Nagy Gergely, Szekeres György, Kvell Krisztián, Berki Tímea, Németh Péter
Department of Immunology and Biotechnology, University of Pécs, Faculty of Medicine, Pécs, H-7643, Hungary.
Pathol Oncol Res. 2002;8(2):115-24. doi: 10.1007/BF03033720.
Recent studies have discovered the existence of water-channel molecules, the so called aquaporins (AQP) presumably involved in active, ATP dependent water transport between the intracellular and extracellular compartments. Both genetic and protein sequences and structures of the AQPs are known and crystallographic analyses of some members of the AQP family have been performed. Specific antibodies are required to examine their histological locations and analyse their roles in physiological and pathological pathways of water transportation and osmotic regulation. Until recently some polyclonal antibodies have been developed against certain members of the AQP family. However, to date highly specific monoclonal antibodies against aquaporins do not exist. We have developed a monoclonal antibody family against the aquaporin 1 (AQP1) and aquaporin 4 (AQP4) molecules. Well-conserved epitop sequences of AQP1 and AQP4 proteins were selected by computer analysis and their synthetic peptide fragments were used as the antigens of immunisation and the following screening. Antibodies were characterised by immunoserological methods (ELISA, dot-blot and immunoblot), flow cytometry and immunohistochemistry of formaldehyde-fixed and paraffin-embedded tissue samples. RT-PCR tests controlled the specificity of the immune reactions. Our monoclonal antibodies recognised AQP1 and AQP4 in all the techniques mentioned above and apparently they are useful both in various quantitative and qualitative measurements of the expressions of AQP1 and AQP4 in several species (human, rat, mouse, invertebrates, even plants). According to preliminary immunohistochemical studies our monoclonal anti-AQP1 and anti-AQP4 antibodies are appropriate tools of patho-morphological examinations on routine formol-paraffin tissue samples.
最近的研究发现了水通道分子的存在，即所谓的水通道蛋白（AQP），推测其参与细胞内和细胞外区间之间活跃的、依赖ATP的水运输。AQP的基因、蛋白质序列及结构均已明确，并且已经对AQP家族的一些成员进行了晶体学分析。需要特异性抗体来检查它们的组织学定位，并分析它们在水运输和渗透调节的生理及病理途径中的作用。直到最近，已经针对AQP家族的某些成员开发了一些多克隆抗体。然而，迄今为止，尚不存在针对水通道蛋白的高度特异性单克隆抗体。我们已经开发了针对水通道蛋白1（AQP1）和水通道蛋白4（AQP4）分子的单克隆抗体家族。通过计算机分析选择了AQP1和AQP4蛋白保守性良好的表位序列，并将其合成肽片段用作免疫和后续筛选的抗原。通过免疫血清学方法（ELISA、斑点印迹和免疫印迹）、流式细胞术以及对甲醛固定和石蜡包埋组织样本进行免疫组织化学来鉴定抗体。RT-PCR测试控制免疫反应的特异性。我们的单克隆抗体在上述所有技术中均能识别AQP1和AQP4，显然它们可用于对几种物种（人类、大鼠、小鼠、无脊椎动物，甚至植物）中AQP1和AQP4表达的各种定量和定性测量。根据初步的免疫组织化学研究，我们的抗AQP1和抗AQP4单克隆抗体是对常规福尔马林-石蜡组织样本进行病理形态学检查的合适工具。
