RECOMBINANT PHAGE ANTIBODIES AS IMMUNOASSAY PROBES FOR LENS PROTEIN MODIFICATION

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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.