title: Monitoring of the non-steroid anti-inflammatory drug indomethacin: development of immunochemical methods for its purification and detection.
abstract: 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.
authors.affiliation: Department of Entomology, Institute of Plant Protection, The Volcani Center, Bet Dagan, Israel.
authors.forename: Nir
authors.initials: N
authors.lastname: Skalka
authors.forename: Alex
authors.initials: A
authors.lastname: Krol
authors.forename: Haim
authors.initials: H
authors.lastname: Schlesinger
authors.forename: Miriam
authors.initials: M
authors.lastname: Altstein
mesh_terms: D000894:Anti-Inflammatory Agents, Non-Steroidal; D000906:Antibodies; D003429:Cross Reactions; D016903:Drug Monitoring / Q000379:methods*; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D007213:Indomethacin / Q000032:analysis / Q000097:blood / Q000302:isolation & purification*; D057230:Limit of Detection; D046228:Microarray Analysis; D052616:Solid Phase Extraction
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D000906:Antibodies; D007213:Indomethacin