Somayeh Zamani, Fatemeh Fotouhi Chahouki, Zahra Nourmohammadi , Saeideh Sadeghi Neshat, Vahideh Mazaheri , Ali Torabi , Behrokh Farahmand ,
Volume 73, Issue 7 (10-2015)
Abstract
Background: The influenza virus is one of the most important factors for higher morbidity and mortality in the world. Recently, researchers have been focused on influenza conserved antigenic proteins such as hemagglutinin stalk domain (HA2) for vaccine production and serological studies. The HA2 plays a major role in the fusion of the virus with host cells membrane. The immunity system enables to produce antibody against HA2. The aim of this study is polyclonal antibody production against influenza HA2. Methods: This study was done in the Influenza Research Lab, Pasteur Institute of Iran, Tehran for one year from September 2013 to October 2014. In the present study, recombinant HA2 protein was produced in prokaryotic system and purified using Nickel affinity chromatography. The purified HA2 was mixed with Freund’s adjuvant (complete and incomplete) and injected into two New Zealand white rabbits by intramuscularly and subcutaneously routes. Immunization was continued for several months with two weeks interval. Before each immunization, blood was drawn by venous puncture from the rabbit ear. Function of rabbit's sera was evaluated using radial immunodiffusion (RID) in both forms, Single RID (SRID) and Double RID (DRID). Finally, antiserum activity against HA2 was evaluated using western blotting as serological assay. Results: Sedimentary line and zone was observed in RID assays (SRID and DRID) represent interaction between HA2 protein and anti- HA2 antibody. As well as, western blotting results was positive for HA2 protein. Therefore, these results showed that polyclonal antibody produced against HA2 protein can identify HA2 protein antigenic sites. Conclusion: These findings show that humoral immune responses have properly been stimulated in rabbits and these antibodies can identify HA2 protein and may be suitable for other serological methods.
Fateme Khosravi Node , Farida Behzadian , Vahideh Mazaheri , Hadiseh Shokouhi , Maryam Saleh , Behrokh Farahmand ,
Volume 75, Issue 8 (11-2017)
Abstract
Background: Each year, Human influenza A (H1N1) virus causes moderate to severe infections with a high prevalence throughout the world. Accordingly, the rapid, sensitive and cost-effective laboratory diagnosis based on viral antigen detection is important. Moreover, the generation of specific antibodies directed against Influenza antigens is essential to the success of both basic and applied research programs. Hemagglutinin (HA) is the major surface envelope glycoprotein of influenza virus, which is subsequently cleaved into two subunits, HA1 and HA2. Since most antigenic sites are in the HA1 domain of HA, HA1 domain of influenza virus was studied as antigen to produce polyclonal antibody.
Methods: In this experimental study we expressed and purified the recombinant HA1 protein in the second half of 2015 at department of influenza and other respiratory viruses, Pasteur Institute of Iran and then prepared the polyclonal rabbit antibody against it. The vector of pET28aHA1 expressing HA1-His tagged protein of H1N1 influenza A/PR/8/34 virus was used for large scale production of HA1 into E. Coli (BL21). By changing expression conditions such as IPTG (Isopropyl β-D-1-thiogalactopyranoside) concentration, time and temperature of incubation, the expression conditions for HA1 were optimized. The total cell protein harvested and purified by nickel affinity chromatography. All above mentioned experiments monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Results: The efficiency of HA1 recombinant protein was high, equal to 400-600 mg/ml of cell lysate. The polyclonal antibody was prepared by immunizing the rabbits using recombinant HA1 with Freund’s adjuvant according to standard protocols. Efficiency of the antiserum evaluated by enzyme linked immunosorbent assay (ELISA). Determination of antibody level in the collected antiserum using serum-based ELISA showed that the specific antibody has risen well through the immunization schedule.
Conclusion: Our data shows that this polyclonal antibody has potential to be produced in rabbit. It will also be used in the future in influenza diagnosis as well as in other immunological applications such as western blot analyses, immunocytochemistry, and immunohistochemistry.
