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PubMed1998

Ultrasensitive and rapid enzyme immunoassay (thin aqueous layer immune complex transfer enzyme immunoassay) for antibody IgG to HIV-1 p17 antigen.

The immune complex transfer enzyme immunoassay for antibody IgG to HIV-1 p17 antigen was performed in two different ways (the present immunoassays I and II) within shorter periods of time than previously reported. In the present (simultaneous) immunoassay I, antibody IgG to HIV-1 p17 antigen in 10 microL of serum samples was incubated simultaneously with 2,4-dinitrophenyl-maltose binding protein-recombinant p17(rp17) fusion protein and rp17-beta-D-galactosidase conjugate in a total volume of 22 microL for 10 min to form the immune complex comprising the three components. The reaction mixture was incubated with a polystyrene bead of 6.35 mm in diameter coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG for 5 min in a styrol test tube (13.3 x 54 mm and 2.1 g) to trap the immune complex. After washing, the polystyrene bead was incubated with 30 microL of epsilonN-2,4-dinitrophenyl-L-lysine solution in a polystyrene tube (12 x 75 mm) coated with affinity-purified (antihuman IgG gamma-chain) IgG for 10 min to transfer the immune complex. In the present (sequential) immunoassay 11, a polystyrene bead of 6.35 mm in diameter coated successively with affinity-purified (anti-2,4-dinitrophenyl group) IgG and 2,4-dinitrophenyl-maltose binding protein-rp17 fusion protein was incubated in a styrol test tube (13.3 x 54 mm and 2.1 g) sequentially with antibody IgG to HIV-1 p17 antigen in 10 microL of serum samples in a total volume of 16 microL for 5 min and subsequently with rp17-beta-D-galactosidase conjugate in a volume of 10 microL for 5 and 10 min. The immune complex formed on the polystyrene bead was transferred to a polystyrene tube coated with affinity-purified (antihuman IgG gamma-chain) IgG for 5 and 10 min in the same way as in the present immunoassay I. During the incubations, the styrol test tubes containing the polystyrene beads and reaction mixtures were shaken, and the polystyrene test tubes were rotated with shaking, so that the polystyrene beads were rotated randomly, and small drops (16 to 30 microL) of the reaction mixtures evenly contacted all parts of the solid phase surfaces during the incubations, though only small parts of the solid phase surfaces were contacted at one time. The intent was to continuously mix thin aqueous layers of the reaction mixtures covering the solid phase surfaces with the rest of the reaction mixtures. (Therefore, these immunoassays were called thin aqueous layer immunoassays.) beta-D-Galactosidase activity bound to the polystyrene tubes was assayed by fluorometry for 30 and 60 min. The present immunoassays I and II, in which only 15 to 25 min were used for the immunoreactions, were as sensitive if not more so than the previous immune complex transfer enzyme immunoassay requiring 150 min for the immunoreactions. In these earlier immunoreactions, the immune complex comprising the three components formed by 30 min incubation was trapped onto two polystyrene beads (3.2 mm in diameter) coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG for 60 min, and was then transferred to two polystyrene beads (3.2 mm in diameter) coated with affinity-purified (antihuman IgG y-chain) IgG for 60 min in a total volume of 150 microL. Furthermore, the present (sequential) immunoassay 11 (and probably I) could become approximately 10 times more sensitive by assaying bound beta-D-galactosidase activity for a longer period of time (10 h), since beta-D-galactosidase activity, bound nonspecifically in the presence of serum samples from HIV-1 seronegative subjects, was considerably low.

2,4-Dinitrophenol↗
PubMed1998

Rapid and ultrasensitive enzyme immunoassay (thin aqueous layer immune complex transfer enzyme immunoassay) for HIV-1 p24 antigen.

The immune complex transfer enzyme immunoassay for HIV-1 p24 antigen was performed in three different ways (in the present immunoassays I, II, and III) within much shorter periods of time than previously reported. In the present (simultaneous) immunoassay I, p24 antigen was incubated simultaneously with 2,4-dinitrophenyl-biotinyl-bovine serum albumin-affinity-purified rabbit anti-p24 Fab' conjugate and monoclonal mouse anti-p24 Fab'-beta-D-galactosidase conjugate in a total volume of 19 microL for 15 min to form the immune complex comprising the three components. The reaction mixture was incubated with a polystyrene bead of 6.35 mm in diameter coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG for 5 min to trap the immune complex. After washing, the polystyrene bead was incubated with 35 microL of epsilonN-2,4-dinitrophenyl-L-lysine for 15 min to elute the immune complex (the first eluate) and, after removing the first eluate, with an additional 35 microL of epsilonN-2,4-dinitrophenyl-L-lysine for 1 min (the second eluate). The first and second eluates were incubated with a polystyrene test tube (12 x 75 mm) coated with streptavidin for 15 min. In the present (sequential) immunoassay II, a polystyrene bead of 6.35 mm in diameter successively coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG and 2,4-dinitrophenyl-biotinyl-bovine serum albumin-affinity-purified rabbit anti-p24 Fab' conjugate was incubated with p24 antigen in a total volume of 20 microL for 5 min and subsequently with monoclonal mouse anti-p24 Fab'-beta-D-galactosidase conjugate in a volume of 5 microL for 20 min. The immune complex formed on the polystyrene bead was transferred to a polystyrene test tube coated with streptavidin as described above. In the present (sequential) immunoassay III, p24 antigen was incubated with monoclonal mouse anti-p24 Fab'-beta-D-galactosidase conjugate in a total volume of 19 microL for 10 min and with a polystyrene bead of 6.35 mm in diameter coated successively with affinity-purified (anti-2,4-dinitrophenyl group) IgG and 2,4-dinitrophenyl-biotinyl-bovine serum albumin-affinity-purified rabbit anti-p24 Fab' conjugate for 20 min. The immune complex formed on the polystyrene bead was transferred as described above. The incubations were performed at room temperature either by shaking the polystyrene beads (one/assay) and the reaction mixtures in styrol test tubes (13.3 x 54 mm and 2.1 g) so as to randomly rotate the polystyrene beads or by rotating the polystyrene test tubes (12 x 75 mm) containing the reaction mixtures, so that small drops (19 to 70 microL) of the reaction mixtures evenly contacted all parts of the solid phase surfaces during the incubations (although they contacted only small parts of the solid phase surfaces at a time) to continuously mix thin aqueous layers covering the solid phase surfaces with the rest of the reaction mixtures. (Therefore, these immunoassays are called thin aqueous layer immunoassays.) The detection limits of p24 antigen by 1 hr assay of bound beta-D-galactosidase activity in the present immunoassays I, II, and III were 0.1, 0.2 and 0.1 amol/assay, respectively, and were slightly higher than or equal to that by the previously reported immune complex transfer enzyme immunoassay, in which the immune complex was formed for 4 hr, was trapped for 16 hr, and was transferred for 3 hr followed by 1-hr assay of bound beta-D-galactosidase activity. By 20-hr assay of bound beta-D-galactosidase activity, the detection limit of p24 antigen was further lowered to 10 zmol/assay in the present (simultaneous) immunoassay I and to 3 zmol/assay in the present (sequential) immunoassay III. However, the nonspecific reaction(s) with serum samples from HIV-1 seronegative subjects hampered the improvement of the detection limit by 20-hr assay of bound beta-D-galactosidase activity.

Acquired Immunodeficiency Syndrome↗
PubMed2002

Ultrafine particles affect experimental thrombosis in an in vivo hamster model.

Particulate air pollution is associated with cardiovascular morbidity and mortality. To investigate this association, we studied the effect of ultrafine (60 nm) polystyrene particles on thrombus formation in a hamster model after intravenous and intratracheal administration of unmodified, carboxylate-polystyrene, or amine-polystyrene particles. Unmodified particles had no effect on thrombosis up to 5 mg/kg. Carboxylate-polystyrene particles significantly inhibited thrombus formation at 500 and 100 microg/kg body weight but not at 50 microg/kg body weight. In contrast, amine-polystyrene particles significantly enhanced thrombosis at 500 and 50 microg/kg body weight but not at 5 microg/kg body weight. Similarly, the intratracheal instillation of 5,000 microg of amine-polystyrene particles significantly increased thrombus formation. The unmodified particles and carboxylate-polystyrene particles had no effect. During platelet aggregation in human platelet-rich plasma, induced with 1.25 microM ADP, unmodified particles had no effect up to 100 microg/ml, and carboxylate-polystyrene particles weakly enhanced platelet aggregation at 25 to 100 microg/ml. However, amine-polystyrene particles (50 and 100 microg/ml) induced platelet aggregation themselves and strongly increased the ADP-induced aggregation. We conclude that the presence of (ultrafine) particles in the circulation may affect hemostasis. The observed in vivo prothrombotic tendency results, at least in part, from platelet activation by positively charged amine-polystyrene particles.

Adenosine Diphosphate↗
PubMed1984

The competitive effects of serum proteins on cell adhesion.

Binding curves for the adsorption of plasma fibronectin, alpha-1-antitrypsin, alpha-2-macroglobulin, ceruloplasmin, transferrin and bovine serum albumin to plain and to hydroxylated polystyrene surfaces were measured. These curves were correlated with the adhesion of BHK cells and leucocytes to these adsorbed protein surfaces in protein-free culture media. Hydroxylated polystyrene adsorbed less of alpha-1-antitrypsin, alpha-2-macroglobulin and albumin than the plain polystyrene. On the other hand the hydroxylated surfaces bound more fibronectin than the plain polystyrene surfaces. Hydroxylated polystyrene surfaces were also more adhesive for both BHK cells and leucocytes than plain polystyrene: a result confirming earlier work. The competition of fibronectin for adsorption to plain polystyrene with alpha-1-antitrypsin, alpha-2-macroglobulin and ceruloplasmin was measured and correlated with effects on cell adhesion. The results suggest that the low adhesiveness of BHK cells and leucocytes on plain polystyrene in sera-containing media is due both to the low binding of fibronectin and to the binding of serum albumin, alpha-1-antitrypsin and alpha-2-macroglobulin. The relative unimportance of fibronectin in adhesion to these surfaces is shown by the finding that cell attachment will not occur to polystyrene surfaces that have bound high levels of the antiadhesive proteins in the presence of fibronectin, even though attachment will occur in the absence of fibronectin provided that the antiadhesive proteins are lacking.

Adsorption↗
PubMed2004

Fundamental characteristics of synthetic adsorbents intended for industrial chromatographic separations.

With the aim of obtaining comprehensive information on the selection of synthetic adsorbents for industrial applications, effect of pore and chemical structure of industrial-grade synthetic adsorbents on adsorption capacity of several pharmaceutical compounds was investigated. For relatively low molecular mass compounds, such as cephalexin, berberine chloride and tetracycline hydrochloride, surface area per unit volume of polystyrenic adsorbents dominated the equilibrium adsorption capacity. On the contrary, effect of pore size of the polystyrenic adsorbents on the equilibrium adsorption capacity was observed for relatively high molecular mass compounds, such as rifampicin, Vitamin B12 and insulin. Polystyrenic adsorbent with high surface area and small pore size showed small adsorption capacity for relatively high molecular mass compounds, whereas polystyrenic adsorbent with relatively small surface area but with large pore size showed large adsorption capacity. Effect of chemical structure on the equilibrium adsorption capacity of several pharmaceutical compounds was also studied among polystyrenic, modified polystyrenic and polymethacrylic adsorbents. The modified polystyrenic adsorbent showed larger adsorption capacity for all compounds tested in this study due to enhanced hydrophobicity. The polymethacrylic adsorbent possessed high adsorption capacity for rifampicin and insulin, but it showed lower adsorption capacity for the other compounds studied. This result may be attributed to hydrogen bonding playing major role for the adsorption of compounds on polymethacrylic adsorbent. Furthermore, column adsorption experiments were operated to estimate the effect of pore characteristics of the polystyrenic adsorbents on dynamic adsorption behavior, and it is found that both surface area and pore size of the polystyrenic adsorbents significantly affect the dynamic adsorption capacity as well as flow rate.

Adsorption↗
PubMed1994

Preparation of nanoparticles bearing high density carbohydrate chains using carbohydrate-carrying polymers as emulsifier.

A novel method of preparing nanoparticles bearing high density carbohydrate chains on their surface is described. Carbohydrate-bearing nanoparticles of poly(lactic acid) or polystyrene were prepared by the solvent evaporation method using a carbohydrate-carrying polystyrene derivative which served as both an emulsifier and a surface coating. The diameter of the obtained nanoparticles ranged from 80 to 300 nm depending on the concentration of the polystyrene derivative. As the concentration of the polystyrene derivatives increased the nanoparticle diameter decreased, indicating that the polystyrene derivatives worked as an emulsifier. The obtained particles were specifically aggregated by carbohydrate-specific lectin, showing that the polystyrene derivative was retained on the particle surfaces and expressed carbohydrate residues. The density of carbohydrates on the particle surfaces was determined to be 3-5 molecules per square nanometre. The particles prepared by the present method were stably dispersed and hardly aggregated in aqueous media during storage and centrifugal treatment compared with the post-coated particles that were prepared by adsorbing polystyrene particles with the polystyrene derivative. In vitro study with isolated rat hepatocytes revealed that surface carbohydrate chains were recognized by hepatocytes.

Animals↗
PubMed1998

Enhancement of immune response to intranasal influenza HA vaccine by microparticle resin.

We evaluated the potential application of ion-exchange resins for the enhancement of intranasal immune response to influenza HA vaccine in mice. Female Balb/c mice were intranasally immunized with inactivated influenza HA vaccine with one of four kinds of resin microparticles: sodium polystyrene sulfonate, calcium polystyrene sulfonate, polystyrene benzyltrimetylammonium chloride, or polystyrene divinylbenzene. Haemagglutinin-inhibiting antibodies were measured in the serum and IgA antibodies in the nasal wash after 4 weeks. The results demonstrated that intranasal administration of influenza HA vaccine in combination with the 20-45 microns sized particles of sodium polystyrene sulfonate resin induced the highest levels of mucosal IgA, and enhanced systemic haemagglutinin-inhibiting antibodies. While the Th2-type cytokine IL-4 was detected in the sera after intranasal immunization with HA vaccine and sodium polystyrene sulfonate, neither IFN-gamma nor IL-2 could be detected. Furthermore, mice intranasally immunized with HA vaccine together with sodium polystyrene sulfonate resin showed higher protection against viral challenge than those that received HA vaccine alone. Intranasal administration of influenza HA vaccine with sodium polystyrene sulfonate resin might be both a safe and an effective means of immunization.

Administration, Intranasal↗
PubMed2006

Characterization of biofilm-forming abilities of antibiotic-resistant Salmonella typhimurium DT104 on hydrophobic abiotic surfaces.

BACKGROUND AND PURPOSE: Salmonella typhimurium DT104 strain has emerged as a global human and veterinary public health concern because of its antibiotic resistance and extensive host range. Although it is thought to be more virulent, to date, factors relevant to its virulence have not been fully elucidated. Thus, understanding how this strain forms biofilms on hydrophobic surfaces will add to current knowledge on its possible virulence mechanism. METHODS: Biofilm-forming abilities of clinical isolates of S. typhimurium DT104 from human and animal sources on hydrophobic inanimate surfaces were assessed by absorbance at 600 nm of crystal violet-bound cells recovered from 96-well tissue culture plates after growth in a nutrient-rich growth medium and various adjusted media; and scanning electron microscopy based on standard procedures. RESULTS: In the nutrient-rich growth medium, Luria-Bertani (LB), biofilms were formed in small quantities, preferentially on polystyrene (p<0.05), and followed different time courses. Significantly lower amounts of biofilms were formed on polystyrene when a nutrient-deficient growth medium (adherence test medium) was used. Inclusion of D-(+)-mannose in LB at a concentration of 100 mM significantly (p<0.05) inhibited biofilm formation on polystyrene. D-(+)-glucose relatively enhanced biofilm formation but D-(-)-mannitol only insignificantly influenced the process. The action of mannose on polyvinly chloride (PVC) was insignificant, suggesting that its action may be surface-dependent. Additionally, glucose significantly reduced biofilm growths of 2 of the isolates and only that of the PVC-loving strain T980021 on polystyrene and PVC, respectively. At the concentration tested, unlike xylose, both D-mannose and D-glucose significantly (p<0.05) inhibited bacterial growth, providing a possible mechanism for their inhibitory action on biofilm formation by S. typhimurium. While stress of starvation resulted in significant reduction in biofilm formation on polystyrene in all but the PVC-loving strain T980021, high osmolarity had little effect on the quantity of biofilm formed on polystyrene. The extent of primary attachment to polystyrene as well as their capacity to form biofilm did not correlate with their cell surface hydrophobicity and exopolysaccharide production. CONCLUSIONS: D-(+)-mannose inhibits biofilm formation by S. typhimurium DT104 on polystyrene but not on PVC. There was also a general lack of correlation between the ability of S. typhimurium DT104 to form biofilm and its physicochemical surface characteristics.

Animals↗
PubMed1992

Novel and ultrasensitive noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay) for alpha-human atrial natriuretic peptide.

A novel and ultrasensitive noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay) for alpha-human atrial natriuretic peptide (alpha-hANP) in plasma is described. alpha-hANP was biotinylated using N-hydroxysuccinimidobiotin and trapped onto an anti-alpha-hANP [6-28] IgG-coated polystyrene ball. After washing, biotinylated alpha-hANP was eluted from the polystyrene ball with HCI and was reacted with 2,4-dinitrophenyl-fluorescein-bovine serum albumin-disulfide-rabbit anti-alpha-hANP [6-28] IgG conjugate. The complex formed was trapped onto (anti-2,4-dinitrophenyl group) IgG-coated polystyrene balls and, after washing, reacted with avidin-beta-D-galactosidase conjugate. The polystyrene balls were washed, and the complex of the three components was eluted with epsilon N-2, 4-dinitrophenyl-L-lysine and transferred to anti-fluorescein IgG-coated polystyrene balls. After washing, the complex was released from the polystyrene balls by reduction with 2-mercaptoethylamine and transferred to (anti-rabbit IgG) IgG-coated polystyrene balls. beta-D-Galactosidase activity bound to the last polystyrene balls was assayed by fluorometry. The detection limit of alpha-hANP [1-28] was 3 fg (1 amol)/tube. Interference by plasma proteins was eliminated by separation of peptides from proteins using a molecular sieve. The assay range of plasma alpha-hANP [1-28] was 0.04-120 ng/L, and plasma levels of hANP in healthy subjects (11-56 ng/L) were measured without concentration.

Atrial Natriuretic Factor↗
PubMed1991

Detection of one attomole of [Arg8]-vasopressin by novel noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay).

One attomole of [Arg8]-vasopressin (AVP) was detected by a novel noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay). AVP was indirectly biotinylated using N-hydroxysuccinimidobiotin and trapped onto an anti-AVP IgG-coated polystyrene ball. After washing, biotinylated AVP was eluted from the polystyrene ball with HCl and was reacted with 2,4-dinitrophenyl-fluorescein disulfide-bovine serum albumin-rabbit anti-AVP IgG conjugate. The complex formed was trapped on [anti-2,4-dinitrophenyl group] IgG-coated polystyrene balls and, after washing, reacted with avidin-beta-D-galactosidase conjugate. The polystyrene balls were washed, and the complex of the three components was eluted with 2,4-dinitrophenyl-L-lysine and transferred to anti-fluorescein IgG-coated polystyrene balls. After washing, the complex was released from the polystyrene balls by reduction with 2-mercaptoethylamine and transferred to [anti-rabbit IgG] IgG-coated polystyrene balls. beta-D-Galactosidase activity bound to the last polystyrene balls was assayed by fluorometry. The detection limit of AVP was 1.1 fg (1 amol)/tube. Interference by proteins in biological fluids was eliminated by separation of peptides from proteins using a molecular sieve. The principle of the present method may be applicable to the measurement of haptens, including peptides, that can be derivatized so as to be bound simultaneously by both anti-hapten antibody and avidin molecules.

Amino Acid Sequence↗
PubMed1997

Electron fluence correction factors for conversion of dose in plastic to dose in water.

In radiation dosimetry protocols, plastic is allowed as a phantom material for the determination of absorbed dose to water in electron beams. The electron fluence correction factor is needed in conversion of dose measured in plastic to dose in water. There are large discrepancies among recommended values as well as measured values of electron fluence correction factors when polystyrene is used as a phantom material. Using the Monte Carlo technique, we have calculated electron fluence correction factors for incident clinical beam energies between 5 and 50 MeV as a function of depth for clear polystyrene, white polystyrene and PMMA phantom materials and compared the results with those recommended in protocols as well as experimental values from published data. In the Monte Carlo calculations, clinical beams are simulated using the EGS4 user-code BEAM for a variety of medical accelerators. The study shows that our calculated fluence correction factor, phi pw, is a function of depth and incident beam energy Eo with little dependence on other aspects of beam quality. However the phi pw values at dmax are indirectly influenced by the beam quality since they vary with depth and dmax also varies with the beam quality. Calculated phi pw values at dmax are in a range of 1.005-1.045 for a clear polystyrene phantom, 1.005-1.038 for a white polystyrene phantom and 0.996-1.016 for a PMMA phantom. Our values of phi pw are about 1-2% higher than those determined according to the AAPM TG-25 protocol at dmax for clear or white polystyrene. Our calculated values of phi pw also explain some of the variations of measured data because of its depth dependence. A simple formula is derived which gives the electron fluence correction factor phi pw as a function of R50 at dmax or at the depth of 0.6R50-0.1 for any clinical electron beam with energy between 5 and 25 MeV for three plastics: clear polystyrene, white polystyrene and PMMA. The study also makes a careful distinction between phi pw and the corresponding IAEA Code of Practice quantity, hm.

Biophysical Phenomena↗
PubMed2004

Adsorption of bovine alpha-lactalbumin on suspended solid nanospheres and its subsequent displacement studied by NMR spectroscopy.

Detailed knowledge of the adsorption-induced conformational changes of proteins is essential to understand the process of protein adsorption. However, not much information about these conformational changes is available. Here, the adsorption of calcium-depleted (APO)- and calcium-containing (HOLO)-bovine alpha-lactalbumin (BLA) on suspended solid polystyrene nanospheres and their subsequent displacement by a surfactant are studied by NMR spectroscopy. To our knowledge, this is the first time that adsorption of proteins on solid nanospheres, with both components present in the NMR sample, is studied by this method. High-quality one-dimensional and two-dimensional 1H NMR spectra of nonadsorbed APO- and HOLO-BLA in the presence of BLA- and/or surfactant-covered solid polystyrene nanospheres in suspension are obtained using standard NMR procedures. BLA and surfactant molecules that are adsorbed on the polystyrene nanospheres give rise to extremely broadened proton resonances. This can be exploited to determine the amount of adsorbed protein and of adsorbed surfactant in a system containing protein, nanospheres, and surfactant, without disturbing the equilibrium of the system. Two-dimensional 1H NMR spectroscopy shows that the chemical shifts of the backbone amide protons of HOLO-BLA after its adsorption and subsequent displacement from polystyrene nanospheres by the surfactant 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) are identical to those of native HOLO-BLA. The adsorption-induced unfolding of BLA to a molten globule state on polystyrene nanospheres is thus fully reversible at the residue level upon CHAPS-induced displacement of BLA. The latter is the now fulfilled essential requirement that enables the future indirect study, at the residue level, of the conformational characteristics of BLA adsorbed on polystyrene nanospheres by hydrogen/deuterium exchange and NMR spectroscopy. The results presented show that NMR spectroscopy is clearly feasible to study the adsorption of BLA on suspended polystyrene nanospheres. This technique should be applicable to the study of the adsorption of other proteins on other surfaces as well.

Adsorption↗
PubMed2001

Binding and orientation of fibronectin on surfaces with collagen-related peptides. Student Research Award in the Masters Science Degree Candidate category, 27th annual meeting of the Society for Biomaterials, St. Paul, MN, April 24-29, 2001.

Although fibronectin (FN) has been used in a variety of in vitro studies to enhance cell and bacteria adhesion, relatively little is known about the molecular interactions of FN with surfaces, particularly the interactions that can control the binding, conformation, and functionality of FN on these surfaces. Even less is known about approaches needed to control binding, orientation, and functionality of FN bound on surfaces. To begin to fill this gap in our knowledge, we hypothesized that functional FN can be bound and specifically oriented on polystyrene surfaces with FN-specific collagen-related peptides (CRPs). We further hypothesized that monoclonal antibodies that react with specific epitopes on FN can be used to quantify both FN binding and orientation on these surfaces. On the basis of these hypotheses, we initiated a systematic investigation of the binding and orientation of FN on polystyrene surfaces with CRPs. To bind FN to surfaces, we used two different CRPs: CRP-I (TLQPVYEYMVGV) and CRP-II (TGLPVGVGYVVTVLT). The binding and orientation of the FN molecule to these immobilized CRPs was quantified with (125)I-FN and monoclonal antibodies. Monoclonal antibodies used for this study were reactive with specific regions of the FN molecule, that is, the amino (N) terminus (anti-N antibodies) and carboxyl (C) terminus (anti-C antibodies). The results of our studies demonstrated that although CRP-I and CRP-II could be bound directly to polystyrene, these directly immobilized CRPs failed to bind (125)I-FN . Thus, to facilitate FN binding to the CRPs, we used bovine serum albumin (BSA) as a spacer to physically elevate the CRPs away from the polystyrene surface. Thus, CRP-I and CRP-II were covalently linked to BSA via the N and C termini of each CRP (CRP-I-BSA and CRP-II-BSA). (125)I-CRP-BSAs were all found to bind to equivalent levels on polystyrene (1.60-2.60 microg/cm2). When CRP-BSAs were immobilized on polystyrene, they all successfully bound (125)I-FN in a range of 34-72 ng/cm2 (mean). Using monoclonal antibodies to FN to characterize the orientation of FN bound to the various CRP-BSAs, we demonstrated that (1) FN consistently bound to either CRP-I-BSA or CRP-II-BSA; (2) bound FN reacted significantly more with anti-C antibodies than with anti-N antibodies; and (3) the increased reactivity of bound FN to anti-C antibodies was consistent, whether FN was bound by CRP-I or CRP-II or the CRPs were bound to BSA by the C or N termini. These data demonstrated an enhanced binding of anti-C antibodies to immobilized CRP-BSA relative to anti-N antibodies. We interpreted the data to be the result of FN binding in an oriented fashion with N termini of FN bound tightly to the BSA-polystyrene surface. In this position, the C termini of FN are exposed and available for binding by the anti-C antibodies. Alternatively, in this orientation the N termini of the FN would not be available to bind the anti-N antibodies, thereby explaining the decreased reactivity of the CRP immobilized FN to the anti-N antibodies. These studies not only demonstrate the utility of peptides in binding and orienting large molecular weight proteins such as FN on surfaces but underscore the need for well-characterized reagents (e.g., monomeric/functional FN and antibodies) to specifically bind, orient, and characterize large molecular weight proteins immobilized on various surfaces.

Animals↗
PubMed1998

Encapsulation of Inorganic Particles by Dispersion Polymerization in Polar Media

Polymer encapsulation of small silica particles, using dispersion polymerization of styrene in aqueous ethanol medium with poly(N-vinyl pyrrolidone) (PVP) as stabilizer, is described. Silica particles, directly synthesized by the Stober process in an aqueous ethanol medium, are either unreacted (hydrophilic character) or coated with 3-(trimethoxysilyl)propyl methacrylate (MPS) (hydrophobic character), which is grafted at the silica particle surface. When the bare silica particles are used as the seed, there is a strong tendency of the silica beads to cover the surface of the polystyrene particles and obviously encapsulation does not occur. On the contrary, when the silica surface is made hydrophobic by coating, the inorganic particles are entirely contained in the polystyrene particles as evidenced by microscopy techniques (TEM, SEM, AFM). It is shown that some polystyrene chains are then chemically bonded to the silica particles, through the coupling agent MPS, and that only a small amount of bonded polystyrene, compared to the total polystyrene synthesized, is sufficient to obtain encapsulation of the silica particles with the entire amount of polystyrene synthesized during the polymerization. Under our experimental conditions, each polystyrene latex particle contains, on average, 4 to 23 silica beads depending, in particular, on the size of the silica. We believe that it is possible to control the composite particle size and morphology by a convenient choice of the composition of the system. Moreover, this new polymer-encapsulation process could be used to synthesize other organic-inorganic composite particles, using, for example, other monomers or minerals. Copyright 1998 Academic Press. Copyright 1998Academic Press

Journal Article↗
PubMed1995

Further simplification of ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-HTLV-I IgG using microplates and fluororeader.

In a previously reported ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-HTLV-I IgG, polystyrene beads were handled with tweezers, and bound beta-D-galactosidase activity was measured with a fluorometer. The use of tweezers was causative of false-positivity by carryover, and testing many samples was difficult. Recently, these drawbacks have been minimized using microplates and a fluororeader. However, tweezers were still required in the initial and final steps. In the present study, the immune complex, comprising 2,4-dinitrophenyl-antigen, anti-HTLV-I IgG, and antigen-beta-D-galactosidase conjugate, was formed in and trapped onto microplate wells coated with (anti-2,4-dinitrophenyl group) IgG. Subsequently, the microplate wells were incubated with epsilon N-2,4-dinitrophenyl-L-lysine and polystyrene beads, modified by attaching to plates through cylindrical bars and coated with (antihuman IgG gamma-chain) IgG, to transfer the immune complex from the microplate wells to the modified polystyrene beads. Alternatively, modified polystyrene beads coated with (anti-2,4-dinitrophenyl group) IgG and microplate wells coated with (antihuman IgG gamma-chain) IgG were substituted for microplate wells coated with (anti-2,4-dinitrophenyl group) IgG and modified polystyrene beads coated with (antihuman IgG gamma-chain) IgG, respectively. The fluorescence intensity for bound beta-D-galactosidase activity was quickly measured with a fluororeader. Thus the modified polystyrene beads were transferred from wells to wells more quickly and easily without tweezers, eliminating false-positivity due to carryover, and it became easy to test many samples with high sensitivity and reliability, although the assay of bound beta-D-galactosidase activity became slightly more time-consuming.

Animals↗
PubMed1976

Atmospheric stability in cell culture vessels.

We have investigated that atmospheric stability in polystyrene and glass cell culture vessels by measuring the dissolved O2 and CO2 in the media of both seeded and unseeded culture vessels incubated at 37 degrees C. There was no diffusion of either O2 or CO2 through glass vessels. At low partial pressures of oxygen (PO2), oxygen diffused into the polystyrene flasks at a rate of 1 to 2 mmHg per 24 hr, and at high PO2, oxygen diffused slowly out of polystyrene flasks. CO2 diffused out of polystyrene flasks with a half-time of 260 hr resulting in a considerable elevation in pH. In seeded polystyrene flasks with the PO2 less than or equal to room air, cellular oxygen consumption was masked by the inward diffusion of oxygen. In addition, the fall in pH due to metabolic CO2 and organic acid production during cell growth in polystyrene flasks was buffered by the diffusion of CO2 out of the vessels.

Carbon Dioxide↗
PubMed2003

Cell surface hydrophobicity and slime production of Staphylococcus epidermidis Brazilian isolates.

The cell surface hydrophobicity of 60 isolates and three reference strains of Staphylococcus epidermidis was assayed by means of bacterial aggregation in liquid broth, phosphate-buffered saline, and in ammonium sulfate, as well as by affinity of the bacteria to n-hexadecane and polystyrene surfaces. In order to better characterize the isolates, the influence of bacterial growth time and enzyme treatment on cell hydrophobicity and the analysis of the slime production were also investigated. The strains presented the following profiles when assayed by the ammonium sulfate aggregation test (SAT): SAT < 1M, SAT 1M - <2M, SAT 2M - <4M, and SAT >or=4M. When SAT < 1M, the strains showed positive results for most of the cell surface hydrophobicity tests. None of the strains belonging to the groups with SAT >or= 1M showed spontaneous aggregation (SA), auto-aggregation (AA), or glass adherence, albeit 32 (62.7%) strains were polystyrene adherent and 42 (82.3%) presented weak adherence to n-hexadecane (>20%). The best correlation of the results was found among the AA and glass adherence tests (100%), followed by SA/ glass adherence (98%) and SA/ AA test (98%). The polystyrene adherence test and microbial adherence to n-hexadecane test (MATH) showed 78% correlation. Proteinase K treatment reduced bacterial adherence to polystyrene, but did not influence the SAT values. Three distinct groups of strains were distinguished by the polystyrene micromethod and glass tube adherence assay: 0.0-0.4 O.D. group, including non-glass adherent isolates; 0.5-0.7 O.D. group, including strains with variable profiles (adherent or non-adherent); and 0.8-1.3 O.D. group, composed of glass-adherent strains. Evaluation by a single method seemed not to reliably determine the surface hydrophobicity characteristics of S. epidermidis clinical isolates. Auto-aggregation properties of the strains that adhered to glass seemed related to slime expression, rather than cell surface hydrophobicity. Data also suggested involvement of protein components in adherence to polystyrene, but not in auto-aggregation properties assayed by SAT.

Adhesiveness↗
PubMed1993

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.

Adsorption↗