ATS 2021 will feature presentations of original research from accepted abstracts. Mini Symposia and Thematic Poster Sessions are abstract based sessions.
Please use the form below to browse scientific abstracts and case reports accepted for ATS 2021. Abstracts presented at the ATS 2021 will be published in the Online Abstract Issue of the American Journal of Respiratory and Critical Care Medicine, Volume 203, May 3, 2021.
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Method Validation for the Measurement of Alpha-1 Antitrypsin Protein in Clinical Samples
Session Title
TP1 - TP001 MECHANISTIC AND TRANSLATIONAL STUDIES IN COPD
Abstract
A1237 - Method Validation for the Measurement of Alpha-1 Antitrypsin Protein in Clinical Samples
Author Block: A. Weber, M. Zimmermann, S. Riedler, E. Minibeck, A. Engelmaier; Pharmaceutical Science, Baxalta Innovations GmbH, a Takeda company, Vienna, Austria.
RATIONALE: The reliability of analytical methods applied for the testing of the clinical samples determines the quality of clinical studies. Validation following for example the European Medicines Agency (EMA) guideline for bioanalytical assay validation is a prerequisite for clinical assays. Alpha-1 antitrypsin (AAT) protein measurement, carried out in different clinical specimens, is an important part of the assay panel for clinical studies addressing AAT deficiency. Here, we present the validation data obtained for two immunological methods to measure AAT protein. METHODS: A nephelometric method (Siemens BN Prospec) and an in-house developed ELISA, using paired commercially available polyclonal antibodies, were applied. Validation samples with AAT concentrations ranging from 0.05 to 2.3 mg/mL in human plasma matrix and from 0.2 to 10 µg/mL in a low protein content matrix were used to determine accuracy, precision (intra- and inter-run) and linearity. Assay calibration was verified by using the international standard for AAT protein ERM-DA470k and the WHO standard 05/612. Sample stability was investigated for repeated freezing-thawing and at room temperature. RESULTS: Both AAT protein assays met all EMA acceptance criteria. For the nephelometric method, mean recoveries of spiked AAT ranged from 100.7% to 107.5%. All individual values were within a 100 ± 14% range. The AAT concentration determined for ERM-DA470k represented 91.5% of its assigned value. Intra- and inter-run precision (n=6), expressed as relative standard deviations, did not exceed 1.8% and 6.5%, respectively. The assay’s lower limit of quantification (LLOQ) was 0.05 mg/mL, thus qualifying the assay to be used as a screening test. The more sensitive ELISA demonstrated mean AAT recoveries ranging from 88.9% to 99.2% with all individual values within a 100 ± 20% range. The mean AAT concentration determined for the AAT WHO standard represented 100.1% of its assigned value. Intra- and inter-run precision did not exceed 3.3% and 10.5%, respectively. The ELISA’s LLOQ was 0.03 µg/mL. Sample stability was shown at low AAT concentrations after repeated freezing thawing for up to three times and at room temperature for up to 4 h. CONCLUSIONS: Both assays for the measurement of AAT protein met the acceptance criteria defined by the EMA guideline for bioanalytical assay validation. This data qualified the assays to be used for the measurement of clinical samples.
Author Block: A. Weber, M. Zimmermann, S. Riedler, E. Minibeck, A. Engelmaier; Pharmaceutical Science, Baxalta Innovations GmbH, a Takeda company, Vienna, Austria.
RATIONALE: The reliability of analytical methods applied for the testing of the clinical samples determines the quality of clinical studies. Validation following for example the European Medicines Agency (EMA) guideline for bioanalytical assay validation is a prerequisite for clinical assays. Alpha-1 antitrypsin (AAT) protein measurement, carried out in different clinical specimens, is an important part of the assay panel for clinical studies addressing AAT deficiency. Here, we present the validation data obtained for two immunological methods to measure AAT protein. METHODS: A nephelometric method (Siemens BN Prospec) and an in-house developed ELISA, using paired commercially available polyclonal antibodies, were applied. Validation samples with AAT concentrations ranging from 0.05 to 2.3 mg/mL in human plasma matrix and from 0.2 to 10 µg/mL in a low protein content matrix were used to determine accuracy, precision (intra- and inter-run) and linearity. Assay calibration was verified by using the international standard for AAT protein ERM-DA470k and the WHO standard 05/612. Sample stability was investigated for repeated freezing-thawing and at room temperature. RESULTS: Both AAT protein assays met all EMA acceptance criteria. For the nephelometric method, mean recoveries of spiked AAT ranged from 100.7% to 107.5%. All individual values were within a 100 ± 14% range. The AAT concentration determined for ERM-DA470k represented 91.5% of its assigned value. Intra- and inter-run precision (n=6), expressed as relative standard deviations, did not exceed 1.8% and 6.5%, respectively. The assay’s lower limit of quantification (LLOQ) was 0.05 mg/mL, thus qualifying the assay to be used as a screening test. The more sensitive ELISA demonstrated mean AAT recoveries ranging from 88.9% to 99.2% with all individual values within a 100 ± 20% range. The mean AAT concentration determined for the AAT WHO standard represented 100.1% of its assigned value. Intra- and inter-run precision did not exceed 3.3% and 10.5%, respectively. The ELISA’s LLOQ was 0.03 µg/mL. Sample stability was shown at low AAT concentrations after repeated freezing thawing for up to three times and at room temperature for up to 4 h. CONCLUSIONS: Both assays for the measurement of AAT protein met the acceptance criteria defined by the EMA guideline for bioanalytical assay validation. This data qualified the assays to be used for the measurement of clinical samples.
