Blood Transfusion Centre of Slovenia, Ljubljana, Slovenia.
Radiol Oncol. 2013 May 21;47(2):128-37. doi: 10.2478/raon-2013-0026. Print 2013 Jun.
Bilitranslocase (TC 2.A.65.1.1) is a bilirubin-specific membrane transporter, found on absorptive (stomach and intestine) and excretory (kidney and liver) epithelia and in vascular endothelium. Polyclonal antibodies have been raised in rabbits in the past, using a synthetic peptide corresponding to AA65-77 of rat liver bilitranslocase, as an antigen. Affinity-purified antibodies from immune sera have been found to inhibit various membrane transport functions, including the bilirubin uptake into human hepatocytes and the uptake of some flavonoids into human vascular endothelial cells. It was described by means of immunohistochemistry using polyclonal antibodies that bilitranslocase expression is severely down-regulated in clear cell renal carcinoma. The aim of our work was development and characterization of high-affinity, specific mAbs against bilitranslocase, which can be used as a potential diagnostic tool in renal cell carcinoma as well as in a wide variety of biological assays on different human tissues.
Mice were immunized with a multi-antigen peptide corresponding to segment 65-75 of predicted primary structure of the bilitranslocase protein. By a sequence of cloning, immune- and functional tests, we aimed at obtaining a specific monoclonal antibody which recognizes a 37 kDa membrane protein, and influences the transport activity of bilitranslocase.
On the basis of previous results, specific IgM monoclonal antibodies were produced in BALB/c mice, in order to further improve and extend the immunological approach to the study of bilitranslocase in renal cancer cells as well as to develop its potential diagnostics use.
In this article we show an immunological approach, based on newly developed monoclonal antibodies, to a detailed biochemical and functional characterization of a protein whose gene and protein structure is still unknown. We were able to demonstrate our novel mAb as a tumor marker candidate of renal cell carcinoma, which may prove useful in the diagnostic procedures.
胆红素转运蛋白（TC 2.A.65.1.1）是一种胆红素特异性膜转运蛋白，存在于吸收（胃和肠）和排泄（肾和肝）上皮细胞以及血管内皮细胞中。过去，人们使用合成的肽段（对应于大鼠肝胆红素转运蛋白的 AA65-77）作为抗原，在兔子中产生了多克隆抗体。从免疫血清中亲和纯化的抗体已被发现可抑制多种膜转运功能，包括人肝细胞内胆红素的摄取以及人血管内皮细胞内某些类黄酮的摄取。通过使用多克隆抗体进行免疫组织化学描述，发现胆红素转运蛋白在透明细胞肾细胞癌中的表达严重下调。我们的工作目的是开发和鉴定针对胆红素转运蛋白的高亲和力、特异性单克隆抗体，这些抗体可作为肾细胞癌以及各种不同人类组织的生物测定中的潜在诊断工具。
用多抗原肽段免疫小鼠，该肽段对应于胆红素转运蛋白预测的一级结构的 65-75 段。通过一系列克隆、免疫和功能测试，我们旨在获得一种特异性单克隆抗体，该抗体可识别一种 37 kDa 的膜蛋白，并影响胆红素转运蛋白的转运活性。
基于先前的结果，我们在 BALB/c 小鼠中产生了特异性的 IgM 单克隆抗体，以进一步改进和扩展对肾癌细胞中胆红素转运蛋白的免疫研究，并开发其潜在的诊断用途。
本文展示了一种基于新开发的单克隆抗体的免疫学方法，用于对一种蛋白进行详细的生化和功能表征，该蛋白的基因和蛋白结构尚不清楚。我们能够证明我们的新型单克隆抗体作为肾细胞癌的肿瘤标志物候选物，这可能在诊断程序中证明有用。
