What you don’t know about how an off-the-shelf Biomarker kit is evaluated and qualified can significantly compromise your biomarker data. Running the kit using the manufacturer’s instructions without systematically testing the accuracy/precision (A/P), range of quantification (ROQ), dilutional linearity (DL), stability, and selectivity can jeopardized the interpretation of the results. The A/P, ROQ, DL, stability, and selectivity are the minimum performance characteristics one should test in order to provide reliable and defendable non-GLP biomarker results.
KCAS is routinely challenged to improve the lower limit of quantification (LLOQ) of an ELISA kit. While it is possible to improve ELISA kits (or any off the shelf kit), the improvement is usually only modest. The alternative is to build the method from scratch, which can often lead one down a rabbit hole and greatly impact the budget.
We are frequently asked, “What will you do differently – when evaluating a kit – than other labs?” Honestly, not much on the surface. Many labs have equipment, SOPs, processes, and experienced scientists who routinely test for biomarkers. Collectively, the KCAS team has more than 50 years of kit and immunoassay experience and we qualify between 30-40 biomarkers per year. These are excellent credentials, but other labs may have similar credentials. The difference is the focus KCAS has on understanding the ever evolving needs of drug development; we are humble, hungry, and intelligent. We have a scientific focused culture, steadfast values of teamwork and recognition that outsourcing partners today must provide expertise, commitment and endurance, to solve problems with tenacity. We have our process fine-tuned and repeatedly provide meaningful biomarker data to our clients.
Our most recent development challenge: A client required a 2-10 fold lowering of the LLOQ. All parties agreed to a timeline and budget for feasibility and 1 week dedicated to optional optimization. 3 weeks later we were able to achieve a 5 fold lowering. The client’s data set was previously ~80% below the limit of quantification (BLOQ) and with the improvement it was reduced to ~20%. So how were we able to improve the LLOQ? The scientist observed that the reaction appeared to continue, so the scientist read the plate a second time about 10 minutes longer than the manufacturer’s instructions. The results they observed was an improvement in OD signal to noise at the LLOQ, and they decided to read it a third time. Follow up experiments confirmed the extended time resulted in a 5-fold lowering of the LLOQ and the change was accurate and precise for multiple users. Additionally, we wanted to try to identify the root cause of the BLQ’s because endogenous levels should have been detectable. Our scientists’ research led us to hypothesis the matrix, specifically the anticoagulant, was interfering with the assay. KCAS confirmed this without impacting the timeline or budget by testing various anticoagulants while confirming the lowering of the LLOQ. The key to our success is the teamwork our Principal Investigators and scientists have when the development objective is clear.
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