Idiotype-anti-idiotype-based noncompetitive enzyme-linked immunosorbent assay of ursodeoxycholic acid 7-N-acetylglucosaminides in human urine with subfemtomole range sensitivity

Norihiro Kobayashi, Kazuyuki Kubota, Hiroyuki Oiwa, Junichi Goto, Toshifumi Niwa, Koju Kobayashi

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15 Citations (Scopus)

Abstract

We have established a noncompetitive enzyme-linked immunosorbent assay (ELISA) for the group-specific determination of 7-N-acetylglucosaminide of ursodeoxycholic acid (UDCA 7-NAG) and its glycine- and taurine-amidated metabolites (UDCA 7-NAGs) in human urine. These metabolites are expected to be a diagnostic marker for patients with primary biliary cirrhosis (PBC). This assay is based on the idiotype-anti-idiotype reaction where the analyte was captured by an excess amount of anti-UDCA 7-NAG antibody, and the unoccupied paratope was blocked with a β-type anti-idiotype antibody. The hapten-occupied antibody was then selectively detected with a biotin-labeled α-type anti-idiotype antibody. The amount of bound biotin, increasing proportionally to the increase in the analyte, was colorimetrically determined using a peroxidase-labeled streptavidin. This assay provided subfemtomole range sensitivity (detection limit 118 amol) and allowed group-specific measurement of the UDCA 7-NAGs in urine without any pretreatment. The present ELISA revealed that significant amounts of UDCA 7-NAGs are excreted even in healthy subjects. Daily excretion rates for healthy males were determined to be 246±184 (S.D.) μg (n=5) as the glycine-amidated UDCA 7-NAG equivalent. Randomly collected urine specimens from patients with PBC (n=7) were also measured, and the assay values (standardized to creatinine excretion) ranged from 1.82 to 13.4 μg/mg Ucre with the average of 5.41±4.53 (S.D.) μg/mg Ucre.

Original languageEnglish
Pages (from-to)1-10
Number of pages10
JournalJournal of Immunological Methods
Volume272
Issue number1-2
DOIs
Publication statusPublished - 2003 Jan 15

Keywords

  • Antiidiotype antibody
  • Bile acid metabolite
  • ELISA
  • Hapten
  • Liver disease
  • Noncompetitive immunoassay

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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