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United States Department of Agriculture

Agricultural Research Service

Title: Heliotropium Europaeum Alkaloids: a Quaternary Pyrrolizidine Alkaloid Approach to Elisa Development

Authors
item Lee, Stephen
item Knill, Andrew - CSIRO
item Michalewicz, Agnieszka - CSIRO
item Stevens, V - CSIRO
item Colegate, Steven - CSIRO

Submitted to: Poisonous Plant Global Research and Solutions
Publication Type: Book / Chapter
Publication Acceptance Date: May 31, 2006
Publication Date: June 20, 2007
Repository URL: http://www.pprl.ars.usda.gov
Citation: Lee, S.T., Knill, A., Michalewicz, A., Stevens, V., Colegate, S.M. 2007. Heliotropium europaeum alkaloids: a quaternary pyrrolizidine alkaloid approach to elisa development. Poisonous Plant Global Research and Solutions,Chpt 81, pp. 476-480.

Interpretive Summary: Heliotropium europaeum contains pyrrolizidine alkaloids and is capable of poisoning and killing livestock. Existing methods for the detection of pyrrolizidine alkaloids present in H. europaeum include ELISA and HPLC-MS methods. The ELISA does not detect the N-oxides. The HPLC-MS method can detect the N-oxides but is not suitable for rapid throughput and is expensive technology. In an attempt to produce antibodies that could be used in an ELISA format to detect both the parent pyrrolizidine alkaloids and their N-oxides simultaneously, the heliotrope alkaloids heleurine and lasiocarpine were each quaternised by reaction with bromotoluic NHS ester. These haptens were linked to appropriate proteins to provide protein conjugates for ELISA plate coating and vaccination respectively. Sera collected from sheep were tested for heleurine and lasiocarpine antibody titres. When the antisera were assessed against lasiocarpine, heliotrine and their N-oxides in an ELISA format, the level of recognition of N-oxides was approximately 50-70% that of the corresponding parent pyrrolizidine alkaloids. This is a considerable improvement on the existing heliotrope pyrrolizidine alkaloid ELISA.

Technical Abstract: Heliotropium europaeum has proven to be an agricultural pest capable of killing stock and severely reducing productivity due to its pyrrolizidine alkaloid content. Existing methods for the detection of pyrrolizidine alkaloids present in H. europaeum include an ELISA and HPLC-MS. The ELISA does not detect the N-oxides very well and thus an extra step is incorporated into the protocol to reduce the N-oxides to their parent tertiary bases. The HPLC-MS method is suitable for the detection of the N-oxides but is not suitable for rapid throughput and is expensive technology. Previous research in which the pyrrolizidine alkaloid riddelliine was quaternised and coupled to an immunogenic carrier protein resulted in an effective ELISA that recognised riddelliine and its N-oxide. This approach was adopted in an attempt to produce antibodies that could be used in an ELISA format to detect the parent pyrrolizidine alkaloids and their N-oxides simultaneously. The heliotrope alkaloids heliotrine, heleurine and lasiocarpine were each quaternised by reaction with N-hydroxysuccinimide (NHS) esters of brominated acids. The only stable quaternary compounds produced were heleurine and lasiocarpine coupled to bromotoluic NHS ester. These haptens were subsequently linked to ovalbumin and to foetal calf serum protein to provide protein conjugates for ELISA plate coating and vaccination respectively. Sera collected from sheep were tested for heleurine and lasiocarpine antibody titres at various time points preceding and following a vaccination protocol. Thus, when the antisera were assessed against lasiocarpine, heliotrine and their N-oxides in solution and the immobilised heleurine and lasiocarpine, the level of recognition of N-oxides was approximately 50-70% that of the corresponding parent pyrrolizidine alkaloids. This is a considerable improvement on the existing heliotrope pyrrolizidine alkaloid ELISA lending support to the general approach.

Last Modified: 12/19/2014
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