Biomedical subjects

Ursula Bilitewski

Publications and source records attributed to Ursula Bilitewski.

8 recordsLinked to original sources

PubMed2005

Development of an enzyme-linked immunoreceptor assay (ELIRA) for quantification of the biological activity of recombinant human bone morphogenetic protein-2.

Human bone morphogenetic protein-2 is a representative of the transforming growth factor-beta (TGF-beta) superfamily of cytokines. It was produced in high-cell-density cultivations of recombinant Escherichia coli leading to the formation of inclusion bodies with aggregated inactive protein so that the protein had to be solubilized and renatured. Thus, the biological activity of the recombinant protein had to be determined. To avoid time-consuming cell-based assays or radioactive labelling of proteins enzyme-linked immunoreceptor assays were developed. They were based on the specific interaction between the biologically active protein and its receptors, of which the extracellular ligand binding domains were tagged with the Fc part of human IgG and expressed in insect cells. The amount of bound ligand, corresponding to the biologically active recombinant protein, was determined via enzyme-labelled antibodies. Application to various batches of protein showed that not only the amount of active protein could be quantified but also the quality of the protein preparations could be evaluated in significantly shorter analysis times than with conventional cell-based assays.

Bone Morphogenetic Protein 2↗

PubMed2005

Application of an SPR-based receptor assay for the determination of biologically active recombinant bone morphogenetic protein-2.

Human bone morphogenetic protein-2 (hBMP-2) is a member of the human transforming growth factor-beta superfamily. Biologically active bone morphogenetic protein-2 (BMP-2) is a dimeric protein that binds in the first step of the signal transduction cascade to specific receptors on the cell-surface. This specific interaction of the dimeric protein with the extracellular ligand-binding domain (ECD) of the receptor was used to develop a receptor-based assay based on an optical biosensor system (Biacore 2000, Biacore AB, Uppsala, Sweden). The ECD of the BMP-receptor type IA, tagged with the Fc part of IgG (BMPR-IA-Fc), was immobilised on the surface of a dextran-protein A-coated sensor chip. Calibration curves were obtained with purified and biologically active recombinant hBMP-2 (rhBMP-2) that showed a linear range from approximately 5 to 250 nM rhBMP-2. Moreover, this assay was used to quantitatively follow the generation of biologically active protein during the renaturation from unfolded and reduced monomers to biologically active dimers. A refolding mixture containing renatured dimeric rhBMP-2 and not correctly folded monomers, was used as the sample solution without any further pre-treatment. It was proven that only the biologically active dimers were recognised by the immobilised receptor, so the generation of biologically active rhBMP-2 during the renaturation process could be monitored directly and rapidly. Furthermore, the results from the optical sensor obtained during the renaturation process showed a good correlation with the data obtained by non-reducing SDS-PAGE analysis carried out at the end of the renaturation process. These data show that the disulphide-bonded dimer corresponds to the biologically active protein capable of binding the BMP-receptor type IA.

Bone Morphogenetic Protein 2↗

PubMed2005

Development of monolithic enzymatic reactors in glass microchips for the quantitative determination of enzyme substrates using the example of glucose determination via immobilized glucose oxidase.

A one-step procedure for the immobilization of glucose oxidase in fused-silica capillaries and in glass microchips was developed based on enzyme entrapment in a polyacrylamide-based monolithic column. The inner capillary surface was silanized with gamma-methacryloxypropyltrimethoxysilane (gamma-MAPS) to allow covalent binding of the gel to the surface. The composition of the polymer was optimized to prevent the formation of bubbles, allow liquid transportation by electroosmotic flow and to maintain the enzymatic activity. These requirements resulted in the addition of polyethylene glycol and poly(acrylic acid) to the acrylamide mixture. The gel containing the enzyme was formed in situ in the capillaries, respectively, in one channel of the microchip. In the microchip, it was limited to the sample injection channel by accordingly controlled silanization of the inner capillary surface. Glucose was detected via the amperometric determination of hydrogen peroxide. A linear correlation between signals and glucose concentration was observed from 0.05 to 1.1 mM glucose with a correlation coefficient of 0.999. The enzymatic monolithic microreactor showed no loss of activity during 8 h of continuous use and during storage in the running buffer at 4 degrees C for about 2 months. Interferents, such as ascorbic acid, were separated from the analyte electrophoretically, so that glucose could be quantified in diluted juices.

Acrylamides↗

PubMed2003

Biochemical analysis with microfluidic systems.

Microfluidic systems are capillary networks of varying complexity fabricated originally in silicon, but nowadays in glass and polymeric substrates. Flow of liquid is mainly controlled by use of electroosmotic effects, i.e. application of electric fields, in addition to pressurized flow, i.e. application of pressure or vacuum. Because electroosmotic flow rates depend on the charge densities on the walls of capillaries, they are influenced by substrate material, fabrication processes, surface pretreatment procedures, and buffer additives. Microfluidic systems combine the properties of capillary electrophoretic systems and flow-through analytical systems, and thus biochemical analytical assays have been developed utilizing and integrating both aspects. Proteins, peptides, and nucleic acids can be separated because of their different electrophoretic mobility; detection is achieved with fluorescence detectors. For protein analysis, in particular, interfaces between microfluidic chips and mass spectrometers were developed. Further levels of integration of required sample-treatment steps were achieved by integration of protein digestion by immobilized trypsin and amplification of nucleic acids by the polymerase chain reaction. Kinetic constants of enzyme reactions were determined by adjusting different degrees of dilution of enzyme substrates or inhibitors within a single chip utilizing mainly the properties of controlled dosing and mixing liquids within a chip. For analysis of kinase reactions, however, a combination of a reaction step (enzyme with substrate and inhibitor) and a separation step (enzyme substrate and reaction product) was required. Microfluidic chips also enable separation of analytes from sample matrix constituents, which can interfere with quantitative determination, if they have different electrophoretic mobilities. In addition to analysis of nucleic acids and enzymes, immunoassays are the third group of analytical assays performed in microfluidic chips. They utilize either affinity capillary electrophoresis as a homogeneous assay format, or immobilized antigens or antibodies in heterogeneous assays with serial supply of reagents and washing solutions.

Chemistry Techniques, Analytical↗

PubMed2003

Application of atomic force microscopy and grating coupler for the characterization of biosensor surfaces.

Atomic force microscopy (AFM) and an optical grating coupler system were used to improve the understanding of the biosensing layer on a Ta(2)O(5)-light-guiding surface. Exemplary, we investigated the immobilization of the protein avidin, the subsequent binding of biotinylated oligonucleotides and hybridization of a complementary 12-mer. The AFM measurements revealed the height of approximately 1.6 nm for a single avidin molecule, while the thickness of the avidin layer on the biosensor surface seemed to be 2.8-3.0 nm. This result lead to the conclusion that the protein was not forming a simple monolayer. However, the thickness of the avidin layer could not be determined directly, but only after shifting of protein by the tip of the AFM leading to grooves of 1 micro m(2) and approximately 3 nm depth. As the height of oxide particles forming the waveguide surface was also in the range of 1.5 nm, the depth of these grooves could also be a result of the deposition of proteins on top of the oxide particles. This was consistent with the increased roughness of the surface after protein binding. Thus, investigations with the grating coupler were used to determine quantitatively the amount of immobilized avidin. On a biotinylated surface the amount of immobilized avidin lead to the assumption of a complete monolayer, whereas simple adsorption proved to be less efficient. A binding ratio of 1:1.3 for avidin and a biotinylated oligonucleotide was achieved. Up to 83% of the bound single strand were accessible for a subsequent hybridization reaction with a 12-mer. These results supported the model of avidin being deposited mainly on top of the oxide particles leading to the picture of a 'rough' complete protein monolayer, which was postulated from the AFM investigations.

Avidin↗

PubMed2002

Mechanistic and molecular investigations on stabilization of horseradish peroxidase C.

The enzyme horseradish peroxidase (HRP) shows a decreasing activity when the enzyme's substrate hydrogen peroxide is present with the degree of inactivation being dependent on the incubation time and the hydrogen peroxide concentration. Incubation times of some minutes do not inactivate the enzyme independent of the H2O2 concentration. After several hours, only 50% of the activity is found for a medium H2O2 excess, and a >100-fold excess of H2O2 completely inactivates the enzyme. Polymeric additives, in particular Gafquat, lead to higher residual activities, whereas stabilizers, such as aminopyrine, preserve the full activity. Circular dichroism (CD) measurements reveal that the enzyme structure remains more or less unchanged when hydrogen peroxide is added. Only when a 1000-fold excess of hydrogen peroxide is present are structural changes observed. UV spectra highlight that the heme group in the enzyme is affected by hydrogen peroxide in a first step. Without any prolonged incubation, a decrease of the Soret band to approximately 50% is found for low hydrogen peroxide concentrations (HRP/H2O2 from 1:1 to 1:100). Higher H2O2 concentrations lead to the formation of catalytically inactive HRP forms. Preincubation of Gafquat, which is a copolymer from vinylpyrrolidone and derivatized methyl methacrylate, with hydrogen peroxide shifts the influence of hydrogen peroxide to higher concentrations, the shift being dependent on the Gafquat concentration. This effect is not observed for other polymers, such as dextrans, but it is also found for the stabilizer aminopyrine. Extended incubation times (24 h) of HRP together with H2O2, however, lead to an at least partial recovery of the Soret band for lower H2O2 concentrations (H2O2/HRP from 1:1 to 1:100). When hydrogen peroxide is used in a >100 fold excess, the heme group is irreversibly destroyed, and even the characteristic band of cpd III is not found. Here, the presence of Gafquat only reduces the degree of destruction. Computer modeling of the interaction between the polymers and the enzyme shows no specific binding sites for the functional groups of the vinylpyrrolidone-methacrylate copolymer Gafquat or of DEAE-dextran on the enzyme, whereas for the only activating polymer, polyethylenimine clustering of binding sites is observed.

Circular Dichroism↗

PubMed2002

Characterization of the vascular endothelial growth factor-receptor interaction and determination of the recombinant protein by an optical receptor sensor.

Vascular endothelial growth factor (VEGF) is one of the most important factors controlling angiogenesis. It is a homodimeric glycoprotein belonging to the family of cysteine-knot proteins. The biological activity is transduced via membrane-spanning receptors of the tyrosine kinase receptor family. Each biologically active VEGF has two receptor binding sites leading to receptor dimerization as first step following ligand binding. The ligand-binding site of the receptor is localized on extracellular Ig-like domains. The extracellular part of the receptor Flt-1 (VEGFR-1) was expressed as soluble protein and was used as receptor in an optical affinity sensor system (BIAcore). Suitable conditions allowed the determination of the association and dissociation rate constants as k(a)=4+/-1.2 x 10(6) M(-1) s(-1) and k(d)=3+/-0.8 x 10(-5) s(-1), respectively, leading to an affinity constant of K(D)=7.5+/-3 pM, which is within the range published already from other investigations and methods. Increasing receptor loadings of the sensor surface decreased the binding efficiency, as the ratio of bound VEGF-molecules to theoretically available binding sites increased from 1:1.5 to 1:2.6. Increasing the surface loading further, allowed the establishment of a quantitative assay with the analytical performance being influenced by the receptor loading and the contact time between sample and immobilized receptor, i.e. sample volume. This assay was used for VEGF determination during the cultivation of a recombinant Pichia pastoris strain.

Binding Sites↗

PubMed2001

Electrochemical characterization of screen-printed carbonaceous electrodes for the determination of peroxidase activity in novel screen-printed flow-through modules.

A novel totally screen-printed flow-through cell for immunoanalysis is presented. It contained screen-printed carbonaceous electrodes, which allowed the determination of peroxidase activity through the electrochemical reduction of p-benzoquinone. As different electrode materials differ strongly in their electrochemical properties, electrodes resulting from various screen-printable carbonaceous pastes were characterized using the hydroquinone/ p-benzoquinone redox couple. For most of the electrodes, cyclic voltammogram peak separations of between 550 and 670 mV were observed indicating only quasi-reversible electrochemical behavior. This was confirmed by variation of the peak separation with scan rate. Heterogeneous electron transfer rates of ca. 0.5 - 1 x 10(-3) cm s(-1) and electrochemical activation energies of ca. 20 kJ mol(-1) were found. These flow-through cells were not only applied to electrochemical peroxidase activity determinations but also, in combination with a separate detector, as affinity reactors. After biotinylation of screen-printed layers, streptavidin and then biotinylated peroxidase could be bound. However, as signals were only 10-20% of those obtained with a column filled with biotinylated glass beads, only the screen-printed electrochemical detector was applied to the detection of antibodies against the African Swine Fever Virus.

Animals↗