preparation of antibody against immunodominant membrane protein (imp) of candidatus phytoplasma aurantifolia

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.