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Immunoassay for determination of trilobolide
L. Huml, M. Jurášek, P. Mikšátková, T. Zimmermann, P. Tomanová, M. Buděšínský, Z. Rottnerová, M. Šimková, J. Harmatha, E. Kmoníčková, O. Lapčík, PB. Drašar,
Language English Country United States
Document type Journal Article
- MeSH
- Apiaceae chemistry MeSH
- Butyrates analysis MeSH
- Enzyme-Linked Immunosorbent Assay MeSH
- Furans analysis MeSH
- Immunoassay methods MeSH
- Rabbits MeSH
- Molecular Structure MeSH
- Antibodies immunology MeSH
- Reproducibility of Results MeSH
- Animals MeSH
- Check Tag
- Rabbits MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Trilobolide (Tb) is a pharmacologically interesting sesquiterpene lactone isolated from Laser trilobum (L.) Borkh. Structural relation to a sarco/endoplasmic reticulum Ca(2+)-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 849pg/mL and 8.89ng/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.
Institute of Experimental Medicine AS CR v v i CZ 142 20 Prague Czech Republic
Institute of Organic Chemistry and Biochemistry CZ 166 10 Prague Czech Republic
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