The problem of digitalis toxicity is one of considerable importance to clinicians and pharmacologists.' Not only is therapy far from satisfactory, but no convenient method is currently available for the specific determination of cardiac glycosides in plasma and tissue fluids.Although antibodies specific for a variety of haptens have been obtained following the classic technique of Landsteiner,2 no record has been found of the use of cardiac glycosides for such studies. Digitalis-specific antibodies could conceivably be an extremely useful tool (1) in the immunological assay of digitalis in plasma and tissue fluids, (2) in the therapy of severe digitalis toxicity, and (3) in the study of the mechanism of action of digitalis. This report describes the coupling of the cardiac glycoside, digoxin,3 to bovine serum albumin and the use of the resulting conjugate as an antigen to induce the formation of antibodies which exhibit digoxin specificity.Materials and Methods.-Reagents: Bovine serum albumin (BSA) was obtained as fraction V powder from Pentex, Inc. "Lanoxin"-brand digoxin and tritiated digoxin (Dig-H3; 112.6 ,.c/ mg; 1 mg/ml in 95% ethanol) were generously supplied by Burroughs Wellcome and Company. Cortisone-1,2-H3 (5.83 mc/mg), hydrocortisone-1,2-H3(50.2 mC/mg), dehydroepiandrosterone7ac-H3 (5.62 mc/mg), and dehydroepiandrosterone-7a-H3 sulfate, ammonium salt (4.23 mc/mg) were obtained from the New England Nuclear Corporation and were chromatographically purified by Dr. William Drucker and then dissolved in absolute methanol. Isotopic compounds were diluted with buffer for use in dialysis experiments.Preparation of protein-digoxin conjugates: Digoxin (Dig) which consists of a steroidal aglycone linked to three digitoxose residues (see Fig. 2) was conjugated to BSA by a periodate oxidation method suggested by Dr. Bernard Erlanger, Columbia University, and based on a technique originally described by Erlanger and Beiser4 5 for other hapten-protein conjugates. One BSA-Dig conjugate was prepared as follows: To 218.8 mg (0.28 mmole) digoxin were added 30 ml 0.1 M sodium periodate, 40 ml absolute ethanol, and 10 ml dioxane. After 30 min at room temperature, 1.8 ml 1 M ethylene glycol was added and the entire reaction mixture was added to 280 mg BSA in 10 ml water which had been adjusted to pH 9.3 with 5% K2CO3. The mixture was stirred for 1 hr at room temperature with dropwise addition of 5% K2CO3 to maintain the pH in the 9.0-9.5 range. After 1 hr, 150 mg sodium borohydride was added and the reaction mixture was set aside for 24 hr at room temperature. Approximately 5.4 ml 1 M formic acid was added to lower the pH to 5.5; at about pH 5.8, considerable precipitation occurred. After 1 hr at room temperature, 1.5 ml 1 M NH40H was added to raise the pH to 8.5. Some cloudiness persisted and the mixture was dialyzed overnight against running tap water. The pH was lowered to 4.8 by the dropwise addition of 1.85 ml 0.1 N HCl with considerable precipitation of protein. After 4 hr at 40C, the suspension was centrifuged 1 hr ...

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