and the anti-NP ScFv for detection of type-specific antibodies to AIV in chicken sera. The ScFv based CI-ELISA was compared with hemagglutination inhibition (HI) test and agar gel immunodiffusion (AGID) test over 850 sera. Sensitivity of the CI-ELISA was 100% with HI and AGID and specificity was 98.7% with HI and 100% with AGID.