Preparation of Antibody Against Immunodominant Membrane Protein (IMP) of Candidatus Phytoplasma aurantifolia

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Background: The witches’ broom disease of lime caused by Candidatus Phytoplasma aurantifolia, is the most devastating disease of acidian lime in the southern parts of Iran. Objectives: At present, no effient method has been developed for controlling the disease, therefore quarantine approaches such as early detection and subsequent eradication of infected trees is very important. Toward this aim, developing a reliable and sensitive detection method would be the fist step to prevent transportation of infected plant materials to other places.   Background: The witches’ broom disease of lime caused by Candidatus Phytoplasma au­rantifolia, is the most devastating disease of acidian lime in the southern parts of Iran.   Objectives: At present, no efficient method has been developed for controlling the dis­ease, therefore quarantine approaches such as early detection and subsequent eradica­tion of infected trees is very important. Toward this aim, developing a reliable and sensi­tive detection method would be the first step to prevent transportation of infected plant materials to other places.   Materials and Methods: In this study, Immunodominant membrane protein (IMP) of the pathogen was selected as a target for detection and preparation of polyclonal anti­body. The IMP is the major protein present on the surface of phytoplasma cells. For this purpose, the DNA region encoding IMP gene was isolated and cloned into pET28a bacte­rial expression vector. The recombinant protein was expressed in a large scale in Escheri­chia coli. Purification was performed under native conditions and the purity and integ­rity of produced recombinant protein were confirmed by western immuno blot analysis using anti His-tag and anti-IMP polyclonal antibodies. The purified recombinant IMP was used for immunization of rabbit. Purification of immunoglobulin was performed by affinity chromatography using protein A column. The purified immunoglobulin was conjugated with the alkaline phosphatase enzyme.   Results: The purified antibodies and conjugates were applied for efficient detection of infected plants in double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and dot immunosorbent assay (DIBA).   Conclusions: These antibodies were proven to be very powerful tools to detect the Can­didatus Phytoplasma aurantifolia in plants.