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Immunochemical detection of lysergic acid diethylamide using a photochemically linked immunogen

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Title: Immunochemical detection of lysergic acid diethylamide using a photochemically linked immunogen
Author: Kerrigan, Sarah
Degree Doctor of Philosophy - PhD
Program Chemistry
Copyright Date: 1997
Abstract: A new immunogen was prepared in which lysergic acid diethylamide (LSD) was photochemically attached to keyhole limpet hemocyanin (KLH) using a heterobifunctional linker. Photoactivation of the aryl azide group in the linker produces a reactive nitrene, which facilitates the covalent attachment of LSD to the protein at a number of different sites on the drug. Multi-site attachment of the drug is advantageous, as this in principle facilitates epitope recognition, which therefore increases the antiserum sensitivity. Using this method, it was possible to attach an average of 35 LSD molecules to each KLH molecule, which is sufficient for immune recognition of the drug. High affinity polyclonal antibodies to LSD were raised against the photochemically linked immunogen. The equilibrium association constant, Ka, was estimated to be 1.3 x 10¹⁰ M⁻¹ using a radiolabeled technique. Comparison of the new antiserum with commercial antibody preparations indicated it had the highest affinity, lowest dissociability and was the most sensitive antiserum by enzyme-linked immunosorbent assay (ELISA). The new antiserum was used for drug detection and extraction purposes. A high affinity immunochromatographic support was prepared, which allowed sub-nanogram quantities of LSD to be selectively extracted from blood and urine with greater than 80% efficiency. An indirect ELISA was developed for the detection of LSD in human urine specimens. The limit of detection was 10 pg/mL when 50 pL urine was used. Analytical recoveries of LSD were between 98 - 106 % and intra and inter-assay precision was good over a wide range of concentrations. Within-run and between-run CVs were 3.7% (n=4) and 6.3% (n=12) for a sample which contained 0.2 ng/mL LSD in urine. The upper and lower limits of quantification were between 7 ng/mL and 50 pg/mL, which is well within the region of forensic interest. The cross-reactivity of the new antiserum with the major metabolite, nor-LSD, was 52%. Other derivatives of interest which cross-react with the antibody included lysergic acid methylproplyamide (34%) and 2-oxo-3-hydroxy-LSD (3.4%). The UBC ELISA, which offered improved sensitivity and precision compared to a number of commercial immunoassays, was used to detect LSD in non-urine matrices, including forensic case samples of post-mortem blood, urine, bile and vitreous fluid.
URI: http://hdl.handle.net/2429/8630
Series/Report no. UBC Retrospective Theses Digitization Project [http://www.library.ubc.ca/archives/retro_theses/]

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