Change of colloidal and surface properties of Mytilus edulis foot protein 1 in the presence of an oxidation (NaIO4) or a complex-binding (Cu2+) agent.

Quartz crystal microbalance with dissipation monitoring (QCM-D) was used to study the viscoelastic properties of the blue mussel, Mytilus edulis, foot protein 1 (Mefp-1) adsorbed on modified hydrophobic gold surfaces. The change in viscoelasticity was studied after addition of Cu2+ and Mn2+, which theoretically could induce metal complex formation with 3,4-dihydroxyphenylalanine (DOPA) moieties. We also used NaIO4, a nonmetal oxidative agent known to induce di-DOPA formation. Reduction in viscoelasticity of adsorbed Mefp-1 followed the order of NaIO4 > Cu2+ > buffer control > Mn2+. We also studied the formation of molecular aggregates of Mefp-1 in solution with the use of dynamic light scattering (DLS). We found that addition of Cu2+, but not Mn2+, induced the formation of larger DLS-detectable aggregates. Minor aggregate formation was found with NaIO4. With the analytical resolution of small angle X-ray scattering (SAXS), we could detect differences in the molecular structure between NaIO4- and Cu2+-treated Mefp-1 aggregates. We concluded from this study that Cu2+ could participate in intermolecular cross-linking of the Mefp-1 molecule via metal complex formation. Metal incorporation in the protein most likely increases the abrasion resistance of the Mefp-1 layer. NaIO4, on the other hand, resulted in mainly intramolecular formation of di-DOPA, but failed to induce larger intermolecular aggregation phenomena. The described methodological combination of surface sensitive methods, like QCM-D, and bulk sensitive methods, like DLS and SAXS, generates high resolution results and is an attractive platform to investigate intra- and intermolecular aspects of assembly and cross-linking of the Mefp proteins.

Adrenoceptor and other pharmacoactive compounds as putative antifoulants.

The search for new antifouling methods, which are non-hazardous for the marine environment, is intense. However, even if several innovations in this field of research have been made, the search for unique molecules with characteristics such as strong biological activity, low residence time in the marine environment and which target special physiological features in marine invertebrate larvae, biofilm forming bacteria or algal spores is still required. This chapter reviews the effects of biogenic amine receptor agonists and antagonists, primarily G protein-coupled receptors, on settling barnacle cypris larvae. Biotechnological research on adrenoceptor compounds as lead molecules in new antifouling technologies is also reviewed.

Conformational epitopes of C3 reflecting its mode of binding to an artificial polymer surface.

The aim of the study was to investigate the incompletely understood mechanisms of complement (C) activation and binding on artificial biomaterials. Polystyrene in the form of microtitre plates was used as target for C binding, detectable by ELISA using monoclonal anti-C3 antibodies specific for conformational epitopes expressed by bound C3 and C3 fragments. C3 binding in whole blood/plasma/serum is maximal at low dilutions and occurs predominantly by C activation. At higher dilutions, C3 binding occurs at approximately 1/3 of maximal levels and is solely an effect of adsorption. C3 adsorption in the lower serum dilution range, occurs at low but clearly detectable levels. Comparative epitope analysis between C3 fragments, actively bound to polystyrene in the presence of serum, and of iC3b bound to sheep erythrocytes, clearly indicates that C3 binding/activation on polystyrene takes place as a C3 convertase-mediated reaction, which in serum/plasma is followed by a secondary factor I-dependent degradation of the bound C3b into iC3b. The neo-epitope analysis of serum-contacting polystyrene revealed that the adsorbed C3, throughout the entire serum dilution range tested, deposits in a state closely similar to that observed for purified C3 at a high packing density. Polystyrene surfaces with adsorbed purified C3 expressing this epitope profile were found to mediate APW dependent deposition of C3b in pig serum, presumably by forming a hybrid convertase with porcine Bb. These data therefore suggest that adsorbed C3 on serum-contacting polystyrene surfaces may initiate complement activation via the APW.

Structure of adsorbed fibrinogen obtained by scanning force microscopy.

It is shown that scanning force microscopy (SFM), operated in the attractive mode, can be used to obtain high resolution pictures of adsorbed fibrinogen molecules on solid surfaces, without the need for staining or special microscope grids. SFM also reveals the three-dimensional structure of the adsorbed molecules. Two forms of adsorbed fibrinogen are demonstrated on hydrophobic silicone dioxide surfaces: a trinodular about 60 nm long and a globular with about a 40 nm diameter. Polymeric networks formed after storage of the surface with adsorbed fibrinogen in PBS for 11 days are also shown. The SFM-results for the trinodular structure suggest the existence of loops or peptide chains extending outside the basic structure of the fibrinogen molecule.

Adsorption to silica nanoparticles of human carbonic anhydrase II and truncated forms induce a molten-globule-like structure.

Human carbonic anhydrase II pseudo-wild type (HCAIIpwt) and two truncated variants were adsorbed to approximately 9 nm silica nanoparticles. Ellipsometry was used as an indirect measure of protein adsorption. The structural changes of adsorbed proteins were investigated with the use of circular dichroism (CD), intrinsic fluorescence, ANS binding ability and inhibitor binding capacity. It was found that the variants that were truncated at positions 5 and 17 in the N-terminal end attain a molten-globule-like state after interaction with the silica nanoparticles. In contrast, the more stable HCAIIpwt retained most of its native structure after 24 h adsorption to silica nanoparticles. The result suggests that surface induced unfolding may give rise to intermediates similar to those for unfolding induced by, for example GuHCl. Thus, the intermediate observed has some features of the molten globule.

Biospecific bimolecular binding reactions - a new ellipsometric method for their detection, quantification and characterization.

A new ellipsometric method for detection, quantification and characterization of bimolecular, specific interactions on solid surfaces, e.g., binding between antigen and antibody and between ligand and receptor, is described. In the method, which we have called diffusion-in-gel (DIG) ellipsometry, one of the binding components is placed in a trough in a gel which has been poured over a solid surface coated with the other binding component. After diffusion, the gel is removed from the surface and ellipsometric measurement of thickness of adsorbed bimolecular layers is performed at different distances from the site of the diffusion trough. Bimolecular binding on the solid surfaces was also studied by wettability determinations with a water condensation technique. Three bimolecular binding systems were studied: bovine serum albumin (BSA)-anti-BSA, ganglioside GM1-cholera toxin, and C-polysaccharide-C-reactive protein. There was no tendency to saturation in the anti-BSA adsorption profile, which was steep with an endpoint thickness of about 16 nm. In contrast, the cholera toxin profile, within a narrow concentration range, rose to a plateau level of about 3 nm thickness of adsorbed cholera toxin. The C-reactive protein profile formed an intermediate pattern. Good agreement was observed between the thickness of the adsorbed ligand layers and wettability as determined by water condensation. Compared with other methods, the DIG ellipsometry technique has several theoretical and practical advantages for the detection and investigation of biospecific bimolecular binding.

Diffusion in gel-enzyme linked immunosorbent assay (DIG-ELISA): a simple method for quantitation of class-specific antibodies.

A new method for quantifying class-specific antibodies is presented. The method has been named Diffusion-In-Gel-Enzyme-Linked-ImmunoSorbentAssay (DIG-ELISA), and is briefly as follows. Antiserum ia allowed to diffuse from wells in a gel layered over an antigen-coated plastic surface. The gel is then removed and the preparation is incubated with enzyme-conjugated anti-immunoglobulin. The enzyme is then visualised in situ by a colour reaction produced by pouring a substrate-containing gel over the plastic surface. Bovine serum albumin and rabbit-anti-BSA were used as a model system, and horseradish peroxidase or alkaline phosphatase as enzymes for visualization.

Diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA): optimal conditions for quantitation of antibodies.

In the diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) the quantitation of antibodies is based on their ability to form a diffusion gradient over an antigen-coated polystyrene surface. The antigen-antibody reaction is then visualized by an enzyme-conjugated anti-immunoglobulin. The enzyme-substrate reaction is finally performed by pouring a substrate-containing gel over the polystyrene surface. In this study with bovine serum albumin as antigen and a corresponding rabbit antiserum, the diffusion time of antiserum was shown to be the most critical variable of the method, while the antigen concentration used for coating, the conjugate binding time and the enzyme-substrate reaction time had a minor influence on the quantitation of antibodies. High antibody levels were measured with greater accuracy than low levels, but the standard deviation was below 10%. It was also shown that different sera containing antibodies to Salmonella typhi O LPS, Klebsiella pneumoniae K1 and O4 LPS, Escherichia coli O2 LPS, Yersinia enterocolitica Y3 LPS, cardiolipin and pneumococcus could be quantitated with the same accuracy.

Diffusion-in-gel thin layer immunoassay (DIG-TIA): optimal conditions for quantitation of antibodies.

A new technique for the quantitation of antigen-antibody reactions, diffusion-in-gel thin layer immunoassay (DIG-TIA), has been developed. The principle of DIG-TIA is that antibodies are allowed to form a concentration gradient by radial diffusion in an agar gel poured on top of an antigen-coated plastic surface. After removal of the gel and visualisation by means of condensation of water vapour on the plastic surface, the antigen-antibody reactions appear as zones of increased wettability. The concentration of antigen used for coating, pH of the agar, incorporation of Tween 20 in the agar, size of diffusion basins, time of diffusion, and reinforcement by anti-immunoglobulin have been studied with regard to their influence on sensitivity and precision in the detection of antibodies with DIG-TIA. A photographic technique for permanent recording of wettability patterns is also described.

A simple spot technique for thin layer immunoassays (TIA) on plastic surfaces.

A simple and sensitive technique for visualization of antigen--antibody reactions is described. The property of many antigens to become adsorbed firmly on to a hydrophobic polystyrene surface while retaining their serological reactivity is taken advantage of. On a surface with adsorbed antigen the corresponding immune serum is applied spot-wise. The antigen--antibody reaction areas on the surface are characterized by a distinct hydrophilic condensation pattern when exposed to water vapour. The results obtained by the immunoassay technique described can be reproduced with great accuracy. The method is well suited for quantitative determination of a wide range of antigens as well as their corresponding antibodies. Antigen concentrations of 0.2--0.8 mg/l and antibody concentrations about 1 mg/l can be detected. By employing an antiimmunoglobulin serum subsequent to the primary antigen--antibody reaction, an increase in sensitivity can be obtained.

A simple chemiluminescence assay for the determination of reactive oxygen species produced by human neutrophils.

We show that phagocyte production of reactive oxygen species can be measured using a microtitre plate based chemiluminescence blotting technique. The production of reactive oxygen species is determined by their ability to catalyze the oxidation of luminol or isoluminol, resulting in light emission which is recorded on a photographic film. The method permits the determination of NADPH oxidase activity from as few as 9000 cells. It could be used to detect NADPH oxidase defects in neutrophils (e.g. from patients suffering from chronic granulomatous disease), and to screen pharmaceuticals with scavenging activity for reactive oxygen species.

Complement factor adsorption on solid surfaces--an ellipsometric method for investigation of quantitative aspects.

An optical method, ellipsometry, has been used for quantification of organic material adsorbed from complement sufficient sera on antibody coated solid surfaces. Maximal adsorption of organic material from complement sufficient human sera occurred at about 0.5 micrograms/cm2 of IgG. C3 but not C5, C8 or C9 was detected on the antibody surface incubated with complement sufficient sera. This may indicate that IgG adsorbed on methylized silicon surfaces lack binding sites for complement factors beyond C3. A modification of the method was also used for quantification of migration inhibition of human polymorphonuclear leucocytes (PMNL). Locomotion inhibition fell in a sharp interval from 0.2 to 0.5 micrograms/cm2 of IgG on the surface. We believe that the suggested type of measurements is important for understanding the quantitative relationships between humoral effects such as antibody dependent complement activation and cellular effects such as migration of PMNL.

Protein absorption and ellipsometry in biomaterial research.

Ellipsometry is an optical surface-sensitive method for the investigation of various aspects of protein adsorption mainly at reflecting metal surfaces and ceramic surfaces. One interesting feature of the method is the possibility of detailed and accurate determination of real-time adsorption kinetics of proteins without labelling of the protein. It is also possible to detect protein adsorption with the use of antibodies that adsorb onto the antigen-coated surfaces and to detect antibodies by their adsorption behaviour with regard to antigen-coated surfaces. Compared to other solid phase methods such as enzyme linked immunosorbent assay (ELISA), immunofluorescence and radioimmunoassay, ellipsometry has the advantage of not involving any labelling of the reactant and it is a relatively inexpensive method to maintain. This review is a current report of 15 years of contributions in biomaterials and biochemical research. Special consideration has been given to biologically related surface phenomena such as protein conformation changes, protein displacement effects, early events in blood clotting and complement activation. Among the technical achievements given prominence are the wettability gradient method and the rational use of silicon as an experimental surface. It is clear that ellipsometry and related methods such as reflectometry and surface plasmon resonance (Biacore) are now being increasingly used in biomaterial research as well as in other areas of research.

Studies of surface activated coagulation: antisera binding onto methyl gradients on silicon incubated in human plasma in vitro.

Human plasma proteins factor XII, high molecular weight kininogen, prekallikrein, factor XI and fibrinogen, participate in surface-initiated coagulation. Antisera binding to methyl gradients made on hydrophilic silicon was studied after immersion in normal and deficient human blood plasma. Scanning ellipsometry was used to quantify the adsorbed organic material. The hydrophilic part of the gradient deposited anti-factor XII and anti-high molecular weight kininogen, but low amounts of anti-fibrinogen. Increased amounts of anti-fibrinogen bound onto the hydrophobic part, and the intermediate gradient region with mixed polar-nonpolar surface characteristics bound low amounts of anti-factor XII, anti-high molecular weight kininogen and anti-fibrinogen. Tentatively, in this gradient region, simultaneous polar and non-polar surface characteristics result in a low-level of surface-activated coagulation. Surfaces immersed in heparinized-and EDTA-plasma indicate different antisera depositions.

In vitro study of monocyte viability during the initial adhesion to albumin- and fibrinogen-coated surfaces.

Surface adherent monocytes and macrophages play a central role in the inflammatory response to biomaterials. In the present study the adhesion, viability and apoptotic changes in material surface adherent monocytes during the first hours of cell-surface interactions in vitro were studied, using tissue culture polystyrene surfaces coated with human albumin and fibrinogen. Human peripheral blood monocytes were enriched by a two-step gradient centrifugation and resuspended (1 x 10(6)/ml) in RPMI with 10% fetal bovine serum. The cells were added to polystyrene surfaces coated with human fibrinogen or albumin and incubated in 37 degrees C (5% CO2, 100% humidity) for 30 min, 1, 2, 3 and 24 h. The adherent cells were stained for early apoptotic changes (exposed phosphatidylserine) and cell death using Annexin-V-fluorescein and propidium iodide staining, respectively. A bi-phasic adhesion was observed on the fibrinogen coated surface, having the highest number of adherent cells after 30 min and 24 h, while the cell number was markedly reduced after 1-3 h. The number of adherent cells on albumin was relatively low after all short time incubations but had reached a high level after 24 h. The number of adherent dead cells was highest after I h on both albumin (approximately 30%) and fibrinogen (approximately 15%). In the 24 h cultures, the viability of adherent cells was high on both surfaces (95-100%). Viable cells staining positive for early apoptotic changes could only be clearly observed on the albumin coated surface, after 30 min of cell-material surface interaction. Cell death, including apoptotic death, thus seems to play an important role during the initial interactions between monocytes and a foreign surface.

Titanium-hydrogen peroxide interaction: model studies of the influence of the inflammatory response on titanium implants.

In vitro studies of titanium and TiO2 as well as other metals were carried out to investigate the role of these metals in the inflammatory response through the Fenton reaction. The TiOOH matrix formed traps the superoxide radical, so that no or very small amounts of free hydroxyl radicals are produced. Ellipsometry and spin trapping with spectrophotometry and electron spin resonance (ESR) were used to study the interaction between Ti and H2O2. Spectrophotometry results indicated that Ti, Zr, Au and Al are low free OH-radical producers. We propose a new model for the titanium-tissue interface where the oxidized titanium surface is covered with a hydrated TiOOH matrix after the inflammatory reaction. This matrix is suggested to possess good ion exchange properties, and extracellular components may interact with the Ti(IV)-H2O2 compound before matrix formation. The TiOOH matrix is formed when the H2O2 coordinated to the Ti(IV)-H2O2 complex is decomposed to water and oxygen. Superoxide (O2-) may be bound therein. The oxide layer initially present may be partly reformed to a TiOOH matrix due to the interaction with hydrogen peroxide.

Interaction between hydrogen peroxide and titanium: a possible role in the biocompatibility of titanium.

Hydroxyl radicals formed from hydrogen peroxide during an inflammatory response are potent agents for cellular deterioration. The behaviour of implanted material in terms of its ability to sustain or stop free radical formation may be therefore very important. In vitro studies of titanium which is known to be biocompatible and osseointegrates into human bone were carried out. In our model studies, the production of free radicals from H2O2 at Ti and TiO2 surfaces was measured by spin trapping techniques. Our findings suggest that there is no sustained hydroxyl radical production at a titanium (oxide) surface. We propose that this is due to the quenching of the Fenton reaction through both trapping and oxidation of superoxide radicals in a TiOOH adduct.

Application of thin layer immunoassay (TIA) for demonstration of antibodies against Entamoeba histolytica.

Thin layer immunoassay (TIA) was used to demonstrate antibodies against Entamoeba histolytica in sera from patients and blood donors. The TIA results agreed well with those obtained by the indirect hemagglutination (IHA) and immunodiffusion (ID) techniques. It is suggested that because of its technical simplicity and low cost TIA may be used alternatively or in addition to the IHA and ID techniques for screening patient sera for antibodies to E. histolytica. However, the new technique must first be evaluated on a goup of clinically well defined patients with different stages of amebiasis.

Effects of Escherichia coli spheroplast formation on assays of H2 and adenosine triphosphate based ampicillin susceptibility tests.

The present study examined the effects of ampicillin on one strain of Escherichia coli in lactose peptone broth with an osmolality of 342 mosm/L under anaerobic conditions. Spheroplast formation occurred at 10 X MIC of ampicillin. The metabolic changes that took place during spheroplast formation disfavored the production of molecular hydrogen. The intracellular bacterial adenosine triphosphate (ATP) level remained normal or slightly elevated during spheroplast formation while viability (cfu/ml) decreased. Thus spheroplast formation did not interfere significantly with ampicillin susceptibility as interpreted by assaying molecular hydrogen and viability. The effect on the ATP assay was, however, pronounced. It was found that the reversion of spheroplasts to bacterial cells for this particular strain (as recorded by cfu/ml) did not occur in quantitative numbers. The ATP assay thus indicated an approximate of the density of cells, while viability studies reported a lower cell density. When using a broth with lower osmolality (50 mosm/L) no spheroplast formation occurred and a close relation between viability and intracellular ATP was observed.

The melanophore aggregating response of isolated fish scales: a very rapid and sensitive diagnosis of whooping cough.

Pertussis toxin (PT) has been found to block noradrenaline-induced pigment aggregation in fish melanophores, and, based on this, a rapid and highly sensitive assay for PT was developed. Some preliminary results have also indicated that it may be possible to detect PT-like activity in saliva samples from patients with clinically suspected pertussis. In the present study the diagnostic value of the fish melanophore method was evaluated in 70 patients suspected of having pertussis; culture, serology and physician diagnosis were used as reference methods. In 60 of the patients, pertussis was verified by at least one of the reference methods. The melanophore test showed PT-like activity in saliva samples from 58 of the patients. Three patients with reference-verified pertussis showed no PT-like activity in the test; among these, one patient had been immunized and had also been treated with erythromycin during 3 days immediately prior to visiting the hospital. The melanophore test has three major advantages: it allows detection of pertussis in the early and curable stage of the disease; it takes only 2 h to perform; and it requires no sophisticated equipment.