RECOMBINANT PHAGE ANTIBODIES AS IMMUNOASSAY PROBES FOR LENS PROTEIN MODIFICATION

超过1000万美国人患有各种类型的视觉障碍。 损伤 视力残疾估计花费400亿美元 每年为视力障碍者提供的护理和服务 受损 白内障是美国第二大致盲原因 据估计，46%的人年龄在 75-85岁的人患有这种疾病导致的视觉障碍。 白内障是最常见的一种白内障， 由于负责维护的机制存在某些缺陷， 透镜透明度。 这些机制知之甚少，但以前 研究表明，透镜蛋白质如α晶体蛋白发挥一些作用， 作用 透镜蛋白经历翻译后修饰，包括 磷酸化、脱酰胺、非酶糖基化和脂质 过氧化作用 这些修饰带来了结构，构象 和功能变化，这些变化与 某些类型的白内障。 实验的广泛目标 该提案中描述的是开发免疫测定试剂， 可以用来探测化学变化的程序， 透镜蛋白。 具体而言，糖基化将在纯化的α晶体蛋白中诱导 和透镜质膜和抗体将产生在细菌 使用重组噬菌体针对修饰的蛋白质进行培养 抗体系统。 还将产生针对磷酸丝氨酸的抗体， 磷酸苏氨酸和丙二醛。 竞争性酶联 将使用免疫吸附测定来确定噬菌体的特异性 然后将其用于探测被修饰的透镜蛋白的抗体 糖基化、磷酸化和脂质过氧化。 检测透镜蛋白中化学修饰的能力将 进一步了解白内障的化学基础， 最终有助于开发药物， 白内障发生的进展。

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.