Biotinylated Steroid Derivatives as Ligands for Biospecific Interaction Analysis with Monoclonal Antibodies Using Immunosensor Devices

• Published in: Analytical biochemistry 2000-07, Vol.282 (2), p.173-185
• Main Authors: Kaiser, Thomas, Gudat, Peter, Stock, Werner, Pappert, Gunter, Grol, Michael, Neumeier, Dieter, Luppa, Peter B.
• Format: Article
• Language: English
• Subjects: anti-steroid antibodies; Antibodies, Monoclonal; Biosensing Techniques - methods; biosensor; biotinylated steroids; Biotinylation; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Estradiol - analogs & derivatives; Estradiol - chemistry; Estradiol - immunology; kinetic analysis; Ligands; quartz crystal microbalance; Streptavidin - chemistry; surface plasmon resonance; Surface Plasmon Resonance - methods; Testosterone - analogs & derivatives; Testosterone - chemistry; Testosterone - immunology
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.2000.4596

Systematic ligand-binding studies of the biospecific interaction between steroids and antisteroid antibodies can be performed in real time using biosensor techniques. In this study, quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) biosensor systems were applied. Different biotinylated testosterone (T) and 17β-estradiol (E2) derivatives were preincubated 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 monoclonal antibodies (mAbs) to these steroidal compounds. The highest immunoreactivity 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 kinetic data confirm that both 3- and 19-specific anti-T mAbs do not discriminate between the 3- and 19-biotinylated T derivatives, whereas the 7α-biotinylated T probe showed no affinity to these two anti-T mAbs. In the case of the 3-specific anti-E2 mAb, comparable interaction data were found for 3- and 6α-biotinylated E2 compounds. The 6-specific anti-E2 mAb showed 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 immunoassay equilibrium measurements.