Kim Sunghee, Zhang Lurong
Department of Biological Sciences, University of Alabama, Tuscaloosa, AL 35487, USA.
J Immunol Methods. 2005 Mar;298(1-2):1-8. doi: 10.1016/j.jim.2004.12.019.
TL1A, a TNF-like ligand, mediates signaling via its cognate receptor DR3, a death receptor whose activation was known to induce both death and survival factors. TL1A, like TNF, is also presumed to circulate as a homotrimeric soluble form. To identify soluble TL1A in the immune system, we have developed a quantitative ELISA by pairing a monoclonal antibody (MAb) with a biotinylated anti-TL1A polyclonal antibody (PAb) as capture and detection antibodies, respectively. The assay had a detection limit of 32 pg/ml. Overnight culture of human umbilical vein endothelial cells (HUVEC) expressed up to 160 pg/ml of TL1A, and that proinflammatory cytokines, IL-1 and TNF, significantly increased TL1A mRNA and soluble protein up to several folds in contrast to IL-6 and IL-11, which induced neither protein nor mRNA in the cells. IL-1 appeared to be a better stimulator of TL1A than TNF-alpha, and the induction of soluble TL1A in HUVEC in response to IL-1 was dose and time-dependent. We have also purified soluble TL1A from a large volume of IL-1 stimulated HUVEC conditioned medium using an anti-TL1A PAb-coupled affinity column. The protein eluted from the column was further reacted with anti-TL1A MAbs in Western blot: 30-kDa and 32-kDa under non-reducing and reducing conditions, respectively. Taken together, our results indicated that ELISA might be useful in studying soluble TL1A regulation in certain inflammatory conditions.
TL1A是一种肿瘤坏死因子样配体，通过其同源受体DR3介导信号传导，DR3是一种死亡受体，已知其激活可诱导死亡和存活因子。与肿瘤坏死因子一样，TL1A也被认为以同源三聚体可溶性形式循环。为了在免疫系统中鉴定可溶性TL1A，我们开发了一种定量酶联免疫吸附测定法（ELISA），分别将单克隆抗体（MAb）与生物素化抗TL1A多克隆抗体（PAb）配对作为捕获抗体和检测抗体。该测定法的检测限为32 pg/ml。人脐静脉内皮细胞（HUVEC）过夜培养表达的TL1A高达160 pg/ml，促炎细胞因子白细胞介素-1（IL-1）和肿瘤坏死因子可使TL1A mRNA和可溶性蛋白显著增加数倍，而白细胞介素-6（IL-6）和白细胞介素-11在细胞中既不诱导蛋白也不诱导mRNA。IL-1似乎比肿瘤坏死因子-α（TNF-α）更能刺激TL1A，并且HUVEC中可溶性TL1A对IL-1的诱导呈剂量和时间依赖性。我们还使用抗TL1A PAb偶联的亲和柱从大量IL-1刺激的HUVEC条件培养基中纯化了可溶性TL1A。从柱上洗脱的蛋白在蛋白质印迹中进一步与抗TL1A MAb反应：在非还原和还原条件下分别为30 kDa和32 kDa。综上所述，我们的结果表明ELISA可能有助于研究某些炎症条件下可溶性TL1A的调节。
