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. 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. 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. Announced in January 2021, it represents the first in a new series, a flagship body positioned above the existing A7 and A9 models. Even a 1.5-times molar excess of alpha 2-macroglobulin over AAT was shown to have no impact, which is not the case for a conventional chromogenic activity assay. The Sony Alpha 1, or A1 for short, is a high-end mirrorless camera with a 50 Megapixel full-frame sensor, built-in stabilisation, 30fps electronic bursts and a wide array of video modes including 4k 120p and 8k 30p. Possible interference caused by the presence of important plasma proteinase inhibitors in the test samples could be excluded for the most abundant inhibitors. Studies addressing the coating procedure demonstrated that adequate and robust conditions had been defined for this essential first step of the ECFISA. 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. This complex is then detected and measured by an anti-AAT peroxidase conjugate. The ECFISA uses plate-bound, still proteolytically active elastase, which attacks functionally active AAT under irreversible formation of a stable stochiometric 1 + 1 complex. 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). 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. Functionally active alpha 1-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.
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