Thesis (M.S.)--Northern Michigan University, 2009. / Includes bibliographical references (leaves 157-171).

A panel of monoclonal antibodies were raised against the 1,4-dihydropyridine sensitive Ca2+ channel of rabbit skeletal muscle. When tested on immunoblot assay of denatured and reduced transverse tubule membranes, four of the antibodies specifically recognized a polypeptide of Mr 140,000. This component co-migrated with the large glycoprotein ?2 subunit of purified Ca2+ channel preparations. On immunoblots of nonreducing gels the antibodies detected a component that migrated more slowly in the gel, with a Mr of 170,000, consistent with the disulphide-linkage of the ?2 subunit to a small component of Mr 30,000. Additionally, three of the antibodies also recognized high molecular weight components of Mr 310,000-330,000 under these conditions. Crossreactive polypeptides of similar apparent molecular weight were detected in immunoblot assays of rabbit heart and brain membranes and of skeletal muscle membranes from different species. Further similarities between the ?2 components of Ca2+ channels from different species were investigated by immunoblot assay, following the limited tryptic digestion of the skeletal muscle membranes. A similar pattern of immunoreactive peptides were detected in each case, suggesting that the ?2 subunits of Ca2+ channels from different species are similar, not only in terms of antibody binding sites but also with respect to similarly positioned trypsin cleavage sites. The extent of glycosylation of the ?2 component was investigated using enzymatic and chemical deglycosylation techniques. Chemical deglycosylation resulted in a core polypeptide of Mr 105,000, consistent with a carbohydrate content of approximately 25%. Enzymatic treatments, although insufficient to completely deglycosylate the ?2 component, reduced the maximal 1,4-dihydropyridine binding capacity of transverse tubule membranes by 73-77%. The co-development of the ?2 subunit with 1,4- dihydropyridine binding activity was shown in rat skeletal muscle. These results indicate that the '2 subunit is an integral structural component of the 1,4-dihydropyridine sensitive Ca2+ channel.

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A physiologic model for phencyclidine disposition in the rat was established. This model was able to accurately predict phencyclidine disposition in most rat tissues. Physiologic models are based on actual physiologic, anatomic, and biochemical considerations. As a result, these models can be used to predict drug disposition under conditions of altered physiology or anatomy. This aspect of physiologic modeling was tested in the present study by examining the ability of the model to predict phencyclidine plasma disposition in dog and man. The model developed in this study was able to accurately predict phencyclidine disposition in these species. A primary goal of this project was to evaluate the effects of the administration of an anti-phencyclidine monoclonal antibody on phencyclidine disposition and toxicity in the rat. The monoclonal antibody was produced in murine ascites fluid. The antibody was purified using a recirculating isoelectric focusing apparatus. This method provided a rapid technique which can be used to purify monoclonal antibody from large quantities of ascites fluid, yielding reasonably good antibody recovery and very high purity. Characterization of the antibody showed only moderate affinity and high cross reactivity. Administration of the monoclonal antibody did not significantly alter either phencyclidine disposition or toxicity. While qualitative differences in recovery from phencyclidine-induced toxicity occurred in rats receiving the anti-phencyclidine antibody, these differences failed to be statistically different from control rats. These results may be explained by the poor qualities (moderate affinity, high cross-reactivity) of the monoclonal antibody.

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I. An analogue of the herbicide atrazine was synthesised and conjugated to carrier proteins for rodent immunisation and monoclonal antibody screening. II. Monoclonal antibodies from hybridoma clone 1G18 specific for the herbicide atrazine were successfully isolated. A suitable ELISA system was optimised and the binding capability and cross reactivity of these antibodies was found to be broadly similar to two other anti-atrazine monoclonal antibodies that were acquired externally. III. The antibody variable region domains of hybridoma 4063-21-1 were cloned and fully sequenced. The variable region domains of hybridoma AM1B5.1 were cloned and partially sequenced. IV. The variable region domains of hybridoma 4063-21-1 were sub-cloned into an expression plasmid and fully functional antibody fragments, specific for the herbicide atrazine, were obtained from <I>E. coli</I> fermentations. The recombinant antibody bound antigen with a binding capability that was 10% that of the whole murine antibody. V. The best yields of anti-atrazine antibody fragment were obtained from fermentations in buffered medium when the fermentation broth pH was maintained above pH 6.0. The recombinant antibody was purified by immobilised metal chelate affinity chromatography and the purified antibody fragment was found to bind antigen 10% as well as the crude recombinant antibody.