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 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.

Salicylic acid‐mediated elicitation of tomato defence against infection by potato purple top phytoplasma

“…Over the past decades, several biotechnological strategies have been explored to mitigate phytoplasmal diseases. These have included tetracycline therapy and transgenic expression of phytoplasma‐specific single‐chain antibodies and antimicrobial peptides (Davis et al ., ; McCoy, ; Lee et al ., ; Le Gall et al ., ; Zhao et al ., ; Du et al ., ; Malembic‐Maher et al ., ; Shahryari, et al ., ). Since these measures require either field applications of antibiotics or generation of genetically modified crops, more environmentally and ecologically favourable approaches are constantly being sought.…”

Generation and Expression in Plants of a Single-Chain Variable Fragment Antibody Against the Immunodominant Membrane Protein of Candidatus Phytoplasma Aurantifolia

“…The IMP gene was previously isolated from the total DNA of lime witches' broom infected Mexican lime plants and cloned in the bacterial expression vector pET-28a [29]. We used this pET28-IMP construct to express recombinant IMP in Escherichia coli.…”

“…Phytoplasma-specific scFvs were isolated by using a purified recombinant IMP expressed in E. coli [29] to screen the Tomlinson I and J scFv phage display libraries, as previously described [27]. Recombinant IMP (50 µg/ml) was immobilized onto immunotubes (Nunc-Maxisorp, Denmark), blocked with 2% (w/v) skimmed milk, and incubated with the phage suspension (10 13 CFU).…”

“…Binding between the bacteria-produced scFv and intact phytoplasma cells was determined by indirect ELISA [17]. Maxisorp 96-well microtiter plates (Nunc-Immuno, Denmark) were coated with an anti-IMP polyclonal antibody diluted 1:500 in PBS, and incubated at 37 o C for 2 h [28]. Extracts from healthy and infected lime plants, or purified recombinant IMP, were added to the plate and incubated overnight at 4 o C. We then added 100 µl of the scFv preparation, and binding was detected using 0.5 µg/ml of the primary mouse anti-Myc monoclonal antibody 9E10 (Abcam, USA) and a secondary goat-anti mouse IgG alkaline phosphatase conjugate (0.16 µg/ml).…”

Production of a phage-displayed single chain variable fragment antibody against infectious bursal disease virus

“…The specificity of produced scFv antibody fragments was analyzed by ELISA as described earlier [14]. Around 100l of IBDV vaccine (50 µg/mL) diluted in 1x PBS was loaded on ELISA plates (Maxisorp, Nunc, Denmark).…”