Background: The accuracy of highly sensitive biomarker methods is often confounded by the presence of various circulating endogenous factors in samples causing matrix effects. Method: This article outlines two different biomarker methods: hepcidin enzyme-linked immunosorbent assay (ELISA) for which an orthogonal assessment of ELISA to liquid chromatography–tandem mass spectrometry was performed to examine the potential matrix effect, and sclerostin ELISA to evaluate the matrix effect. Results: Although the potential interfering effects of the endogenous hepcidin variants (prohepcidin and clipped) showed that these proteins had >30% immunoreactivity in ELISA, the hepcidin ELISA preferentially measures full-length hepcidin when the molar ratios of full-length to variants remain >1. The correlation of ELISA to liquid chromatography–tandem mass spectrometry results showed full-length hepcidin as the major form in diseased populations. Conclusion: A fit-for-for-purpose assessment of matrix effect/selectivity was also performed for each method. This article demonstrates the utility of a fit-for-purpose approach to assess the validity of biomarker methods in evaluating the interconnected parameters of matrix effects, sensitivity and selectivity.
Methods to measure soluble ligands as disease- or drug-related biomarkers are commonly used in both predictive/diagnostic and drug development applications . The use of relevant biomarkers could provide proof of mechanism for target effect, determine proof of concept showing downstream effect, and facilitate patient stratifications or dose escalation [2–5]. Development and validation of biomarker methods by using a ligand-binding assay platform impose distinct challenges relative to methods intended for PK assessment and a comparative analysis of which has been laid out thoroughly in the review articles by Lee [5,6]. Traditionally cited challenges in developing biomarker methods compared with that of PK are the general lack of gold standards or universal references making the selection of a most appropriate reference material an important process and the presence of matrix effects.
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