Quantification and detection of DcR3, a decoy receptor in TNFR family
Quantification and detection of DcR3, a decoy receptor in TNFR family
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DOI：
10.1016/j.jim.2003.11.004
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发表时间：
2004-02-01
影响因子：
2.2
通讯作者：
Kim, S
中科院分区：
文献类型：
--
作者：
Chen, JG;Zhang, LR;Kim, S
可溶性诱饵受体DcR3属于肿瘤坏死因子受体（TNFR）家族，已知该受体可结合三种TNF家族配体，即Fas配体（FasL）、LIGHT和TL1A。为了帮助我们理解DcR3在免疫系统中的作用，我们开发了定量酶联免疫吸附试验（ELISA）来检测人体生物体液中的可溶性DcR3。选择识别DcR3分子上不同表位的两种单克隆抗体MD3E2和MD3B1分别作为捕获抗体和检测抗体，在ELISA中配对。检测限为36 pg/ml，动态范围为0.25 ~ 16 ng/ml。细胞培养上清回收率为91 ~ 112%，人血清回收率为90 ~ 108%。检测内CVs和检测间CVs分别小于7.2%和11.2%。在一组被测试的细胞系中，结肠癌细胞系SW480分泌的DcR3水平最高，为3.2 ng/ml。从人类血清样本的筛选中，我们发现39名健康个体，59名肿瘤患者和46名肾功能衰竭患者平均表达（平均+/- sd）。分别为0.56 +/- 0.52、2.3 +/- 1.6和4.6 +/- 2.8 ng/ml DcR3。为了证实ELISA的特异性，我们从SW480细胞培养上清中纯化了天然DcR3，并通过免疫沉淀在临床血清中鉴定了一种天然DcR3。综上所述，我们的数据表明，本研究开发的ELISA对定量多种人体生物体液中可溶性DcR3具有特异性和敏感性，该检测方法将有助于研究DcR3在某些病理生理条件下的调节。(C) 2004 Elsevier B.V.版权所有
A soluble decoy receptor, DcR3, belongs to the tumor necrosis factor receptor (TNFR) family, and this receptor is known to bind to three TNF family ligands, namely Fas ligand (FasL), LIGHT, and TL1A. To aid our understating of the role of DcR3 in the immune system, we have developed quantitative enzyme-linked immunosorbent assay (ELISA) to detect soluble DcR3 in human biological fluids. Two monoclonal antibodies, MD3E2 and MD3B1, that recognized different epitopes on the DcR3 molecule were selected as capture and detection antibodies, respectively, to be paired in ELISA. The assay had a detection limit of 36 pg/ml with a dynamic range of 0.25-16 ng/ml. The recovery range was 91-112% for cell culture supernatant and 90-108% for human sera. Intra- and inter-assay CVs were less than 7.2% and 11.2%, respectively. Among a panel of cell lines tested, colon adenocarcinoma cell line, SW480, secreted the highest levels of DcR3 at 3.2 ng/ml. From the screening of human sera samples, we discovered that 39 healthy individuals, 59 tumor patients, and 46 patients with renal failure expressed an average (mean +/- S.D.) 0.56 +/- 0.52, 2.3 +/- 1.6, and 4.6 +/- 2.8 ng/ml DcR3, respectively. To confirm the specificity of ELISA, we have purified native DcR3 from SW480 cell culture supernatants and identified a native DcR3 in a clinical serum by immunoprecipitation. Taken together, our data demonstrated that the ELISA developed in this study was specific and sensitive to quantify soluble DcR3 in a variety of human biological fluids and that the assay would be useful for studying the regulation of DcR3 in certain pathophysiological conditions. (C) 2004 Elsevier B.V. All rights reserved.