As shown in Table 1, antisera raised against dihydroouabain demonstrated cross-reactivities of only 1.4% with ouabain and <0.05% with its aglycone, ouabagenin. Furthermore, the antisera did not detect (<0.01%) digoxin or its aglycone, digoxigenin, and only slightly detected dihydrodigoxin (<0.05% cross-reactivity).
(a) Cross-reactivity, % Substance Anti-ouabain Anti-dihydroouabain Dihydroouabain 18 100 Ouabain 100 1.40 Ouabagenin 10 <0.05 Digoxin 0.66 <0.01 Dihydrodigoxin 6.70 <0.05 Digoxigenin 3.30 <0.01 Cholesterol 0.03 0.70 Bufalin 10 0.07 (a) Cross-reactivity is expressed as the molar concentration of the calibrator (ouabain or dihydroouabain) at 50% c/[c.sub.o] over the molar concentration of the compound at 50% c/[c.sub.o] multiplied by 100%.
Ouabain (G-strophanthin) octahydrate, ouabagenin, dihydroouabain, digoxin, digitoxin, digoxigenin, and other related steroids (aldosterone, bufalin, cortisone, hydrocortisone, progesterone, [beta]-estradiol, estrone, testosterone, and dehydroepiandrosterone), bovine thyroglobulin, and sodium cyanoborohydride were obtained from Sigma.
Ouabagenin showed the highest cross-reactivity (52%), followed by digoxin (1.7%) and digitoxin (0.6%), whereas the cross-reactivity of all steroids was <0.001%.
This is supported by the cross-reactivity profile of the ouabain antiserum (a-ouabain-199) raised against the ouabain-thyroglobulin conjugate, which demonstrated the highest cross-reactivity (52%) to the aglycone ouabagenin. The steroid nucleus with its hydroxyl groups and apparently also the lactone ring thus form the antigenic determinant.
The rhamnose moiety, which is absent from ouabagenin, is less important, leading to only a partial reduction in immunoreactivity.
Compound Cross-reactivity, % Cardenolide Digoxin 5.3 Digitoxin 78 Ouabagenin 55 Strophanthidin 47 Dihydroouabain 0.3050 Endogenous Corticosterone 0.0011 Progesterone 0.0150 Testosterone 0.0140 Cortisol 0.0005 17[alpha]-Hydroxyprogesterone 0.0035 18-Hydroxydeoxycorticosterone 0.0217 Aldosterone 0.0022 Dehydroepiandrosterone sulfate 0.0003 [beta]-Estradiol 0.0003 Results were obtained at 50% displacement from the zero standard calculations (see text).