Immunoassay for determination of trilobolide

0474379 - ÚOCHB 2018 RIV US eng J - Článek v odborném periodiku
Huml, L. - Jurášek, M. - Mikšátková, P. - Zimmermann, T. - Tomanová, P. - Buděšínský, Miloš - Rottnerová, Z. - Šimková, M. - Harmatha, Juraj - Kmoníčková, Eva - Lapčík, O. - Drašar, P. B.
Immunoassay for determination of trilobolide.
Steroids. Roč. 117, Jan (2017), s. 105-111. ISSN 0039-128X. E-ISSN 1878-5867.
[Conference on Isoprenoids /23./. Minsk, 04.09.2016-07.09.2016]
Institucionální podpora: RVO:61388963 ; RVO:68378041
Klíčová slova: trilobolide * avidin-biotin * ELISA * Laser trilobum * synthesis * immunoassay
Obor OECD: Biochemical research methods; Pharmacology and pharmacy (UEM-P)
Impakt faktor: 2.523, rok: 2017

Trilobolide (Tb) is a pharmacologically interesting sesquiterpene lactone isolated from Laser trilobum (L.) Borkh. Structural relation to a sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin bring promising prospects for Tb to be used in the development of new anti-cancer drugs. As long as there are still unanswered questions regarding its investigation, a need for novel analytical tools emerge. Since immunoassays serve as one of powerful tools within the investigation of natural products, the development of indirect competitive enzyme-linked immunosorbent assay (ELISA) utilizing coating based on avidin-biotin technology is described. In our set-up of ELISA, newly synthesized biotinylated Tb served as immobilized competitor. Tb-carboxymethyloxime-bovine serum albumin (BSA) and Tb-succinoyl-BSA conjugates were used separately for immunization of rabbits. Two sets of polyclonal antibodies (RAbs) were obtained. Antibodies against Tb-succinoyl-BSA conjugate (RAb No. 206) were chosen as the best. Under optimized conditions, limit of detection and 50% intercept of our ELISA were 849 pg/mL and 8.89 ng/mL, respectively. The cross-reactivity (CR) was tested on 10 structurally related compounds and CR did not exceed 6.1%. The reproducibility of the system is expressed as intra- and inter-assay coefficients of variation (9.7% and 11.4%, respectively). Based on conducted experiments, we proposed the use of ELISA for quantification of Tb in complex biological matrices such as plant extracts. A method was applied to analyze three extracts obtained from different parts of L trilobum. Data obtained were compared to those acquired by UHPLC-MS/MS. The concordance between the methods (103-87%) showed the ability of ELISA to quantify Tb.
Trvalý link: http://hdl.handle.net/11104/0271443