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一种对晚期糖基化终产物具有广泛表位特异性的多克隆抗体的研发以及这些表位在衰老眼睛的布鲁赫膜中的定位。

Development of a polyclonal antibody with broad epitope specificity for advanced glycation endproducts and localization of these epitopes in Bruch's membrane of the aging eye.

作者信息

Farboud B, Aotaki-Keen A, Miyata T, Hjelmeland L M, Handa J T

机构信息

Departments of Ophthalmology, UC Davis, Davis, CA, 95616-8794, USA.

出版信息

Mol Vis. 1999 Jul 14;5:11.

Abstract

PURPOSE

To develop an antibody that recognizes a variety of advanced glycation endproduct (AGE) epitopes.

METHODS

Glycolaldehyde was used to modify bovine serum albumin and HPLC analysis was used to measure pentosidine formation as an indicator of AGE formation. A polyclonal anti-AGE antibody was synthesized by injecting glycolaldehyde-incubated keyhole limpet hemocyanin into rabbits, affinity purified using AGE modified bovine serum albumin coupled to an affinity resin column, and characterized by immunoblot analysis.

RESULTS

HPLC analysis of glycolaldehyde treated bovine serum albumin detected high levels of pentosidine formation, suggesting that glycolaldehyde is a potent precursor for pentosidine. By immunoblot analysis, our antibody recognized carboxymethyllysine and pentosidine, two well-characterized AGEs, as well as other AGE epitopes. Immunohistochemical evaluation showed evidence of AGEs in Bruch's membrane (including basal laminar deposits and drusen), choroidal extracellular matrix, and vessel walls in an 82 year old nondiabetic globe. A similar staining pattern was observed in an age-matched diabetic control. In contrast, no staining was seen with the antibody in a 20 month old nondiabetic globe.

CONCLUSIONS

A unique anti-AGE antibody was synthesized that recognizes a variety of AGE epitopes including carboxymethyllysine and pentosidine. Its best use might be in broad surveys of the age-dependent accumulation of a large number of AGE epitopes that might not be revealed by antibodies to pentosidine or CML.

摘要

目的

开发一种能识别多种晚期糖基化终产物(AGE)表位的抗体。

方法

用乙醇醛修饰牛血清白蛋白,并采用高效液相色谱分析来测定戊糖苷的形成,以此作为AGE形成的指标。通过将经乙醇醛孵育的钥孔血蓝蛋白注射到兔子体内来合成多克隆抗AGE抗体,使用与亲和树脂柱偶联的AGE修饰牛血清白蛋白进行亲和纯化,并通过免疫印迹分析对其进行表征。

结果

对经乙醇醛处理的牛血清白蛋白进行高效液相色谱分析,检测到高水平的戊糖苷形成,表明乙醇醛是戊糖苷的有效前体。通过免疫印迹分析,我们的抗体识别羧甲基赖氨酸和戊糖苷这两种特征明确的AGEs以及其他AGE表位。免疫组织化学评估显示,在一位82岁非糖尿病患者的眼球中,布鲁赫膜(包括基底膜沉积物和玻璃膜疣)、脉络膜细胞外基质和血管壁中有AGEs存在的证据。在年龄匹配的糖尿病对照中观察到类似的染色模式。相比之下,在一个20个月大的非糖尿病眼球中,该抗体未显示染色。

结论

合成了一种独特的抗AGE抗体,它能识别多种AGE表位,包括羧甲基赖氨酸和戊糖苷。其最佳用途可能是广泛调查大量AGE表位的年龄依赖性积累情况,而这些情况可能无法通过针对戊糖苷或CML的抗体揭示出来。

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