Sensitive and specific measurement of alpha₁-antitrypsin activity with an elastase complex formation immunosorbent...

Functionally active alpha₁-antitrypsin (AAT) is measured predominantly with a chromogenic elastase inhibition assay, where the concentration of AAT activity inversely correlates with the levels of residual elastase. This standard assay has moderate sensitivity as it hardly allows the measurement of samples containing less than 10 µg of functionally active AAT per mL. To overcome this drawback, we developed a new assay format for the measurement of functionally active AAT, which we termed the elastase complex formation immunosorbent assay (ECFISA). The ECFISA uses plate-bound, still proteolytically active elastase, which attacks functionally active AAT under irreversible formation of a stable stochiometric 1 + 1 complex. This complex is then detected and measured by an anti-AAT peroxidase conjugate. Using three different approaches for the preparation of functionally inactive AAT - heating, oxidation, and complex formation with elastase - we confirmed beyond doubt that the ECFISA exclusively measures functionally active AAT and that these measurements are unimpaired by the presence of high concentrations of functionally inactive AAT. Studies addressing the coating procedure demonstrated that adequate and robust conditions had been defined for this essential first step of the ECFISA. Possible interference caused by the presence of important plasma proteinase inhibitors in the test samples could be excluded for the most abundant inhibitors. Even a 1.5-times molar excess of alpha₂-macroglobulin over AAT was shown to have no impact, which is not the case for a conventional chromogenic activity assay. Functional activities determined with the ECFISA and validated chromogenic elastase inhibition assay matched well with a mean absolute bias of 0.64% calculated for the 25 samples measured. The results of the bioanalytical assay validation complied with the acceptance criteria for ligand-binding assays as given by current guidelines on validation of bioanalytical methods. Overall, the data obtained demonstrated the ECFISA as an accurate, precise, selective, and very sensitive method for AAT activity measurement at low levels previously inaccessible for direct measurement.