A monoclonal antibody-based enzyme-linked immunosorbent assay of glycolithocholic acid sulfate in human urine for liver function test
A monoclonal antibody-based enzyme-linked immunosorbent assay of glycolithocholic acid sulfate in human urine for liver function test
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DOI：
10.1016/s0039-128x(02)00036-3
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发表时间：
2002-09-01
期刊：
影响因子：
2.7
通讯作者：
Mizuuchi, Y
中科院分区：
文献类型：
--
作者：
Kobayashi, N;Katsumata, H;Mizuuchi, Y
尿中石胆酸（LCA）的硫酸化代谢产物水平有望成为肝功能的有用指标。因此，一个敏感的，具体的，和可行的酶联免疫吸附试验（ELISA），这些硫酸化LCA代谢产物（LCA-Suls）应建立。通过第二抗体将新产生的对乙醇石胆酸硫酸盐（甘氨酸酰胺化LCA-Sul（GLCA-Sul））具有特异性的单克隆抗体固定在微量滴定板上。将尿样和碱性磷酸酶标记的抗原加入平板中，然后在室温下孵育3 h。在该竞争性反应之后，使用磷酸对硝基苯酯作为底物比色测量结合的酶活性。GLCA-Sul的检测限为0.4 pg/测定。非酰胺化LCA-Sul和牛磺酸结合LCA-Sul分别显示40%和11%的交叉反应性，而胆酸（CA; 0.02%）、鹅脱氧胆酸（CDCA; 0.63%）和脱氧胆酸（DCA; 2.2%）的3-硫酸盐显示非常低的交叉反应性。通过平行性、回收率试验和试验内/试验间方差充分验证了ELISA系统对临床样品的适用性。与胆汁酸硫酸酯酶的酶解缀合导致尿水平显著降低，支持ELISA对GLCA-Sul的特异性。健康志愿者清晨尿液中的平均GLCA-Sul水平为314 ng/mg Ucre（男性：n = 16）和507 ng/mg Ucre（女性：n = 9）。慢性肝炎（CH）和肝硬化（LC）等肝病患者Ucre值明显增高（平均5222 ng/mg，n = 2.1）。本'单克隆ELISA'被预测是有用的，作为一种新的非侵入性的诊断工具，肝功能和肝胆疾病。(C)2002年由Elsevier Science Inc.出版
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 3 h. 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 = 2 1). The present 'monoclonal ELISA' is predicted to be useful as a novel noninvasive diagnostic tool for liver function and hepatobiliary diseases. (C) 2002 Published by Elsevier Science Inc.