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.”
Thinking Outside the Plate
A new wave of multiplexing technologies is paving the way for their use not only in biomarker discovery and validation but also for a host of applications that range from diagnostics to prognostics. “Multiplexing assays provide faster, more efficient analyses that are less costly than using multiple singleplex methods,” said Paul Rhyne, Ph.D., associate director, pharmaceutical candidate optimization, Bristol-Myers Squibb (www.bms.com).
Dr. Rhyne uses several technologies for identifying and validating biomarkers. “We routinely use three platforms. Luminex’ (www.luminexcorp.com) xMAP® consisting of color-coded beads that can be coated with a reagent specific to a particular bioassay, allowing the capture and detection of up to 100 different analytes at the same time. Soon that will go to 500. This provides great flexibility because you can control the bead set to mix and match analytes.”
The other platforms Dr. Rhyne uses are plate-based multiplexing. “These are planar arrays that use antibodies as capture reagents. Meso Scale’s (www.meso-scale.com) Discovery MultiArray Technologies use a combination of electrochemiluminescent detection and patterned arrays. Another technology we use is Pierce’s (www.piecenet.com) SearchLight that quantifies up to 16 chemiluminescent or 24 infrared proteins per well. All three multiplexing platforms are sensitive and reliable, however each has different advantages and disadvantages relating to application, high throughput, automation, and equipment.”
Dr. Rhyne advised investigators to consider thinking outside the “plate” regarding multiplexing. “Applications for multiplexing are wide open. Traditionally, they were used for cytokine analysis, but now we see they can be used for much more such as validating biomarker assays and for diagnostics. For example, it is now possible to take a patient’s blood sample and use a multiplexed assay such as Luminex’ respiratory viral panel to confirm viral infection, identify the virus, and rapidly adjust the care needed. Multiplexing is a technology that is amenable to many more applications.”
Most investigators agree that in the future biomarker development and validation will become a more organized and industrialized process. Predictive biomarkers will progress from single measurement to multianalyte assays. All in all, advances should provide an improved decision tree for steadily moving biomarkers through the pipeline.