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Title: Cloning and expression of herbicide-specific single chain antibody fragments in Escherichia coli and Nicotiana tabacum
Author: Learmonth, Dianne
ISNI:       0000 0001 3606 6448
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 1997
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An anti-diuron scAb fragment was produced and expressed in E. coli. Characterization by ELISA demonstrated that the scAb displayed a similar binding profile to the parent monoclonal antibody, although its binding sensitivity appeared to be 10-50 times lower. Analysis of purified samples by HPLC showed the presence of scAb in both monomeric and dimeric forms. Expression yield of the anti-diuron scAb was low (0.05 mg/l culture volume), and attempts to reduce the toxicity of production in E. coli, using alternative expression vectors, were not successful. Production in an E. coli strain which allowed cytoplasmic folding resulted in an increase in yield of soluble scAb, but this antibody material showed reduced stability. The anti-atrazine scAb was expressed in tobacco. Although the levels of scAb produced were lower than might have been expected (0.014% total soluble protein), the scAb was fully functional and exhibited a binding affinity similar to that of bacterially-produced scAb. BIAcore analysis of affinity purified samples indicated a binding affinity comparable to that of the parental Mab. In competition ELISA, the scAb showed a concentration-dependent reduction in antigen binding in the presence of free atrazine. Binding sensitivity of the scAb for atrazine, and for the closely related triazine propazine, were very similar, while binding sensitivity for simazine was 10 times lower. Bacterially-produced anti-atrazine scAb demonstrated similar binding sensitivities for the atrazine analogues.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Tobbaco; Pesticide detection Botany Molecular biology Cytology Genetics