Building Robust Bioassays
To fill the gap between biomarker discovery and clinical utility, robust assays must be developed, said Philip M. Hemken, Ph.D., principle scientist, cancer research diagnostics, Abbott Laboratories (www.abbott.com). “Investigators need to be rigorous in validating biomarkers early in the process. There are several key points for developing a robust assay.
“First, there needs to be suitable product definition to clarify intended use and define product requirements (e.g., sample precision, both analytical and clinical specificity and sensitivity),” Dr. Hemken pointed out. “Secondly, there are preanalytical considerations such as accounting for specimen tube type, specimen stability, biorhythm variability, male versus female, and age.”
It’s important to verify the suitability of the assay design, Dr. Hemken added. “For example, it’s crucial to determine the best format such as competitive versus sandwich assays, the range of the curve required, and buffer components. Design of experiments can be used to optimize the reagent design.”
Another consideration for making the assay robust is to carefully characterize all reagents in the assay. “Questions to ask include: How pure are my antibody and antigen preparations? Is there any aggregation? How stable are they? What is the specificity of the antibody? Does the recombinant antigen behave similarly to the native antigen?”
Finally, Dr. Hemken advised carefully scrutinizing the analytical performance of the assay. “Important aspects include determining reagent stability, precision, lot-to-lot uniformity, dilution linearity, causes of interference, high-dose hook effect, and spike recovery.”
Dr. Hemken’s take-home message was that, “in order to produce robust biomarker assays, it’s important to increase analytical performance testing and stringency. The challenge is to be much more diligent in preanalytical testing, assay design selection, and reagent characterization. Only then will you be able to more confidently address the clinical utility of a biomarker.”