Biomedical Engineering Reference
In-Depth Information
TABLE 10.1 Recommended Studies, Listed by Assay Phase, to be Included During
Feasibility, Development, and Validation of a Flow Cytometry-Based Neutralizing
Antibody Assay
Assay Phase
Study Type
Optimization
Development
Validation
Select cell line
Identify appropriate controls
Prepare cell banks: master and working
banks
Effect of FBS on assay performance
Human matrix reactivity/interference
Drug dose response
NAb positive control dose response
Detector reagent specificity, concentration,
and incubation time
Characterize cell line
Demonstrate method specificity
Effect of cell passage on assay
performance
Effect of cell confluence on assay
performance
Minimum required matrix dilution
Determine necessity of gating cells
Inter- and intra-assay variability
Stability of signal poststaining
(time and temperature)
Test for prozone effect
Define assay response range
Drug interference
Evaluate different lots of critical reagents
Test for Instrument variation
Determine assay cut point
Analyst variation
Sample and reagent stability
Use of plate loader
Studies that are specific to flow cytometry are shown in bold.
Changes that could affect the assay performance using flowcytometry are related
to cell surface receptor endocytosis (capping) or shedding, anomalous changes to
the membrane and/or receptor expression, or architecture caused by adherence to
plastics or cells, cell confluency, or biological sample matrix causing stimulation or
inhibition of protein expression or signaling. These effects on assay performance
may be minimized by standardization of cell culturing, sample handling, and
staining procedures. The effect of paraformaldehyde fixation on the stability of
the signal poststaining can be evaluated during assay development and confirmed in
validation by testing stained cells with and without the fixation step.
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