Protein-coated Surfaces Research Articles

Evidence for autolysin-mediated primary attachment of Staphylococcus epidermidis to a polystyrene surface.

Biofilm formation on a polymer surface which involves initial attachment and accumulation in multilayered cell clusters (intercellular adhesion) is proposed to be the major pathogenicity factor in Staphylococcus epidermidis foreign-body-associated infections. We have characterized two distinct classes of biofilm-negative Tn917 mutants in S. epidermidis affected in initial attachment (class A) or intercellular adhesion (class B). mut1 (class A mutant) lacks five surface-associated proteins with molecular masses of 120, 60, 52, 45 and 38 kDa and could be complemented by transformation with a 16.4 kb wild-type DNA fragment. The complemented mutant was able to attach to a polystyrene surface, to form a biofilm, and produced all of the proteins missing from mut1. Subcloning experiments revealed that the 60 kDa protein is sufficient for initial attachment. Immunofluorescence microscopy using an antiserum raised against the 60 kDa protein showed that this protein is located at the cell surface. DNA-sequence analysis of the complementing region revealed a single open reading frame which consists of 4005 nucleotides and encodes a deduced protein of 1335 amino acids with a predicted molecular mass of 148kDa. The amino acid sequence exhibits a high similarity (61% identical amino acids) to the atl gene product of Staphylococcus aureus, which represents the major autolysin; therefore the open reading frame was designated atlE. By analogy with the S. aureus autolysin, AtlE is composed of two bacteriolytically active domains, a 60 kDa amidase and a 52 kDa glucosaminidase domain, generated by proteolytic processing. The 120 kDa protein missing from mut1 presumably represents the unprocessed amidase and glucosaminidase domain after proteolytic cleavage of the signal- and propeptide. The 45 and 38kDa proteins are probably the degradation products of the 60 and 52 kDa proteins, respectively. Additionally, AtlE was found to exhibit vitronectin-binding activity, indicating that AtlE plays a role in binding of the cells not only to a naked polystyrene surface during early stages of adherence, but also to plasma protein-coated polymer surfaces during later stages of adherence. Our findings provide evidence for a new function of an autolysin (AtlE) in mediating the attachment of bacterial cells to a polymer surface, representing the prerequisite for biofilm formation.

Self-consistent-field modelling of adsorbed casein Interaction between two protein-coated surfaces

E. Dickinson, V. J. Pinfield, D. S. Horne and F. A. M. Leermakers, J. Chem. Soc., Faraday Trans., 1997, 93, 1785 DOI: 10.1039/A608417F

Culture of human vascular endothelial cells on an RGD-containing synthetic peptide attached to a starch-coated polystyrene surface: comparison with fibronectin-coated tissue grade polystyrene

A synthetic peptide, Gly-Arg-Gly-Asp-Ser-Pro-Lys (GRGDSPK), which includes the cell-adhesive region of fibronectin, Arg-Gly-Asp (RGD), was covalently bound to a dialdehyde starch (DAS) coating on a polymer surface by reductive amination. The GRGDSPK/DAS-coated surface was characterized by atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). AFM and SEM revealed a uniform, roughened, textured surface, much more so than standard polymer or adhesive protein-coated polymer surfaces. XPS showed that GRGDSPK binding to DAS occurred in dose-dependent fashion in the 0-200 micrograms ml-1 GRGDSPK concentration range, with a plateau happening in the 200-400 micrograms ml-1 range. AFM revealed a uniform peptide layer on the DAS surface with a maximum separation distance of 50 nm between peptides. Angle-dependent XPS showed that the peptide is present in nearly constant amounts to at least 10 nm depth of the DAS coating. The attachment, spreading and growth properties of anchorage-dependent human umbilical vein endothelial cells (EC) on the GRGDSPK/DAS-coated polystyrene surface were compared with a standard fibronectin-coated polystyrene surface. EC adhesion, spreading and growth properties were similar for cells plated on polystyrene surfaces coated with fibronectin (5 micrograms cm-2) and GRGDSPK (25-50 micrograms ml-1)/DAS. In contrast, EC adhesion, spreading and growth performance significantly increased for cells plated on GRGDSPK (100-200 micrograms ml-1)/DAS compared with the fibronectin-coated surface. These findings support the conclusion that the GRGDSPK/DAS-coated surface can be substituted for an adhesive protein-coated surface in the culture of anchorage-dependent cells.

Adsorption/desorption of amine fluorides to hydroxyapatite

This study concerned the adsorption and desorption of commercial amine fluoride (AmF) preparations to hydroxyapatite (HA). The influence of pH, ionic strength, temperature, saliva and albumin, the latter as a gingival crevicular fluid analogue, on adsorption/desorption was investigated. AmF levels were determined using a surfactant electrode. AmFs 297 and 335 were found to bind immediately and irreversibly to HA in water over a range of pH values, ionic strengths and temperatures, the amounts increasing with concentration. More monovalent AmF 335 was adsorbed than divalent AmF 297. Any AmF desorbed by water from HA was at the lowest end of the minimum inhibitory concentration for oral bacteria. AmF 297 was desorbed by CaCl 2, and to a lesser extent by H +, OH −, NH 4 +, La 3 +, EDTA, Triton X100 and ethanol, whereas AmF 335 was only slightly desorbed by ethanol. Preadsorption of proteins on HA had little effect on subsequent adsorption or desorption of either AmF. It is postulated that both AmF 297 and AmF 335 are inactivated by an excess of proteins in the surrounding medium, supra- or subgingivally, and not by such proteins preventing or altering the mode or rate of adsorption, or interfering with antibacterial activity, when the AmFs contact a protein-coated tooth surface.

Antibodies that activate β2 integrins can generate different ligand binding states

A human erythroleukemic cell line (K562) that does not normally express beta 2 integrins has been transfected with the genes encoding these integrins. The resulting cell lines show minimal background adhesion but can be stimulated to bind to appropriate substrates when activated with either of two different antibodies to CD18. The two antibodies appear to generate different ligand binding states in LFA-1 such that different members of the ICAM family are recognized. Antibody-activated complement receptor type 3 and p150,95-transfected cells bind protein-coated surfaces, although they require slightly different activation conditions for optimal binding.

Surfactant protein A modulates release of reactive oxygen species from alveolar macrophages.

The production and release of reactive oxygen species (the respiratory burst) is a common metabolic pathway linked to several macrophage-related reactions. The most abundant surfactant protein A (SP-A) binds to alveolar macrophages (AM) through a specific surface receptor with high affinity. Because such binding might initiate or modulate the respiratory burst, we wanted to know whether and how SP-A affects the oxygen radical release from AM. To answer these questions, we measured the release of reactive oxygen species from rat AM under various in vitro conditions using enhanced chemiluminescence systems. We prepared SP-A from pulmonary surfactant isolated either from silica-treated rats or adult dogs. Resident AM were harvested from pathogen-free Wistar rats by lung lavage. Adhered and nonadhered AM were assessed on protein-free or protein-coated surfaces of 96-well microtiter plates. On protein-free surfaces, the sole addition of SP-A failed to induce measurable oxygen radical release from 2 x 10(5) adhered or nonadhered AM, while zymosan opsonized with SP-A induced a marked increase over control. On protein-coated surfaces, AM respond differently depending on the coated protein: on SP-A-coated surfaces, a dose-dependent enhancement of oxygen radical release with a mean effective concentration of approximately 1.15 micrograms/ml was found. No such enhancement was seen on plates coated with similar amounts of either human fibronectin or collagen, and the enhancement with serum albumin was not dose related. Our data demonstrate that SP-A only enhances oxygen radical release from AM if SP-A is fixed to zymosan or the surface of the reaction vial in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative Estimation of the Degree of Cell Spreading on Different Surfaces and Cell Monolayers Using a Fluorescent Plate Scanner

A simple, rapid, and quantitative method for estimation of the degree of spreading of fluorescently labeled cells adherent to an artificial or biological surface has been designed, based on the principle that the medium between adherent cells and the surface of adhesion absorbs fluorescent light emitted by the adherent cells. Using the Beer-Lambert equation for extinction coefficient of monochromatic light, we have calculated the factor by which fluorescence signal actually emitted from cells adherent to the bottom of the microtiter well would differ from the fluorescence of these cells detected by a bottom-scanning plate reader. The physical nature of cell-cell ligand-receptor bridges depends on many parameters, such as the type of the receptor(s), the intensity and direction of a stress force, blockade of the receptors with monoclonal antibodies (MAb), and transfer of adhesion mediation to different ligand-receptor pairs. When employed in various adhesion models, this new technique has demonstrated quantitative changes of cellular spreading of human neutrophils adherent to different extracellular matrix protein-coated surfaces and to endothelial cells after neutrophil/endothelial cell activation or in the presence of MAb directed against neutrophil integrins. These measurements of spreading of adherent cells appear more sensitive than visual observation of cellular morphology on biological surfaces or cells.

Priming of human monocyte superoxide production and arachidonic acid metabolism by adherence to collagen- and basement membrane-coated surfaces.

Monocytes (m phi s) come into intimate contact with basement membranes and extracellular matrix proteins as they extravasate from the blood to the interstitium or to sites of tissue injury. We examined the in vitro effects of m phi adherence to an endothelial cell-derived basement membrane or to purified extracellular matrix proteins on phorbol myristate acetate (PMA)-stimulated superoxide production and prostanoid secretion. Elutriation-purified human peripheral blood m phi s were adhered to tissue culture wells that were precoated with the following purified proteins: bovine serum albumin (BSA), collagen type I (COL I), collagen type IV (COL IV), fibronectin (FN), or laminin (LM). To model the provisional matrix at sites of tissue injury, m phi s were also adhered to wells coated with either denatured collagen type I or gelatin (GEL) or coated with basement membrane (BM) derived from endothelial cell monolayers. The m phi s were adhered to the protein-coated surfaces for 1 h at 37 degrees C in serum-free medium and washed to remove nonadherent cells, and the number of adherent m phi s was measured. Monolayers of m phi s were also incubated for an additional 18 h, at which time both adherence and cell spreading were measured. PMA-stimulated superoxide production by adherent m phi s was determined after 1 and 18 h of adherence to the protein-coated surfaces. PMA-stimulated release of two prostanoids, prostaglandin E2 (PGE2) and thromboxane B2 (TxB2) was measured after 18 h of m phi adherence to the surfaces. Following 18 h of adherence, PMA-stimulated superoxide anion secretion and secretion of PGE2 and TxB2 were augmented of primed by m phi s adherent to COL I, GEL, or BM. In contrast, no such priming effects were observed by m phi s adherent to COL IV, FN, or LM. The results suggest that adherence to basement membranes, collagen type I-containing surfaces in the interstitium, or denatured collagen at sites of tissue injury primes m phi respiratory burst and arachidonate metabolism to inflammatory agonists. Induction of priming events by substrate-specific adherence may be an important factor regulating host defense functions of m phi s in the extracellular matrix.

Albumin inhibits neutrophil spreading and hydrogen peroxide release by blocking the shedding of CD43 (sialophorin, leukosialin).

Spreading of neutrophils on protein-coated surfaces is a pivotal event in their ability to respond to soluble, physiologic agonists by releasing large amounts of hydrolases and oxidants. Using neutrophils plated on serum-, fibrinogen- or fibronectin-coated surfaces, we investigated the effect of human serum albumin (HSA) on spreading-dependent neutrophil responses. HSA suppressed the respiratory burst of neutrophils in response to tumor necrosis factor-alpha (TNF), complement component C5a or formylated peptide, but not phorbol myristate acetate. HSA was suppressive only if added before the onset of the respiratory burst, and suppression was reversed when HSA was removed. Likewise, HSA selectively and reversibly inhibited TNF-induced cell spreading and the associated fall in cAMP. However, HSA did not hinder TNF-induced cell adherence to the same protein-coated surfaces. We investigated cell surface sialoproteins as modulators of cell spreading and as targets for the anti-spreading action of HSA. Oxidation of the cell surface with periodate followed by reduction with 3H-borohydride and immunoblotting with specific mAbs helped identify the predominant sialoprotein on human neutrophils as CD43 (sialophorin, leukosialin). Treatment of neutrophils with C. perfringens sialidase desialylated CD43, markedly enhanced the ability of the cells to respond to TNF by spreading and undergoing a respiratory burst, and antagonized the ability of HSA to inhibit these responses. TNF-treated, adherent neutrophils shed CD43, and this was blocked by HSA, but not by ovalbumin. Exogenous neutrophil elastase removed CD43 from the neutrophil surface. HSA blocked the actions of both sialidase and elastase on CD43. In contrast, ovalbumin did not block the action of sialidase on CD43, and HSA did not inhibit the ability of sialidase to hydrolyze a synthetic substrate. These results suggested that HSA might bind CD43. In fact, the extracellular portion of CD43 bound to HSA-Sepharose, but not to ovalbumin- or glycylglycine-Sepharose. Finally, two mAbs recognizing different epitopes on CD43 mimicked HSA's inhibitory effects on neutrophil function. Thus, HSA can dissociate attachment of neutrophils from spreading. This dissociation may help neutrophils migrate along a chemotactic gradient, while decreasing their release of oxidants. CD43, a long, rigid molecule with a markedly negative charge, antagonizes neutrophil spreading. HSA appears to inhibit spreading-dependent neutrophil functions by binding to CD43 and interfering with the ability of neutrophils to shed it.

Activated platelets form protected zones of adhesion on fibrinogen and fibronectin-coated surfaces.

Leukocytes form zones of close apposition when they adhere to ligand-coated surfaces. Because plasma proteins are excluded from these contact zones, we have termed them protected zones of adhesion. To determine whether platelets form similar protected zones of adhesion, gel-filtered platelets stimulated with thrombin or ADP were allowed to adhere to fibrinogen- or fibronectin-coated surfaces. The protein-coated surfaces with platelets attached were stained with either fluorochrome-conjugated goat anti-human fibrinogen or anti-human fibronectin antibodies, or with rhodamine-conjugated polyethylene glycol polymers. Fluorescence microscopy revealed that F(ab')2 anti-fibrinogen (100 kD) did not penetrate into the contact zones between stimulated platelets and the underlying fibrinogen-coated surface, while Fab antifibrinogen (50 kD) and 10 kD polyethylene glycol readily penetrated and stained the substrate beneath the platelets. Thrombin- or ADP-stimulated platelets also formed protected zones of adhesion on fibronectin-coated surfaces. F(ab')2 anti-fibronectin and 10 kD polyethylene glycol were excluded from these adhesion zones, indicating that they are much less permeable than those formed by platelets on fibrinogen-coated surfaces. The permeability properties of protected zones of adhesion formed by stimulated platelets on surfaces coated with both fibrinogen and fibronectin were similar to the zones of adhesion formed on fibronectin alone. mAb 7E3, directed against the alpha IIb beta 3 integrin blocked the formation of protected adhesion zones between thrombin-stimulated platelets and fibrinogen or fibronectin coated surfaces. mAb C13 is directed against the alpha 5 beta 1 integrin on platelets. Stimulated platelets treated with this mAb formed protected zones of adhesion on surfaces coated with fibronectin. These protected zones were impermeable to F(ab')2 antifibronectin but were permeable to 10 kD polyethylene glycol. These results show that activated platelets form protected zones of adhesion and that the size of molecules excluded from these zones depends upon the composition of the matrix proteins to which the platelets adhere. They also show that formation of protected zones of adhesion by platelets requires alpha IIb beta 3 integrins while the permeability properties of these zones of adhesion are regulated by both alpha IIb beta 3 and alpha 5 beta 1 integrins.

Fibronectin and proteolytic fragments of fibronectin interfere with the adhesion of Staphylococcus epidermidis to plastic.

The adhesion of five strains of slime-positive Staphylococcus epidermidis to plastic microwells was significantly diminished (P < 0.005) in a concentration-dependent fashion when wells were previously coated with increasing concentrations (1.6-13.1 micrograms cm-2) of human fibronectin (FN). The adhesion of four of five strains was significantly reduced when wells were coated with 3.2 micrograms cm-2 of FN and at concentrations > or = 6.5 micrograms cm-2 the adhesion of all slime-positive strains was significantly reduced. The coating of microwells with chymotryptic fragments of FN containing the heparin-binding, gelatin-binding, or cell-binding domains also reduced bacterial adhesion but none of the fragments exceeded the anti-adhesive activity of intact FN. A comparison of FN-coated or albumin-coated microwells showed that both proteins caused a significant reduction in the adhesion of test strains to plastic but that the anti-adhesive activity of FN was greater than albumin at all concentrations tested. The adhesion of the slime-negative phase variant of one of the test strains to plastic was neither enhanced nor reduced by FN coating indicating that the production of an exopolysaccharide by Staph. epidermidis influences interactions with protein-coated surfaces. These results support the contention that FN does not mediate the adhesion of all strains of Staph. epidermidis to plastic surfaces.

Human vascular endothelial cell attachment and growth inhibition by type V collagen

Purpose: Human vascular prostheses develop a pseudointima that after time is devoid of a complete endothelial lining. The composition of this pseudointima consists of both cellular and noncellular components such as coagulation proteins and extracellular matrix proteins. Of these extracellular molecules, type V collagen has been reported to be localized to surfaces of vascular prostheses. We have hypothesized that type V collagen may be involved in the mechanisms of inhibition of endothelialization on vascular prostheses. In this study, interactions of human protein and cells with clinically used material and reference surfaces were analyzed in vitro.Methods and Results: Human collagen types IV and V or human fibronectin was coated on disks punched from expanded polytetrafluoroethylene, bacteriologic polystyrene, and tissue culture-treated polystyrene. Fibronectin adsorbed equally to these surfaces, but differential adsorption of type V collagen occurred. The attachment and growth of human saphenous vein and umbilical vein endothelial cells and of human skin fibroblasts were also evaluated on protein-coated or uncoated surfaces. Type IV collagen and fibronectin promoted the attachment of these cells, but type V collagen reduced cellular adhesion. Growth of endothelial cells was significantly inhibited on surfaces coated with type V collagen even when additional growth substances such as serum, retinal-derived growth factor, and heparin were present in the medium. Human adult dermal fibroblast adhesion and cell growth were not affected by coating the surfaces with type V collagen.Conclusions: The components of the extracellular matrix of the pseudointima may directly influence endothelial cells by inhibiting cell proliferation, migration from within the graft or from anastomoses, or both. Type V collagen, a matrix protein found at luminal surfaces of vascular prostheses, may be one protein responsible for control of endothelial responses.

Adhesion-dependent protein tyrosine phosphorylation in neutrophils treated with tumor necrosis factor.

Human neutrophils (PMN) respond to tumor necrosis factor (TNF) by releasing their granules, reorganizing their cytoskeleton, and massively secreting hydrogen peroxide. This response is dependent on adhesion to extracellular matrix proteins and expression of CD11b/CD18 integrins (Nathan, C., S. Srimal, C. Farber, E. Sanchez, L. Kabbash, A. Asch, J. Gailit, and S. D. Wright. 1989. J. Cell Biol. 109:1341-1349). We investigated the role of tyrosine phosphorylation in the response of PMN to TNF. PMN adherent to protein-coated surfaces but not suspended PMN showed tyrosine phosphorylation of several proteins (approximately 150, approximately 115, approximately 75, and approximately 65 kD) in response to TNF. Tyrosine phosphorylation was evident 5 min after addition of TNF and lasted at least 2 h. The tyrosine kinase inhibitors K252a, genistein and ST638 suppressed tyrosine phosphorylation and blocked hydrogen peroxide production in a reversible manner at low concentrations. Tyrosine kinase inhibitors also blocked the spreading of PMN in response to TNF. Dihydrocytochalasin B did not inhibit tyrosine phosphorylation, but in its presence phosphorylation was rapidly reversed. By immunocytochemistry, the majority of tyrosine phosphoproteins were localized to focal adhesions. Thus TNF-induced tyrosine phosphorylation depends on adhesion of PMN to extracellular matrix proteins, and participates in the transduction of the signals that direct the cells to spread on a biological surface and undergo a respiratory burst.

Recruitment of CD11b/CD18 to the neutrophil surface and adherence-dependent cell locomotion.

Chemotactic stimulation of neutrophils results in translocation of CD11b/CD18 (Mac-1) from intracellular storage pools to the cell surface. Though results from several laboratories indicate that the newly arrived surface Mac-1 is not involved in the adherence induced by the initial stimulus, the present study addresses the hypothesis that this Mac-1 plays a role in subsequent adherence-dependent functions. The response of human neutrophils to changing concentrations of a chemotactic stimulus was evaluated by determining the amount of newly arrived surface Mac-1, and Mac-1-dependent adhesion and locomotion. Small step-wise increases in the concentration of f-Met-Leu-Phe (FMLP) resulted in proportional stepwise increases in surface Mac-1 that plateaued within 2-4 min. This newly arrived Mac-1 supported adhesion to protein-coated surfaces only when the cells were exposed to an additional increase in the FMLP stimulus level. Adherence-dependent cellular locomotion was evaluated in chambers that allowed rapid changes in the stimulus concentration. Repeated small increments in the stimulus level at 200-s intervals resulted in significantly longer migration paths than a single-step increase in the stimulus. The results support the hypothesis that small increments in the chemotactic stimulus bring Mac-1 to the cell surface, and this newly mobilized Mac-1 is available for adherence-dependent locomotion with subsequent increases in the concentration of the stimulus.

Reflectance method for simple determination of proteinase activity in microliter samples of a complex serum-like fluid

A technique using an optical instrument, a reflectometer, for quantitative determination of proteinase activity in microliter samples of complex serum-like fluids, e.g., crevicular exudate from single sites, was developed. The technique allowed the use of various proteins as enzyme substrate. The reflectometer measures the mass of a layer, such as protein, adsorbed to a reflecting surface. This is done by measuring the reflected light intensity of the p-polarized light beam on a surface. We used methylized silicon surfaces that were coated with fibrinogen, α 2-macroglobulin, or hemoglobin as enzyme substrates. The test solution was incubated overnight in a basin made in an agar gel applied on the top of the protein-coated surface. In 82 exudates from periodontitis sites, with pocket depths ≥ 6 mm, fibrinogenolytic activity corresponding to 1 μg ml −1 of trypsin and pronase P was found in 20% of the samples.

Postadsorptive transitions in fibrinogen adsorbed to polyurethanes: changes in antibody binding and sodium dodecyl sulfate elutability.

Residence time-dependent changes in fibrinogen after adsorption to six different polyurethanes were examined by measuring polyclonal antifibrinogen binding to the adsorbed protein. The amount of adsorbed fibrinogen that could be eluted by sodium dodecyl sulfate (SDS) was also measured. Baboon fibrinogen was first adsorbed from dilute plasma to the polymers, which were then stored in either buffer or buffered albumin solution prior to testing. Subsequently, the amount of antifibrinogen bound by the adsorbed fibrinogen was measured using a direct enzyme linked immunosorbent assay (ELISA). Alternatively, the surface with the adsorbed fibrinogen was soaked in a 3% SDS solution, and the amount of retained 125I-radiolabeled fibrinogen was measured. With increasing residence time, decreases in both antibody binding and the SDS elutability of the adsorbed fibrinogen occurred, but the rate of change was dependent on the polyurethane to which the fibrinogen was adsorbed. In addition, the antibody binding per unit of adsorbed fibrinogen, when measured immediately after the adsorption step, varied by approximately a factor of 3 among the various polyurethanes. When the protein-coated surfaces were stored in buffered albumin solution rather than buffer, the decrease in the reactivity of fibrinogen with residence time did not occur on some of the surfaces. This study shows that the chemical properties of the adsorbing surface influence the rate at which adsorbed fibrinogen undergoes change. The significance of the polymer-dependent changes in adsorbed fibrinogen with respect to blood reactions with polymers is discussed.

A PC-based imaging system for automated platelet identification.

In this communication, a PC-based imaging system was developed for automatically identifying fluorescence-labeled individual platelets adherent to protein-coated surface under flow conditions. It is to eliminate the laborious and time-consuming task, and the subjective error of manual measurements. Based upon the features of adherent platelets, three passes of the image processing were developed for platelet identification. From the results, 90-95% accuracy could be routinely obtained. The platelet distribution and other related parameters could be easily extracted and investigated.

Optical biosensor assay (OBA)

We describe a new biosensor immunoassay involving optical diffraction to detect clinically important analytes in human body fluids. A silicon wafer is used as a support for immobilization of antigen or antibody. The protein-coated surface is illuminated through a photo mask to create distinct periodic areas of active and inactive protein. When the surface is incubated with a positive sample, antigen-antibody binding occurs only on the active areas. Upon illumination with a light source such as a laser, the resulting biological diffraction grating diffracts the light. A negative sample does not result in diffraction because no antigen-antibody binding occurs to create the diffraction grating. The presence or absence of a diffraction signal differentiates between positive and negative samples, and the intensity of the signal provides a quantitative measure of the analyte concentration. The technique is demonstrated with a quantitative assay of choriogonadotropin in serum.

Postadsorptive transitions in fibrinogen adsorbed to Biomer: Changes in baboon platelet adhesion, antibody binding, and sodium dodecyl sulfate elutability

Residence-time-dependent changes in fibrinogen after its adsorption to Biomer were examined by measuring platelet adhesion and antibody binding to the adsorbed protein, and the amount of adsorbed fibrinogen which could be eluted by sodium dodecyl sulfate (SDS). Baboon fibrinogen was first adsorbed (from either pure solution or dilute plasma) to Biomer, which was then stored in either buffer or buffered albumin solution prior to testing. Subsequently, the adherent protein layer was either probed for fibrinogen capable of mediating platelet adhesion using 111In radiolabeled, washed platelet suspensions under both static and shearing conditions, or for fibrinogen capable of binding antibody using a direct enzyme linked immunosorbent assay (ELISA). Alternatively, the surface with the adsorbed protein layer was soaked in a 3% SDS solution, and the amount of 125I radiolabeled fibrinogen retained was measured. Decreases in platelet and antibody binding, and in the SDS elutability of the adsorbed fibrinogen after it was stored in buffer were detected, although different rates of decrease were observed for each method. When the protein-coated surfaces were stored in buffered albumin solution rather than buffer, the decrease in the reactivity of fibrinogen was prevented. While each of the three assays measures a different property of adsorbed fibrinogen, this study suggests that the adherent protein undergoes time dependent conformational changes which render it less reactive toward platelets and antibodies, and more resistant to elution by SDS.

Measurement of Cell Adhesion and Migration on Protein-Coated Surfaces

ABSTRACTThe performance of biomaterials forin vivoandin vitroapplications can depend critically on tissue cell adhesion and migration. We have been investigating the role that specific reversible interactions between cell adhesion receptors and complementary substratum-bound ligands play in the regulation of cell adhesion and migration. With an axisymmetric radial flow detachment assay (RFDA) [1] we measured cell-substratum adhesive strength for human smooth muscle cells (HSMCs) on surfaces coated with type IV collagen (CIV). We found that the critical shear stress for detachment increased linearly with increasing CIV coating concentration. Using time-lapse videomicroscopy and image analysis we tracked the movement of individual HSMCs over similar CIV-coated surfaces. Cell speed and persistence were determined for variations in CIV coating concentration by applying a persistent random walk model for individual cell movement. Cell speed reached a maximum at an intermediate concentration of CIV, supporting the hypothesis that an optimal cell-substratum adhesiveness exists for HSMC movement. This combination of techniques for measuring adhesion and motility provides a valuable tool to examine the role of cell-biomaterial interactions on cell behavior.