RECOMBINANT PHAGE ANTIBODIES AS IMMUNOASSAY PROBES FOR LENS PROTEIN MODIFICATION

1000多万美国人患有各种类型的视觉障碍 减损。据估计，视力障碍花费400亿美元。 每年为视障人士提供护理和服务 受伤了。白内障是美国第二大致盲原因 据估计，46%的年龄在 75-85岁的人患有这种疾病引起的视力障碍。 老年性白内障是这种疾病最常见的形式，据信 由负责维护的机制中的某些缺陷造成 镜头透明度。人们对这些机制知之甚少，但之前 研究表明，晶状体蛋白，如α晶体蛋白，起到了一定的作用。 角色。晶状体蛋白质经历翻译后修饰，包括 磷酸化、脱酰胺化、非酶糖基化和脂质 过氧化。这些修饰带来了结构、构象 和功能改变，已被牵连在发病机制中 某些类型的白内障。这些实验的总体目标是 在这项建议中描述的是开发免疫分析试剂和 可以用来探测发生在 晶状体蛋白质。 具体地说，在纯化的α晶体蛋白中将诱导糖基化 晶状体质膜和抗体会在细菌中产生 利用重组噬菌体培养抗修饰蛋白 抗体系统。也会产生针对磷酸丝氨酸的抗体， 磷酸苏氨酸和丙二醛。竞争性酶连锁 免疫吸附试验将用于确定噬菌体的特异性 抗体将被用来探测经过修饰的晶体蛋白 糖基化、磷酸化和脂质过氧化。 检测晶状体蛋白质中化学修饰的能力将 进一步了解白内障的化学基础，并可能 最终有助于开发可用于逆转或延缓的药物 白内障发病机制的研究进展。

More than 10 million Americans suffer from various types of visual impairment. Visual disabilities cost an estimated $40 billion dollars annually for the care and services provided to people who are visually impaired. Cataract is the second leading cause of blindness in the United States and it has been estimated that 46% of persons between the ages of 75-85 years suffer from visual disorders resulting from this disease. Age-related cataract, the most common form of the disease, is believed to result from certain defects in the mechanisms responsible for maintaining lens transparency. These mechanisms are poorly understood but previous studies have shown that lens proteins such as alpha crystallins play some role. Lens proteins undergo posttranslational modifications including phosphorylation, deamidation, non-enzymatic glycosylation and lipid peroxidation. These modifications bring about structural, conformational and functional changes which have been implicated in the pathogenesis of certain forms of cataract. The broad objective of the experiments described in this proposal is to develop immunoassay reagents and procedures that can be used to probe the chemical changes that occur in lens proteins. Specifically, glycosylation will be induced in purified alpha crystallin and lens plasma membrane and antibodies will be produced in bacterial cultures against the modified proteins using the Recombinant Phage Antibody system. Antibodies will also be produced against phosphoserine, phosphothreonine and malondialdehyde. Competitive Enzyme Linked Immunosorbent Assay will be used to determine the specificity of the phage antibodies which will then be used to probe lens proteins modified by glycosylation, phosphorylation and lipid peroxidation. The ability to detect the chemical modifications in lens protein will further the understanding of the chemical basis of cataract and may ultimately help in developing drugs that can be used to reverse or retard the progress of cataractogenesis.