In the Phase I of the SBIR, we developed antibody probes to synthetic peptides deduced from the sequence of beta2AR. Additionally, we expressed the complete sequence of beta2AR in three expression vectors: pEXB beta- galactosidase fusion protein), pINHB (a """"""""secretory"""""""" vector) and in human kidney cell line (293). The receptor expression and antibody reactivity have been extensively validated using binding studies, photolabeling, EIA, immunoblotting, immunoprecipitation and flow cytometry methods. In the present proposal, these materials are utilized to assemble the following products/services. Two assay systems will be developed for autoantibody screening. The recombinant receptors will be utilized as antigens on Western blots to screen for autoantibodies to the receptor which might recognize linear epitopes. A complete panel of positive and negative controls will assist in definitive identification of the reactive band as the receptor. Purified beta2AR protein will be utilized in an EIA to capture autoantibodies that react with discontinuous epitopes which may not be picked up in other assay systems. A flow cytometry-based assay is also being developed using whole cell expressing the BAR to. screen for autoantibodies. Quantitation of the receptor on the cell surface will be carried out using EIA and flow cytometric methods which employ monoclonal and polyclonal antibodies to synthetic peptides and to the purified receptor. Clinical validation of the assays will include screening for autoantibodies in Chagas disease, dilated cardiomyopathy and subsets of asthma patients and monitoring of altered receptor levels associated with disease states or beta agonist treatment as occurs in asthma.
