Calibration Curves in Quantitative Ligand Binding Assays: Recommendations and Best Practices for Preparation, Design, and Editing of Calibration Curves

Abstract

Abstract.The accuracy of reported sample results is contingent upon the quality of the assay calibration curve, and as such, calibration curves are critical components of ligand binding and other quantitative methods. Regulatory guidance and lead publications have defined many of the requirements for calibration curves which encompass design, acceptance criteria, and selection of a regression model. However, other important aspects such as preparation and editing guidelines have not been addressed by health authorities. The goal of this publication is to answer many of the commonly asked questions and to present a consensus and the shared views of members of the ligand binding assay (LBA) community on topics related to calibration curves with focus on providing recommendations for the preparation and editing of calibration curves.

Quality Controls in Ligand Binding Assays: Recommendations and Best Practices for Preparation, Qualification, Maintenance of Lot to Lot Consistency, and Prevention of Assay Drift

“…Monitor the concentration-response relationship to ensure that calibrator responses and, in particular that of the zero and high calibrators, have not changed from those observed during pre-study validation. This ensures that LLOQ and ULOQ of the assay are primarily intact and their variability consistent with the that observed in validation (1,2,4). Ensure that curve slope and asymptotes are consistent with the variability observed during validation (4).…”

“…Ensure that curve slope and asymptotes are consistent with the variability observed during validation (4). Select replacement calibrator stocks/reference standards with performance characteristics most similar to the existing calibrators.Ensure previously established equivalence criteria are met.Additional recommendations regarding performance of calibration curves can be found in DeSilva et al , Viswanathan et al , and Azadeh et al (1,2,4). …”

“…A recommended approach to mitigate matrix volume issue would be to prepare 2 of the 3 QC levels in a surrogate matrix and only one level in the study matrix. If a surrogate matrix is used, its equivalence to the study matrix should be demonstrated (4). It is preferable to use the same lot of the matrix as used for the preparation of calibrators, to generate QCs to enhance consistency and reproducibility if selectivity has been demonstrated during pre-study validation.…”

Thyroid Hormones-Thyrotropin Interrelationships in Thyroid Dysfunction States

“…As suggested by Bortolotto et al (2015) 15 and Azadeh et al (2018) 16 for the ligand binding assays, the sigmoid equation of 4PL model that supports the non-linear relationship between FT4 and TSH is given by…”

A qPCR Method for AAV Genome Titer with ddPCR-Level of Accuracy and Precision

“…Many analytical assays rely on a standard curve to translate raw signals into reportable values, based on the assumption that test samples resemble the reference standard within the analytical range. 16 Plasmid DNA is widely used as a qPCR standard because of the convenience to produce and characterize. However, it significantly differs from AAV in a number of ways: First, plasmid DNA is double-stranded while AAV genome is single-stranded.…”