Monitoring of progestins: Development of immunochemical methods for purification and detection of levonorgestrel |
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Authors: | Moran Shalev Michal Bardugo Alex Krol Alisa Bronshtein |
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Affiliation: | a Department of Entomology, Institute of Plant Protection, the Volcani Center, Bet Dagan 50250, Israel b Analyst Research Laboratories, Rabin Park, Hamada St. 12, Rehovot, 76703, Israel |
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Abstract: | A polyclonal antibody (Ab) for the progestin levonorgestrel (LNG) was generated, and immunochemical assays for its detection, clean-up and concentration were developed. A highly specific microplate diagnostic assay for the detection of LNG was developed that used the enzyme linked immunosorbent assay (ELISA) method. The LNG ELISA developed was sensitive and reproducible; it exhibited I50 and I20 values of 3.3 ± 1.8 ng mL−1 and 0.6 ± 0.4 ng mL−1, respectively, and the Abs did not cross react with any of the tested steroid hormones. The above Abs were used to develop a sol-gel-based immunoaffinity purification (IAP) method for concentration and clean-up of LNG that is compatible with subsequent immunochemical or instrumental chemical analytical procedures, such as liquid chromatography followed by mass spectrometry (LC-MS/MS). Development of the columns included successful entrapment of Abs within a tetramethoxysilane (TMOS)-based SiO2 polymer network. The Abs could bind the free analyte from solution, and the bound analyte could be easily eluted from the sol-gel matrix at high recoveries. The Ab selectivity towards the antigen was high, in both ELISA and the sol-gel columns, but the entrapped Abs cross-reacted with two other steroid hormones - ethynylestradiol (EE2) and nortestosterone (NT) - which share similar epitopes with LNG, despite the lack of cross reactivity in the ELISA. The validity of the method was investigated by LC-MS/MS and a good analytical correlation was obtained. |
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Keywords: | ACN, acetonitrile BSA, bovine serum albumin CMA, (carboxymethoxy)amine hemihydrochloride CMO, carboxymethyloxime DCC, N&prime ,N&prime -dicyclohexylcarbodiimide DCM, methylene chloride DDW, double distilled water DMF, dimethylformamide EE2, ethynylestradiol ELISA, enzyme linked immunosorbent assay FLX, fluoxetine GC, gas chromatography HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HRP, horseradish peroxidase IAP, immunoaffinity purification IMT, indomethacin KLH, Keyhole Limpet Hemocyanin LC, liquid chromatography LNG, levonorgestrel LOD, limit of detection MPA, medroxyprogesterone acetate MRM, multiple reaction monitor MS, mass spectrometry NHS, N-hydroxysuccinimide NSAIDs, non-steroid anti-inflammatory drugs NT, nortestosterone ON, overnight PBS, phosphate-buffered saline PBST, sodium phosphate containing Tween PPs, pharmaceutical products OVA, ovalbumin PEG, polyethylene glycol Py, pyridine RIA, radioimmunoassay SPE, solid-phase extraction SSRIs, selective serotonin reuptake inhibitors TMB, 3,3&prime ,5,5&prime -tetramethyl benzidine TMOS, tetramethylsilane TMP, thrimethoprim |
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