The influence of binding capacity and affinity on the improved performance of N-terminally extended hCG peptides,...

The improvement of peptide-ELISA responses by the use of small synthetic peptides elongated at the N-terminus with an Ata-group or a (Lys)7 extension has been analyzed. For this purpose, binding capacity and affinity were evaluated by specific ELISA procedures. The ELISA experiments on binding capacity, performed with saturating antibody concentrations, revealed a difference of more than three orders of magnitude in binding capacity between the parent peptides and the N-terminally linked peptides, in favor of the latter peptides. Antibody affinity values were determined by a liquid-phase equilibrium method as well as by a solid-phase equilibrium method. N-terminal extension of the peptides had almost no effect on the affinity when equilibrium between the peptide and the antibody was reached in solution. In contrast, solid-phase affinity was greatly enhanced when the N-terminally linked peptides were adsorbed to the polystyrene surface. This enhancement was determined by the N-terminal extension and the peptide amino acid sequence (40 to 600 times higher). Thus, the use of N-terminally extended peptides can greatly increase the performance of a peptide-ELISA through improved surface effects, resulting in higher binding capacity and functional affinity.