A monoclonal antibody-based enzyme-linked immunosorbent assay of glycolithocholic acid sulfate in human urine for liver function test

Norihiro Kobayashi, Hiroshi Katsumata, Yoshiaki Uto, Junichi Goto, Toshifumi Niwa, Koju Kobayashi, Yoshiaki Mizuuchi

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Urinary levels of sulfated metabolites of lithocholic acid (LCA) are expected to be a useful index of liver function. Thus, a sensitive, specific, and feasible enzyme-linked immunosorbent assay (ELISA) of these sulfated LCA metabolites (LCA-Suls) should be established. A newly generated monoclonal antibody specific to glycolithocholic acid sulfate (glycine-amidated LCA-Sul (GLCA-Sul)) was immobilized on microtiter plates via a second antibody. A urine specimen and an alkaline phosphatase-labeled antigen were added to the plate, which was then incubated at room temperature for 3h. After this competitive reaction, bound enzyme activity was measured colorimetrically using p-nitrophenyl phosphate as a substrate. The detection limit for GLCA-Sul was 0.4pg/assay. Nonamidated LCA-Sul and taurine-conjugated LCA-Sul showed 40 and 11% cross-reactivities, respectively, while 3-sulfates of cholic acid (CA; 0.02%), chenodeoxycholic acid (CDCA; 0.63%), and deoxycholic acid (DCA; 2.2%) exhibited very low cross-reactivities. Applicability of the ELISA system to clinical samples was well validated by parallelism, recovery test, and intra/inter-assay variance. Enzymatic deconjugation with bile acids sulfatase resulted in dramatically decreased urinary levels, supporting the specificity of the ELISA toward GLCA-Sul. The mean GLCA-Sul levels in early morning urine from healthy volunteers were 314ng/mg Ucre (males: n=16) and 507ng/mg Ucre (females: n=9). Patients with liver diseases, including chronic hepatitis (CH) and liver cirrhosis (LC) exhibited significantly higher values (mean 5222ng/mg Ucre: n=21). The present 'monoclonal ELISA' is predicted to be useful as a novel noninvasive diagnostic tool for liver function and hepatobiliary diseases.

Original languageEnglish
Pages (from-to)827-833
Number of pages7
JournalSteroids
Volume67
Issue number10
DOIs
Publication statusPublished - 2002 Sep 1

Keywords

  • Bile acid sulfate
  • ELISA
  • Glycolithocholic acid sulfate
  • Human urinary levels
  • Liver function test
  • Monoclonal antibody

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Endocrinology
  • Pharmacology
  • Clinical Biochemistry
  • Organic Chemistry

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