Author/Authors :
Shibaei، Naeimeh نويسنده Department of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran Shibaei, Naeimeh , Majidi، Jafar نويسنده Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. , , Razavi، Khadijeh نويسنده National Institute of Genetic Engineering and Biotechnology, P.O. Box 14965/161, Tehran, I.R. Iran , , Karkhane، Ali Asghar نويسنده Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14965/161, Teh , , Sokhandan Bashir، Nemat نويسنده Plant Protection Department, University of Tabriz, P.O. Box 345, Tabriz, I.R. Iran , , Aghebati Maleki، Leili نويسنده Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ,
Abstract :
There are many studies related to the production of a ELISA kit for diagnosing virus infections. However, production of most kits depends on purification of whole virus particles, which involves the use of costly equipment and reagents. The purpose of this study was to check out if the anti-CP42 antibodies could be used as a diagnostic assay for detection of Grapevine fanleaf Virus (GFLV). In this study, recombinant GFLV coat protein gene related to selected antigenic determinants was inserted into pET-28a bacterial expression vector and the construct (pET-28a CP42) was cloned into E. coli strain (DE3). Expressed protein was verified with western blotting assay by the use of commercially available anti-GFLV antibody. The recombinant protein was purified using nickel–nitrilotriacetic acid (Ni–NTA) resin. Balb/c mice were immunized with purified protein and splenocytes of hyperimmunized mice were fused with murine myeloma Sp2/0 cells. Positive hybridomas were selected by ELISA using CP42 as coating antigen. The results showed that monoclonal antibody (MAb) specific to CP42 has been successfully generated. Efficiency of produced antibody was analyzed by ELISA and western blotting assay using some confirmed grapevine samples. The infection was confirmed previously based on morphological features and ELISA assay, performed using commercial anti-GFLV antibody. The monoclonal antibody reacted with antigen in ELISA and immunoblot method. Our results demonstrated that anti recombinant CP42 monoclonal antibodies are able to diagnose whole virus in infected grapevine sample using ELISA test.
