For measurement of MBG and endogenous ouabain, samples of plasma and urine were extracted on SepPak C-18 cartridges (Waters, Milford, Massachusetts, USA) as described previously in detail [11 (link)]. The MBG DELFIA fluoroimmunoassays based on anti-MBG 3E9 and 4G4 mAbs were performed as previously described for rabbit anti-MBG polyclonal antibodies [11 (link)]. The assay is based on competition between immobilized antigen (MBG-glycoside-thyroglobulin) and MBG, other cross-reactants, or endogenous CTS within the sample for a limited number of binding sites on an anti-MBG mAbs. Secondary (goat antimouse) antibody labeled with non-radioactive europium was obtained from Perkin-Elmer (Waltham, Massachusetts, USA).
The endogenous ouabain assay was based on a similar principle utilizing an ouabain–ovalbumin conjugate and ouabain antiserum (anti-OU-M-2005; 1 : 20 000) obtained from rabbits immunized with a ouabain-BSA conjugate [20 (link)]. The cross-reactivity of this ouabain antibody is (%) ouabain, 100; ouabagenin, 52, digoxin, 1.8; digitoxin, 0.47; progesterone, 0.002; prednisone, 0.001; proscillaridin, 0.03; bufalin, 0.10; aldosterone, 0.04; telocinobufagin, 0.02; resibufagin, 0.15; marinobufotoxin, 0.06; cinobufagin, 0.02; and MBG, 0.036.
The endogenous ouabain assay was based on a similar principle utilizing an ouabain–ovalbumin conjugate and ouabain antiserum (anti-OU-M-2005; 1 : 20 000) obtained from rabbits immunized with a ouabain-BSA conjugate [20 (link)]. The cross-reactivity of this ouabain antibody is (%) ouabain, 100; ouabagenin, 52, digoxin, 1.8; digitoxin, 0.47; progesterone, 0.002; prednisone, 0.001; proscillaridin, 0.03; bufalin, 0.10; aldosterone, 0.04; telocinobufagin, 0.02; resibufagin, 0.15; marinobufotoxin, 0.06; cinobufagin, 0.02; and MBG, 0.036.
