Journal of Lipid Research, Vol 20, 960-965, Copyright © 1979 by Lipid Research, Inc.
Development of a solid-phase enzyme immunoassay for ursodeoxycholic acid: application to plasma disappearance of injected ursodeoxycholic acid in the rabbit
Y Maeda, T Setoguchi, T Katsuki and E Ishikawa
A bile acid disappearance test using an enzyme immunoassay for
ursodeoxycholic acid (UDCA) is presented. The immunoassay employs an
antiserum produced in rabbits with UDCA coupled by amide linkage to egg
albumin. An antigen (UDCA)-enzyme (beta-D-galactosidase) complex was
prepared by adding the N-hydroxy-succinimide ester of UDCA to beta-D-
galactosidase in a molar ratio of 5000:1. The anti-UDCA serum was coupled
to glass beads and a competitive reaction between bile acids and UDCA
coupled to the enzyme on the glass beads was measured by determining enzyme
activity. One bead was used for each test tube. Thus it was convenient to
wash and transfer the bead to a fresh test tube after incubation. The
procedure requires 2.5 hr at 30 degrees C for the competitive reaction and
enzyme assay. Using a 1:100 dilution of anti- serum, the intensity of
fluorescence of 4-methylumbelliferone produced from
4-methylumbelliferyl-beta-D-galactoside by the enzyme decreased linearly
with a logarithmic increase of UDCA concentration over a range of from 0.1
to 10 pmnd taurine conjugates, and good recovery data were obtained. The
development of the enzyme immunoassay using glass beads shortens analysis
time; furthermore, the method makes it possible to detect obstructive
jaundice in rabbits before the serum bilirubin level is elevated.
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