Alpha-1-antitrypsin is the major protease inhibitor in human serum, and plays a vital role in prevention of tissue damage by proteases released during infections and inflammatory processes. Several of the genetic variants of alpha-1-antitrypsin have been associated with disease, including pulmonary disease, neonatal and adult liver disease, renal disease, inflammatory eye disease, and rheumatic diseases. Determination of alpha-1-antitrypsin phenotypes (termed Pi-typing) is therefore of considerable clinical interest. Currently, Pi-typing is done by isoelectric focusing on thin layer polyacrylamide gels, a difficult technique performed by few clinical laboratories. The first objective of the proposed research is to raise monoclonal antibodies, by the technique of Kohler and Milstein, capable of distinguishing the clinically significant genetic variants of alpha-1-antitrypsin: M1, M2, M3, S and Z. Although the structural differences between these variants are slight - in some cases only a single amino acid - the differences should be distinguishable by monoclonal antibodies. The second objective is the development of antibody based methods of Pi-typing, both a radioimmunoassay (RIA) and an enzyme linked immunosorbent assay (ELISA). An antibody-based method would revolutionize Pi-typing, and bring it within the technical repertoire of almost all clinical laboratories. It would circumvent the present difficulty of distinguishing the M subtypes electrophoretically, and would accelerate the investigation of clinical aspects of this important genetic polymorphism. Variant-specific ELISA or RIA techniques could also be used to ask questions about the physiology and function of alpha-1-antitrypsin variants that cannot be addressed with existing methods. The third objective is to use the variant-specific antibodies and assays to conduct several prototype studies illustrating their utility. These studies will use the ability of variant-specific monoclonal antibodies to distinguish between allelic molecules of alpha-1-antitrypsin in bodily fluids - serum, bronchoalveolar lavage, and synovial fluids - of heterozygous individuals. The results may elucidate the reasons for the associations of certain genetic variants with disease.
