Development and evaluation of a practical ELISA for human urinary lipocalin-type prostaglandin D synthase.
Development and evaluation of a practical ELISA for human urinary lipocalin-type prostaglandin D synthase.
复制标题
人尿脂质运载蛋白型前列腺素 D 合酶实用 ELISA 的开发和评估。
作者：
H. Oda;Y. Shiina;K. Seiki;N. Sato;N. Eguchi;Y. Urade
背景
慢性肾功能衰竭患者尿中脂质运载蛋白型前列腺素D合成酶（L-PGDS）的排泄显著增加，但其在较晚期肾脏疾病中的诊断潜力仍有待阐明。
方法
针对重组人L-PGDS产生了六种小鼠单克隆抗体（MAb）。我们构建了一个夹心ELISA与两个单克隆抗体，识别不同的表位具有高亲和力，并评估其检测性能和临床实用性与尿液样本健康对照，糖尿病患者，和各种肾脏疾病的患者。
结果
使用NH（2）末端截短的L-PGDS进行的蛋白质印迹分析将表位定位为Ala（23）-瓦尔（28）（MAb-7 F5和-10 A3）、Ser（52）-Ala（73）（MAb-9A 6）、Tyr（107）-瓦尔（120）（MAb-1B 7和-6 F5）和Gly（140）-Pro（155）（MAb-6 B 9）。单克隆抗体1B 7和7 F5对天然L-PGDS的K（d）值分别为3.6和3.9 nmol/L，建立了双抗体夹心ELISA法。回收率为91- 111%，批内和批间CV分别<6%和<9%。ELISA显示标准品和尿液样品的平行性，各种尿液成分没有显着干扰。即使血清肌酸酐没有增加，糖尿病肾病、伊加肾病和慢性肾小球肾炎患者的尿L-PGDS排泄量也显着增加。肾脏疾病患者尿L-PGDS与尿白蛋白（r = 0.64; P <0.0001）、N-乙酰-β-D-氨基葡萄糖苷酶（r = 0.43; P <0.001）和血肌酐（r = 0.66; P <0.0001）相关。在284 mg/mol肌酐的临界值下，该测定对糖尿病肾病的敏感性为74%，对慢性肾小球肾炎的敏感性为83%，特异性为93%。
结论
该ELISA系统适用于临床常规检测尿L-PGDS，并可用于检测晚期肾脏疾病。
BACKGROUND
Urinary excretion of lipocalin-type prostaglandin D synthase (L-PGDS) is significantly increased in patients with chronic renal failure, but its diagnostic potential in less advanced stages of renal diseases remains to be elucidated.
METHODS
Six mouse monoclonal antibodies (MAbs) were raised against recombinant human L-PGDS. We constructed a sandwich ELISA with two MAbs that recognized different epitopes with high affinities and assessed its assay performance and clinical utility with urine samples from healthy controls, diabetic patients, and patients with various renal diseases.
RESULTS
Western blot analyses with NH(2)-terminus-truncated L-PGDS mapped the epitopes to Ala(23)-Val(28) (MAb-7F5 and -10A3), Ser(52)-Ala(73) (MAb-9A6), Tyr(107)-Val(120) (MAb-1B7 and -6F5), and Gly(140)-Pro(155) (MAb-6B9). A sandwich ELISA was constructed with MAb-1B7 and -7F5, the K(d) values of which were 3.6 and 3.9 nmol/L, respectively, for native L-PGDS. Recoveries were 91-111%, and intra- and interassay CVs were <6% and <9%, respectively. The ELISA showed parallelism of standard and urine samples and no significant interference by a variety of urinary constituents. Urinary L-PGDS excretion was significantly increased in patients with diabetic nephropathy, IgA nephropathy, and chronic glomerulonephritis even when serum creatinine was not increased. In patients with renal diseases, urinary L-PGDS was correlated with urinary albumin (r = 0.64; P <0.0001), N-acetyl-beta-D-glucosaminidase (r = 0.43; P <0.001), and serum creatinine (r = 0.66; P <0.0001). At a cutoff value of 284 mg/mol creatinine, the assay had sensitivities of 74% for diabetic nephropathy and 83% for chronic glomerulonephritis and a specificity of 93%.
CONCLUSIONS
This ELISA system is suitable for measurement of urinary L-PGDS in a routine clinical assay and may be useful to detect less advanced stages of renal diseases.