Biotinylated steroid derivatives as ligands for biospecific interaction analysis with monoclonal antibodies using immunosensor devices

Systematic ligand-binding studies of the biospecific interaction between st eroids and antisteroid antibodies can be performed in real time using biose nsor techniques. In this study, quartz crystal microbalance (QCM) and surfa ce plasmon resonance (SPR) biosensor systems were applied. Different biotin ylated testosterone (T) and 17 beta-estradiol (E2) derivatives were preincu bated with streptavidin and immobilized on the sensor surfaces. We obtained low matrix densities of antigen enabling the investigation of the binding kinetics and position specificities of various anti-E2 and anti-T monoclona l antibodies (mAbs) to these steroidal compounds. The highest immunoreactiv ity of anti-E2 and anti-T mAbs is not necessarily for the specific modified steroid that was used as a protein-coupled hapten for immunization, The ki netic data confirm that both 3- and 19-specific anti-T mAbs do not discrimi nate between the 3- and 19-biotinylated T derivatives, whereas the 7 alpha- biotinylated T probe showed no affinity to these two anti-T mAbs, In the ca se of the 3-specific anti-E2 mAb, comparable interaction data were found fo r 3- and 6 alpha-biotinylated E2 compounds. The 6-specific anti-Ea mAb show ed comparable ligand binding, but a significant higher dissociation rate to the position-specific antigen. The QCM and SPR results correspond well to the data from cross-reactivity studies in solution as well as to enzyme imm unoassay equilibrium measurements. (C) 2000 Academic Press.