Articles
PRELIMINARY DATA ON AN ANTIBODY FOR AN ISOXANTHOHUMOL ELISA
Article number
848_26
Pages
247 – 252
Language
English
Abstract
Isoxanthohumol is a weak estrogenic prenylflavanone from hop that possesses anti-carcinogenic effects.
It is also a precursor of 8-Prenylnaringenin.
A carboxylic acid hapten of prenylflavanone was synthesized with a spacer arm of 2 carbons at the C4 position.
The hapten was characterized by 1H-NMR, 13C-NMR, IR, MS (ESI+) and HR-MS (ESI+). It was coupled to Bovine Serum Albumin (BSA) by the activated ester method.
The coupling efficiency was assessed by MALDI-MS. It was found that the average coupling ratio was 26.5±1. Two antibodies were raised in two rabbits.
For the preliminary tests, competitive ELISA was chosen as the testing method.
To undertake such tests, H-prenylflavanone was coupled to swine thyroglobulin with a 50:1 ratio in order to perform the coating step.
Antibodies were tested for titres and the best was retained for further investigations.
A first standard curve was obtained with the following conditions: coating 0.03 μg/ml, Ac1: 1/250,000; Ac2: 1/10,000. Specificity tests were undertaken using various flavones and flavanones.
Cross-reactions were: Xanthohumol: 0.09%, 8-Prenylnaringenin: 2.71%, Naringenin: 12.6%, Liquiritigenin: 3.2%, Eriodictyol 1.6%. A sample of Lifenol® was assayed after extraction with ethanol:water 75:25 or enzyme hydrolysis and extraction with ethyl-acetate.
The average concentration was 6.3±2.0% or 6.0±2.1% after one or the other extraction.
It is also a precursor of 8-Prenylnaringenin.
A carboxylic acid hapten of prenylflavanone was synthesized with a spacer arm of 2 carbons at the C4 position.
The hapten was characterized by 1H-NMR, 13C-NMR, IR, MS (ESI+) and HR-MS (ESI+). It was coupled to Bovine Serum Albumin (BSA) by the activated ester method.
The coupling efficiency was assessed by MALDI-MS. It was found that the average coupling ratio was 26.5±1. Two antibodies were raised in two rabbits.
For the preliminary tests, competitive ELISA was chosen as the testing method.
To undertake such tests, H-prenylflavanone was coupled to swine thyroglobulin with a 50:1 ratio in order to perform the coating step.
Antibodies were tested for titres and the best was retained for further investigations.
A first standard curve was obtained with the following conditions: coating 0.03 μg/ml, Ac1: 1/250,000; Ac2: 1/10,000. Specificity tests were undertaken using various flavones and flavanones.
Cross-reactions were: Xanthohumol: 0.09%, 8-Prenylnaringenin: 2.71%, Naringenin: 12.6%, Liquiritigenin: 3.2%, Eriodictyol 1.6%. A sample of Lifenol® was assayed after extraction with ethanol:water 75:25 or enzyme hydrolysis and extraction with ethyl-acetate.
The average concentration was 6.3±2.0% or 6.0±2.1% after one or the other extraction.
Publication
Authors
S. Shinkaruk, V. Lamothe, C. Bennetau-Pelissero, B. Bennetau, P. Babin, J.-M. Schmitter
Keywords
isoxanthohumol, Hapten, ELISA, specificity test
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