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Comparative studies on the interaction of proteins with a polydimethylsiloxane elastomer. II. The comparative antigenicity of primary and secondarily adsorbed IgG1 and IgG2a and their non-adsorbed counterparts.

The antigenicity of bovine IgG1 and IgG2a adsorbed on a polydimethysiloxane (PEP) elastomer, on a widely used polystyrene (Imm 2, Dynatech) or immobilized as biotinylated proteins to streptavidin covalently bound to polystyrene (SA-PS) was compared using various monoclonal (mAbs) and polyclonal antibodies (pAb) to bovine IgG. The IgGs were either adsorbed as native proteins or pre-denatured with 6M Guanidine-HCl (Gu-HCl) or 6 M Gu-HCl/0.1% 2-mercaptoethanol. In special situations, bovine and human IgG was immobilized by secondary adsorption to an albumin monolayer adsorbed on either PEP or Imm 2. Results indicate that pre-denaturation of IgGs with 6 M Gu-HCl/2-mercaptoethanol destroys all antigenicity whereas those IgGs pretreated with 6 M-GuHCl are indistinguishable in their antigenicity from the IgGs adsorbed to either PEP or Imm 2 without such treatment. When immobilized on SA-PS, Gu-HCl-treated IgGs were significantly less detectable, especially when tested using mAbs. In general, IgGs adsorbed on PEP or Imm 2 were less antigenic than when immobilized on SA-PS. However, two monoclonals specific for the IgG2a(A2) allotypic variant, favored the adsorbed protein and one polyclonal best recognized the IgG2a(A1) variant adsorbed on Imm 2 rather than when adsorbed on PEP or immobilized on SA-PS. Both IgG1 and IgG2a, bound by apparent protein-protein interactions to an albumin monolayer, were significantly more detectable than when directly adsorbed on either Imm 2 or PEP. Using 125I-antibody or its Fab fragment to reduce steric hindrance in detection, we observed the same differences in detectability as when measured by enzyme-linked immunosorbent assay. Failure to identify a steric hindrance effect and the preference of some antibodies for adsorbed allotypic variants, support the concept of adsorption-induced conformational change (AICC). We conclude that proteins adsorbed as a monolayer on the PEP elastomer used to form the envelope of silicone breast implants are conformationally altered, but not necessarily to the same extent or the same manner as when adsorbed on polystyrene. The significantly great antigenicity of secondarily adsorbed IgG suggests that it may be present in near native conformation.

Adsorption↗

Characteristics of the production of active oxygen species from adherent and non-adherent neutrophils.

Our objective was to evaluate the characteristics of the production of AOS from the neutrophils that had adhered to the endothelial cells, fibronectin or polystyrene, using the method of electron paramagnetic resonance (EPR) spin trapping. Neutrophils and endothelial cells were isolated from human venous blood and umbilical veins, respectively. AOS production from neutrophils was not elicited only by adhesion. The stimulation of adherent neutrophils with phorbol myristate acetate (PMA) induced the production of AOS. The production of AOS from adherent neutrophils to endothelial cells, but not to fibronectin or polystyrene, decreased with the interval time between the adhesion and the stimulation by PMA. The amount of AOS produced by the neutrophils adherent to fibronectin or polystyrene was maintained for one hour after stimulation with PMA, whereas that by suspended neutrophils gradually decreased with the time after stimulation. Results indicate that adherent and non-adherent neutrophils exhibit differing time course of AOS production.

Cell Adhesion↗

The initial stages of adsorption of plasma derived proteins on artificial surfaces in a controlled flow environment.

Surfaces of amorphous carbon, polystyrene, and polycarbonate were exposed to solutions of fibrinogen, a modified fibrinogen which lacked the alpha chain protuberance, and serum albumin. The results were studied by electron microscopy. The exposures occurred in a well characterized flow environment at a shear rate of 135/s. All three protein species formed a film when adsorbed to carbon films. When the proteins were adsorbed to polystyrene surfaces, formation of a network was observed. Polycarbonate surfaces adsorbed the proteins both as a network and as a continuous film. It was observed that the nature of the adsorption process depended upon the specific combination of molecule and material. For example, on carbon, individual fibrinogen molecules retain their trinodular structure and adsorb randomly until a monolayer forms. On polystyrene, the individual fibrinogen molecules appear as globules and a network forms before complete coverage occurs.

Adsorption↗

Mechanisms of biomaterial-induced superoxide release by neutrophils.

Biomaterial-centered infection is an important cause of the failure of prosthetic implants and organs. Because neutrophils mediate host defense against infection, the effect of biomaterials on neutrophil superoxide release and the mechanism of that effect were investigated using three materials commonly employed in surgical practice. The graft materials were expanded polytetrafluorethylene (PTFE), polyurethane and woven dacron. Polystyrene, a commonly used laboratory support vessel, was also studied. Both polystyrene and polyurethane were activating, but serum inhibitable, whereas PTFE was nonactivating, and woven dacron was not activating unless serum was present. The signaling mechanisms used by these materials demonstrated time and material dependency. Pertussis toxin inhibition of G protein-dependent activation had little or no effect on biomaterial induced activation, whereas FMLP-induced activation of the same biomaterial-associated cells was inhibited. Protein kinase C inhibition with staurosporine greatly inhibited polystyrene-induced activation, but had only a partial effect with polyurethane and even less effect with the activation associated with serum-treated woven dacron. These studies demonstrated that biomaterial contact-induced neutrophil activation differed from that described for cells in suspension, and showed that activation mechanisms on one material cannot be extrapolated to mechanisms on other materials.

Alkaloids↗

Modulation of adhesive properties of DEAE-dextran with laminin.

Human squamous epithelial cells produce lower amounts of laminin and fibronectin when cultured on DEAE-dextran than when cultured on gelatin-coated polystyrene (Biotechnol. Bioeng., 33:1235). The epithelial cells also spread much more slowly on DEAE-dextran than they do on gelatin-coated polystyrene. To determine if the low level of matrix production by cells grown on DEAE-dextran contributed to the slowness of cell spreading on this substrate, microcarriers made from DEAE-dextran were treated with exogenous laminin (10 micrograms/cm2 of surface area) and then examined for ability to support cell adhesion. Squamous epithelial cells spread as rapidly on the laminin-treated DEAE-dextran as they did on gelatin-coated polystyrene (much more rapidly than on untreated DEAE-dextran). This indicates 1) that laminin can bind to DEAE-dextran in a fashion that is biologically usable by anchorage-dependent cells, and 2) that when laminin is bound to DEAE-dextran, the failure of squamous epithelial cells to rapidly spread is overcome. These data support the hypothesis that failure of the cells to synthesize an intact extracellular matrix on DEAE-dextran is responsible, at least in part, for the slowness with which cells spread on this substrate.

Adhesives↗

Temperature-induced cell detachment on immobilized pluronic surface.

The Pluronic F68 and F127, a triblock copolymer of ethylene oxide and propylene oxide, was activated using carbonyldiimidazole (CDI), and CDI-activated Pluronic F68 and F127 was subsequently immobilized on the surface of a poly-L-lysine-coated polystyrene tissue culture flask. Cell culture was performed on the Pluronic-immobilized flask. The morphology of fibroblasts (L929 cells) on the Pluronic F127-immobilized flask was mainly spherical, and showed less spreading behavior than that on the Pluronic F68-immobilized flask and conventional tissue culture flask. This observation indicates that L929 cells on Pluronic F127-immobilized flasks were cultured in a bio-inert environment. L929 cells were successively detached from both Pluronic F127-immobilized flask and Pluronic F68-immobilized flask by cooling the flask to 4-15 degrees C. This detachment is due to the hydration and dehydration properties of Pluronic, depending on the temperature. Umbilical cord blood was cultured in the Pluronic F127-immobilized and conventional polystyrene tissue culture flasks at 37 degrees C. The expression ratio of surface markers on hematopoietic stem cells (CD34 and CD133) cultured in the Pluronic F127-immobilized flask was significantly higher than that of the cells in polystyrene tissue culture flask.

AC133 Antigen↗

Differential phosphorylation of paxillin in response to surface-bound serum proteins during early osteoblast adhesion.

An early signaling event during the adhesion and spreading of cells is integrin-mediated tyrosine phosphorylation of the cytoskeletal adaptor protein paxillin and the non-receptor tyrosine kinase pp125(FAK) at focal contacts. To determine the influence of surface-charge and -adsorbed adhesion proteins on this signaling pathway, paxillin phosphorylation was examined during attachment of MC3T3-E1 osteoblast-like cell onto charged and uncharged polystyrene, and on adsorbed layers of serum proteins, fibronectin (Fn), vitronectin (Vn), a mixture of Fn and Vn, and albumin. Paxillin phosphorylation was induced 2.4-fold (P < 0.05) on charged vs uncharged polystyrene only in the presence of serum proteins. Activation of paxillin via Fn or Vn alone, or in combination, resulted in significantly lower phosphorylation signals compared to whole serum (41 +/- 6.9%, P < 0.05, 45 +/- 5.9%, P < 0.05, and 76 +/- 9.8%, P < 0.075, respectively). Confocal laser microscopy confirmed increased co-localization of phosphotyrosine and paxillin at protruding lamellopodia of spreading osteoblasts on charged vs uncharged serum-pretreated polystyrene. Taken together, these data suggest that subtle differences in surface characteristics mediate effects on adhering cells via adsorbed serum proteins involving the cytoskeletal adaptor protein paxillin.

3T3 Cells↗

Distribution of blood-borne particles of two different sizes in rat spleens.

Polystyrene microspheres 5 micron in diameter and Indian ink, consisting of particles 0.03 micron in diameter, were injected into the splenic artery in rats. The distribution of the polystyrene microspheres and the ink particles in the spleen were examined microscopically and morphometrically. The polystyrene microspheres appeared mainly in the red pulp, and the Indian ink particles mostly in the marginal zone, which functions as an immunological filter. (No arteries opened into the lymphoid follicles.)

Animals↗

High molecular weight neoglycoconjugates for solid phase assays.

Adsorption of a carbohydrate on solid phase is the necessary stage of the immunosorbent assay (ELISA) and analogous methods of the study of carbohydrate-protein interaction. Usually physical adsorption on polystyrene requires a high concentration of conjugated carbohydrate and, thus, enormous consumption of it. In this study, we explored two approaches allowing more rational use of oligosaccharide (Glyc). The first of them is based on the covalent immobilization of neoglycoconjugates on the NH(2)-modified polystyrene; the second one is based on the elevated adherence of high m.w. neoglycoconjugates to polystyrene. Covalent immobilization of polyacrylamide conjugates, Glyc-PAA, provided a possibility to solve the problem, but the nonspecific binding of antibodies in ELISA proved to be unacceptably high. At the same time, the increase of the Glyc-PAA m.w. from 30 kDa to 2,000 kDa allowed a 10-20 fold decrease of its consumption, when using physical adsorption, whereas the assay background remained at the low level. The amount of 2,000 kDa Glyc-PAA that is sufficient for the coating of a standard 96-well plate corresponds to the nanomole level of oligosaccharide, this providing a possibility to use saccharides that are available in a very limited amount when studying the carbohydrate-protein interaction with solid-phase techniques.

Acrylic Resins↗

In vitro tagging of embryos with nanoparticles.

PURPOSE: To develop an in vitro method for tagging embryos and to compare the development of the embryos after nanoparticles injection versus externally-applied nanoparticles derived from either polystyrene or polyacrylonitrile. METHODS: Each mouse 1-cell embryo (the selected test-model) was either: (a) injected by intracytoplasmic injection or (b) co-incubated with different nanoparticles at 37 degrees C, 5% CO2 in air. The embryos were assessed after 2 and 6 days of culture. RESULTS: Embryo development was similar for externally-applied polystyrene nanoparticles and control (97.6 +/- 2.7 versus 100.0 +/- 0%) but different for polyacrylonitrile nanoparticles (90.0 +/- 2.8 %) on day 2. However, the results were similar on Day 6. Injected embryos were linked to lower percent development on Day 2. Few injected embryos reached blastocyst stage on Day 6 after a brief UV-fluorescence exposure. CONCLUSIONS: Tagging embryos by external polystyrene-based nanoparticles was the better method when compared with injected nanoparticles. Larger nanoparticles in microsphere range were easier to qualitate. Inhibited hatching limited their use beyond the blastocyst stage.

Animals↗

An enzyme-linked immunosorbent assay for zein and other proteins using unconventional solvents for antigen adsorption.

An enzyme-linked immunosorbent assay (ELISA) performed in polystyrene microtiter plates that can detect and quantitate the maize prolamin zein is described. The assay yields positive reactions with as little as 1 ng of antigen and uses solvents not ordinarily employed in ELISA methods. A systematic investigation of zein adsorption to polystyrene in various solvents supports the hypothesis that antigen binding occurs through nonpolar interactions. The method was also used to determine structural relationships among three zein polypeptides differing in size and charge. Additional experiments indicate that a number of soluble proteins are absorbed to polystyrene in the denaturing agent urea and retain immunological reactivity. The retention of antigen reactivity after solubilization in 6-8 M urea suggests that ELISA methods may be applicable to other proteins which are insoluble, or rendered insoluble, in aqueous buffers.

Cross Reactions↗

A system for electron therapy dosimetry surveys with thermoluminescence dosimeters.

Radiation-therapy dosimetry surveys employing thermoluminescence dosimeters (TLDs) are now being considered for high-energy electron beams. Using a system of individually calibrated pressed LiF TLDs in a water and a polystyrene phantom, we established that the distortions of depth-dose distributions in non-conducting materials previously observed at high absorbed doses and high dose rates were not detectable in the present geometry at doses and dose rates as much as 40 times higher than those employed in radiation therapy. The system was then used to measure TLD response in water and in polystyrene in the nominal electron-energy range from 7 to 18 MeV. In the water phantom, the well-known trend for TLD response to decrease with increasing electron energy was observed. In the polystyrene phantom, TLD response was found to be independent of electron energy.

Electrons↗

Antigen attachment in ELISA.

The amount of antigen on the solid phase was studied during various steps of an enzyme-linked immunosorbent assay (ELISA) for chicken anti-bovine serum albumin (BSA) antibodies. Three materials for the solid phase were used: polystyrene, nylon and cyanogen bromide activated paper. There was noticeable leakage of antigen during the assay from both polystyrene (30%) and nylon (60%). This occurred during all steps of the assay: during incubation with the sample serum, incubation with the enzyme conjugated antibodies and during the washing procedures. During incubation with the sample serum detachment of antigen was quite rapid and could result in competition between bound and free antigen for antibodies. The presence of antigen-antibody complexes in reaction mixtures could also lead to erroneous results in the antibody assay. With covalent coupling of antigen to solid phase (cyanogen bromide activated paper), batch-to-batch variation in the surface concentration of antigen was strikingly less than in the assay using plastics. Detachment of antigen from activated paper during the assay was less than with polystyrene or nylon. ELISA with activated paper as the solid phase thus seems suitable as a reference method for standardisation of the antigen phase in ELISA systems.

Animals↗

New experimental criteria for optimization of solid-phase antigen concentration and stability in ELISA.

A peroxidase saturation technique for the determination of coating antigen concentration necessary to saturate polystyrene plates with a wide range of different antigens is described. The same technique has also been used to compare the stability of antigen-polystyrene bonds for native and denaturated antigens. Furthermore, the inappropriate selection of solid-phase antigen concentration and its influence on ELISA results is analyzed and an experimental criterion to select the optimum antigen concentration is proposed. Two different antigens, BSA-Ar and hydatid antigen, were used for ELISA determination of specific antibodies as a model system. Optimum solid-phase antigen concentration was determined by two different methods: peroxidase saturation, in which binding of peroxidase to non-antigen-occupied polystyrene surface sites was used to evaluate the degree of coating by antigen; chequer-board titration, using several immune sera of different affinity. Optimum antigen concentration selected by chequer-board titration using low affinity sera was similar to that selected with peroxidase saturation. On the other hand, lower antigen concentration would be selected by chequer-board titration using high affinity sera. For this reason, the concentrations of low affinity antibodies would be underestimated using the chequer-board titration. These results indicate that peroxidase saturation should be used to avoid avidity-dependent artifacts in ELISA.

Animals↗

Quantitation of vitronectin and clusterin. Pitfalls and solutions in enzyme immunoassays for adhesive proteins.

Vitronectin (S protein) and clusterin (SP-40,40/cytolysis inhibitor) are non-homologous, multifunctional proteins which both inhibit complement lysis. Vitronectin is an adhesive protein which binds strongly to polystyrene by hydrophobic interactions. The current study demonstrated that clusterin adsorbed even more efficiently to polystyrene than did vitronectin. This adsorption increased in the presence of Tween 20 and was not abolished by blocking or by the use of other detergents. In double antibody enzyme immunoassays such non-specific binding might invalidate the results. However, the non-specific binding of both proteins was efficiently abolished by the following experimental format: Dynatech Immulon 2 microtiter plate, acidic sample buffer (pH 6.0) containing 0.2% Tween 20 and high sample dilution. Vitronectin was successfully quantitated using this approach, but the measurement of clusterin was not reliable because of high inter-well variation of binding. However, since few serum proteins adsorb to polystyrene in the presence of detergents, clusterin was successfully quantitated in a single antibody enzyme immunoassay in which samples were coated directly onto Nunc Maxisorp plates in the presence of 0.2% Tween 20. In normal blood donors the serum concentration (median and 2.5-97.5 percentile) of vitronectin was 0.34 g/l (0.24-0.53) and of clusterin 0.34 g/l (0.25-0.42).

Adsorption↗

Growth of endothelial and HeLa cells on a new multipurpose microcarrier that is positive, negative or collagen coated.

A new cell culture microcarrier that can be covalently bonded by cell attachment proteins and can be thin-sectioned for electron microscopy was synthesized. It was easily made by sulfonating cross-linked polystyrene beads for a negative surface charge followed by covalent attachment of polyethylenimine for a positive charge. Cell attachment proteins, e.g. collagen was covalently bonded directly to the microcarrier using a carbodiimide or after activating the microcarrier surface with glutaraldehyde. HeLa-S3 cells attached, spread and grew to confluence more efficiently on the positive microcarriers and those coated with collagen than on the negative ones. Endothelial cells, grew best on those with a negative surface charge. The nature of the microcarrier surface was not the only aspect involved in cell adhesion but also the type of serum proteins absorbed. Qualitatively different proteins coated the microcarriers depending upon whether the carrier was negative, positive or coated with collagen. Comparison of various types of available microcarriers indicated that the modified cross-linked polystyrene beads used here were best for transmission and scanning electron microscopy. Endothelial cells grown on the microcarriers had the same ultrastructure as cells grown in monolayers in culture dishes. Of a variety of microcarriers tested the modified cross-linked polystyrene beads were the only ones that could be used for both ultrastructural and biochemical techniques.

Animals↗

Human serum albumin as a probe for surface conditioning (opsonization) of block copolymer-coated microspheres.

The adsorption of human serum albumin to polystyrene microspheres sterically stabilized with block copolymers, was investigated using photon correlation spectroscopy and laser doppler anenometry. The block copolymers used were non-ionic surfactants of the poloxamer and poloxamine series made of polyoxyethylene and polyoxypropylene chains. Photon correlation spectroscopy and laser doppler anenometry showed that the coating reduced the adsorption of the protein to the polystyrene microspheres surface. Quantitative studies using 125I-labelled human serum albumin and sodium dodecyl sulphate-polyacrylamide gels (in combination with densitometry), were employed to evaluate the adsorption of human serum albumin to uncoated and coated polystyrene microspheres. They confirmed that the hydrophilic polyoxyethylene steric layer, created by coating with the block copolymers, reduced the adsorption of human serum albumin. Moreover, the amount of human serum albumin adsorbed was related to the polyoxyethylene content of the block copolymers.

Absorption↗

Heparin-like functionalized polymer surfaces: discrimination between catalytic and adsorption processes during the course of thrombin inhibition.

Thrombus formation on blood-contacting artificial surfaces is a major problem. Antithrombogenic polymer surfaces have been obtained either by heparin binding, or by grafting sulphonate and/or amino acid sulphonamide groups on insoluble polystyrene. In addition to their capacity to adsorb thrombin, such surfaces were shown to be able to catalyse its inhibition by antithrombin III (AT), i.e. they are endowed with heparin-like activity. The results were mainly obtained by using clotting assays. In many cases, delineating adsorption and catalytic processes by such assays is not possible when evaluating anticoagulant polymer surfaces. To overcome this problem, the kinetics of thrombin adsorption and inhibitions by AT and heparin cofactor II (HC) in the presence of such surfaces have been measured by using an assay performed with a thrombin-specific chromogenic substrate. A simple kinetic model of thrombin consumption is proposed. The relevant calculations, carried out with the help of a computer program, lead to determination of relative second order rate constants of thrombin adsorption and inhibitions by AT and HC in the presence of the polymers. In addition to thrombin adsorption, polystyrene surfaces bearing only sulphonate groups catalyse inhibition by AT, whereas polystyrene surfaces bearing either aspartate, glycinate or isophthalate sulphonamide groups catalyse both inhibitions by AT and HC.

Adsorption↗