Biological assay validation is the cornerstone for robust drug discovery and development. Considering the abundance of analytes that are identified and characterized using bioanalytical techniques such as ELISA assays, validating these methods can be complex. Validation of ELISA analysis and ELISA monoclonal antibody testing includes several challenges. Hence, a thorough discussion around biological assay validation, such as the ELISA method, is critical for correlating biomarkers and drug concentrations with physiological endpoints.
ELISA assay method is a gold standard bioanalytical technique. However, the traditional ELISA method lacks multiplexing abilities. Hence, researchers are increasingly relying on multiplexed biological assays for simultaneous analysis of multiple analytes in a single assay volume. Multiplexing saves time, resources, and precious biological samples. Validating multiplex biological assays has unique challenges and requires innovative solutions. Therefore, the current article discusses emerging trends in multiplexed biological assay validation.
Multiplexed biological assay validation
Today, the Luminex and Meso Scale multiplex platforms are increasingly being employed in clinical and biomedical research. The Luminex technology is a bead-based assay where individual beads are conjugated with distinct capture molecules. This molecule can be a peptide, receptor, or an antibody. The antibody is internally dyed with distinct fluorophores, and hence, each has a unique spectral signature.
On the other hand, the Meso Scale Discovery platform combines electrochemiluminescence detection with multi-array technology to offer highly dense biological information. The microplates consisting of high-binding carbon electrodes have capture antibodies that hold the analytes of interest. Detection antibodies labeled with Sulfo-tag labels are then added to this combination. Both the Meso Scale and Luminex platform can develop customized assays. Reagent qualities will determine assay performance.
Multiplex assays have several advantages over singleplex assay systems. They conserve biological samples and can be easily miniaturized. Most importantly, these assay systems can simultaneously detect multiple analytes in a single assay volume. However, multiplex biological assays face unique assay validation challenges.
A single multiplex assay plate is equivalent to numerous individual ELISA assays. Hence, data analysis takes longer and is more complex. Validating multiple parameters in the same panel will not be optimal. Validation requires a more extensive method validation. These unique challenges require adequate regulatory considerations.
The US FDA provides guidelines for bioanalytical method validation. These guidelines are not specific for multiplexed nonclinical studies. Analytical method validation should address parameters such as accuracy, selectivity, precision, range, stability, and reproducibility. Method validation for biomarker testing should also include pharmacokinetic assays. Additionally, individual biomarker assays may require unique considerations for validating biomarker assays. Specifically, the validation recommendations provided by the US FDA guidance document for the pharmacokinetic assay may not necessarily apply to biomarker testing. Hence, each bioanalytical assay may have unique method validation requirements.
Today, sponsors are increasingly using fit-for-purpose approaches for validating multiplex assays. The fit-for-purpose strategy includes validating bioanalytical assay to fulfill the intended application of the method. Hence, the extent of method validation will depend on the analyte of interest and the stage of drug discovery and development. Due to its applicability and advantages, the fit-for-purpose approach has now become the mainstay strategy in multiplex biological assay validation.
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