Diagnostic Potential of Recombinant scFv Antibodies Generated Against Hemagglutinin Protein of Influenza A Virus

Abstract

Human influenza A viruses have been the cause of enormous socio-economic losses worldwide. In order to combat such a notorious pathogen, hemagglutinin protein (HA) has been a preferred target for generation of neutralizing-antibodies as potent therapeutic/diagnostic agents. In the present study, recombinant anti-HA single chain variable fragment antibodies were constructed using the phage-display technology to aid in diagnosis and treatment of human influenza A virus infections. Spleen cells of mice hyper-immunized with A/New Caledonia/20/99 (H1N1) virus were used as the source for recombinant antibody (rAb) production. The antigen-binding phages were quantified after six rounds of bio-panning against A/New Caledonia/20/99 (H1N1), A/California/07/2009 (H1N1)-like, or A/Udorn/307/72(H3N2) viruses. The maximum phage yield was for the A/New Caledonia/20/99 (H1N1), however, considerable cross-reactivity was observed for the other virus strains as well. The HA-specific polyclonal rAb preparation was subjected to selection of single clones for identification of high reactive relatively conserved epitopes. The high-affinity rAbs were tested against certain known conserved HA epitopes by peptide ELISA. Three recombinant mAbs showed reactivity with both the H1N1 strains and one (C5) showed binding with all the three viral strains. The C5 antibody was thus used for development of an ELISA test for diagnosis of influenza virus infection. Based on the sample size in the current analysis, the ELISA test demonstrated 83.9% sensitivity and 100% specificity. Thus, the ELISA, developed in our study, may prove as a cheaper alternative to the presently used real time RT–PCR test for detection of human influenza A viruses in clinical specimens, which will be beneficial, especially in the developing countries.

The emerging influenza virus threat: status and new prospects for its therapy and control

“…A recent study demonstrated the diagnostic potential of recombinant scFv antibodies generated against the hemagglutinin protein of influenza A virus for diagnosis and treatment of human influenza A virus infections. In that study, an ELISA was developed that demonstrated 83.9% sensitivity and 100% specificity for H1N1 influenza A viruses and promised to be a cheaper alternative to the costly RRT-PCR test [68]. At research institutes and in reference or hospital laboratories, where sophisticated equipment is available, electron microscopy, cytology and histology may also be used to diagnose influenza virus infections.…”

DNA and protein-generated chimeric molecules for delivery of influenza viral epitopes in mouse and humanized NSG transfer models

“…Numerous scFvs have been constructed against haptens, 53 proteins, 54 carbohydrates, 55 receptors, tumor antigens, 56 and viruses. 57 All of these scFvs are good for use in many areas, such as medical therapies and diagnostic applications. In our studies, we used a B/T cell conserved influenza viral epitope to target activating cellular receptors.…”

Editorial: Influenza Virus Vaccines and Immunotherapies

“…Carnell et al reviewed comprehensive methods for influenza virus neutralization assays, based on the pseudotype viral particles, which utilize chimeric viruses bearing influenza glycoproteins, and depict how such assays can replace the traditional HA neutralization assays for the evaluation of new age influenza vaccines ( 18 ). To detect the incidence of Influenza virus with high efficiency and accuracy, Rajput et al generated HA specific high affinity recombinant single chain variable fragment (scFv) antibodies showing high sensitivity (83.9%) and specificity (100%) for three different strains of influenza virus ( 19 ).…”