Polyclonal Antibody against Recombinant Nucleoprotein of the Influenza A Virus (H1N1); Production and Purification

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.
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.
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.
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.