If you’ve been following the other posts in this series, you know that the use of commercial biomarker assay kits is on the rise as a lower cost, less resource-intensive option to support drug development programs. But commercially available immunoassay kits are created for a larger market, so even when procured from the optimal vendor, it is unlikely that a kit in its purchased state will be a perfect fit for the needs of your study. That being said, it is still important to be critical and thorough during kit selection in order to choose the kit that most closely aligns with your biomarker assay demands. In this final post we share tips to help you make the best kit selection, and discuss how to then use method adaptation to customize your chosen kit to fit the exact aims and goals of your biomarker study.
Optimizing the Kit Selection Process
After you determine your preferred kit vendor, their kit options should be compared and screened for suitability to your study via a feasibility evaluation. There are several tests that need to be considered as a part of this decision, and they can be evaluated using acceptance criteria published in the Draft 2013 FDA Guidance on Bioanalytical Method Validation for ligand binding assays.
The first test involves calibration range verification and algorithm selection and will help you evaluate if the kit can achieve your desired sensitivity and range of quantification. It typically involves using several levels of spiked controls, preferably in the sample matrix, to fit the calibration curve with an appropriate algorithm.
The next step is an assay selectivity and matrix interference test, in which several biological samples are spiked with biomarker standard material to evaluate recovery of the added material.
Lastly, a dilutional linearity and parallelism test should be conducted by assaying several samples at various dilutions, using a minimum of three test samples containing endogenous biomarker and one or more spiked diluent samples in the defined matrix. The parallelism observed will demonstrate the immunological similarities between calibrators and the endogenous analyte, and will evaluate the potential for matrix effects and prozone.
If all of these tests yield satisfactory results, it is recommended you confirm kit specificity by evaluating method specificity for your analyte of interest, or by cross-checking its performance against another vendor’s kit for comparison. You can also choose to obtain calibration material from a reliable source and evaluate the calibration curve using kit reagents. Although work intensive, kit specificity confirmation is important as determining specificity, or lack thereof, at this early point in the process can save you a significant amount of time and money in the long run.
Optimizing Your Kit through Method Adaptation
Selecting the closest-fit commercial kit for your needs helps to simplify your method adaptation process. Method adaptation is used to find solutions to attributes that are missing from the standard format of your selected kit. There are many alterations that can be made to customize it for your unique study; I’ve called out a few below:
Method adaption could include creating additional calibrators, as most kits come with four or more levels, but draft 2013 FDA guidance recommends the inclusion at least six non-zero calibrators. The level of method validation required will often drive calibration adaptation.
You may also need to determine an appropriate curve-fitting algorithm, since the fitting of a curve suggested by your kit vendor will not include specific recommendations for the curve-fitting model or weighting parameters. You must determine a model that lines up with the sensitivity and precision you are looking for in your assay.
Given that calibrators and controls are typically recombinant proteins in proprietary matrices, you may want to include biological controls with endogenous biomarkers at two or more levels when available.
If you are substituting reagents, you’ll need to evaluate their suitability through assay performance prior to making the change. And, if you are using automation for the method, you can use bulk quantities of critical reagents, but the automated process must be included and validated in the test procedure.
Remember that however you modify your test method for your selected kit, it must be validated according to its intended use as described in part 2 of our series.
Working Toward a Common Goal
All of the recommendations described here contribute to the common goal of standardization and harmonization of commercial kit applications to better support biomarker quantification for biopharmaceutical development. I believe that taking a systematic approach to selecting and adapting your kit will allow for the generation of the high-quality data that you undeniably need to successfully progress your biomarker study.
If you’re interested in learning more about how BioAgilytix works with commercial immunoassay kits, don’t hesitate to reach out to me with questions.