Virella Gabriel, Thorpe Suzanne R, Alderson Nathan L, Stephan Elias M, Atchley Daniel, Wagner Francesco, Lopes-Virella Maria F
Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC 92425, USA.
J Lipid Res. 2003 Mar;44(3):487-93. doi: 10.1194/jlr.M200370-JLR200. Epub 2002 Dec 1.
Advanced glycosylation end-products (AGEs) are believed to play a significant role in the development of vascular complications in diabetic patients. One such product, AGE-LDL, has been shown to be immunogenic. In this report, we describe the isolation and characterization of human AGE-LDL antibodies from the sera of seven patients with Type 1 diabetes by affinity chromatography using an immobilized AGE-LDL preparation that contained primarily the AGE N epsilon (carboxymethyl)lysine (CML, 14.6 mmol/mol lysine), and smaller amounts of N epsilon (carboxyethyl)lysine (CEL, 2.7 mmol/mol lysine). The isolated antibodies were predominantly IgG of subclasses 1 and 3, and considered proinflammatory because of their ability to promote Fc gamma R-mediated phagocytosis and to activate complement. We determined dissociation constants (Kd) for the purified antibodies. The average Kd values (4.76 +/- 2.52 x 10(-9) mol/l) indicated that AGE-LDL antibodies are of higher avidity than oxidized LDL antibodies measured previously (Kd = 1.53 +/- 07 x 10(-8) ml/l), but of lower avidity than rabbit polyclonal LDL antibodies (Kd = 9.34 x 10(-11)). Analysis of the apolipoprotein B-rich lipoproteins isolated with polyethylene glycol-precipitated antigen-antibody complexes from the same patients showed the presence of both CML and CEL, thus confirming that these two modifications are recognized by human autoantibodies. A comparative study of the reactivity of purified AGE-LDL antibodies with CML-LDL and CML-serum albumin showed no cross-reactivity.
晚期糖基化终末产物（AGEs）被认为在糖尿病患者血管并发症的发生发展中起重要作用。其中一种产物，AGE-LDL，已被证明具有免疫原性。在本报告中，我们描述了通过亲和色谱法从7名1型糖尿病患者的血清中分离和鉴定人AGE-LDL抗体的过程，该亲和色谱使用的固定化AGE-LDL制剂主要含有AGE Nε（羧甲基）赖氨酸（CML，14.6 mmol/mol赖氨酸）和少量的Nε（羧乙基）赖氨酸（CEL，2.7 mmol/mol赖氨酸）。分离出的抗体主要是1和3亚类的IgG，由于它们能够促进FcγR介导的吞噬作用并激活补体，因此被认为具有促炎作用。我们测定了纯化抗体的解离常数（Kd）。平均Kd值（4.76±2.52×10⁻⁹ mol/l）表明，AGE-LDL抗体的亲和力高于先前测定的氧化LDL抗体（Kd = 1.53±0.7×10⁻⁸ mol/l），但低于兔多克隆LDL抗体（Kd = 9.34×10⁻¹¹）。用聚乙二醇沉淀的抗原-抗体复合物从同一患者中分离出的富含载脂蛋白B的脂蛋白分析显示同时存在CML和CEL，从而证实这两种修饰可被人自身抗体识别。纯化的AGE-LDL抗体与CML-LDL和CML-血清白蛋白反应性的比较研究显示无交叉反应性。
