title: 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.
authors.affiliation: Department of Respiratory Virology, Vallabhbhai Patel Chest Institute, University of Delhi , Delhi , India.
authors.forename: Roopali
authors.initials: R
authors.lastname: Rajput
authors.affiliation: Project Directorate on Foot and Mouth Disease, Indian Veterinary Research Institute Campus , Mukteswar , India.
authors.forename: Gaurav
authors.initials: G
authors.lastname: Sharma
authors.affiliation: Department of Respiratory Virology, Vallabhbhai Patel Chest Institute, University of Delhi , Delhi , India.
authors.forename: Varsha
authors.initials: V
authors.lastname: Rawat
authors.affiliation: Department of Respiratory Virology, Vallabhbhai Patel Chest Institute, University of Delhi , Delhi , India.
authors.forename: Anju
authors.initials: A
authors.lastname: Gautam
authors.affiliation: Department of Respiratory Virology, Vallabhbhai Patel Chest Institute, University of Delhi , Delhi , India.
authors.forename: Binod
authors.initials: B
authors.lastname: Kumar
authors.affiliation: Project Directorate on Foot and Mouth Disease, Indian Veterinary Research Institute Campus , Mukteswar , India.
authors.forename: B
authors.initials: B
authors.lastname: Pattnaik
authors.affiliation: Former National Consultant (Avian Influenza), WHO-India Office, Bharatiya Kala Kendra , New Delhi , India.
authors.forename: H K
authors.initials: HK
authors.lastname: Pradhan
authors.affiliation: Department of Respiratory Virology, Vallabhbhai Patel Chest Institute, University of Delhi , Delhi , India.
authors.forename: Madhu
authors.initials: M
authors.lastname: Khanna
keywords: ELISA; HA; antibodies; diagnosis; influenza; phage display; recombinant; scFv