Monitoring of progestins: development of immunochemical methods for purification and detection of levonorgestrel.
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 I(50) and I(20) 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 SiO(2) 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.
Shalev
Moran
M
Department of Entomology, Institute of Plant Protection, the Volcani Center, Bet Dagan 50250, Israel.
Bardugo
Michal
M
Nudelman
Ayelet
A
Krol
Alex
A
Schlesinger
Haim
H
Bronshtein
Alisa
A
Altstein
Miriam
M
D000906:Antibodies / Q000276:immunology; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003280:Contraceptives, Oral, Synthetic / Q000032:analysis* / Q000302:isolation & purification; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D016912:Levonorgestrel / Q000032:analysis* / Q000302:isolation & purification; D012821:Silanes / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry; D052616:Solid Phase Extraction; D021241:Spectrometry, Mass, Electrospray Ionization
D000906:Antibodies; D003280:Contraceptives, Oral, Synthetic; D012821:Silanes; D016912:Levonorgestrel; C061205:tetramethoxysilane; D012822:Silicon Dioxide