Radioimmunoassay for the determination of digoxin and related compounds in Digitalis lanata

A radioimmunoassay technique has been developed for the measurement of digoxigenin glycosides in crude extracts from both fresh and dried leaf material of Digitalis lanata, The antibody, obtained by immunizing rabbits against a conjugate of digoxin with human serum albumin, had a high affinity (K_a = 0.8 × 10¹⁰ l/mol) for digoxin and permitted detection of as little as 60 fmol digoxin (45 pg) per 0.1 ml of sample. The antiserum was highly specific for digoxigenin and its glycosides, with only diginatin showing a substantial cross reactivity (3?0%). The use of ³H]-labelled and ¹²⁵I]-labelled digoxin as tracer and of dextran-coated charcoal or ammonium sulfate for separation did not change the specificity of the assay nor the properties of the standard curve. This method has been found to correlate with the usual fluorimetric determination of digoxin, but is more sensitive by a factor of 10⁴. A correlation analysis of 8 and 30 different D. lanata plants (leaf discs and drugs analysed with both methods) gave correlation coefficients of r = 0.989 and r = 0.907 respectively. The analysis of a single leaf disc, 3 mm in diameter (obtained from a fresh leaf), gave an exact measure of the digoxin content found in the dried leaf drug (r = 0.973). With a semi-automated technique, about 2000 quantitative analyses per week can be performed by one person, thus providing the potential to screen plants for use in breeding or tissue culture work. The distribution of digoxigenin equivalents in single seeds, seedlings and plants of different ages has also been investigated.