A modified double antibody sandwich enzyme-linked immunosorbent assay for measurement of alpha-1-antitrypsin in biologic fluids

Abstract

Alpha-1-antitrypsin ( α 1 AT) is the major protease inhibitor in human serum, and plays an important role protecting tissues from potentially harmful enzymes released during inflammatory reactions. Proteolytic enzymes such as leukocyte elastase are usually released and inactivated locally at the site of inflammation, so there has been much recent interest in measuring local α 1AT concentrations in biologic fluids. In this study, we developed a modified double antibody sandwich enzyme-linked immunosorbent assay (ELISA) and used it to measure α 1AT concentrations in several biologic fluids. The assay was sensitive to as little as 20 ng/ml of α 1AT. Serum concentration measured by the ELISA correlated well with levels determined by radial immunodiffusion (RID) and the ELISA was far more sensitive than RID. In synovial fluid, higher concentrations determined by the ELISA compared with RID probably reflect interference of diffusion of α 1AT in the RID gel by hyaluronic acid and protease-inhibitor complexes. Synovial fluid did not interfere with the detection of added α 1AT by ELISA, but it did reduce the amount detected by RID by about 30% in 2 fluids. In saliva, α 1AT concentrations of less than 1 μg/ml were easily quantified. Bronchoalveolar lavage fluids have been extensively studied because of the important role of α 1AT in pulmonary inflammatory processes. We found concentrations of 1–3 μg/ml in most samples with our assay. These levels were comparable to those previously reported with assays that required up to 50-fold concentration of the fluid. Neither saliva nor bronchoalveolar fluid significantly interfered with detection by ELISA of added α 1AT. This modified double antibody sandwich ELISA may have broad applications for studies of the role of α 1AT in health disease.