The case for calibration-free concentration analysis.

Accurate measurement of protein concentration is crucial in biological research, where protein-based reagents play a key role in assay performance and reliability. Traditional methods of protein quantification often measure total protein concentration, failing to account for the active portion capable of binding to its intended target. Calibration-free concentration analysis (CFCA), which uses surface plasmon resonance (SPR) technology, offers a solution by specifically measuring the active protein concentration of the sample. CFCA leverages binding under partially mass-transport limited conditions to directly quantify the functional protein in a sample, overcoming variability associated with recombinant protein production. This article provides a background on CFCA and the case for its more widespread use in protein reagent characterization, as it offers a way to reduce lot-to-lot and vendor-to-vendor variability while improving reproducibility and standardization of assays. This perspective was informed by searching and selecting pertinent articles from PubMed (Nov 2024-March 2025) and by examining the reference lists of key papers.