A naïve human antibody fragment phage display library was screened against conjugated microcystin-LR (MCLR). Phage antibodies were eluted with free MCLR to promote the isolation of free antigen binders. Isolated antibody fragments were cloned into a soluble expression vector and single-chain antibody (scAb) expressed in a bacterial host. All scAbs bound free antigen in an indirect competition ELISA and levels of sensitivity were determined. The most responsive clone 3A8 had an 800-fold increase in sensitivity to MCLR than previously documented scAbs and was able to detect levels below guidelines for MCLR in drinking water set by the World Health Organisation (WHO) at 1 μg/L. Despite its sensitivity to MCLR, full characterisation revealed low levels of cross-reactivity with other microcystin variants. <i>In vitro</i> affinity maturation was employed to increase cross-reactivity to other microcystin variants in scab 3A8. A chain-shuffled library was constructed where a naïve V<sub>L</sub> repertoire was shuffled with clone 3A8 V<sub>H</sub> chain. The library was screened and isolated phage antibodies cloned and expressed as scAbs. Chain-shuffled clone CSB-D9 displayed a 50-fold increase in cross-reactivity for a single microcystin variant and was capable of detecting all variants available below the guideline levels of 1 μg/L. The highly cross-reactive scab was used for the determination of total microcystin content in cyanobacterial extracts and results showed good correlation with HPLC quantification. Immunoaffinity chromatography utilising scab CSB-D9 was used for the concentration of trace amount of microcystin from large volumes of water, out-performing columns generated with anti-microcystin whole antibody molecules. The ability of scab CSB-D9 to protect cultured hepatocytes against microcystin-induced apoptosis was also determined. Results indicated a possible therapeutic application for human antibody fragments isolated from phage display libraries.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:445137 |
| Date | January 2007 |
| Creators | Drever, Matthew |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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