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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,

. 2017 ; 117 (-) : 105-111. [pub] 20160904

Language English Country United States

Document type Journal Article

Persistent link   https://www.medvik.cz/link/bmc17031458

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.

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$a Trilobolide (Tb) is a pharmacologically interesting sesquiterpene lactone isolated from Laser trilobum (L.) Borkh. Structural relation to a sarco/endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin bring promising prospects for Tb to be used in the development of new anti-cancer drugs. As long as there are still unanswered questions regarding its investigation, a need for novel analytical tools emerge. Since immunoassays serve as one of powerful tools within the investigation of natural products, the development of indirect competitive enzyme-linked immunosorbent assay (ELISA) utilizing coating based on avidin-biotin technology is described. In our set-up of ELISA, newly synthesized biotinylated Tb served as immobilized competitor. Tb-carboxymethyloxime-bovine serum albumin (BSA) and Tb-succinoyl-BSA conjugates were used separately for immunization of rabbits. Two sets of polyclonal antibodies (RAbs) were obtained. Antibodies against Tb-succinoyl-BSA conjugate (RAb No. 206) were chosen as the best. Under optimized conditions, limit of detection and 50% intercept of our ELISA were 849pg/mL and 8.89ng/mL, respectively. The cross-reactivity (CR) was tested on 10 structurally related compounds and CR did not exceed 6.1%. The reproducibility of the system is expressed as intra- and inter-assay coefficients of variation (9.7% and 11.4%, respectively). Based on conducted experiments, we proposed the use of ELISA for quantification of Tb in complex biological matrices such as plant extracts. A method was applied to analyze three extracts obtained from different parts of L. trilobum. Data obtained were compared to those acquired by UHPLC-MS/MS. The concordance between the methods (103-87%) showed the ability of ELISA to quantify Tb.
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$a Jurášek, Michal $u University of Chemistry and Technology Prague, Faculty of Food and Biochemical Technology [342], CZ-166 28 Prague, Czech Republic.
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$a Zimmermann, Tomáš $u University of Chemistry and Technology Prague, Faculty of Food and Biochemical Technology [342], CZ-166 28 Prague, Czech Republic.
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$a Tomanová, Pavla $u University of Chemistry and Technology Prague, Faculty of Food and Biochemical Technology [342], CZ-166 28 Prague, Czech Republic.
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$a Buděšínský, Miloš $u Institute of Organic Chemistry and Biochemistry, CZ-166 10 Prague, Czech Republic.
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$a Rottnerová, Zdeňka $u University of Chemistry and Technology Prague, Faculty of Food and Biochemical Technology [342], CZ-166 28 Prague, Czech Republic.
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$a Šimková, Markéta $u University of Chemistry and Technology Prague, Faculty of Food and Biochemical Technology [342], CZ-166 28 Prague, Czech Republic.
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$a Harmatha, Juraj $u University of Chemistry and Technology Prague, Faculty of Food and Biochemical Technology [342], CZ-166 28 Prague, Czech Republic; Institute of Organic Chemistry and Biochemistry, CZ-166 10 Prague, Czech Republic.
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$a Kmoníčková, Eva $u Institute of Experimental Medicine, AS CR, v.v.i., CZ-142 20 Prague, Czech Republic; Charles University in Prague, Faculty of Medicine and Biomedical Center in Pilsen, CZ-301 66 Pilsen, Czech Republic.
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$a Lapčík, Oldřich $u University of Chemistry and Technology Prague, Faculty of Food and Biochemical Technology [342], CZ-166 28 Prague, Czech Republic. Electronic address: oldrich.lapcik@vscht.cz.
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$a Drašar, Pavel B $u University of Chemistry and Technology Prague, Faculty of Food and Biochemical Technology [342], CZ-166 28 Prague, Czech Republic. Electronic address: drasarp@vscht.cz.
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