A monoclonal antibody-based enzyme-linked immunosorbent assay of glycolithocholic acid sulfate in human urine for liver function test.
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 0.4 pg/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 314 ng/mg Ucre (males: n=16) and 507 ng/mg Ucre (females: n=9). Patients with liver diseases, including chronic hepatitis (CH) and liver cirrhosis (LC) exhibited significantly higher values (mean 5222 ng/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.
Kobayashi
Norihiro
N
Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Aoba-ku, 980-8578, Sendai, Japan.
Katsumata
Hiroshi
H
Uto
Yoshiaki
Y
Goto
Junichi
J
Niwa
Toshifumi
T
Kobayashi
Koju
K
Mizuuchi
Yoshiaki
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006000:Glycocholic Acid / Q000031:analogs & derivatives* / Q000276:immunology / Q000652:urine*; D006521:Hepatitis, Chronic / Q000175:diagnosis / Q000652:urine; D006801:Humans; D008103:Liver Cirrhosis / Q000175:diagnosis / Q000652:urine; D008107:Liver Diseases / Q000175:diagnosis / Q000652:urine; D008111:Liver Function Tests / Q000379:methods*; D008297:Male; D051379:Mice; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; C018154:sulfolithocholylglycine; D006000:Glycocholic Acid