A calibration curve is used to know the instrumental response to an analyte and determine the analyte’s concentration in a sample; it is a plot of how the response changes with the analyte concentration. The linear part of this plot can then be used to guess the concentration of a sample of the analyte, by linking its reaction to said concentration. It is made by preparing a set of standard solutions with known concentrations of the analyte.
Calibration standards are used to create a calibration curve, from which the concentrations of analytes in Quality controls and unknown study samples are determined. Each calibration standard should meet the pre-defined criteria for accuracy, precision, and selectivity. The precision and reproducibility of the bioanalytical assays are ensured by monitoring the calibration curve over repeated batch assays.
Ligand Binding Assays (LBAs) and chromatographic assays
There are significant differences between calibration curves in Ligand Binding Assays (LBAs) and chromatographic assays. In chromatographic assays, the calibration curve is prepared by calculating the response from a set of standards where it is usually in a linear function of the concentration.
Unlike LBAs, the instrument response may be directly or inversely linked to the analyte concentration, depending on the assay’s format. LBAs depend on the analyte’s interaction with a binding agent like an antibody or a receptor component; this is in divergence to traditional chromatographic assays. Detecting the analyte is free of its binding to a macromolecule. The dynamic equilibrium nature of protein-protein interaction leads to a non-linear response in LBAs and needs more complex mathematical fitting.
Assay sensitivity
The definition of a clinical trial’s assay sensitivity states the trial’s ability to differentiate between an effective treatment and a less effective or an ineffective one. It is a crucial consideration when designing, conducting, and analyzing a clinical trial. The lack of adequate assay sensitivity has different indications. A trial is neither internally valid nor is proficient in comparing the effectiveness of two interventions.
Analytical sensitivity
Analytical sensitivity is simply the ability of a test to detect a target analyte such as an antibody or antigen, which is typically expressed as the smallest detectable concentration of the analyte in a sample that can accurately be calculated by an assay.
Quality controls
Quality controls are employed to assess the precision and accuracy of an assay and the samples’ stability. Quality control standard (QC) is a spiked sample used to observe a bioanalytical method’s performance and evaluate the integrity and validity of the results of the unknown samples analyzed in an individual batch. QCs should be prepared by applying a similar matrix that is as close as attainable to the study matrix. A recommendation is that the preparation of QCs should be free of calibrators to prevent systemic spiking errors. They should also be spiked separately at each level instead of through serial dilutions of the high QC.
In LBA methods, a calibration curve is utilized to interpolate anonymous samples and assay quality control concentrations. The calibrators and Quality controls should be made in lots of blank matrices free of interference or matrix effects.
