蛍光偏光免疫測定法におけるジゴキシン様物質の発生と光散乱の関与

Abstract

Digoxin-like factor (DLF) is known to interfere with a digoxin assay and falsely elevate the digoxin concentration. DLF has been reported in patients with liver and renal diseases, terminal pregnant women and infants. In this study, cholestasic rats induced by a surgical bile-duct ligation were used to clarify the appearance of DLF in a fluorescence polarization immunoassay (FPIA), and finally a precipitation method was improved. DLF increased immediately and reached about 0.4 ng/mL at 48 hrs after the bile-duct ligation. After centrifugal deproteinization with a precipitation reagent, 6 % sulfosalitylic acid aq. solution : MeOH mixture (1 : 1) (SSA), 69 and 202μg of a residue were detected while using a membrane filter (cellulose acetate ; pore size, 0.45 μm) at room temperature for 20 and 60 min, respectively. The scattering-light intensity (480-490 nm) of the cholestasic plasma was 5.5 times higher than that of the normal plasma and moreover increased by 20% after 60 min of incubation at room temperature. However, this high scattering-light intensity diminished when kept at 4°C for 60 min after adding the precipitation reagent, and DLF could thus be reduced. Porphyrin, a mixture of bilirubin and urobirin, can easily be aggregated by increasing the ionic strength and acidity, therefore, the addition of NaCl into cholestasic plasma in order to elevate the ionic strength potentiated the scattering-light intensity and DLF. These findings suggested that DLF in the cholestasic plasma may arise as a result of the appearance of some large particles after centrifugation because of insufficient deproteinization, which can scatter excited light due to polarization.