Michael Grol

Structure of the IGF-binding domain of the insulin-like growth factor-binding protein-5 (IGFBP-5): implications for IGF and IGF-I receptor interactions.

Binding proteins for insulin‐like growth factors (IGFs) IGF‐I and IGF‐II, known as IGFBPs, control the distribution, function and activity of IGFs in various cell tissues and body fluids. Insulin‐like growth factor‐binding protein‐5 (IGFBP‐5) is known to modulate the stimulatory effects of IGFs and is the major IGF‐binding protein in bone tissue. We have expressed two N‐terminal fragments of IGFBP‐5 in Escherichia coli; the first encodes the N‐terminal domain of the protein (residues 1–104) and the second, mini‐IGFBP‐5, comprises residues Ala40 to Ile92. We show that the entire IGFBP‐5 protein contains only one high‐affinity binding site for IGFs, located in mini‐IGFBP‐5. The solution structure of mini‐IGFBP‐5, determined by nuclear magnetic resonance spectroscopy, discloses a rigid, globular structure that consists of a centrally located three‐stranded anti‐parallel β‐sheet. Its scaffold is stabilized further by two inside packed disulfide bridges. The binding to IGFs, which is in the nanomolar range, involves conserved Leu and Val residues localized in a hydrophobic patch on the surface of the IGFBP‐5 protein. Remarkably, the IGF‐I receptor binding assays of IGFBP‐5 showed that IGFBP‐5 inhibits the binding of IGFs to the IGF‐I receptor, resulting in reduction of receptor stimulation and autophosphorylation. Compared with the full‐length IGFBP‐5, the smaller N‐terminal fragments were less efficient inhibitors of the IGF‐I receptor binding of IGFs.

Biosensor technology for the detection of illegal drugs II: antibody development and detection techniques

In a joint project of Deutsche Aerospace, Boehringer Mannheim and the University of Potsdam portable devices for the detection of illegal drugs, based on biosensor technology, are being developed. The concept enrichment of the drug from the gas phase and detection by immunological means. This publication covers the development of specific antibodies and various detection procedures. Antibodies with a high affinity for cocaine have been developed with the aid of specially synthesized immunogens. A competitive detection procedure with biosensors based on optical grating couplers and applying particulate labels has been established, showing a lower detection limit of 10-10 mol/l for cocaine. Additionally, a combination of a displacement-immunoreactor and an enzymatically amplified electrode was investigated, which at present still suffers from insufficient sensitivity of the immunoreactor. An alternative, fleece-matrix based test procedure, where enrichment and detection steps are integrated in a single unit, is promising in terms of simplicity and sensitivity. A simple swab-test for the detection of cocaine at surfaces has been developed, which has a lower detection limit of about 10 ng and which can be performed within one minute.

Biosensor technology for the detection of illegal drugs I: objectives, preparatory work, and drug enrichment

In a joint project of Deutsche Aerospace, Boehringer Mannheim and the University of Potsdam portable devices for the detection of illegal drugs, based on biosensor technology, are being developed. The concept enrichment of the drug from the gas phase and detection by immunological means. This publication covers the description of our objectives, preparatory work and results concerning enrichment of drugs from the gas phase. Vapor pressures of cocaine and cannabinoids have been determined. A test gas generator has been constructed which allows for reproducible preparation of cocaine concentrations between 2 ng/l and 2 pg/l. Coupling of a thermodesorption unit with GC/MS has been established for reference analysis. As another analytical tool, an ELISA with a lower detection limit of about 0,5 pg cocaine/assay has been developed. Applying fleece-type adsorbers, enrichment factors for cocaine in the range of 105 have been realized. No significant interference was found with potentially disturbing substances.