Immunoassay for determination of trilobolide.
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 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.
University of Chemistry and Technology Prague, Faculty of Food and Biochemical Technology [342], CZ-166 28 Prague, Czech Republic.
Lukáš
L
Huml
University of Chemistry and Technology Prague, Faculty of Food and Biochemical Technology [342], CZ-166 28 Prague, Czech Republic.
Michal
M
Jurášek
University of Chemistry and Technology Prague, Faculty of Food and Biochemical Technology [342], CZ-166 28 Prague, Czech Republic.
Petra
P
Mikšátková
University of Chemistry and Technology Prague, Faculty of Food and Biochemical Technology [342], CZ-166 28 Prague, Czech Republic.
Tomáš
T
Zimmermann
University of Chemistry and Technology Prague, Faculty of Food and Biochemical Technology [342], CZ-166 28 Prague, Czech Republic.
Pavla
P
Tomanová
Institute of Organic Chemistry and Biochemistry, CZ-166 10 Prague, Czech Republic.
Miloš
M
Buděšínský
University of Chemistry and Technology Prague, Faculty of Food and Biochemical Technology [342], CZ-166 28 Prague, Czech Republic.
Zdeňka
Z
Rottnerová
University of Chemistry and Technology Prague, Faculty of Food and Biochemical Technology [342], CZ-166 28 Prague, Czech Republic.
Markéta
M
Šimková
University of Chemistry and Technology Prague, Faculty of Food and Biochemical Technology [342], CZ-166 28 Prague, Czech Republic; Institute of Organic Chemistry and Biochemistry, CZ-166 10 Prague, Czech Republic.
Juraj
J
Harmatha
Institute of Experimental Medicine, AS CR, v.v.i., CZ-142 20 Prague, Czech Republic; Charles University in Prague, Faculty of Medicine and Biomedical Center in Pilsen, CZ-301 66 Pilsen, Czech Republic.
Eva
E
Kmoníčková
University of Chemistry and Technology Prague, Faculty of Food and Biochemical Technology [342], CZ-166 28 Prague, Czech Republic. Electronic address: oldrich.lapcik@vscht.cz.
Oldřich
O
Lapčík
University of Chemistry and Technology Prague, Faculty of Food and Biochemical Technology [342], CZ-166 28 Prague, Czech Republic. Electronic address: drasarp@vscht.cz.
Pavel B
PB
Drašar
D000818:Animals; D000906:Antibodies / Q000276:immunology; D019661:Apiaceae / Q000737:chemistry; D002087:Butyrates / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D005663:Furans / Q000032:analysis*; D007118:Immunoassay / Q000379:methods*; D015394:Molecular Structure; D011817:Rabbits; D015203:Reproducibility of Results
D000906:Antibodies; D002087:Butyrates; D005663:Furans; C086352:trilobolide
Avidin-biotin; ELISA; Immunoassay; Laser trilobum; Synthesis; Trilobolide