Tehran University of Medical Sciences Iranian Journal of Allergy, Asthma and Immunology 1735-1502 16 1 2017 02 25 60 71 EN Naeimeh Shibaei Department of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran Jafar Majidi Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran AND Immunology Research Center (IRC), Tabriz University of Medical Sciences, Tabriz, Iran Khadijeh Razavi Department of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran Ali Asghar Karkhane Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran Nemat Sokhandan-Bashir Department of Plant Protection, University of Tabriz, Tabriz, Iran Leili Aghebati-Maleki Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran 2016 03 09 2016 07 05 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. https://ijaai.tums.ac.ir/index.php/ijaai/article/view/841 https://ijaai.tums.ac.ir/index.php/ijaai/article/download/841/698