This research was undertaken in order to design techniques and processes that enable the manufacture of effective antivenoms. To prepare a broad specificity anti venom for European vipers from chicken yolk it was first necessary to develop a simple effective method to extract avian immunoglobulin (lgY). A specific fluoroimmunoassay was developed to monitor IgY recovery and serum IgY levels in immunised hens. The most promising extraction methods from the literature were compared using a triglyceride kit to monitor lipoprotein removed and SDS-PAGE and ELISA to monitor purity and activity respectively. Caprylic acid followed by ammonium sulphate proved the best method. Unfortunately only low levels of specific IgY were achieved and it was necessary to include an affinity purification step to demonstrate their effectiveness in an EDso test. Pepsin, papain and trypsin all produced Fab' fragments from IgY but only pepsin digested the resultant Fc fragments. Pepsin could also digest other proteins in egg yolk, thereby avoiding the need to salt fractionate IgY prior to its digestion with a consequent improvement in the recovery of Fab'. A small scale affinity purification (SSAP) assay was developed, characterised and used to determine specific antibody levels in ovine antisera. Small doses (l5IAg) of venom produced significant specific levels but larger doses produced a better response and were used to produce anti venom. Binding studies with SSAP demonstrated a high concentration of specific antibodies in V.latastei antisera that bind to components in the venoms of other European vipers. A specific ovine F(ab')2-based V.latastei antivenom approximately twice as potent as the anti venom used currently in Spain was prepared from the ovine antisera. Evidence is presented that SSAP should supersede manual ELISA for assessing specific antibody levels in antisera. No major gain in recovery and purity resulted from processing whole blood rather than serum for preparing antivenom.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:413414 |
| Date | January 2004 |
| Creators | Harrison, Kenneth Louis |
| Publisher | Queen Mary, University of London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://qmro.qmul.ac.uk/xmlui/handle/123456789/28570 |
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