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An enzyme-linked inummosorbent assay (ELISA) of pregnenolone sulfate (PREGS) has been developed for examining whether it is a veritable neurosteroid. 11alpha-Hemiglutaryloxy-PREGS was newly synthesized and conjugated with bovine serum albumin (BSA), which was injected to rabbits for the production of anti-PREGS antibodies. A bridge-heterologous ELISA system employing the sequential saturation method exhibited a high sensitivity with a midpoint of 30 pg. Although the antibody showed some cross-reactivity with PREG (4.4%), it easily discriminated other related steroids reported to exist in the mammalian brain. The rat brain homogenate was treated with hexane and subjected to an OASIS HLB cartridge, which was washed with AcOEt to remove the unconjugated steroids, and then the desired sulfate was eluted with EtOH. The recovery rate of PREGS through the pretreatment was satisfactory, but its brain levels in the preliminary experiments were much lower than those previously measured by gas chromatography (GC)-mass spectrometry (MS) after solvolysis. These results practically agreed with our previous results by liquid chromatography (LC)-electrospray ionization (ESI)-MS without deconjugation. (C) 2002 Elsevier Science B.V. All rights reserved.