Efficient Conjugation of Chelating Agents to IgG and Accurate Colorimetric Determination of the Chelate-to-IgG Molar Ratio.

Background Chelate conjugation is an essential step for metal-ion labelling of proteins, and the chelate-to-protein (chelate/protein) molar ratio of the resulting protein-chelate conjugate critically influences the conjugate’s functions. Therefore, both controlling and measuring the chelate/protein molar ratio are important technical considerations. Among available methods for chelate/protein determination, a colorimetric method using the Y(III)–Arsenazo III reagent is convenient because it requires neither large-scale facilities nor expensive instruments. We examined this assay method for three representative chelating agents (DOTA-NHS, DTPA-di, and CHX-A”-DTPA). Concerning the conjugation reactions of chelating agents, high and reproducible reaction yields are desired. However, the reported yields have varied widely, and no standardized, high-yield, and reproducible procedure has been established. Results We established new measurement methods to determine the chelate-to-protein molar ratio for DOTA-NHS and CHX-A”-DTPA using human IgG as the target protein for conjugation. We also established a conjugation procedure that affords high conjugation-reaction yields for these chelating agents. We achieved these outcomes both by preparing stock solutions of the chelating agents in dehydrated DMSO and by employing the “dry-handling technique” throughout the conjugation procedure. Through the combination of these two practices, we successfully obtained high reproducibility and high yields in the reactions. Conclusions The establishment of the two measurement methods for the chelate-to-protein molar ratio and the conjugation procedure delivering the above yields may substantially contribute to research and clinical applications of metal ion-labeled proteins in many fields of science and medicine, particularly in the field of nuclear medicine.