针对晚期氧化蛋白产物的抗体制备与应用

Preparation and application of antibodies against advanced oxidation protein products

目的:制备特异性识别晚期氧化蛋白产物(Advanced oxidation protein products,AOPP)的多克隆抗体,为研究AOPP的致病机理及与临床病情相关性提供有效工具。方法:用次氯酸氧化家兔血清白蛋白以获得AOPP-RSA,以此为免疫原免疫家兔,亲和层析纯化免疫血清,ELISA鉴定多克隆抗体的效价及特异性,Western blot检测慢性肾脏病(chronic kidney disease,CKD)患者血浆中AOPP,免疫组织化学方法检测肾组织中AOPP分布与定位。结果:获得效价达10-6的抗AOPP免疫血清,纯化抗体可特异地与不同种属来源的氧化修饰白蛋白结合,而与正常白蛋白及糖基化蛋白终产物无交叉反应。该抗体可识别人血浆中的AOPP,可特异地与大鼠CKD模型及各种炎症性CKD患者肾组织成份反应。结论:成功制备了可与不同种属AOPP结合的特异性多克隆抗体,并首次用于检测存在于CKD患者血浆、肾组织及模型动物肾组织中的AOPP,为深入研究AOPP的病理作用及其在组织定位提供了有效工具。

Objective：To prepare the polyclonal antibodies against advanced oxidation protein products （AOPP）, and to provide an effective agent for research on the pathogenesis of AOPP and assess exaetly the relationship between AOPP and relative diseases. Methods： AOPP-rabbit serum albumin （AOPP-RSA） was prepared by treating RSA with hypochloric acid. The rabbit anti-AOPP-RSA polyclonal antibodies were generated and purified by affinity chrolnatogyaphy. The titers and the specificity of the antibodies were measured by ELISA. The plasma AOPP and the localization of AOPP in nephridial tissues of some patients with chronic kidney disease （CKD） were determined using rabbit anti- AOPP-RSA. Results：Titers of the antiboches were 10- 6. Purified antibedies reacted specifically with oxidized albumin from different genus, but could not react with normal albumin and glycosylated albumin. The high level of AOPP in plasma from CKD patients was confirmed by Western blot. The antibedies could be used to immunostain AOPP deposition in different regions of kidney lissues from both CKD patients and CKD rat models. Conclusion：We successfully generate rabbit anti-AOPP polyclonal antibedies with high titers and striking specificity. The presence of plasma AOPP and localizations of AOPP in kidney tissues of CKD patients can be demonstrated using the antibodies. The development of anti- AOPP polyclonal antibodies may provide a new tool to explore the pathogensis of AOPP and assess exaetly the relationship between AOPP and relative diseases.