糖基化终产物抗血清的制备及应用

The preparation and application of antiserum against advanced glycated end products

目的 以糖基化终产物（ Ａ Ｇ Ｅｓ） 的免疫学检测方法，检测血清或组织 Ａ Ｇ Ｅｓ 水平，为深入研究糖尿病并发症的机制打下基础。方法 用纯化的 Ａ Ｇ Ｅｓ牛血清白蛋白（ Ｂ Ｓ Ａ） 免疫新西兰白兔，获取 Ａ Ｇ Ｅｓ 抗血清，用酶联免疫吸附法（ Ｅ Ｌ Ｉ Ｓ Ａ） 测定其效价，鉴定特异性；并用以检测糖尿病小鼠和糖尿病患者血清 Ａ Ｇ Ｅｓ 水平。结果 抗血清效价达１∶１０５ ，不同蛋白、氨基酸与不同还原糖孵育而形成的 Ａ Ｇ Ｅｓ 均能与该抗血清发生交叉免疫反应；非糖基化蛋白质或氨基酸、脱氧吗啉果糖（ Ａｍａｄｏｒｉ 产物） 不能竞争性地抑制 Ａ Ｇ Ｅｓ人血清白蛋白与抗体结合；糖尿病小鼠、糖尿病患者及尿毒症患者血清 Ａ Ｇ Ｅｓ水平均显著高于其对照组，差异有显著和非常显著意义（ Ｐ＜ ００５ ～００００１） 。结论 成功地获得了高效、特异的抗 Ａ Ｇ Ｅｓ 抗体，用于临床 Ａ Ｇ Ｅｓ 血清学检测；结果提示， Ａ Ｇ Ｅｓ 可能在糖尿病血管病变的发生发展中起重要作用。

Objectives To prepare polyclonal antiserum against advanced glycation end products (AGEs) and establish an ELISA system to measure serum or tissue AGEs.Methods The antiserum was obtained by immunizing New Zealand white rabbits against purified AGEs-BSA, and titered by a non-competitive ELISA. A competitive ELISA was established to identify the specificity of the antibody and determine the serum samples from patients and experimental animals.Results The titer of antiserum was 1∶10 5. AGEs formed by incubation of proteins and amino acids with reducing sugars had cross reactivity with the obtained antiserum. However, unmodified proteins and amino acids, Amadori product, BSA incubated with glucose and aminoguanidine simultaneously failed to compete with AGE HSA binding the antiserum. Furthermore, serum AGEs levels were significantly elevated in the patients with DM, ESRD and diabetic mice ( P <0.05～0.0001) compared with the controls.Conclusion We have successfully obtained a specific antiserum against AGEs and established a method to measure the serum AGEs levels by ELISA.