An enzyme-linked immunosorbent assay for quassin and closely related metabolites

A microtitration plate enzyme-linked immunosorbent assay has been developed for quassin and closely related seco-triterpenes. The method is much more sensitive than conventional chromatographic techniques and enables the routine analysis of large numbers of samples with a detection limit of 5 ng quassin. Antibody production was elicited by injecting a conjugate of iso-quassinic acid linked to bovine serum albumin into rabbits. Important features of the technique are (a) the use of a double-antibody, noncompetitive enzyme-linked immunosorbent assay for a low-molecular-weight, nonantigenic compound using microtitration plates; and (b) that the initial incubation of samples with primary antiserum can be carried out in buffer containing up to 20% (v/v) methanol with minimal loss of sensitivity. The structural requirements for recognition of the hapten by the antiserum are considered based on the levels of cross-reaction found with a wide range of other quassinoids.