A Radioimmunoassay for the Cholinesterase Inhibitory Alkaloid Galanthamine

Abstract

Galanthamine is a naturally occurring alkaloid in some plants of the species Amaryllidaceae. It is frequently used in clinical practice because of its anticholinesterase activity on both the central and peripheral nervous systems (1).A radioimmunoassay (RIA) procedure for quantitative determination of galanthamine was developed and ethanol extracts of about 200 different plants were subjected to RIA. The highest content of galanthamine was found in Leucojum aestivum L., Amaryllidaceae. The average content of leaves of this plant is 0.45%. OH CR3 galanthamineThe procedure was carried out as follows: Since substances with low molecular masses do not possess antigenic properties, a hapten-conjugate had to be synthesised in order to be applied to an animal species to build up antibodies. Galanthamine itself does not have a suitable group for coupling to a protein molecule, so at first galanthamine hemisuccinate was synthesised. The galanthamine hemisuccinate was then coupled to bovine serum albumine by the carbodiimide method. The conjugate obtained was then used as an antigen, having been applied to rabbits to build up galanthamine antibodies. A dilution from the rabbit blood serum was then used as an antibody solution for the assay.As a radioactive labelled tracer ['Hl-galanthamine acetate was synthesised from galanthamine and [3111-acetic anhydride. The specificity of the antibodies was determined and the cross-reactivity with some other amaryllidaceous alkaloids was investigated. Different assay parameters, like the right concentration of the (NH4)2504 solution for the precipitation of the antigen-antibody complexes and the correct pH value of the reaction medium were optimised.For each trial, a standard curve is plotted with percentage bound vs. galanthamine content in the standard solutions, which number was optimised to ten. The detection limit was found to I ng/ml galanthamine, which makes this method useful especially with work and investigations of plant tissue and cell cultures. Acknowledgements