Generation and Characterization of Monoclonal Antibodies against the Pyrrolizidine Alkaloid Retrorsine

Abstract

Monoclonal antibodies against retrorsine were established using a retrorsine-hemisuccinate::bovine-thyroglobuline conjugate as antigen for immunization. In competitive enzyme-linked immunoassays the specificity of the obtained antibodies was tested with 20 different pyrrolizidine alkaloids. Among these the alkaloids acetylgynuramine, gynuramine, integerrimine, neoplatyphylline, platyphylline, rosmarinine, senecionine, and seneciphylline were able to bind the antibody. There was no cross-reactivity with the alkaloids senkirkine, angeloyl-heliotridine, heliotridine, axillarine, anacrotine, monocrotaline, madurensine, otosenine, retronecine, tussilagine, triangularine, and jacoline as well as with seneciphylline N-oxide. From this antigen spectrum it can be concluded that besides the necic structure itself the dominant and discriminative epitope consists of the exocyclic ethylidene group of the various diesters.

Scientific Opinion on Pyrrolizidine alkaloids in food and feed

“…Even though such cross reactivities were seen, the second system proved to be well suited for the quantification of riddelliine plus riddelliine-N-oxide in a mixture of Senecio riddellii and alfalfa plant materials. Finally, it should be mentioned that producing monoclonal antibodies against retrorsine conjugated to bovine-thyroglobine, Zündorf et al (1998) obtained antibodies with a certain affinity to 8 different pyrrolizidine alkaloids and no cross reaction to 12 other such compounds. The authors concluded that beside the necic structure itself the dominant and discriminative epitope consists of the exocyclic ethylidene group of the various diesters.…”

“…Most were based on polyclonal antibodies. Only one of the retrorsine-protein conjugate assays was established with monoclonal antibodies (Zündorf et al, 1998). A major disadvantage is that most established antibodies so far only detect closely related PAs (e.g.…”

Generation and Characterisation of Monoclonal Antibodies to Oleanolic Acid

“…For immunisation about 50 mg (determined by Bradford protein assay) of each of the thyroglobulin conjugates in a final volume of 200 mL according to Zündorf et al [2] were injected i. p. into Balb/C mice.…”

“…For further characterisation of the antibody binding via ELISA, we first had to determine the optimal ratio of coated antigen to antibody solution. Therefore, a checkerpoint titration was performed as described [2]. Increasing concentrations of cell culture supernatant (0.1 ± 40 mL) or of purified antibody were incubated with 0.2 ± 1.0 mg of the protein conjugates of oleanolic acid or succinyloleanolic acid.…”

“…Monoclonal antibodies (mABs) offer a highly specific and sensitive detection method for defined target molecules. Their use in diagnosis, therapy, and drug monitoring is increasing [2], [3]. Basis for the generation of mABs is the cell-fusion technique of Köhler and Milstein [1], which allows the generation of unlimited amounts of a specific monoclonal antibody against a welldefined target out of a single cell line.…”