Immunochemical recognition of A2E, a pigment in the lipofuscin of retinal pigment epithelial cells
Immunochemical recognition of A2E, a pigment in the lipofuscin of retinal pigment epithelial cells
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DOI：
10.1073/pnas.0706806104
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发表时间：
2007-09-11
影响因子：
11.1
通讯作者：
Sparrow, Janet R.
中科院分区：
文献类型：
--
作者：
Abeywickrama, Chandirna;Matsuda, Hiroko;Sparrow, Janet R.
随着年龄的增长，自发荧光脂褐质色素 A2E 在视网膜色素上皮细胞中积累，在某些视网膜疾病中尤其丰富。为了生成识别这种吡啶鎓双维A酸分子的多克隆抗体，我们用牛血清白蛋白 (BSA) 缀合物对兔子进行免疫，其中该蛋白质通过其吡啶鎓乙醇胺部分与 A2E 分子连接。通过基质辅助激光解吸电离/飞行时间质谱 (MALDI-TOF MS) 对 A2E-BSA 缀合物进行分析表明存在五个与 BSA 共价连接的完整 A2E 分子，因此认为它是适合免疫的抗原。通过免疫细胞化学染色，兔多克隆抗体识别了培养细胞中积累的 A2E，而斑点印迹分析显示与 A2E 和 A2E-兔血清白蛋白 (A2E-RSA) 缀合物结合，但与各种类维生素A没有交叉反应。免疫前血清无反应性。在荧光光谱研究中，抗体-A2E 结合通过荧光增加和发射最大值蓝移来证明，这与抗体结合时 A2E 环境的变化一致。抗体结合时荧光发射的变化可以反映几个过程，包括对反式顺式异构化和光激发 A2E 的系统间交叉的限制。
The autofluorescent lipofuscin pigment A2E accumulates in retinal pigment epithelial cells with age and is particularly abundant in some retinal disorders. To generate a polyclonal antibody that recognizes this pyridiniurn bisretinoid molecule, we immunized rabbits with bovine serum albumin (BSA) conjugates in which the protein was linked to the A2E molecule via its pyridinium ethanolamine moiety. Analysis by matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS)of the A2E-BSA conjugate indicated the presence of five intact A2E molecules covalently linked to BSA, thus deeming it a suitable antigen for immunization. By immunocytochemical staining, the rabbit polyclonal antibody recognized A2E that had accumulated in cultured cells, whereas dot-blot analysis revealed binding to both A2E and A2E-rabbit serum albumin (A2E-RSA) conjugate but no cross-reactivity with various retinoids. Preimmune serum was non-reactive. In fluorescence spectroscopy studies, antibody-A2E binding was evidenced by a fluorescence increase and by a blue-shift in the emission maximum consistent with a change in A2E milieu upon antibody binding. The changes in fluorescence emission upon antibody binding could reflect several processes including restrictions on trans-cis isomerization and intersystem crossing of photoexcited A2E.