Bioanalytical Method Validations of Three Alpha1-Antitrypsin Measurement Methods Required for Clinical Sample Analysis

Background/Objectives: The quality of clinical studies is largely determined by the bi-oanalytical methods used for testing study samples. Rigorous assay validation following the criteria defined for example by the European Medicines Agency guideline for bio-analytical method validation is a prerequisite for such assays. Alpha1-antitrypsin (AAT) measurement, i.e., the specific measurement of AAT protein and its associated elastase inhibitory activity, is an integral part of assay panels for clinical studies addressing AAT deficiency. Specifically, AAT must be measured in the matrix of citrated human plasma as well as in diluted solutions with high salt concentrations obtained through bron-choalveolar lavage (BAL). Sensitive and selective measurement methods are required as BAL has a low level of AAT. Methods: We present the validation data obtained for three AAT measurement methods. Two of them, nephelometry and the enzyme-linked im-munosorbent assay, which clearly differ in their sensitivity, provide AAT protein con-centrations, while the highly sensitive elastase complex formation immunosorbent as-say specifically measures the inhibitory activity of AAT against its pivotal target pro-tease neutrophil elastase. Using samples with relevant AAT concentrations, we ad-dressed the assay characteristics accuracy, precision, linearity, selectivity, specificity, limit of quantification and short-term analyte stability Results: Overall, the three methods demonstrated low total errors even at low analyte concentrations, adequate linearity over the required assay range and acceptable selectivity and specificity, while the short-time stability of the analyte could be shown. Conclusions: All three AAT measurement methods met the acceptance criteria defined by current guidelines on bioanalytical assay validation, which qualifies them to be used for the measurement of clinical samples.