Biomedical subjects

H Nygren

Publications and source records attributed to H Nygren.

At least 37 records · Page 2Linked to original sources

PubMed1996

Exposure of blood to biomaterial surfaces liberates substances that activate polymorphonuclear granulocytes.

Human whole blood, anticoagulated or not, was exposed to hydrophilic glass surfaces or methylated hydrophobic glass surfaces under saline cover. Platelet-poor plasma or serum was prepared after 10 minutes of exposure, measured in respect to complement activation, and transferred to a suspension of granulocytes, which acted as bioprobes. The granulocytes were prepared from blood, anticoagulated with ethylenediaminetetraacetic acid, and evaluated regarding intracellular Ca2+ concentration (Calcium Green-1 fluorescence), integrin expression (CD-11b immunohistochemistry), respiratory burst (chemiluminescence), and priming (increase in N-formyl-methionyl-leucyl-phenylalanine-induced respiratory burst). The results indicate that humoral factors formed during the surface exposure of blood were able to activate the probe granulocytes. The exposure to hydrophilic surfaces led to a calcium transient three times the magnitude of that of hydrophobic surfaces. This response could be blocked by the presence of heparin during the blood-surface exposure but was not affected by the addition of heparin to the probe granulocytes. Hirudin, a specific thrombin blocker, had no effect. The exposure to hydrophobic surfaces led to complement activation in serum that induced priming and respiratory burst of the probe granulocytes. In conclusion, the study provides evidence that hydrophilic-hydrophobic surface treatment significantly affects the immediate inflammatory response of a blood-biomaterial interaction that is moderated by the presence of heparin.

Antithrombins↗

PubMed1995

Logarithmic growth in surface adsorption.

A review is made of experimental data on surface adsorption of particles and polymers from water solutions, their analysis and interpretation in terms of general theoretical models of surface adsorption. A characteristic isotherm and kinetics is found and defined as logistic growth. The discussion is focused on literature indicating a possibility of describing logistic growth by using statistical (probabilistic) models based on the mean stay time of molecules on the surface. The statistical approach is further elaborated as follows: ligands arriving at a surface have a binary choice-to bind or to become reflected. Since the number of attempts to bind, n, will be high we can use the true mean of the binomial distribution to describe the reaction and write: S = n a; where S is the number of successful attempts and a is the probability of binding. The probability, a, will depend on the site density and on the sticking probability of the ligand at the binding site. Several experimental studies show that surface reactions have a nonlinear time- and concentration dependence and can be described by a Boltzmann factor of the form, I(1-e-t/tau); where I is the flux of ligands to the surface and tau = stay-time. The exponential form indicates that the reactions are self-dependent, and a statistical model for description of such reactions will be of the form: S(t) = N(o)(1-2-alpha t) (2-beta t); where N(o) is the number of molecules present in the system, alpha relates to the probability of positive cooperativity or t-dependent binding, and beta relates to the probability of desorption.

Adsorption↗

PubMed1995

Discontinuous formation and desorption of clusters during particles adsorption at surfaces.

A theoretical model was derived to describe the discontinuous formation and desorption of clusters during particle adsorption at surfaces. Two steps were investigated: (1) time-dependent adsorption, where we found that the initial slope and the limiting magnitude of an adsorption isotherm depend on the clusters' distribution. A higher magnitude of both the adsorption and desorption rates appear to contract the time scale and hence increase the initial slope. Decreasing the geometrical parameter, q, which represents the shape of an adsorbed cluster, enhances the growth of large clusters on the surface. (2) A concentration dependence model shows that the number of adsorbed molecules increases with increases in the value of n (nucleation capacity). Furthermore, higher rates of adsorption provide steeper initial slopes (higher affinity of, molecules to surface). Decreasing q from 2 to 1, i.e. from a circular to a linear cluster formation, slightly decreases the magnitude of the isotherms.

Journal Article↗

PubMed1994

Kinetics of antibody binding to surface-immobilized antigen. Analysis of data and an empiric model.

The kinetics of the reaction between monoclonal antibodies and surface-immobilized hapten (dinitrophenyl, DNP) was measured with in situ ellipsometry. A flow cuvette with a small volume (50 microliters) and a high flow rate (3 ml/min) was used in order to avoid mass-transport limitation of the reaction. An initial linear phase was recorded by off-null ellipsometry with a time resolution of 0.1 s. The linear phase was followed by an accelerated rate of antibody binding. The time dependence of the rapid reaction exhibited an exponential form. The apparent rate of antibody binding then decreased continuously with time and the surface concentration of bound antibodies was proportional to the logarithm of time. An empiric model is suggested that describes the time course and the concentration-dependence of the reaction.

Antibodies, Monoclonal↗

PubMed1993

Cooperativity in the antibody binding to surface-adsorbed antigen.

The binding to surface-adsorbed antigen of monoclonal mouse IgG-antibodies (mAbs), with two different affinities to dinitrophenyl (DNP), was measured by a calibrated ELISA. The concentration-dependence of antibody binding to surface-bound antigen of different epitope densities was analysed using Scatchard plots. The dissociation of bound tritium-labelled antibodies was measured in the presence of unlabelled antibodies in the bulk. At low surface concentration of bound anti-DNP, both high-affinity mAb and low-affinity mAb show a positive cooperativity in the binding reaction to antigen of high epitope density. Using antigen of lower epitope densities, the positive cooperativity is more pronounced for low-affinity clones. At higher surface concentrations of bound anti-DNP, the Scatchard plots indicate a negative cooperativity of binding, which is also implied by the increased dissociation found in the presence of antibodies in solution. The study confirms previous findings that the binding of antibodies to surface-bound antigen not only depends on intrinsic antibody affinity measured in solution. Other factors, such as self-interaction, also affect the heterogeneous binding reaction.

Adsorption↗

PubMed1993

Pre-adsorption of a cellulose ether onto polymer surfaces: adsorption of adhesins and platelet activation.

The effect of pre-adsorbed cellulose ethers upon the adsorption of plasma proteins at polymer surfaces was measured by an enzyme-linked immunosorbent assay. The resulting adsorption of plasma proteins was correlated with the sticking and activation of platelets at the blood-material interface, measured by scanning electron microscopy and release of beta-thromboglobulin. Retained Ca2+ activity was made possible by hirudin, a specific thrombin inhibitor, from medicinal leeches as an anticoagulant drug, thereby keeping the initial coagulation cascade and calcium-dependent cell-protein interactions intact during the experiments. The results show that pre-adsorption of a hydrophobic cellulose ether with a flocculation temperature < 37 degrees C, on polyurethane and polytetrafluoroethylene, decreased the adsorption of fibrinogen, fibronectin and vitronectin. A corresponding decrease was found in the number of cells sticking to the surface and in the release of beta-thromboglobulin from platelets.

Adsorption↗

PubMed1993

Nonlinear kinetics of ferritin adsorption.

The adsorption of ferritin at a methylized quartz surface was measured with off-null ellipsometry and transmission electron microscopy. An initial lag-phase was seen, followed by an accelerating adsorption leading to mass transport limitation of the reaction. The rate of adsorption then decreased at a surface concentration far below monolayer coverage, and a continuously decreasing rate of binding was seen. The slope of the binding rate was linear with the logarithm of time (fractal kinetics). The adsorbed ferritin molecules were distributed in clusters as seen by transmission electron microscopy. Clusters grown during the mass transport limited adsorption had crystalline structure at short range and low fractal dimensions (df = 0.89) over long range. Clusters grown during adsorption with fractal kinetics showed random structure at short range and a high fractal dimension df = 1.86 over all ranges. These findings indicate some new important mechanisms responsible for the complex kinetics of macromolecular reactions at solid-liquid interfaces. The results are discussed in relation to recently developed theories of self-organized criticality.

Adsorption↗

PubMed1992

Kinetics supramolecular structure and equilibrium properties of fibrinogen adsorption at liquid-solid interfaces.

Adsorption of fibrinogen onto hydrophobic and hydrophilic quartz surfaces was studied by ellipsometry and transmission electron microscopy (TEM) of negatively stained proteins. The initial adsorption at the hydrophobic surface, measured by ellipsometry, can be described by an apparent forward rate constant k1 of 2 x 10(4) M-1 s-1. This constant was time-dependent and is therefore considered as a rate coefficient. The apparent forward rate coefficient of adsorption to a hydrophilic surface was both time-dependent and concentration-dependent, indicating a history-dependent process of adsorption. Plateau levels of adsorption were concentration-dependent and lower at the hydrophilic quartz surface (1.2 pmol/cm2) than at the hydrophobic surface (1.8 pmol/cm2). These surface concentrations correspond to rather tight-packed monolayers of molecules adsorbed end-on. The initial desorption can be described by a first order rate constant (k-1 approximately 10(-4) s-1), down to 80-90% of the initial surface concentration. The dissociation rate then decreased (k-1 approximately 10(-6) s-1) resulting in an apparently stable level of adsorbed protein. Slow changes of the binding strength of adsorbed proteins was seen during 24-72 h adsorption time. Deviations from an ideal equilibrium isotherm were seen both in the time dependence and as concavities in a Scatchard plot, suggesting intermolecular cooperativity. At low bulk concentrations a heterogeneous distribution of fibrinogen molecules was found at the surface below monolayer coverage. The supramolecular structure was characterized by the formation of end-to-end dimers and trimers laying down at the surface. At higher surface concentration adsorbed molecules showed polycrystalline structure with repeated nearest neighbor distances at 16 nm. The distribution of adsorbed fibrinogen molecules indicates that surface-adsorbed fibrinogen may form a two-phase system, containing significant amounts of water. The atypical kinetics and concentration dependence of fibrinogen adsorption may thus be due to properties of a two-dimensional phase separation from a three-dimensional liquid bulk.

Adsorption↗

PubMed1992

Adsorption of coagulation proteins from whole blood on to polymer materials: relation to platelet activation.

A combination of methods, immunoassays of plasma proteins and platelet release of beta-thromboglobulin and chromogenic substrates for enzymatically active coagulation factors, was used to measure the reactions of coagulation proteins upon contact between whole blood and artificial surfaces as a function of time and surface material. Four types of well-known polymer surfaces, polyvinylchloride, polytetrafluoroethylene, polyurethane and silicone rubber, were investigated to elucidate if a simple and fast in vitro experimental set-up can be of guidance in the selection of materials for use in vivo. Platelets were activated at the polymer surfaces whereas the coagulation enzymes showed little activity on the polymer surfaces tested. There was a correlation between the adsorption of adhesins (fibrinogen, fibronectin and factor VIII-related antigen) at the surfaces and the release of beta-thromboglobulin from platelets, suggesting that adsorption of adhesins is a major determinant of blood compatibility of polymer materials. Significant differences between the surfaces were seen--polyurethane being the surface with the least protein adsorbed and least platelet activation initiated. This study shows that it is possible to make a first in vitro choice of possible blood compatible artificial surfaces before expensive and cumbersome in vivo experiments.

Adsorption↗

PubMed1991

Computer simulation of surface-induced aggregation of ferritin.

Models are presented describing the transient mass-transport limited adsorption and cluster growth of ferritin at a solid surface. Computer simulations are carried out on a hexagonal lattice using a computer model that can be characterized as a two-dimensional stochastic cellular automaton allowing different rules regarding association, lateral interaction and dissociation to be incorporated in the model. The fractal dimensions of individual clusters were extracted from simulated aggregates and for similar rules found to be consistent with literature values on reversible diffusion-limited aggregation in two dimensions. The distribution of clusters versus free surface were shown to be affected by neighbor-dependent association probability. Low fractal dimension clusters were generated by a combination of strong lateral cohesion and neighbor-dependent dissociation to the bulk. By comparing computer simulated aggregation to experimental electron micrographs of adsorbed ferritin layers it is suggested that neighbor-dependent association, neighbor-dependent dissociation and lateral interactions are important factors in the complex dynamics of adsorbed protein layers.

Computer Simulation↗

PubMed1990

Surface-induced aggregation of ferritin. Kinetics of adsorption to a hydrophobic surface.

The adsorption of ferritin from a water solution to a hydrophobic methylised quartz surface was studied by transmission electron microscopy, allowing direct examination of the iron core of the molecule without further preparation. The initial adsorption was seen to result in small clusters of molecules, the number of sites/cm(2) being concentration dependent. The adsorption process continued via cluster growth. The rate of adsorption increased and the process became mass transport limited. The clusters formed initially had low fractal dimensions (D approximately 1.0) and a coordination number, cn of 2.6-2.8, which increased with time. These clusters were abruptly restructured at a coordination number of 3.5, and the apparent rate of adsorption decreased during the reorganisation of the adsorbed layer. Finally, an equilibrium level was reached which was stable for at least 24 h. The distribution of ferritin molecules at equilibrium was in clusters with a fractal dimension of D = 1.14 +/- 0.16 and D= 1.33 +/- 0.08, respectively, for ferritin concentrations in the bulk of 10 and 100 microg/ml. Rinsing of adsorbed ferritin layers with buffered salt solution resulted in a rapid transient condensation of the clusters, but the net dissociation of protein was slow with the rate of dissociation being proportional to the logarithm of time. The condensed clusters were slowly restructured to linear polymers of ferritin molecules with a coordination number of 1.9 after 24 h of rinsing. The dissociation of protein molecules continued slowly for more than 3 days of rinsing. The results of the present study indicate that the rate of protein adsorption and desorption is strongly related to the supramolecular structure of the adsorbed protein film. Dense clusters of protein are not stable and this phenomenon may explain the formation of a dynamic equilibrium in spite of the fact that protein adsorption to a solid phase may appear to be practically irreversible.

Journal Article↗

PubMed1990

Surface-induced aggregation of ferritin. Concentration dependence of adsorption onto a hydrophobic surface.

The isotherm of ferritin adsorption onto a hydrophobic surface was studied by transmission electron microscopy. Adsorbed ferritin was found to be distributed in molecular clusters. The adsorption process was diffusion-rate-limited after 20 h adsorption time at bulk concentrations below 1 mg/1. The clusters formed during the diffusion-rate-limited adsorption had a fractal dimension D approximately 1.0 when averaged over all clusters. The pair distribution function g(r) showed an increased probability of finding nearest neighbours at distances less than 30 nm. The surface concentration of adsorbed ferritin was weakly dependent on the bulk concentration of ferritin in the range 10 mg/1-10 g/1 and the average number of nearest neighbour molecules was constant in this concentration range. The mass distribution of adsorbed ferritin c(r) had a fractal dimension D = 1.8 at a bulk concentration of 10 g/l and a surface concentration corresponding to theta = 0.45 +/- 0.05. The pair correlation function g(r) showed decreasing probability of finding nearest neighbour molecules over long distances as in percolating clusters. The results indicate that ferritin adsorbs strongly to the surface at low surface concentrations and weakly at high surface concentrations. The stability of ferritin adsorption was correlated to the average number of nearest neighbour molecules, indicating a possibility that desorption is a critical supramolecular phenomenon.

Journal Article↗

PubMed1989

Immunochemistry at interfaces.

The immunochemistry of antibody binding to solid-phase immobilized antigen is reviewed. Experimental data are compared with different theoretical models of reaction mechanisms at solid-liquid interfaces. It was found that reactions at the solid-liquid interface can become limited by the diffusion rate due to depletion of reactants close to the surface, even though the intrinsic bimolecular reaction at the surface is reaction-rate limited. The forward reaction-rate constant decreases with increasing concentration of bound antibodies at the surface, and when not limited by diffusion the forward reaction rate can be more than 1000-fold slower than the corresponding reaction in a liquid solution. Possible explanations for this phenomenon are discussed. The dissociation of bound antibodies is a slow process at solid phases. The antigen-antibody complexes formed are practically irreversible. Some evidence is presented which indicates that the stability of these complexes can be due to attractive lateral interactions between bound antibodies.

Antigen-Antibody Reactions↗

PubMed1988

Effect of antibody affinity on the isotherm of antibody binding to surface-immobilized antigen.

The binding of monoclonal antibodies to surface-adsorbed antigen was studied. Mouse IgG antibodies directed against dinitrophenyl groups (DNP) and O6-ethyl-2'-deoxyguanosine with known affinity for the antigen were used. The hapten was coupled to a protein, bovine serum albumin (BSA) or keyhole limpet hemocyanin, and adsorbed to polystyrene or silicone surfaces. Four different DNP-BSA epitope densities were used. Antibodies were incubated with the antigen-coated surface overnight. The bound antibodies were detected either optically by ellipsometry or by enzyme-conjugated anti-mouse IgG antibodies in the common ELISA technique. Absorbance values from ELISA measurements were transformed to surface density through calibration by ellipsometry. The experimental data showed that the binding of a high affinity antibody (Ka = 2.0 X 10(10] was diffusion rate limited after 24 h incubation time. Identical binding isotherms were found for high and low affinity clones of anti-DNP antibodies (Ka = 4.1 X 10(7) and 3.5 X 10(5] when antigen of high epitope density was used. At low epitope density the amount of bound low affinity antibodies decreased. Electron microscopy was used for studies of the distribution of colloidal gold-antibody complexes bound to surface-immobilized antigen. The results of the experiment showed that low affinity antibodies were bound in clusters whereas high affinity antibodies bound as single particles. These findings were related to the ELISA measurements. The results indicate that the binding isotherm of antibody to surface adsorbed antigen is not merely a reflection of the intrinsic antibody affinity measured in solution. Other macromolecular properties of antibodies, e.g., lateral intermolecular interactions and phase separation, affect the heterogeneous binding reaction.

Animals↗

PubMed1988

Experimental demonstration of lateral cohesion in a layer of adsorbed protein and in layers of gold-antibody complexes bound to surface immobilised antigen.

The spatial distribution of ferritin molecules adsorbed on quartz surfaces and the spatial distribution of antibody-coated colloidal gold particles over an antigen-coated surface was studied by electron microscopy. Ferritin molecules were found to initially adsorb in small clusters at 2.5 X 10(9) sites/cm2. The initial nucleation was followed by growth of the clusters. Gold antibody complexes were initially bound to the antigen-coated surface as single particles, and formation of clusters was a secondary event at higher particle densities. The results indicate that lateral cohesion between macromolecules may play a role in the stability of adsorbed layers of protein and surface immobilised antigen-antibody complexes.

Animals↗

PubMed1988

Kinetics of antigen-antibody reactions at solid-liquid interfaces.

The kinetics of antigen-antibody reactions is reviewed with special attention paid to the specific properties at solid-liquid interfaces. Theories of possible diffusion limitation in forward reaction rates are compared to experiments. It is found that the intrinsic forward reaction rate in the bimolecular antigen-antibody reaction is normally not limited by diffusion either in solution or at the solid-liquid interface. However, reactions at the solid-liquid interface can be diffusion limited due to depletion of reactants close to the surface. This effect depends on geometry, intrinsic reaction rate and surface concentration of receptor molecules. Normally cell surface reactions are not diffusion limited whereas reactions at artificial surfaces often are limited by diffusion. When not limited by diffusion it is also found that the intrinsic forward and reverse reaction rates are lower for surface reactions compared to reactions in solution. Antigen-antibody reactions at solid-liquid interfaces can often be considered as practically irreversible and limited by mass transport or steric interactions.

Antigen-Antibody Reactions↗

PubMed1988

A diffusion limited reaction theory for a microtiter plate assay.

Calculations are presented describing the diffusion limited kinetics of a solid-phase immunoassay in which reactants are immobilized at the inner surface of a cylindrical well. The calculations refer to an unstirred situation and simplified expressions are presented which can be used for calibration and optimization of the assay.

Animals↗

PubMed1988

Use of cellulose ethers as peptide antigen carriers in the ELISA.

A procedure for surface immobilisation of peptides is described. Insulin, a model for peptide antigens, was covalently coupled to alkyl-hydroxyalkyl-cellulose ethers. The cellulose-insulin conjugate was then adsorbed to the plastic surface of microtitre wells and was used as antigen in an ELISA assay. The adsorbed conjugate was shown to be stable in undiluted plasma or serum whereas adsorbed insulin was removed from the surface by incubation in undiluted serum or plasma. Adsorption of serum albumin or cellulose ether polymers to the microtitre plates followed by incubation with whole blood, showed that adsorbed albumin but not the cellulose ether was exchanged by fibrinogen at the surface. The results indicate that coupling of peptides to alkyl-hydroxyalkyl-cellulose ethers is an efficient means of immobilising peptide antigens to hydrophobic surfaces.

Antigens↗