Biomedical subjects

H Nygren

Publications and source records attributed to H Nygren.

At least 19 recordsLinked to original sources

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↗

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↗

PubMed1988

Molecular and supramolecular structure of adsorbed fibrinogen and adsorption isotherms of fibrinogen at quartz surfaces.

The adsorption of fibrinogen to quartz surfaces was measured by ellipsometry, ELISA, and electron microscopy. The initial adsorption at low concentrations was diffusion rate limited as determined by the ELISA and by counting the number of adsorbed molecules at electron micrographs. From ellipsometry, ELISA, and electron microscopy measurements it was found that the surface concentration of adsorbed fibrinogen increased continuously over four decades in bulk concentration of fibrinogen. At a hydrophilic quartz surface a plateau level of the adsorption isotherm was found at a surface concentration of 0.1 pmol/cm2 where the adsorbed molecules had a mean intermolecular distance of 10 +/- 5 nm between neighbors. At higher surface concentrations the molecules were densely packed and formed a layer where single molecules could not be identified. Adsorbed fibrinogen showed different structure at hydrophobic and hydrophilic quartz surfaces. At a hydrophilic surface, the fibrinogen molecules appeared as a 46 nm nodose rod consisting of 6-7 nodes with a diameter of 4 nm. At a hydrophobic surface, the molecule appeared as a binodular or trinodular rod with a node diameter of 5-9 nm, connected with a thin filament to form a 40-nm rod. Adsorption from higher concentrations of fibrinogen in solution resulted in adsorbed spheric structures with a diameter of 18-24 nm at the hydrophobic surface and in end-to-end polymers at the hydrophilic quartz membrane.

Adsorption↗

PubMed1988

Adsorption of coagulation proteins and adhesion and activation of platelets at the blood-solid interface. An experimental study of human whole blood.

A procedure is presented allowing detailed studies of the adsorption of coagulation factors from whole blood on to surface. Anticoagulant (citrate or hirudin) was added to fresh venous blood. The blood was incubated in hydrophilic or hydrophobic glass tubes without contact with air. The adsorption of fibrinogen, fibronectin and factor IX was measured with an enzyme immunoassay using specific antibodies directed against these proteins. Adsorption of enzymically active kallikrein was measured using a chromogenic peptide substrate. Adhesion and activation of platelets was measured by direct examination in a scanning electron microscope and by measurement of release of beta-thromboglobulin. The results show that the adsorption of plasma proteins at the blood-solid interface is dependent on the anticoagulant used, surface energy of the test surface and incubation time. In experiments using hirudin a specific inactivator of thrombin, as anticoagulant, we found dynamic changes of the adsorbed protein film which could not be studied using citrated blood.

Adsorption↗

PubMed1988

Immunological suppression of delayed hypersensitivity responses in mouse lungs as reflected by numbers of mononuclear cells, mast cells and mucus-producing cells.

The suppression of delayed hypersensitivity (DH) in the lung as reflected by the appearance of mononuclear cells, mast cells and mucus-producing cells was studied in Balb/c mice. Immunosuppression was induced by intravenous and peroral administration of picrylsulfonic acid (PSA) in mice subsequently sensitized with picrylchloride (PiCl). The animals exhibited a decreased DH reactivity as assessed by ear thickness increase compared with mice sensitized but not exposed to PSA pretreatment. In mice exposed to PSA intravenously (suppressed) and sensitized with PiCl there was a decrease in the number of mononuclear cells in the lung after challenge, compared to mice sensitized and challenged only. Similarly, the mast cell and mucus-producing cell numbers were slightly lower in animals immunosuppressed intravenously with PSA compared with sensitized mice. Such a decrease in the numbers of mononuclear cells, mast cells and mucus-producing cells in the lung was not seen in animals treated with PSA perorally, although these animals exhibited suppressed DH reaction in the ears. The present results indicate that induction of mononuclear cells and to some extent mast cells and mucus-producing cells in the lung relates to the DH reaction and its regulation.

Animals↗

PubMed1988

Regulation by T cells of delayed hypersensitivity reaction in mouse lung as reflected by mononuclear cells, mast cells and mucus-producing cells.

The appearance of mononuclear cells, mast cells and mucus-producing cells in the lung and their linkage to the development of delayed hypersensitivity (DH) reactions were studied. Adoptive transfer of immune lymph node cells, spleen cells and serum and in vivo treatment with monoclonal antibodies to L3T4-positive T cells in Balb/c mice were performed to investigate the cellular regulation of the number of mononuclear cells, mast cells and mucus-producing cells in the lung. Immune lymph node cells and, to a lesser extent, immune spleen cells from mice sensitized epicutaneously with picrylchloride transferred DH reactions to the recipients as assessed by ear thickness increase after challenge. Serum from sensitized mice was not able to transfer a DH reaction. Cyclophosphamide treatment of donor mice increased the DH reaction in the recipient mice. Adoptive transfer of immune lymph node cells and spleen cells gave a slight increase in the number of mononuclear cells in the lung of recipient mice compared with controls. This weak accumulation of mononuclear cells in the lungs of recipient mice, however, was not accompanied by a consistent increase in the number of mucus-producing cells and mast cells. The number of spleen cells expressing the L3T4 antigen decreased after in vivo treatment with the monoclonal GK1.5 (anti-L3T4) antibody as assessed by immunohistochemistry. This antibody treatment also resulted in an inhibition of the DH reaction and a decrease in the number of mononuclear cells and mucus-producing cells, but not in mast cells in the lung of sensitized and challenged mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Kinetics of antibody binding to solid-phase-immobilised antigen. Effect of diffusion rate limitation and steric interaction.

The binding of monoclonal antibodies to surface-immobilised antigen was studied. Antibodies against dinitrophenyl-benzene and O6-ethyl-2'-deoxyguanosine with a known affinity for the antigen were used. The amount of bound antibodies was measured by ellipsometry with an accuracy of +/- 0.15 pmol/cm2, and a sensitivity of 0.11 pmol/cm2. The binding rate of the initial antibody binding could become diffusion rate limited, and the binding rate at surface concentrations above 1 pmol/cm2 was affected by steric interaction between bound antibodies. Bound antibodies did not dissociate when rinsed with saline for up to 20 h, but dissociated in the presence of antigen (0.1 mM). The dissociation rate did not follow any identifiable rate constant. The results are discussed in relation to theoretical models of the kinetics of antigen-antibody reactions at solid-liquid interfaces.

Antibodies, Monoclonal↗

PubMed1987

Regulation of the delayed hypersensitivity reaction in the lung reflected as mononuclear, mast cell and mucus cell appearance after T helper cell depletion and adoptive transfer.

Epicutaneous sensitization with picrylchloride (PiCl) induced a strongly delayed hypersensitivity (DH) reaction in mice. Local challenge in the airways of these mice resulted in increased numbers of mononuclear cells, mast cells and mucus cells. Depletion of T helper cells in vivo by treatment with monoclonal antibody (GK 1.5) inhibited the DH reaction. This treatment also resulted in a decrease in the number of mononuclear and mucus cells in the lung after intranasal challenge. The DH reaction was transferred to recipients with immune lymph node cells and spleen cells from mice sensitized epicutaneously with PiCl. The recipient mice also showed a slight increase in the number of mononuclear cells in the lung after intranasal challenge. These results indicate that T cells are not only involved in the DH reaction but also in the accompanying lung reaction.

Animals↗

PubMed1987

An improved negative staining technique using a thin quartz membrane as sample support.

A negative staining technique is presented based on the use of 40-60 nm quartz membrane supported by a silicon grid. The quartz membrane is fabricated by thermal growth of silicon dioxide on a silicon substrate followed by an anisotropic silicon etching step giving rectangular holes in the silicon substrate. The hydrophilic membrane is shown to be ideally suited for negative staining due to its spreading characteristics, homogeneity, heat resistance and mechanical stability. Micrographs of phage lambda are presented showing the detailed structure of the tail. A simple method of calculating the number of adsorbed particles based on diffusion limited association is also presented.

Microscopy, Electron↗

PubMed1986

Determination by ellipsometry of the affinity of monoclonal antibodies.

The reaction between monoclonal antibodies and surface-immobilised hapten was studied by ellipsometry, a method allowing absolute measurement of the surface concentration of proteins. Monoclonal antibodies against 2-phenyloxazolone were used and their affinity for the antigen in solution was determined by calculations of the equilibrium constant from data obtained by measuring fluorescence quenching of the hapten due to antibody binding. The binding rate of antibody to surface-immobilised hapten and the dissociation rate of the complex were measured by ellipsometry. The equilibrium constant of the heterogeneous antigen-antibody reaction was determined by a Scatchard plot. The affinity of the antibodies for the antigen was found to be higher in the heterogeneous than in the homogeneous reaction by a factor which varied between different monoclonal antibodies.

Animals↗

PubMed1986

External diffusion in solid-phase immunoassays.

Calculations are presented describing the influence of external diffusion in the kinetics of solid-phase immunoassays. The analysis is concerned with systems where one reactant is immobilized at the surface of a sphere of arbitrary radius. The solution for a plane surface is found as a limiting case. The factors determining whether the reaction is diffusion or reaction controlled are found to be sphere radius, surface concentration of binding sites, forward reaction rate and diffusion constant of reacting species. Means of determining whether the reaction is diffusion or reaction controlled from observable quantities are described. When applied to heterogeneous antibody-antigen binding it is found that normally the binding to cell-size spheres is not limited by external diffusion. However, when applied to solid-phase assays with high surface concentrations of binding sites immobilized at plane surfaces or macroscopic spheres the binding is found to be diffusion limited. The importance of a mass transfer analysis in this case is also discussed.

Antigen-Antibody Complex↗