Accuracy of Serial Dilutions Tested Over a Large Dilution Range


The process of serially diluting solutions in microplates is common for many assays including preparation of dose response curves for IC50, drug efficacy testing, etc. There are many critical steps to this type of assay that are carefully controlled, such as contaminant quantitation and prospective drug concentration. Related to these assay steps, yet equally important to the success of the assay, is the ability of the liquid handler to accurately dispense the drug compound during the dose response analysis. Absent reproducible volume control of the liquid handler the volume of any solution dispensed by it, and therefore the concentration of drug candidate, are questionable.

Measurement of the dilution accuracy is not easily accomplished using traditional quality control methods.  In widely accepted single dye absorbance QC methods, the dynamic range of the chomophore is too narrow to evaluate all of the dilution steps required to make the dose response curve.  In contrast, fluorescent methods provide the required sensitivity, but lack the traceability pedigree associated with absorbance methods.  The limitations of both single dye absorbance and fluorescence methods require users to measure the accuracy of a subset of the total number of dilution steps forcing assumptions to be made about the repeatability of the liquid handler over multiple aspiration and dispense cycles.  Herein is presented the proof-of-concept for an approach which measures the accuracy of all dilution steps in a microplate using one multi-dye solution.  Furthermore, this absorbance-based method provides the NIST traceability necessary for a standard method.

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