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(1) Human neuron specific enolase (NSE) was purified from human brain tissue and used as an immunogen in the production of murine monoclonal antibodies. It was also used as a standard preparation of NSE in the screening of monoclonal antibodies and in the investigations of an enzyme-linked immunosorbent assay (ELISA) for NSE. (2) Four monoclonal antibodies were raised to NSE. These did not cross react with the alpha isoenzyme of enolase (NNE). Approximately 76% of positive well supernatants reacted with both NSE and NNE. (3) All four monoclonal antibodies reacted in an ELISA but only two of the four reacted on an immunoblot. (4) The monoclonal antibodies were purified by precipitation by ammonium sulphate and by Protein A-affinity column chromatography. The latter resulted in immunoglobulin which was pure by SDS-PAGE. (5) The purified monoclonal antibodies were labelled with horseradish peroxidase by the periodate oxidation method, with ¹²⁵I using the Chloramine T and the Bolton-Hunter methods, and with biotin. (6) Labelling with biotin was the most effective method. (7) The labelled monoclonal antibodies were used in the investigation of a sandwich ELISA for NSE. These experiments showed that high binding occurred with no antigen present. It was surmised that monoclonal antibody-monoclonal antibody binding may be occurring. (8) The high no-antigen control was investigated in a number of ways including altering the assay pH, the detergent concentration, the type of solid support and by using Fab fragments in the assay instead of whole molecule.

Typescript. / Bibliography: leaves 87-94. / ix, 94 l illus

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Thesis (Ph. D.)--University of Wisconsin, 1966. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

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In the studies described in this thesis the ability of muscarinic and ?1-adrenoceptor, as well as depolarising stimuli to initiate phosphoinositide metabolism in various regions of rat brain were examined. Furthermore, the ability of these stimuli to initiate phosphoinositide hydrolysis in developing brain was observed. Both muscarinic and ?1-adrenoceptor-induced phosphoinositide hydrolysis have marked regional distributions in rat brain. This regional distribution of functional responsiveness seems to correlate reasonably well with measurements of known receptor density. It is also clear that there is no variability in the coupling of both of these receptors in rat brain, thus implying a relationship between the functional responses and receptor occupancy. Pirenzepine appears to be able to differentiate between muscarinic receptor-induced phosphoinositide responses in the hindbrain from those in the forebrain regions. Both elevated K+ and veratrine can initiate phosphoinositide hydrolysis in rat brain. The regional responses to elevated K+ seem, at least, in part to be due to transmitter release, although a role for voltage-sensitive Ca++ channels in such responses is indicated by the effects produced by dihydropyridine Ca++ channel antagonists and activators. Muscarinic and ?1-adrenoceptors show different developmental patterns of phosphoinositide responsiveness. The ?1-adrenoceptor seems to be more efficiently coupled during the first two weeks of postnatal development whereas the muscarinic receptor shows no variability in coupling. Instead, carbachol produces supramaximal responses in young rats. Lithium also potentiates [3H]-InsP1 and [3H]-InsP2 accumulations more in young rats. Moreover lithium produces a time-dependent inhibition of [3H]-InsP3 and [3H]-InsP4 in both young and adult rats. Physostigmine produces no enhancement of the response to elevated K+ in young rats. Furthermore, brain slices from young rats seem to be more sensitive to the Ca++ channel activator BAY-K8644. The significance of these results are discussed in the text.