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

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 0.4 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.