Analytical validation and harmonization of activity-based protease assays: requirements for clinical translation.

Abstract

Activity-based (catalytic) measurement of proteases in body fluids has emerged as a diagnostic modality distinct from antibody-based concentration measurement, supported by proof-of-concept evidence in oncologic and non-oncologic disease. In analytical chemistry terms, the open question is how to characterize and control a reaction-rate measurement made in a complex biological matrix. This review sets out the analytical validation, standardization, and quality assurance requirements that would allow a research-grade catalytic protease assay to become a laboratory-developed test or an approved in vitro diagnostic. We delimit the assay class across its reporter formats, explain why the metrological framework of clinical chemistry must be adapted for activity measurement in complex matrices, and operationalize the core analytical parameters for a kinetic, matrix-dependent readout: analytical specificity, precision, detection and quantification limits, linearity, trueness, robustness, and interference. We address the unresolved problem of defining and tracing a unit of proteolytic activity, the absence of quality control materials and external quality assessment schemes, and the regulatory pathway under current US and European frameworks. Catalytic protease measurement is analytically plausible and biologically informative but remains research-grade. Its translation depends less on new biology than on analytical validation, harmonization, quality control, and regulatory conformity. We propose a staged minimum validation-and-reporting framework, condensed into a checklist, as a starting point for the field to develop by consensus.