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Stanazolol derived ELISA as a sensitive forensic tool for the detection of multiple 17α-methylated anabolics

L. Huml, D. Havlová, O. Longin, E. Staňková, B. Holubová, M. Kuchař, E. Prokudina, Z. Rottnerová, T. Zimmermann, P. Drašar, O. Lapčík, M. Jurášek

. 2020 ; 155 (-) : 108550. [pub] 20191205

Language English Country United States

Document type Journal Article, Research Support, Non-U.S. Gov't

Persistent link   https://www.medvik.cz/link/bmc21012844

Two valuable forensic tools based on enzyme-linked immunoassays (ELISAs) for the analysis of 17α-methylated steroids were developed using haptens of stanazolol and its conjugates with biotin. Haptens containing terminal carboxylic group were conjugated to bovine serum albumin (BSA), rabbit serum albumin (RSA) or ovalbumin (OVA). Eight batches of antisera (RAbs) obtained by immunization of rabbits were tested in an indirect competitive ELISA system using immobilization of RSA conjugate (RSA/hapten) and competitor immobilization of the biotinylated conjugate (AB-ELISA) to avidin (avidin/hapten). The best results were achieved with the RAb 212 antibodies in RSA/ST-3 and avidin/ST-10 assembled variants. For the RSA/ST-3 system, an IC50 of 0.3 ng/mL and a detection limit of 0.02 ng/mL were measured. In case of avidin/ST-10 variant, IC50 was of 3.9 ng/mL and a detection limit of 0.57 ng/mL were obtained. The effect of solvent was tested as well as the stability of coated microtiter plates over four-month period. The cross-reactivity of the developed assays with other anabolic steroids was tested and high sensitivity towards 17α-methylated steroids was observed. RSA/ST-3 assay showed significant cross-reactivity with 17α-methyltestosterone (81.2%), oxymetholone (30.4%), methandienone (10.0%) and methyl dihydrotestosterone (7.7%). Similarly, in the avidin/ST-10 assay, 17α-methyltestosterone (34.5%), mestanolone (32.1%), oxymetholone (22.7%), methandienone (14.2%), 9-dehydromethyltestosterone (12.5%) and oxandrolone (1.2%) exhibited high cross-reactivity. The functionality of the developed systems was verified by the successful identification of a series of 17α-methylated anabolic steroids in a set of real samples including pharmaceutical preparations seized by the Police of the Czech Republic on the black market.

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$a Two valuable forensic tools based on enzyme-linked immunoassays (ELISAs) for the analysis of 17α-methylated steroids were developed using haptens of stanazolol and its conjugates with biotin. Haptens containing terminal carboxylic group were conjugated to bovine serum albumin (BSA), rabbit serum albumin (RSA) or ovalbumin (OVA). Eight batches of antisera (RAbs) obtained by immunization of rabbits were tested in an indirect competitive ELISA system using immobilization of RSA conjugate (RSA/hapten) and competitor immobilization of the biotinylated conjugate (AB-ELISA) to avidin (avidin/hapten). The best results were achieved with the RAb 212 antibodies in RSA/ST-3 and avidin/ST-10 assembled variants. For the RSA/ST-3 system, an IC50 of 0.3 ng/mL and a detection limit of 0.02 ng/mL were measured. In case of avidin/ST-10 variant, IC50 was of 3.9 ng/mL and a detection limit of 0.57 ng/mL were obtained. The effect of solvent was tested as well as the stability of coated microtiter plates over four-month period. The cross-reactivity of the developed assays with other anabolic steroids was tested and high sensitivity towards 17α-methylated steroids was observed. RSA/ST-3 assay showed significant cross-reactivity with 17α-methyltestosterone (81.2%), oxymetholone (30.4%), methandienone (10.0%) and methyl dihydrotestosterone (7.7%). Similarly, in the avidin/ST-10 assay, 17α-methyltestosterone (34.5%), mestanolone (32.1%), oxymetholone (22.7%), methandienone (14.2%), 9-dehydromethyltestosterone (12.5%) and oxandrolone (1.2%) exhibited high cross-reactivity. The functionality of the developed systems was verified by the successful identification of a series of 17α-methylated anabolic steroids in a set of real samples including pharmaceutical preparations seized by the Police of the Czech Republic on the black market.
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$a Havlová, Dominika $u Department of Chemistry of Natural Compounds, University of Chemistry and Technology Prague, CZ-166 28 Prague, Czech Republic
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$a Jurášek, Michal $u Department of Chemistry of Natural Compounds, University of Chemistry and Technology Prague, CZ-166 28 Prague, Czech Republic. Electronic address: jurasekm@vscht.cz
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