Antibody-based detection and quantification of pectobacterium carotovorum SSP. Carotovorum

Bassoriello, Melissa Maria Ivana
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University of Guelph

'Pectobacterium carotovorum' ssp. 'carotovorum ' ('Pcc') is implicated in the destruction of ornamental plants in greenhouse recirculating systems. PCR-based detection and quantification of 'Pcc' requires expensive instrumentation and knowledgeable users. This thesis describes the production of polyclonal antibodies and a single-domain antibody fragment (VHH) against 'Pcc' lipopolysaccharide (LPS), and the development of user-friendly diagnostic assays for detection and quantification of the pathogen. Polyclonal ELISAs against heat-killed (HK) 'Pcc' (limit of detection (LOD) = 81 CFU/ml; limit of quantitation (LOQ) = 216 CFU/ml) and 'Pcc' LPS (LOD = 23 ng/ml; LOQ = 76 ng/ml) were developed. A preliminary user-friendly dipstick assay was also developed (>= 105 CFU/ml). A phage display library was constructed (6.0 x 105 clones/ml), yielding one unique anti-'Pcc' LPS VHH. Using the ' Pcc' LPS-specific VHH to produce affordable, user-friendly diagnostic assays is feasible since antibody fragments can be produced on a large scale through expression in 'Escherichia coli' or ' Piccia pastoris'.

Ornamental plants, Greenhouses, Recirculating systems, Pectobacterium caratovorum ssp. caravorum, Polyclonal antibodies