Submitted to: Journal of Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/2/2008
Publication Date: 2/8/2008
Citation: Iracheta-Cardenas, M.M., Sandoval-Alejo, B.D., Roman-Calderon, M.E., Keremane, M.L., Lee, R.F., Rocha-Pena, M.A. 2008. Production of Polyclonal Antibodies to the Recombinant Coat Protein of Citrus tristeza virus and Their Effectiveness for Virus Detection. Journal of Phytopathology, 156: 243-250 Interpretive Summary: Double antibody sandwich indirect enzyme-linked immunosorbent assays (DASI-ELISA) are often used for large scale testing for detection of plant viruses, such as Citrus tristeza virus (CTV). The DASI-ELISA requires an antibody for trapping of non-denatured virus particles, and a second (intermediate) antibody specific for the target virus for detection and quantization of the trapped virus particles; the antibodies are usually raised in different animal species to enable use of commercially available anti-species conjugates. It is often difficult to highly purify the virus away from host proteins in sufficient quantity to produce antibodies useful for detection. We report here the development of antibodies raised using antigens from Escherichia coli expressed recombinant CTV coat protein gene from several CTV isolates, and demonstrate their usefulness for trapping antibodies and as intermediate antibodies in the DASI-ELISA for detection of CTV. Antibodies raised against E. coli expressed recombinant genes usually are very poor as trapping antibodies; this procedure may be useful for development of trapping antibodies for use with other viruses.
Technical Abstract: The p25 coat protein gene of three Citrus tristeza virus (CTV) isolates, two from Mexico and one from India, was amplified by RT-PCR and further cloned and expressed in Escherichia coli cells. The recombinant coat protein (rCP) of the three CTV isolates was injected into rabbits and goats for antibody production. Western blotting assays with the rCP CTV specific antibodies showed a positive reaction with the homologous and heterologous rCP of the three CTV isolates and with the corresponding native coat protein present in crude sap extractions of CTV infected citrus tissue but not with extractions from healthy tissue. The rCP antibodies from goat and rabbit reacted as both plate trapping and intermediate antibodies in double sandwich indirect ELISA, discriminating efficiently healthy and CTV infected citrus, with optical density (OD405) values in the range of 0.151-2.415 for CTV infected samples and less than 0.100 for healthy tissue. Commercially available anti-CTV antibodies were used as a reference.