Conjugation to preactivated proteins using divinylsulfone and iodoacetic acid

Two methods for the preactivation of proteins and conjugation of peptides to proteins under mild conditions are presented. Preactivation of proteins with divinylsulfone (DVS) permits peptide conjugation through either amino, hydroxyl or sulphydryl groups depending on the coupling pH used, while preactivation with iodoacetic acid (IAA) N-hydroxy-succinimide ester permits selective conjugation through sulphydryl groups. In addition, the latter method allows quantitation of the conjugation ratio through determination of carboxymethyl cysteine after acid hydrolysis. The divinylsulfone activated proteins can be stored for extended periods of time at -20 degrees C until required for conjugation, while the iodoacetic acid activated protein can be stored for a few days at -20 degrees C. These conjugation methods were investigated with respect to obtaining peptide/protein conjugates for immunization purposes and for use as reagents in immunoassays. The DVS activated proteins permitted direct conjugation of luteinizing releasing hormone (LHRH) through its tyrosine side chain and allowed synthesis of well defined conjugates. The DVS derivatives of bovine serum albumin (BSA), reduced and carboxymethylated BSA and purified protein derivative (PPD) were compared with respect to their potential value as carriers for obtaining antibodies to LHRH (M(r) 1000) and epidermal growth factor (EGF, M(r) 5000). IAA-PPD was evaluated as a carrier for the conjugation of glutathione specifically through its cysteine side chain and for obtaining antibodies to glutathione. The antisera obtained were specific and of high titer, and the methods described here will thus allow the convenient synthesis of carrier conjugates with well defined characteristics.