Maryam Hashemi; Rasool Madani; Mahmoudreza Aghamaali; Tara Emami; Fariba Golchinfar
Volume 21, Issue 10 , 2019, Pages 1-8
Abstract
Background: In recent years, various poultry diseases have posed risks to this industry. Respiratory and infectious diseases are the most common diseases. According to previous findings, influenza disease is recognized as a significant life-threatening disease in the industry of poultry worldwide. Influenza ...
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Background: In recent years, various poultry diseases have posed risks to this industry. Respiratory and infectious diseases are the most common diseases. According to previous findings, influenza disease is recognized as a significant life-threatening disease in the industry of poultry worldwide. Influenza virus type A, which belongs to the family Orthomyxoviridae, is responsible for a serious infectious disease. H9N2 AVI subtype circulates in the poultry worldwide, causing significant economic losses and infections in humans, also domestic and wild animals.Objectives: The purpose of the present experimental research was monoclonal antibodies (MAbs) production in contradiction of the NP of avian influenza virus (AIV) H9N2 subtype to detect AIV antigens and antibodies in the Department of Proteomics and Biochemistry, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran. in 2017.Methods: The conserved protein of NP from H9N2A/Chicken/Iran/259/2014 virus, with 60 kDa molecular weight, was isolated using the electroelution method. The purified protein was applied for Australian BALB/c mice immunization. After evaluating immuniza- tion by ELISA assay, the spleen of immunized mice was isolated and hybridized with SP2/0 myeloma cell. Next, the hybridized cell was cultured, and clone soups were collected after 15 days to examine antibodies via ELISA assay. The produced antibodies using Western blotting and antibody isotype kits were characterized.Results: Thirty antibody-producing clones were examined for reactivity against Nucleoprotein (NP), the antigen, at 1 μg/mL con- centration. According to the ELISA assay of antibody titers, two (3/F10 and 2/D7) out of 30 antibodies were bound to the antigen with titer 0.863 and 1.641, respectively. Two hybrid clones, 2D7 and 3F10, which produced anti-NP antibodies, were isolated and cultured. Characterizing of the produced antibodies using Western blotting was performed using H9N2 virus; finally, two clone soups (3/F10 and 2/D7) reacted with the NP virus protein. According to the isotyping of antibodies produced by 3F10 and 2D7 clones, 3F10 clone produced IgG1 with a κ chain, and IgG1 concentration was 1.997, based on ELISA assay. Also, 2D7 clone produced IgG2a with a κ chain, and IgG2a concentration was obtained 1.951.Conclusions: According to the findings of our study, the produced antibody might be used in the diagnosis of influenza.
Maryam Hashemi; Mahmoudreza Aghamaali; Rasool Madani; Tara Emami
Volume 21, Issue 10 , 2019, Pages 1-5
Abstract
Background: Avian influenza virus (AIV) belongs to the family of Orthomyxoviruses type A and causes avian influenza (AI) infectious disease. Currently, serological diagnostic techniques such as agar gel propagation (AGP), hemagglutination inhibition, and enzyme- linked immunosorbent assay (ELISA) are ...
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Background: Avian influenza virus (AIV) belongs to the family of Orthomyxoviruses type A and causes avian influenza (AI) infectious disease. Currently, serological diagnostic techniques such as agar gel propagation (AGP), hemagglutination inhibition, and enzyme- linked immunosorbent assay (ELISA) are considered as important tools for the antibodies detection against viral antigens. Due to antigenic variation in the surface of AIV glycoproteins (hemagglutinin and neuraminidase), these proteins cannot be used in serological tests. Development of assays to detect AI surface glycoproteins is problematic because a great variety of combinations of these subtypes are found in nature. The internal antigen determinants on the nucleoprotein (NP) are highly conserved within influenza viruses, making this protein more appropriate for a serological test.Objectives: In the experimental present study, an effectual method was expanded to purify NP of H9N2 AIV based on Electroelution method.Methods: AIV strain A/flash chicken/Iran/772/1998 (H9N2) was acquired from the Department of Avian Influenza Reference Labora- tory, Razi Vaccine and Serum Research Institute, Iran, about 2 cc, in 2017. Nucleoprotein of AIV (H9N2) was purified by an efficient and simple modified method directly from native polyacrylamide gel electrophoresis (PAGE) according to the Electroelution method. The purified protein concentration was defined by the Lowry method, and the purified NP protein (60 KDa) was examined by Tricine- SDS-PAGE.Results: The protein concentration of the virus solution was 4.62 mg/mL by the Lowry method. The purified Nucleoprotein con- centration was 0.296 mg/mL by Lowry method and the Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis results showed only a 60-KDa protein band in the gel.Conclusions: The current technique was simple and rapid and made it possible to isolate NP from the H9N2 virus. The Nucleo-protein antigen is an appropriate candidate potential to detect antibodies against all subtypes of AIV and used as the main target antigen for the diagnosis of influenza virus due to its very high scale of sequence preserved among exist strains.
