Procedures for the preparative purification of human acetylcholinesterase (AChE; EC 3.1.1.7) have been examined. Chromatofocussing showed caudate nucleus and cerebrospinal fluid (CSF) AChE eluting between isoelectric points (pi) of 5.2 - 5.57, whilst isoelectric focussing (IEF) revealed heterogeneous AChE isoforms migrating between pI 4.5 - 6. Affinity chromatography proved to be better in yielding highly pure samples. The ligands used were either acridinium or procainamide, which yielded brain AChE recoveries of 10.1% and 42.8%, respectively. Various other methods were used such as gel filtration and ion-exchange chromatography on a Pharmacia SMART System. Purified brain AChE was used as antigen to generate anti-AChE monoclonal antibodies (mAbs) by in vivo immunization. BMS-3E4, -7G10, and -9F4 reacted with erythrocyte (G2) AChE, whilst BMS-6D6 bound to brain (G4) AChE in dot blot, titration, and sedimentation experiments. Conversely, the four mAbs recognized only the G4 form and not the G<sub>2</sub> form on immunoblotted IEF and non-denaturing Clarke gels. Deglycosylation studies suggest that the four mAbs recognize a carbohydrate epitope linked to AChE. As a preparative step, tissue culture media containing these mAbs were ammonium sulphate precipitated and purified on a gel filtration column. Under reducing conditions, two bands migrating at 80 and 25 kDa were seen, corresponding to the heavy and light chains of Immunoglobulin M (IgM). Four additional mAbs were raised by the novel method of in vitro immunization, using a synthetically-produced C-terminus peptide as antigen. BMS-5, -6, -7, and -8 are all IgMs which recognize soluble brain and not erythrocyte nor membrane-bound AChE in dot blots, IEF, and sedimentation analyses. Although immunocytochemistry of the above-mentioned mAbs did not demonstrate any positive cholinergic binding, the present mAbs may still offer potential clinical and biochemical applications.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:358696 |
| Date | January 1993 |
| Creators | Novales-Li, Philipp |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://ora.ox.ac.uk/objects/uuid:8f0d7a87-9df9-40be-89a5-bc9953f27f03 |
Page generated in 0.0019 seconds