The performance of immunodiagnostic assays such as ELISA is governed by many different factors. Reflectometry was used to monitor peptide adsorption and the resulting antibody binding activity on a polystyrene surface, Surface plasmon resonance was used to analyze affinity and kinetic parameters of the (immobilized) peptide-antibody interaction. We demonstrate the capability of both the reflectometer and the BIAcore instrument to determine these immunoassay factors independently. When peptidic antigens other than the parent protein antigen were applied, reduced antibody binding activity (10 times lower) and faster dissociation (100 times faster) rather than poor adsorption proved to be the critical factors determining immune reactivity. When the peptides were modified chemically or when their molecular size was increased, antibody binding activity as well as affinity could be improved or even restored.