Radioimmunoassay determination of scopolamine yields in tissue cultures of Datura

Author

Brett J. Savary

Date of Award

12-1987

Degree Type

Thesis

Degree Name

Master of Science

Major

Botany

Major Professor

Donald K. Dougall

Committee Members

Beth C. Mullin, Thomas T. Chen, Karen W. Hughes

Abstract

The purpose of this study was to establish a radioimmunoassay (RIA) and determine Its applicability for quantifying scopolamlne yields In plant cell cultures of Datura. Scopolamine binding antisera were obtained using the Immunogen described by Weller et al. (1981). A RIA was established based on the commercially prepared (N-methyl[³H])-RIA scopolamine methylchloride as labelled antigen. The linear range of the scopolamine standard curve extended from 0.15 to 1.5 ng In the assay mixture. The standard curve was routinely reproduced, and the precision of triplicates, measured as the coefficient of variation for B/B_o, was ca. 2.3%. The specificities of two selected antisera were characterized for the cross-reactivities of metabolically related and structurally similar tropane alkaloids.

Callus tissue cultures were established from leaf explants of five species of Datura. Simple ethanol extracts were prepared and assayed with the RIA. Antigen activity was detected In all culture lines. Cultures derived from plants of D. stramonium consistantly averaged the highest yields of antigen activity by the RIA when grown on B5 medium (Gamborg, 1970) supplemented with 1 mg/L 2,A-dlchlorophenoxyacetlc acid. Variations In activity yields were observed between species and between lines established from different Individual plants of a species.

An extract of D. stramonium callus tissue was separated by reverse phase HPLC and the collected fractions were assayed with the RIA to determine If the antlgenlc activity measured was specifically due to scopolamine. Five antlgenic activity peaks were recognized. Correspondence of elution times of the activity peaks with both internal and external spikes of alkaloid standards gave tentative identification for scopolamine, 6-hydroxyhyoscyamine, hyoscyamine, and littorine as components of the extract. Hyoscyamine was determined to be the major antigenic component and was present in the callus extract at a 25-fold quantity greater than scopolamine. Thus it was determined the RIA cannot be used to selectively quantitate scopolamine in simple extracts of callus tissue of D. stramonium because a low cross-reacting compound was accumulated in an excess quantity sufficient to overwhelm the specificity of the antiserum. These results indicate the application of any "specific" RIA to tissue culture extracts needs a verification to determine if low cross-reacting compounds are accumulated in excess of the compound of interest at levels sufficient to invalidate the specificity of the antiserum.

The KLH-10 RIA described here can potentially be applied to simple extracts of callus tissue if a solid-phase extraction purification step (e.g., by a Sep Pak) is introduced to the preparation of extracts before RIA analysis. Purification by a solid-phase extraction cartridge can be optimized for the selective removel of hyoscyamine from D. stramonium callus extracts before RIA analysis. The HSA-12 RIA can be applied to unpurified extracts for the determination of scopolamine-type tropane alkaloids as a group. Alternatively, if an RIA is required that must specifically quantitate scopolamine in unpurified tissue culture extracts, then an new immunogen must be prepared with scopolamine conjugated to carrier protein through the tropic acid moiety, and antisera must be produced and screened to obtain an antiserum with a higher specificity for scopolamine.

Recommended Citation

Savary, Brett J., "Radioimmunoassay determination of scopolamine yields in tissue cultures of Datura. " Master's Thesis, University of Tennessee, 1987.
https://trace.tennessee.edu/utk_gradthes/13582