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

Abstract

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.