Idiotype-anti-idiotype-based noncompetitive enzyme-linked immunosorbent assay of ursodeoxycholic acid 7-N-acetylglucosaminides in human urine with subfemtomole range sensitivity.
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-antiidiotype reaction where the analyte was captured by an excess amount of anti-UDCA 7-NAG antibody, and the unoccupied paratope was blocked with a beta-type antiidiotype antibody. The hapten-occupied antibody was then selectively detected with a biotin-labeled alpha-type antiidiotype 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.) microg (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 microg/mg Ucre with the average of 5.41+/-4.53 (S.D.) microg/mg Ucre.
Kobayashi
Norihiro
N
Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai 980-8578, Japan.
Kubota
Kazuyuki
K
Oiwa
Hiroyuki
H
Goto
Junichi
J
Niwa
Toshifumi
T
Kobayashi
Koju
K
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000276:immunology* / Q000652:urine*; D000888:Antibodies, Anti-Idiotypic; D015415:Biomarkers / Q000652:urine; D016022:Case-Control Studies; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D006801:Humans; D007130:Immunoglobulin Idiotypes; D008105:Liver Cirrhosis, Biliary / Q000175:diagnosis / Q000652:urine; D008297:Male; D012016:Reference Values; D012680:Sensitivity and Specificity; D014580:Ursodeoxycholic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000276:immunology* / Q000652:urine*
D000888:Antibodies, Anti-Idiotypic; D015415:Biomarkers; D007130:Immunoglobulin Idiotypes; C060582:ursodeoxycholic acid N-acetylglucosaminide; D014580:Ursodeoxycholic Acid; D000117:Acetylglucosamine