RESUMEN
The present research focused on the development of an immunoassay and an immunochemical sol-gel-based immunoaffinity purification (IAP) method for purification and detection of the non-steroid anti-inflammatory drug (NSAID) indomethacin (IMT). A polyclonal antibody (Ab) for IMT was generated, and two sensitive microplate assays for the detection of IMT were developed (termed OV and HRP formats), based on the enzyme-linked immunosorbent assay (ELISA) method. The limits of detection of the assays were 15 ± 1.25 ng mL(-1) (n = 50) and 12 ± 0.17 ng mL(-1) (n = 4) for the OVA and HRP formats, respectively. The Abs exhibited slight cross-reactivity with other NSAIDs. The Abs were also used to develop a sol-gel-based IAP method for clean-up and concentration of IMT. Several sol-gel formats with various amounts of antibodies were examined; the best and most reproducible format was at a TMOS:HCl molar ratio of 1:6 in which 120 µL of IMT Abs was entrapped. The binding capacity under these conditions was ca. 100 to 250 ng of IMT with very low non-specific binding (less than 5% of the applied amount). The sol-gel IAP method, combined with solid-phase extraction, successfully eliminated serum interference to a degree that enabled analysis of spiked serum samples by ELISA. The method was also found to be fully compatible with subsequent chemical analytical methods, such as liquid chromatography followed by mass spectrometry. The approaches developed in this study form a basis for analysis of IMT in biological samples in order to monitor their pharmacokinetic properties, and may be further used to study population exposure to IMT, and to monitor the occurrence of IMT contamination in water samples.
Asunto(s)
Monitoreo de Drogas/métodos , Inmunoensayo/métodos , Indometacina/aislamiento & purificación , Antiinflamatorios no Esteroideos , Anticuerpos , Reacciones Cruzadas , Inmunoensayo/instrumentación , Inmunoensayo/normas , Indometacina/análisis , Indometacina/sangre , Límite de Detección , Análisis por Micromatrices , Extracción en Fase SólidaRESUMEN
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.