Monitoring of progestins: Development of immunochemical methods for purification and detection of levonorgestrel

Shalev, Moran; Bardugo, Michal; Nudelman, Ayelet; Krol, Alex; Schlesinger, Hadassa; Bronshtein, Alisa; Altstein, Miriam

doi:10.1016/j.aca.2010.03.029

Cited by 9 publications

(5 citation statements)

References 41 publications

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“…In the past few years, we have developed highly effective and reproducible IAP technology, based on the entrapment of Abs in a ceramic SiO 2 matrix termed sol-gel, which facilitates efficient, singlestep cleanup and concentration of analyzates from large volumes of crude samples. The applicability of this approach was tested with Abs raised in our laboratory against a variety of PPs such as the steroid hormones levonorgestrel, ethinylestradiol, and nortestosterone [24], medroxyprogesterone acetate [25], the nonsteroid anti-inflammatory drug indomethacin [26], several other pesticides (pyrethroids and atrazine) [34,35], forensic compounds (TNT) [36], and environmental contaminants [37,38]. Implementation of the Abs for the development of a sol-gel based ATL IAP method is under development.…”

Section: Discussionmentioning

confidence: 99%

“…In previous studies, we developed ELISAs and immunoaffinity purification methods for some PPs: steroid hormones, levonorgestrel, ethinylestradiol and nortestosterone [24], medroxyprogesterone acetate [25], and the nonsteroid antiinflammatory drug indomethacin [26]. In the present study, we used two different approaches for ATL monitoring: an immunochemical approach based on ELISA, and an innovative diagnostic environmental assay based on a receptor-binding assay that can monitor the interaction between ATL and its target receptor b1AR.…”

Section: Introductionmentioning

confidence: 99%

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Development of an enzyme-linked immunosorbent assay and a beta-1 adrenergic receptor–based assay for monitoring the drug atenolol

Sapir

Shalev

Skalka

et al. 2012

Environmental Toxicology and Chemistry

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Abstract-Two approaches for monitoring atenolol (ATL) were applied: an immunochemical assay and a competitive-binding assay, based on the interaction between ATL and its target receptor, b1 adrenergic receptor (b1AR). Polyclonal antibodies (Abs) for ATL were generated, and a highly specific microplate immunochemical assay, that is, an enzyme-linked immunosorbent assay (ELISA), for its detection was developed. The ATL ELISA exhibited I50 and limit of detection (I20) values of 0.15 AE 0.048 and 0.032 AE 0.016 ng/ml, respectively, and the Abs did not cross-react with any of the tested beta-blocker drugs. Furthermore, a human b1AR (h-b1AR) was stably expressed in Spodoptera frugiperda cells (Sf9). The receptor was employed to develop a competitive-binding assay that monitored binding of ATL in the presence of isoproteranol by quantification of secondary messenger, cyclic adenosine monophosphate (cAMP), levels in the transfected cells. The assay showed that the recombinant h-b1AR was functional, could bind the agonistic ligand isoproterenol as well as the antagonist ATL, as indicated by a dose-dependent elevation of cAMP in the presence of isoproteranol, and decrease after ATL addition. The highly efficient and sensitive ELISA and the receptor assay represent two methods suitable for efficient and cost-effective large-scale, high-throughput monitoring of ATL in environmental, agricultural, and biological samples. Environ.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Development of an enzyme-linked immunosorbent assay and a beta-1 adrenergic receptor–based assay for monitoring the drug atenolol

Sapir

Shalev

Skalka

et al. 2012

Environmental Toxicology and Chemistry

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“…In previous studies we developed enzyme-linked immunosorbent assays (ELISA) and immuno-affinity purification methods for the steroid hormone, LNG [11,12] and the NSAID, IMT [13]. In the present study we focused on the progestin drug, MPA.…”

Section: Introductionmentioning

confidence: 99%

“…This method has been successfully applied by many laboratories, including ours, for purification of many compounds in the analysis of a variety of environmental, food, and biological samples. For review see [11][12][13][32][33][34][35][36][37][38][39][40][41][42][43][44]) and references therein.…”

Section: Introductionmentioning

confidence: 99%

Development of Immunochemical Methods for Purification and Detection of the Steroid Drug Medroxyprogesterone Acetate

Bronshtein¹,

Krol²,

Schlesinger³

et al. 2012

JEP

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An immunochemical sol-gel-based immunoaffinity purification (IAP) method for purification and detection of the progestin drug medroxyprogesterone acetate (MPA) was developed. A polyclonal antibody (Ab) for MPA was generated, and two competitive (indirect and direct) sensitive enzyme-linked immunosorbent assays (ELISAs) for its detection were developed and implemented to determine the recovery and efficiency of the sol-gel based IAP method. The detection limits of the assays were 1.4 ± 0.2 ng·mL^-1 (n = 4) and 4.0 ± 0.4 ng·mL^-1 (n = 25) for the indirect and direct ELISAs, respectively. The Abs did not exhibit cross-reactivity with any other progestin or steroid hormone, with the exception of megestrol acetate, with which the Ab exhibited 76% cross-reactivity. The sol-gel IAP method successfully eliminated serum interference to a degree that enabled ELISA analysis of spiked serum samples. This method was also found fully compatible with subsequent chemical analytical methods, such as liquid chromatography followed by mass spectrometry (LC-MS/MS). The approaches developed in this study form a basis for analysis of MPA in biological samples and may be further used to study population exposure to MPA and to monitor MPA contamination in water samples

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“…In the present study, we tested some of the above parameters by using sol-gel-entrapped Abs generated against the steroid hormone levonorgestrel (LNG) [16], which belongs to the group of endocrine disruptors [17]. We examined the effects of changes in the sol-gel matrix format on the activity of the entrapped Ab and on matrix structure, as it relates to the properties of the porogen PEG at various molecular weights and concentrations, and under diverse hydrolysis conditions ( i.e.…”

Section: Introductionmentioning

confidence: 99%

Sol-Gel Entrapped Levonorgestrel Antibodies: Activity and Structural Changes as a Function of Different Polymer Formats

Shalev

Altstein

2011

Materials

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The paper describes development of a sol-gel based immunoaffinity method for the steroid hormone levonorgestrel (LNG) and the effects of changes in the sol-gel matrix format on the activity of the entrapped antibodies (Abs) and on matrix structure. The best sol-gel format for Ab entrapment was found to be a tetramethoxysilane (TMOS) based matrix at a TMOS:water ratio of 1:8, containing 10% polyethylene glycol (PEG) of MW 0.4 kDa. Addition of higher percentages of PEG or a higher MW PEG did not improve activity. No activity was obtained with a TMOS:water ratio of 1:12, most likely because of the very dense polymer that resulted from these polymerization conditions. Only minor differences in the non-specific binding were obtained with the various formats. TMOS was found to be more effective than tetrakis (2-hydroxyethyl)orthosilicate (THEOS) for entrapment of anti-levonorgestrel (LNG) Abs. However, aging the THEOS-based sol-gel for a few weeks at 4 °C stabilized the entrapped Abs and increased its binding capacity. Confocal fluorescent microscopy with fluorescein isothiocyanate (FITC) labeled immunoglobulines (IgGs) entrapped in the sol-gel matrix showed that the entrapped Abs were distributed homogenously within the gel. Scanning electron microscopy (SEM) images have shown the diverse structures of the various sol-gel formats and precursors.

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Monitoring of progestins: Development of immunochemical methods for purification and detection of levonorgestrel

Cited by 9 publications

References 41 publications

Development of an enzyme-linked immunosorbent assay and a beta-1 adrenergic receptor–based assay for monitoring the drug atenolol

Development of an enzyme-linked immunosorbent assay and a beta-1 adrenergic receptor–based assay for monitoring the drug atenolol

Development of Immunochemical Methods for Purification and Detection of the Steroid Drug Medroxyprogesterone Acetate

Sol-Gel Entrapped Levonorgestrel Antibodies: Activity and Structural Changes as a Function of Different Polymer Formats

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