Author/Authors :
Alikhani, M Influenza and other Respiratory Viruses Department - Pasteur institute of Iran, Tehran - Department of Genetic Engineering - Research Center for Science and Biotechnology - P.O. Box 19395-1949, Tehran , Behzadian, F Department of Genetic Engineering - Research Center for Science and Biotechnology - P.O. Box 19395-1949, Tehran , Mehrbod Influenza and other Respiratory Viruses Department - Pasteur institute of Iran, Tehran , Khosravi Node, F Influenza and other Respiratory Viruses Department - Pasteur institute of Iran, Tehran - Department of Genetic Engineering - Research Center for Science and Biotechnology - P.O. Box 19395-1949, Tehran , Shokouhi Targhi, H Influenza and other Respiratory Viruses Department - Pasteur institute of Iran, Tehran , Farahmand, B Influenza and other Respiratory Viruses Department - Pasteur institute of Iran, Tehran
Abstract :
Background and Aims: Influenza is an acute respiratory illness that is caused by a virus
belonging to Orthomyxoviridae family. This virus spreads rapidly every year in cold season
and leads to morbidities and mortalities especially in adults and children, which causes
billions of dollars of economic losses. Accordingly, development of a rapid, sensitive and
inexpensive laboratory diagnosis based on antigen detection to distinguish this infection from
other respiratory tract viruses is important. In addition, specific anti-influenza antibody
production against influenza virus antigen is essential for basic and applied research
programs. Influenza A virus nucleoprotein (NP) is a structural protein and a major component
of the ribonucleoprotein complex. It has a high expression level during infection. NP consists
of 498 amino acids with molecular weight of 57 KD. The aim of this study was to produce
and purify polyclonal antibody against recombinant nucleoprotein of the influenza A virus.
Materials and Methods: Rabbit immunization was performed based on a specific program
by NP purified recombinant antigen and Freund's adjuvant. Serum immunoglobulin was
separated by ammonium sulfate and IgG purification was conducted by ion exchange
chromatography (DEAE-cellulose). To evaluate the reaction between antigens and purified
antibodies, SRID and ELISA serological tests were applied. Results: The results obtained from SDS-PAGE and Western blot showed a dense band of
purified NP. The results of ELISA confirmed an increase in NP antibody titer after one
month. Antibody levels detection by ELISA showed a sensitivity of 1 to 50. In SRID,
sedimentary areola was observed due to the interaction of NP antigen and antiserum. Western
blot results were also positive for the NP protein.
Conclusions: The NP antigen purified in this study, as well as the produced and purified
antibodies, had the ability to be used in serological tests to detect influenza A virus. It can
also be used in basic research methods such as Western blot, immunohistochemistry and
immunocytochemistry.
Keywords :
antigen purification , polyclonal antibody production , antibody purification , Western blot
