Monoclonal Antibody-Based Enzyme Immunoassay for the Quantitative Determination of the Tropane Alkaloid, Scopolamine

A monoclonal antibody (SP1-4-A2) against scopolamine was produced, characterized, and used to develop a sensitive and selective, competitive enzyme immunoassay for the quantitation of the alkaloid. The assay uses nor-scopolamine-N-β-propionic acid coupled to alkaline phosphatase as tracer and is linear from 10 pg to 10 ng of scopolamine. As little as 10 pg of scopolamine can be quantitated in an unprocessed plant extract or in human serum after suitable dilution, corresponding to detection limits of 0.1 ng scopolamine/ml of plant extract or 0.5 ng/ml of serum. The assay is more selective for scopolamine (percent cross-reactions for hyoscyamine = 0.21%, 6-hydroxy-hyoscyamine = 0.17%) than previously reported immunoassays. The assay format was designed to minimize intra- and inter-plate variabilities which are, on an average, all below 3% (coefficients of variation). The assay reported here has been validated against an HPLC-based technique using plant samples and was shown to correlate closely (y = 0.959 × + 0.14, r = 0.982).