Nagai Ryoji, Fujiwara Yukio, Mera Katsumi, Motomura Keita, Iwao Yasunori, Tsurushima Keiichiro, Nagai Mime, Takeo Kazuhiro, Yoshitomi Makiko, Otagiri Masaki, Ikeda Tsuyoshi
Department of Medical Biochemistry, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto 860-8556, Japan.
Ann N Y Acad Sci. 2008 Apr;1126:38-41. doi: 10.1196/annals.1433.001. Epub 2007 Dec 13.
Polyclonal and monoclonal antibodies have been widely applied to demonstrate the presence of advanced glycation end products (AGEs) in vivo. However, our previous study showed that monoclonal anti-AGE antibody (6D12) and polyclonal anti-N epsilon-(carboxymethyl)lysine (CML) antibody recognize not only CML but also N epsilon-(carboxyethyl)lysine (CEL), thus indicating that we should pay attention to the specificity of the antibodies. As a result, we prepared specific monoclonal antibodies against CML, CEL, N omega-(carboxymethyl)arginine (CMA), and S-(carboxymethyl)cysteine (CMC). Our immunochemical study using anti-CMA antibody demonstrated that the CMA content increased in a time-dependent manner when collagen was incubated with glucose, indicating that immunological quantification using the specific antibody is especially useful for measuring an acid-labile AGE structure, such as CMA. Monoclonal antibody is also applied to identify a novel biological marker in pathological lesions. We prepared antibody libraries against proteins modified with aldehydes, such as glyoxal, methylglyoxal, and glycolaldehyde (GA), and one antibody, GA5, which specifically reacts with the GA-modified protein that is recognized in human atherosclerotic lesions. Following successive high-performance liquid chromatography purification, the GA5-reactive compound was isolated and its chemical structure was found to be 3-hydroxy-4-hydroxymethyl-1-(5-amino-5-carboxypentyl) pyridinium cation, which was named GA-pyridine. Taken together, these results demonstrate that a specific antibody is a powerful tool for analyzing novel biomarkers, formation pathways, and the efficacy of AGE inhibitors.
多克隆抗体和单克隆抗体已被广泛应用于证明体内晚期糖基化终产物（AGEs）的存在。然而，我们之前的研究表明，单克隆抗AGE抗体（6D12）和多克隆抗Nε-（羧甲基）赖氨酸（CML）抗体不仅能识别CML，还能识别Nε-（羧乙基）赖氨酸（CEL），因此表明我们应注意抗体的特异性。结果，我们制备了针对CML、CEL、Nω-（羧甲基）精氨酸（CMA）和S-（羧甲基）半胱氨酸（CMC）的特异性单克隆抗体。我们使用抗CMA抗体进行的免疫化学研究表明，当胶原蛋白与葡萄糖孵育时，CMA含量呈时间依赖性增加，这表明使用特异性抗体进行免疫定量对于测量酸性不稳定的AGE结构（如CMA）特别有用。单克隆抗体也被用于识别病理病变中的新型生物标志物。我们制备了针对用醛修饰的蛋白质（如乙二醛、甲基乙二醛和乙醇醛（GA））的抗体文库，以及一种抗体GA5，它能与在人类动脉粥样硬化病变中识别的GA修饰蛋白特异性反应。经过连续的高效液相色谱纯化，分离出了GA5反应性化合物，其化学结构被发现是3-羟基-4-羟甲基-1-（5-氨基-5-羧基戊基）吡啶阳离子，命名为GA-吡啶。综上所述，这些结果表明特异性抗体是分析新型生物标志物、形成途径和AGE抑制剂疗效的有力工具。
