Aim: Ruthenium-labeled antibodies are commonly used detection reagents in bioanalysis assays and must be characterized to ensure quality. The aim of this work was to develop a method to determine the concentration and incorporation ratio (the degree of labeling [DOL]) of ruthenium-labeled antibodies by UV/VIS spectroscopy. Materials & methods: Free SULFO-TAG compound was scanned using UV/VIS and showed an absorbance peak at 292 nm. In contrast, antibodies demonstrate UV absorbance at 280 nm. After experimentally determining the extinction coefficients at 280 and 292 nm of free ruthenium and antibody, we generated a formula based on the Beer–Lambert law that calculates both concentration and DOL of these ruthenium-labeled antibodies. Conclusion: The concentration and DOL values determined by our method were comparable to those determined from bicinchoninic acid and LC/MS for the same reagents. This method creates a faster and more accessible reagent characterization process that uses far less reagent than the more traditional alternatives.
Keywords:
- characterization
- critical reagents
- incorporation ratio
- ruthenium