Coupling antibody with DTPA: An alternative to the cyclic anhydride

The authors have previously shown that the bicyclic anhydride of DTPA may be used to attach this chelator to several proteins, including IgG antibody, rapidly and efficiently and that the coupled proteins may be labeled with In-111. As an alternative to the cyclic anhydride, we have now investigated the N-hydroxysuccinimide pentaester of DTPA for this purpose. The ester was prepared by reacting the imide with the cyclic anhydride and, like the cyclic anhydride, the ester is stable to storage at room temperature in moisture-free environments. The conditions under which IgG may be coupled with the ester were compared to that of the anhydride. Whereas maximum coupling with the anhydride is achieved in 20 seconds, about 10 minutes is required at room temperature with the ester, although the rate of coupling is increased at 370C. The effect on coupling efficiency of pH, protein concentration, and molar ratios is quantitatively different than that observed earlier with the anhydride although the trends are similar. Under optimum conditions, coupling efficiency with the ester is 90 +- 5% (pH=7.0) vs. 80 +- 5% (pH=8.4) for the anhydride. Antibody coupled with the ester showed a greater degree of dimer and polymer formation under identical coupling conditions and significantly greater instability during serum incubations