Abstract
In recent years, the undeclared presence of various anabolic androgenic steroids (AAS) in commercial supplements has been confirmed. This fact can be a potential threat to all athletes using these supplements, and therefore, there is of increased interest in the implementation of rapid methods for the detection of AAS. The presented study describes the development of an immunostrip test for the detection of multiple 17α-methylated AAS based on direct and indirect competitive principle using gold nanoparticles as a label. As a capture reagent on test lines conjugated stanazolol to rabbit serum albumin (RSA/ST-3) was used, the intensity of color formed in the test line of the AAS-positive sample was visually distinguishable from that of negative sample within 10 min. The optimized closed direct and indirect format of the test provided a similar visual detection limit (0.7 and 0.9 ng/mL, respectively). The most commonly orally abused AAS (17α-methyltestosterone, methandienone, methyldihydrotestosterone, oxandrolone and oxymetholone) showed a strong cross-reaction. Developed immunostrips were successfully applied to analysis of artificially contaminated dietary supplements with 17α-methylated AASs. The developed immunostrips offer potential as a useful user-friendly method for capturing suspicious dietary supplement samples with different contents of AAS at levels far below the usually used concentrations of AAS.
Highlights
In recent years, the use of anabolic androgenic steroids (AAS) has been increasing, despite their proven negative effects
In our recent work by Huml et al [7], we have described the development of a group-selective immunoassay method based on stanazolol (ST)-derived polyclonal rabbit antibodies (RAb)
IInCTthme ceuthrroednst whaovrek,awleowdeesrcrsiebnestihtieviotyptcimomizpatairoendotfoththeecoEmLpISoAsi,tiuosnuoaflltyheinsctrreipastiensgt, itthseapdpetleiccatbioilnitylimonitthbeydoenteectoirodneorfoAfAmSa, gannditusudcec[e2s1s,f3u6l,t3e7s]t.inTghoefraerftoifriec,iainllythcoisnwtamorikn,atthede dcoiemtapreytistiuvpeptleesmt wenastsawrriatnhg1e7di-nminetdhiyrelactte(dFigAuAreS.2C) and direct format (Figure 2D), because the method of arrangement could have the effect of reducing the detection limit of the 2d.eMvealotepreiadlsmaentdhoAdp. pTahreatduisfference is in the labeled antibodies
Summary
The use of anabolic androgenic steroids (AAS) has been increasing, despite their proven negative effects. IInCTthme ceuthrroednst whaovrek,awleowdeesrcrsiebnestihtieviotyptcimomizpatairoendotfoththeecoEmLpISoAsi,tiuosnuoaflltyheinsctrreipastiensgt, itthseapdpetleiccatbioilnitylimonitthbeydoenteectoirodneorfoAfAmSa, gannditusudcec[e2s1s,f3u6l,t3e7s]t.inTghoefraerftoifriec,iainllythcoisnwtamorikn,atthede dcoiemtapreytistiuvpeptleesmt wenastsawrriatnhg1e7d́i-nminetdhiyrelactte(dFigAuAreS.2C) and direct format (Figure 2D), because the method of arrangement could have the effect of reducing the detection limit of the 2d.eMvealotepreiadlsmaentdhoAdp. pTahreatduisfference is in the labeled antibodies. GAR tertiary antibody diluted in redistilled water to a concentration of 1 mg/mL, AuNPs and 5 mmol/L borate buffer was used to prepare the conjugate (direct arrangement). The ratio of these reactants in the mixture was as follows: 2 mL of 5 mmol/L borate buffer; 1 mL of AuNPs; 100 μg of antibody.
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