Quantification and detection of DcR3, a decoy receptor in TNFR family

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.