DEVELOPMENT AND APPLICATION OF AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR LUPIN ALKALOIDS

An enzyme-linked immunosorbent assay (ELISA) has been developed for the determination of total alkaloids in lupin seed. Polyclonal antialkaloid antibodies from sheep antisera raised against a conjugate of (+)-13-hydroxylupanine hemisuccinate with keyhole limpet hemocyanin were used to construct the indirect ELISA. A bovine serum albumin-hexahydrophthalate-hydroxylupanine conjugate was used as the plate-coating antigen. Different bridge moieties and conjugation techniques were used to overcome unwanted cross-reactivity associated with the production of heterologous antisera. Antisera showed high specificity for (+)-lupanine and (+)-13-hydroxylupanine, the major alkaloids found in the commercial variety of lupin seed grown in Western Australia (Lupinus angustifolius). The most sensitive ELISA had a range of 0.1-1000 nmol/mL with 50% inhibition at 8 nmol/mL alkaloid in solution. Comparison between ELISA and capillary gas chromatographic methods of analysis had a correlation coefficient of 0.94 (p < 0.001) over the range 10-900-mu-g/g alkaloid in the seed. Inhibition studies on the alkaloids found in different species of lupin seed were also performed by ELISA.