Biomedical subjects

Sten Ohlson

Publications and source records attributed to Sten Ohlson.

10 recordsLinked to original sources

PubMed2006

Screening for transient biological interactions as applied to albumin ligands: a new concept for drug discovery.

The notion that many biological interactions are based on transient binding (dissociation constants (K(d)) in the range of 10-0.01 mM) is familiar, yet the implications for biological sciences have been realized only recently. An important area of biological sciences is drug design, where the traditional "lock and key" view of binding has prevailed and drug candidates are usually selected on their merits as being tight binders. However, the rationale that transient interactions are of importance for drug discovery is slowly gaining acceptance. These interactions may relate not only to the desired target interaction but also to unwanted interactions creating, for example, toxicity problems. Here we demonstrate, in a high-throughput screening format, affinity selection of weak binders to a model target of albumin by zonal retardation chromatography. It is perceived that this approach can define the "transient drug" as a complement to current drug discovery procedures.

Albumins↗

PubMed2006

A label-free continuous total-internal-reflection-fluorescence-based immunosensor.

In this study, we continuously monitored, second-by-second, concentration changes of two different carbohydrates (maltose and panose) by using monoclonal antibodies in an optical immunosensor based on total internal reflection fluorescence. Earlier studies have demonstrated that these antibodies increase their intrinsic tryptophan fluorescence upon binding of carbohydrate antigens. Using the four immobilized monoclonal antibodies with low affinities (K(d)>10(-6)M), fast kinetics (k(off)>1s(-1)), and high reversibility gave opportunities for developing a continuous immunosensor without any need for regeneration. Since intrinsic fluorescence was used, no extrinsic labeling was necessary. Sensitivity was in the range of 1-5 microM for panose, and 10-15 microM for maltose and the loss of intensity was as low as 3.5% per hour during measurements. Calculations of DeltaH degrees and DeltaS degrees from the temperature dependence of K(d) indicated an enthalpic driven antigen-antibody binding event that is diminished upon antibody immobilization. We feel certain that weakly interacting antibodies can be used in future applications for continuous monitoring where there is a need to achieve instantaneous information on the concentration of an analyte.

Antibodies, Monoclonal↗

PubMed2006

Transiently binding antibody fragments against Lewis x and sialyl-Lewis x.

Biomolecular recognition is often characterised by low affinity where many weak interactions work either alone or in concert, resulting in an inherent dynamic situation. For example the well-studied weak binding of cell-cell interactions is predominantly based on a range of carbohydrates that interact with numerous (protein) ligands. Finding appropriate binders to these carbohydrate structures may pave the way for new analytical strategies based on low affinity, and recombinant antibody technology is a promising approach to the development of such reagents. We have in the present study characterised two low affinity human single chain antibody fragments (scFv) by surface plasmon resonance for use in such applications. The two clones, LeX1 and sLeX10, had been selected from a naive phage display library against Lewis x (Le(x)) and sialyl Le(x) (sLe(x)), respectively. Both LeX1 and sLeX10 showed low affinity, with K(D) values of 3.5+/-0.7 x 10(-5) M for Le(x) and 2.6+/-0.7 x 10(-5) M for sLe(x), respectively. Kinetic studies revealed the scFvs to be associated with fast dissociation rates, with Kd values higher than 0.1 s(-1) for both LeX1 and sLeX10. Apart from the Lewis structures Le(x) and sLe(x), we investigated the conformational isomers Lewis a and sialyl-Lewis a together with the monosaccharide units of the Lewis structures, and both scFvs showed high specificity for their respective carbohydrate. Taking these observations together we have demonstrated that scFv with fast reaction kinetics and low affinity have the necessary characteristics for further development as specific tools in analytical strategies, e.g. differentiation of cells based on the various configurations of carbohydrate epitopes.

Antibodies↗

PubMed2005

HPLC analysis of carbohydrate deficient transferrin isoforms isolated by the Axis-Shield %CDT method.

BACKGROUND: Carbohydrate-deficient transferrin (CDT) is elevated during prolonged overconsumption of alcohol and CDT is considered to be the most specific biochemical marker for alcohol overconsumption. However, an accurate method for analysing CDT is necessary because the test is frequently used for example in legal matters. METHODS: Patient serum samples were analysed with the Axis-Shield %CDT and eluates were pooled together. Transferrin was purified from the pool by affinity chromatography and further analysed with HPLC to determine the ratios of different transferrin isoforms. RESULTS: In the eluates using the Axis-Shield %CDT method, a substantial amount of trisialo transferrin was found, which is generally not considered a CDT isoform. CONCLUSIONS: The fact that trisialo transferrin is present may generate falsely elevated CDT results and it could at least partly explain the discrepancy between results of the Axis-Shield %CDT assay and HPLC in routine analysis.

Chromatography, Affinity↗

PubMed2005

Analysis of the specificity and thermodynamics of the interaction between low affinity antibodies and carbohydrate antigens using fluorescence spectroscopy.

The purpose of this work has been to examine whether fluorescence spectroscopy can be used to investigate weak or transient binding between monoclonal antibodies and carbohydrate antigens. In earlier studies we have demonstrated that the three monoclonal antibodies 39.4 (IgG2b), 39.5 (IgG2b) and 61.1(IgG3) bind weakly to the glycosidic alpha(1-4) bond present in e.g. maltose and panose. In this study these antibodies showed an enhancement in the fluorescence intensity of tryptophan upon binding in solution to these two carbohydrate antigens. Using a structural analog to maltose, cellobiose, no fluorescence intensity change was induced. Dissociation constants for these antibodies at different temperatures (5-40 degrees C) were obtained in the range of 0.003-0.2 mM and they were in accordance with earlier data from studies on affinity chromatography and surface plasmon resonance. Almost a doubling of the dissociation constants was observed for every 10 degrees C increase in temperature, giving an exothermal reaction with standard enthalpy change of -51 kJ/mol, for the association between antibody and carbohydrate antigen. It was seen that the extra glycosyl ring in panose increased the affinity more than eight times for the monoclonal antibody 39.5. A standard entropy increase of 21%, probably due to hydrophobic effects, is introduced by the extra glycosyl ring, while the enthalpy stays unaffected. This direct fluorescence approach to measure the binding and thermodynamics of an interacting antigen-antibody pair is simple and accurate since measurements are performed in solution and no immobilization or fluorophore labeling of the components is required. Introduction of fluorescence techniques will be a useful complement to current procedures to measure interaction of antibody with antigen and in particular they will offer solutions to detect transiently binding antigens.

Animals↗

PubMed2004

Lectin affinity capillary electrophoresis in glycoform analysis applying the partial filling technique.

The study of protein glycosylation and its significance in biological interactions is a field of growing interest. This work demonstrates a lectin-based separation of protein glycoforms of alpha1-acid glycoprotein (AGP or orosomucoid) with capillary electrophoresis. Glycoform analysis was performed with a "partial filling technique" with the lectin Concanavalin A (Con A) as affinity ligand. Con A separated human AGP into two peaks; the first peak included AGP glycoforms without biantennary glycans, and the second peak represented the fraction that had one or more biantennary glycans. The applicability of the method was demonstrated with the analysis of AGP from clinical samples and AGP treated with N-glycosidase F. The AGP separation was also used as a reporter system to estimate the dissociation constant (KD) between Con A and a competing sugar.

Electrophoresis, Capillary↗

PubMed2004

Determination of protein-ligand affinity constants from direct migration time in capillary electrophoresis.

A simple method to calculate dissociation constants for protein-ligand interactions by partial-filling capillary electrophoresis is demonstrated. The method uses raw migration time data for the ligand and needs only additional information about capillary inner radius and the absolute amount of protein loaded. A theoretical study supported by experimental data also demonstrates that the retention of analyte in affinity capillary electrophoresis (ACE) using the partial-filling technique depends linearly on the absolute amount of selector added but is independent of both selector zone length and selector mobility. Factors such as field strength and electroosmotic flow are also cancelled out if they are kept constant. The theory is confirmed and the usefulness of the method is demonstrated by enantioseparations using alpha-acid glycoprotein (AGP) and cellulase (Cel 7A) as chiral selectors.

Algorithms↗

PubMed2004

Binding interactions of Escherichia coli with globotetraosylceramide (globoside) using a surface plasmon resonance biosensor.

Surface plasmon resonance with an alkane L1 chip was used to investigate the binding of uropathogenic Escherichia coli, carrying adhesion receptors, to globotetraosylceramide (globoside; GbO4). The immobilization of globoside was reproducible and resulted in a stable globoside layer on the L1 chip. The data indicated that the globoside-immobilized L1 chip could be used for studying interactions with live or chemically fixed E. coli. The results indicated that the dissociation time was significantly reduced in glutaraldehyde-fixed E. coli as compared to living cells. Overall, the report demonstrates the significance of the L1 chip in terms of sensitivity, specificity, handling, and speed when studying globoside/E. coli interactions. This model may assist in screening for compounds that can inhibit the binding of uropathogenic E. coli to glycolipid ligands on target cells.

Bacterial Adhesion↗

PubMedDate not supplied

Detection of weakly interacting anti-carbohydrate scFv phages using surface plasmon resonance.

Methods to characterise and confirm specificity of scFv displayed on phages are important during panning procedures, especially when selecting for antibody fragments with weak affinities in the millimole to micromole range. In this report the surface plasmon resonance (SPR) biosensor was used to study and verify specificity of phages displaying weak anti-carbohydrate scFvs. The variable immunoglobulin light (VL) and heavy (VH) chain genes of the weak monoclonal antibody 39.5 were amplified and cloned into a phagemid and displayed as a scFv-pIII fusion protein on filamentous phage. This monoclonal antibody recognises with weak affinity the structural sequence Glcalpha1-4Glc present in a variety of carbohydrate molecules. Injection of the 39.5 phages over a biosensor chip immobilised with a (Glc)4-BSA conjugate confirmed selective binding of the scFv to its antigen. Inhibition studies verified the specificity. These results clearly show that SPR technology can be used to evaluate in terms of binding and specificity weakly interacting scFv displayed on the phage surface.

Animals↗

PubMedDate not supplied

Thermostable carbohydrate-binding modules in affinity chromatography.

Affinity chromatography is routinely used mostly on a preparative scale to isolate different biomolecules such as proteins and carbohydrates. To this end a variety of proteins is in common use as ligands. To extend the arsenal of binders intended for separation of carbohydrates, we have explored the use of carbohydrate-binding modules (CBM) in affinity chromatography. The thermostable protein CBM4-2 and two variants (X-6 and A-6) thereof, selected from a newly constructed combinatorial library, were chosen for this study. The CBM4-2 predominantly binds to xylans but also crossreacts with glucose-based oligomers. The two CBM-variants X-6 and A-6 had been selected for binding to xylan and Avicel (a mixture of amorphous and microcrystalline cellulose), respectively. To assess the ability of these proteins to separate carbohydrates, they were immobilized to macroporous microparticulate silica and analyses were conducted at temperatures ranging from 25 to 65 degrees C. With the given set of CBM-variants, we were able to separate cello- and xylo-oligomers under isocratic conditions. The affinities of the CBMs for their targets were weak (in the mM-microM range) and by adjusting the column temperature we could optimize peak resolution and chromatographic retention times. The access to thermostable CBM-variants with diverse affinities and selectivities holds promise to be an efficient tool in the field of affinity chromatography for the separation of carbohydrates.

Amino Acid Sequence↗