Optimization of the modification of carrier proteins with aminated haptens (1)
Haptens of molecular weight under 1000 Da are not immunogenic by themselves and need to be conjugated to macromolecules (e.g. proteins, peptides or synthetic poly-amino acids) in order to stimulate a potent immune response. One of the methods most commonly used to perform these modifications, when the hapten has primary amino groups, is activation of the carboxylic acids of the protein with 1-Ethyl-3-(3-Dimethylaminopropyl) carbodiimide (EDC) and further reaction between this activated group and the amino group in the hapten. In order to increase the reactivity of the carrier proteins, some proteins (bovine serum albumin (BSA), mouse serum albumin (MSA) or Hemocyanin (KLH)) are commercialized with their external primary amino groups modified with succinic anhydride. This treatment increases the number of reactive groups (Lys may be 20–40% of the total number of aspartic and glutamic groups), increasing the possibilities of introducing the hapten.
At pH 8, 85% of the primary amino groups were modified.
Only between 8% (at pH 5) and 5% (at pH 9) of the natural carboxylic groups were modified using 10 mM octyldiamine over the pH range studied. This maximum at acidic pH is probably a consequence of the complex mechanism of the reaction. The first step conjugation was promoted by the activation of carboxylic groups by EDC. The improvement in immuno-response using succinylated proteins derived from the higher reactivity of the succinyl groups is greater than that which can be attributed to any increase in the number of reactive groups. The percentage modification of the protein with octyldiamine was much higher (by a factor of 6–8 times depending on the pH studied) when using succinyl-BSA-agarose rather than native BSA-agarose.
The presence of dioxane improved the modification of the degree of the hyper-succinylated protein at all pH values. Moreover, the pH at which the maxi- mum modification was achieved was shifted towards more alkaline values with the addition of growing concentrations of dioxane.
when the immunization was performed using ET-hypersuccinylated BSA conjugates, a potent immune response was observed (positive even when using 1 / 64,000 dilution). The low immunogenicity of the ET-637-NH2 molecule makes it necessary to develop a conjugate with a high ratio of hapten molecules per protein carrier.
