Optimization of the modification of carrier proteins with aminated haptens

In this report we show that succinic groups are far more reactive to amino compounds than the carboxylic groups derived from Asp and Glu on the protein when using coupling via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) (even by an 8 fold factor). Accordingly, a new carrier-protein was designed where both natural amino and carboxylic moieties were transformed into succinic residues. To prepare this hypersuccinylated carrier, all exposed carboxylic acids were first transformed into amino groups by reaction with ethylendiamine after activation with EDCI. Secondly, all these residues together with the ones from Lys were succinylated to prepare a fully succinylated protein. This was even more relevant considering that the amount of Lysine was 2-4 fold lower than Asp and Glu in most of the proteins. These "hyper-succinylated" proteins (KLH or BSA) offer significant improvements in protein reactivity compared to the native proteins (by a factor of 8-10). The optimization of the reaction, in which the presence of dioxane was found to be influential, permitted further improvements in the modification of the protein. Finally, this new strategy was successfully used to develop antibodies against the commercial anti-tumor molecule, ET-637-NH2. Using native KLH no response was found, whereas 1/64,000 serum dilutions gave very high values in ELISA procedures when immunization was performed using the hyper-succinylated KLH.