Efficient conjugation of chelator agents to IgG and accurate colorimetric determination of the chelator-to-IgG molar ratio.

Chelator conjugation is an essential step for metal-ion labelling of proteins, and the chelator-to-protein (chelator/protein) molar ratio of the resulting protein-chelator conjugate critically influences the conjugate's functions. Therefore, both controlling and measuring the chelator/protein molar ratio are important technical considerations. Among available methods for chelator/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 chelator agents (DOTA-NHS, DTPA-di, and CHX-A″-DTPA). Concerning the conjugation reactions of chelator 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. We established new measurement methods to determine the chelator-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 chelator agents. We achieved these outcomes both by preparing stock solutions of the chelator agents in anhydrous 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.