尿Ⅳ型胶原酶免疫测定法的建立及其在糖尿病肾病中的应用

Development of an enzyme immunoassay for urinary collagen Ⅳ and its application in diabetic nephropathy

目的 通过建立一种简便、灵敏的双抗体夹心酶联免疫吸附法 (ELISA)检测尿Ⅳ型胶原 (ⅣC) ,探讨糖尿病 (DM )患者肾小球基底膜代谢状况以及尿ⅣC对糖尿病肾病 (DN)的诊断价值。方法 检测 5 0名正常人和 12 7例DM患者的血清ⅣC及尿ⅣC。并将 12 7例DM患者根据其 2 4h尿白蛋白 (Alb)排泄率 (UAE)分成 3组 :UAE <30mg/ 2 4h(A组 )、30mg/ 2 4h <UAE <30 0mg/ 2 4h(B组 )、UAE >30 0mg/ 2 4h(C组 )。 结果 双抗体夹心ELISA检测尿ⅣC ,灵敏度为 1.2ng/ml;批内变异系数 (CV)为 5 .4 %～ 10 .6 % ,批间CV为 7.8%～18.5 %。在 3组DM患者中 ,A组患者血清、尿ⅣC的阳性率分别为 2 8.3%、31.7% ,明显高于对照组 (P <0 .0 1) ;C组 19例DM患者中有 18例已发展为DN ,尿ⅣC的阳性率 (6 3.2 % )显著高于A组 (2 8.3% ) (P <0 .0 1) ,C组血清ⅣC的阳性率 (36 .8% )与A组 (31.7% )比较差异无显著性 (P >0 .0 5 )。结论 本研究建立了一种简便、灵敏的检测尿ⅣC的ELISA方法 ,该法对DN的诊断具有较高的敏感性和特异性。

Objective To develop an enzyme immunoassay for urinary collagen Ⅳ(ⅣC) in order to evaluate the metabolism of renal glomerular basement membranes and for the diagnosis of diabetic nephropathy(DN). Methods To determine the levels of serum ⅣC and urinary ⅣC in 50 normal peoples and 127 type Ⅱ diabetes mellitus(DM) patients divided into three groups according to the urinary albumin excretion(UAE): UAE<30 mg/ml(group A), 30 mg/ml <UAE<300 mg/ml(group B), UAE>300 mg/ml(group C). Results The detection limit of ELISA was 1.2 ng/ml; The within run CV was 5.4%-10.6%, the between run CV was 7.8%- 18.5% . The positive rates of urinary ⅣC(28.3%) and serum ⅣC(31.7%) were obviously higher in group A than that in control group( P < 0.01 ); The positive rate of urinary ⅣC(63.2%) was significantly higher in group C (18 DM patients were complicated by DN) than that in group A (28.3%) ( P < 0.01 ). The positive rate of serum ⅣC had no difference between two groups(36.8%, 31.7%) ( P >0.05). Conclusions We have developed a sensitive and simple ELISA for ⅣC. The increased urinary ⅣC level in DM patients could be a sensitive and specific marker of DN.