Author
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YUAN, QING - Luzhou Medical College |
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Jordan, Ramon |
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BRLANSKY, RONALD - University Of Florida |
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MINENKOVA, OLGA - Sigma Tau Pharmaceutical |
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Hartung, John |
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Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 7/25/2015 Publication Date: 7/30/2015 Publication URL: http://handle.nal.usda.gov/10113/61475 Citation: Yuan, Q., Jordan, R.L., Brlansky, R.H., Minenkova, O., Hartung, J.S. 2015. Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display. Journal of Microbiological Methods. 117:148-154. Interpretive Summary: Xylella fastidiosa subsp. pauca is a bacterium that is difficult to grow in the laboratory, is transmitted by insects and causes citrus variegated chlorosis (CVC) in Brazil. CVC is a very serious disease of sweet orange and causes yield losses as high as 90% in Brazil. Other forms of Xylella fastidiosa cause important diseases on grapevine, peaches and almond in the United States. The citrus form of Xylella fastidiosa does not occur in the United States, and USDA APHIS would very much like to keep it that way. Antibody based detection assays are commercially available for Xylella fastidiosa, but they can’t tell the difference between strains of Xylella that are native to the United States and the foreign forms that attack citrus. We have made a special ‘library’ of antibodies against the citrus strain of Xylella fastidiosa using recombinant DNA technology. The library contained more than 10 million different antibodies, and we selected several antibodies that recognized the citrus strain of Xylella fastidiosa but not any of the strains native to the United States. These antibodies have been used in a standard antibody based assay, and recognized the citrus strain of the Xylella fastidiosa, but not the native strains that cause diseases of grapevine, peach, and almond. These antibodies will be useful for regulatory scientists that want to identify the pathogen in plant samples. Research scientists may also find them useful for experimental applications. Technical Abstract: Xylella fastidiosa is a member of the gamma proteobacteria. It is fastidious, insect-vectored and xylem-limited and causes a variety of diseases, some severe, on a wide range of economically important perennial crops, including grape and citrus. Xylella fastidiosa subsp pauca causes citrus variegated chlorosis in Brazil and was included on the USDA APHIS Agriculture Bioterrorism Agent and Toxin list. Antibody based detection assays are commercially available for Xylella fastidiosa, and are effective at the species, but not at the subspecies level. We have made a library of scFv antibody fragments directed against Xylella fastidiosa subsp pauca strain 9a5c (citrus) by using phage display technology. Single chain variable fragments (scFv), made by the linkage of the variable heavy and light chain regions retain the binding properties of classical antibodies. BALB/c mice were immunized with 9a5c bacteria at a concentration of 108 cfu/100 µl buffer. mRNA from the spleens of the immunized mice was purified and converted into cDNA. Antibody gene repertoires were PCR-amplified using 23 primers for the heavy chain variable region (VH) and 21 primers for the light chain variable region (VL). The VH and VL were joined by overlap extension PCR, and then the genes of the scFv library were ligated into the phage vector pKM19. The library contained 1.2×107 independent clones with full-length scFv inserts. In each of 3 cycles of affinity-selection with 9a5c, about 1.0×1012 phage were used for panning with 4.1×106, 7.1×106, 2.1×107 phage recovered after the first, second and third cycles, respectively. Sixty-six percent of clones from the final library bound Xylella fastidiosa 9a5c in an ELISA. Some of these scFv antibodies recognized strain 9a5c and did not recognize Xylella fastidiosa strains that cause Pierce’s disease of grapevine. |
