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,E Ishikawa

doi:10.1016/s0022-2275(20)39997-1

Abstract

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.

Highlights

A bile acid disappearance test using an enzyme this method has limited sensitivity
Succinimide ester of ursodeoxycholic acid (UDCA) to P-D-galactosidase in a molar ratio of 5000: 1. T h e 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
A radioimmunoassay for the determination of bile acids has been reported by Simmonds et al [7] using an anti-glycocholic acid serum. This radioimmunoassay satisfies the criteria of specificity, sensitivity, reproducibility, and technical ease [7,8,9,10,11,12]; its applicability is limited to a few laboratories because of the use of radioisotopes

Summary

Preparation of glycine and taurine conjugates of UDCA

The reaction products of the mixed anhydride procedure were isolated by preparative TLC on 20 X 20 cm glass plates coated with 1.0-mm thick layers of silica gel using the solvent system chloroformmethanol-acetic acid-water 65:20: 10:5 (v/v). The mixture was added to 7 ml of distilled water containing 16 mg of egg albumin, stirred in the cold for 2 hr, and emulsified with an equal volume of Freund’s adjuvant (Difco Laboratories, Detroit, MI) One ml of this mixture was injected subcutaneously into the legs and foot pads of four New Zealand white rabbits. After washing with acetone and drying, the beads were immersed in a 1% aqueous solution of glutaraldehyde for 1 hr and washed with 0.25 M sodium phosphate buffer, pH 7.5. They were placed in a rabbit antiserum which had been diluted with 0.25 M sodium phosphate buffer, pH 7.5 (1: 100 dilution) overnight at 4°C. One unit of enzyme activity was defined as that which hydrolyzed 1 pmol of the substrate per min under the assay conditions

RESULTS

Plasma tolerance test in rabbits

CVd nglml

DISCUSSION