Novel Monoclonal Antibody and Peptide Binders for Mycobacterium avium subsp. paratuberculosis and Their Application for Magnetic Separation

The generation of novel Mycobacterium avium subsp. paratuberculosis (MAP)-specific monoclonal antibodies and phage-display derived peptide binders, along with their application for the magnetic separation (MS) of MAP cells, is described. Our aim was to achieve even greater MAP capture capability than is possible with peptide-mediated magnetic separation (PMS) using a 50:50 mix of biotinylated-aMp3 and biotinylated-aMptD peptide-coated beads. Gamma-irradiated whole MAP cells and ethanol extracted antigens (EEA) from these cells were used to elicit an immune response and as phage-display biopanning targets. A range of novel binders was obtained and coated onto paramagnetic beads, both individually and in various combinations, for MS evaluation. IS900 PCR was employed after MS to provide quick results. Capture sensitivity was assessed using a range of MAP concentrations after which the most promising beads were tested for their specificity for MAP, by performing MS followed by culture using 10 other Mycobacterium species. Magnetic beads coated with the biotinylated EEA402 peptide demonstrated a greater level of MAP capture than the current PMS method, even when low numbers of MAP (<10 cfu/ml) were present; however these beads also captured a range of other mycobacteria and so lacked capture specificity. Magnetic beads coated with monoclonal antibodies 6G11 and 15D10 (used as a 50:50 mix or as dually coated beads) also demonstrated improved MAP capture relative to the current PMS method, but with little cross-reactivity to other Mycobacterium spp. Therefore, two new MS protocols are suggested, the application of which would be dependent upon the required endpoint. Biotinylated EEA402-coated beads could potentially be used with a MAP-specific PCR to ensure detection specificity, while beads coated with 6G11 and 15D10 monoclonal antibodies could be used with culture or the phage amplification assay.     

Sulfasalazine release from alginate-N,O-carboxymethyl chitosan gel beads coated by chitosan

In the present study, spherical beads were prepared from a water-soluble chitosan (N,O-carboxymethyl chitosan, NOCC) and alginate with ionic gelation method. Then, swollen calcium–alginate–NOCC beads were coated with chitosan. To prepare drug loaded beads, sulfasalazine (SA) was added to the initial aqueous polymer solution. The effect of coating, as well as drying procedure, on the swelling behavior of unloaded beads and SA release of drug loaded ones were evaluated in simulated gastrointestinal tract fluid. The rate of swelling and drug release were decreased for air-dried and coated beads in comparison with freeze-dried and uncoated ones, respectively. No burst release of drug was observed from whole tested beads. Chitosan coated beads released approximately 40% of encapsulated drug in simulated gastric and small intestine tract fluid. Based on these results, the chitosan coated alginate–NOCC hydrogel may be used as potential polymeric carrier for colon-specific delivery of sulfasalazine.     

Biodegradable polymer based encapsulation of neem oil nanoemulsion for controlled release of Aza-A

Azadirachtin a biological compound found in neem have medicinal and pesticidal properties. The present work reports on the encapsulation of neem oil nanoemulsion using sodium alginate (Na-Alg) by cross linking with glutaraldehyde. Starch and polyethylene glycol (PEG) were used as coating agents for smooth surface of beads. The SEM images showed beads exhibited nearly spherical shape. Swelling of the polymeric beads reduced with coating which in turn decreased the rate of release of Aza-A. Starch coated encapsulation of neem oil nanoemulsion was found to be effective when compared to PEG coated encapsulation of neem oil nanoemulsion. The release rate of neem Aza-A from the beads into an aqueous environment was analyzed by UV-visible spectrophotometer (214nm). The encapsulated neem oil nanoemulsion have the potential for controlled release of Aza-A. Neem oil nanoemulsion encapsulated beads coated with PEG was found to be toxic in lymphocyte cells.     

Transfer of Fas (CD95) protein from the cell surface to the surface of polystyrene beads coated with anti-Fas antibody clone CH-11

Mouse monoclonal anti-Fas (CD95) antibody clone CH-11 has been widely used in research on apoptosis. CH-11 has the ability to bind to Fas protein on cell surface and induce apoptosis. Here, we used polystyrene beads coated with CH-11 to investigate the role of lipid rafts in Fas-mediated apoptosis in SKW6.4 cells. Unexpectedly, by treatment of the cells with CH-11-coated beads Fas protein was detached from cell surface and transferred to the surface of CH-11-coated beads. Western blot analysis showed that Fas protein containing both extracellular and intracellular domains was attached to the beads. Fas protein was not transferred from the cells to the surface of the beads coated with other anti-Fas antibodies or Fas ligand. Similar phenomenon was observed in Jurkat T cells. Furthermore, CH-11-induced apoptosis was suppressed by pretreatment with CH-11-coated beads in Jurkat cells. These results suggest that CH-11 might possess distinct properties on Fas protein compared with other anti-Fas antibodies or Fas ligand, and also suggest that caution should be needed to use polystyrene beads coated with antibodies such as CH-11.Key words: Fas, CD95, CH-11, apoptosis, Fas ligand, polystyrene beads.     

Synexin binds in a calcium-dependent fashion to oriented chromaffin cell plasma membranes

Oriented plasma membrane fragments from chromaffin cells, isolated on polylysine-coated polyacrylamide beads, bind synexin in a calcium dependent manner. Synexin binding was also detected on beads coated with chromaffin granule membranes, but not to beads coated with erythrocyte membranes or to uncoated beads. Synexin binding to plasma membrane coated beads showed a specific requirement for calcium (K1 2 = 200 microM) and was insensitive to other divalent cations such as magnesium, strontium and barium. Synexin binding to either plasma membrane or granule membrane coated beads was saturable, was partially reversible by EGTA and was directly observed by SDS-polyacrylamide gel electrophoresis.     

Selective binding of mouse and human spermatozoa to beads coated with extracellular matrix components

The ability of mouse epididymal and human ejaculated spermatozoa to bind to beads coated with various extracellular matrix components was examined. Mouse spermatozoa preferentially bound to beads coated with heparin (average values ranging between 6.2 and 8.8 sperm per bead were obtained in different experiments) and with chondroitinsulfate (6.2-7.0), and also, although with significant differences across replicate experiments, to beads coated with laminin (7.9-15.6 sperm per bead) and with collagen type I (6.1-18.5). Human spermatozoa bound to collagen-coated beads (15.4-22.6 sperm per bead) and, to a much lower extent, to chondroitin-sulfate-coated beads (3.2-4.7); they were also able to bind heparin-coated beads, although with ample differences between individual sperm donors (ranging between 0.8 and 18.7 sperm per bead). Very few human and mouse sperm bound fibronectin-coated beads; beads coated with albumin, hyaluronic acid, and chondronectin were always totally free of adhering sperm. The possible physiological role of the interactions between spermatozoa and extracellular matrix components are discussed.     

Immobilization of thermoalkalophilic recombinant esterase enzyme by entrapment in silicate coated Ca-alginate beads and its hydrolytic properties

Thermoalkalophilic esterase enzyme from Bal?ova (Agamemnon) geothermal site were aimed to be immobilized effectively via a simple and cost-effective protocol in silicate coated Calcium alginate (Ca-alginate) beads by entrapment. The optimal immobilization conditions of enzyme in Ca-alginate beads were investigated and obtained with 2% alginate using 0.5mg/ml enzyme and 0.7 M CaCl(2) solution. In order to prevent enzyme from leaking out of the gel beads, Ca-alginate beads were then coated with silicate. Enzyme loading efficiency and immobilization yield for silicate coated beads was determined as 98.1% and 71.27%, respectively and compared with non-coated ones which were 68.5% and 45.80%, respectively. Surface morphologies, structure and elemental analysis of both silicate coated and non-coated alginate beads were also compared using Fourier Transform Infrared Spectroscopy (FT-IR) and Scanning Electron Microscope (SEM) equipped with Energy-dispersive X-ray spectroscopy (EDX). Moreover, silicate coated alginate beads enhanced reusability of esterase in continuous processes compared to non-coated beads. The hydrolytic properties of free and immobilized enzyme in terms of storage and thermal stability as well as the effects of the temperature and pH were determined. It was observed that operational, thermal and storage stabilities of the esterase were increased with immobilization.     

Effects of different fragments of the fibronectin molecule on latex bead translocation along neural crest migratory pathways

Previous studies from this laboratory have utilized latex beads as probes of embryonic migratory pathways. After microinjection into embryos at the time of neural crest migration, uncoated latex polystyrene beads were found to translocate to ventral sites and to settle in the vicinity of endogenous neural crest derivatives. However, latex beads coated with fibronectin did not translocate ventrally, but remained associated with cells surrounding the implantation site. Fibronectin is a large glycoprotein with a variety of biological activities and multiple binding domains. Here, the binding activities which might be responsible for immobilization of the fibronectin-coated beads are examined. Latex beads were coated with three types of fragments of the fibronectin molecule representing different functional domains: (i) a 66-kDa fragment containing collagen-binding activity; (ii) a mixture of 45- and 32-kDa fragments containing heparin-binding activity; and (iii) a 120-kDa fragment containing cell-binding activity. The beads coated with fibronectin fragments were injected into the newly formed trunk somites of avian embryos. After injection, beads coated with either the heparin- or the collagen-binding domain translocated ventrally and distributed analogously to uncoated latex beads. In contrast, the majority of beads coated with the fibronectin cell-binding domain did not translocate but remained associated with dermamyotomal cells surrounding the injection site. The cell-binding fragment, however, was not as effective as the intact fibronectin molecule in preventing translocation of the beads. The results suggest that the cell-binding domain is primarily responsible for restriction of fibronectin beads from the ventral neural crest pathway. Because intact fibronectin is more effective at immobilizing beads than is the cell-binding fragment, other binding domains of fibronectin, more efficient coating with intact fibronectin, or crosslinking of intact fibronectin molecules may also play some role in immobilization of the beads at the implantation site.     

Improved method for isolation of plasma membrane on cationic beads. Membranes from Dictyostelium discoideum

The plasma membrane from Dictyostelium discoideum was routinely purified 35-fold by an improved technique using beads coated with positively charged polymers. Cells were attached to the beads and bare regions between the cells were neutralized with a polyanion. The neutralization decreased contamination of the bare regions by intracellular proteins released when cells were disrupted to leave behind beads coated by plasma membrane. The neutralization increased the purification as measured by membrane-bound ¹²⁵I-labeled concanavalin A. Contamination by markers for various intracellular components was markedly decreased. Various bare-site neutralization reagents were evaluated and gave different results depending upon their charge density and molecular weight. The pH of the neutralization was critical. The optimum pH for cell attachment to beads, 5.0, had little effect as regards bare-site neutralization. A new procedure is given that optimizes the essential features for the plasma membrane isolation on beads.     

Influence of ibuprofen as a solid-state plasticizer in eudragit® RS 30 D on the physicochemical properties of coated beads

The purpose of this study was to investigate the physicochemical properties of nonpareil beads coated with Eudragit RS 30 D containing ibuprofen as a multifunctional agent. The influence of the concentration of ibuprofen in the film coating and the effect of the coating level on drug release from coated beads was determined in pH 7.2 phosphate buffer solution. The influence of storage time at 23 degrees C and 60 degrees C on the release of ibuprofen from coated beads was also investigated. The thermal properties of the films were determined using a differential scanning calorimeter. Scanning electron microscopy was employed to image the surface morphology of the coated beads. Infrared spectroscopy was used to study the interaction of Eudragit RS 30 D and ibuprofen. Results from the dissolution studies demonstrated that increasing the amount of ibuprofen in the polymeric film reduced the rate of drug release, mainly because of a more complete coalescence of the polymeric particles of the latex dispersion. The glass transition temperature (Tg) of Eudragit RS 30 D films decreased and the surface of the coated beads became smoother as the concentration of ibuprofen was increased. Hydrogen bonding between the polymer and ibuprofen was demonstrated by Fourier transform infrared spectroscopy. No significant differences were found in drug dissolution between the coated beads stored at 23 degrees C for 12 months and those stored at 60 degrees C for 12 hours. The results of this study demonstrated that the ibuprofen plasticized the Eudragit RS 30 D. Furthermore, the dissolution rate of ibuprofen can be controlled and changes in the drug release rate can be minimized by using the drug-induced plasticization technique with this polymer.     

Rapid determination of bovine lactoferrin in dairy products by an automated quantitative agglutination assay based on latex beads coated with F(ab′)2 fragments

This study evaluated an automated immunoassay for bovine lactoferrin (LF) in dairy products based on latex beads coated with F(ab')2 fragments. Methods: F(ab')2 fragments were obtained by pepsin digestion of rabbit anti-bovine LF (IgG fraction) and polystyrene latex beads were coated with the F(ab')2 fragments. We used the beads to develop a rapid and homogeneous light scatter immunoassay employing an autoanalyzer (the Automated Latex assay). The Automated Latex assay was easy to perform and could rapidly determine bovine lactoferrin in lactoferrin-supplemented products. It was sensitive enough for testing products and showed good precision.     

Fibronectin-coated beads are endocytosed by cells and align with microfilament bundles

Carboxylated latex beads coated with fibronectin bind to the surfaces of NIL8 hamster cells. Similar beads coated with bovine serum albumin, gelatin or normal rabbit immunoglobulin do not bind to the cells, whereas beads coated with polylysine or rabbit anti-hamster immunoglobulin bind, as do fibronectin-coated beads. Surface-bound beads are endocytosed by the cells. This endocytosis is inhibited by N-ethyl maleimide or low temperatures. The endocytosed beads form arrays aligned with the microfilament bundles inside the cells and after extended incubations (18–24 h) become concentrated in the perinuclear region. These results suggest that, after phagocytosis of large particles, the endocytic vesicles align with microfilament bundles, as previously reported for coated pits involved in receptor-mediated endocytosis. The system described here offers possibilities for the analysis of membrane and transmembrane interactions involved in cell-substratum binding and phagocytosis.     

Chitosan coated alginate beads for the survival of microencapsulated Lactobacillus plantarum in pomegranate juice

This work studied the effect of multi-layer coating of alginate beads on the survival of encapsulated Lactobacillus plantarum in simulated gastric solution and during storage in pomegranate juice at 4°C. Uncoated, single and double chitosan coated beads were examined. The survival of the cells in simulated gastric solution (pH 1.5) was improved in the case of the chitosan coated beads by 0.5-2 logs compared to the uncoated beads. The cell concentration in pomegranate juice after six weeks of storage was higher than 5.5logCFU/mL for single and double coated beads, whereas for free cells and uncoated beads the cells died after 4 weeks of storage. In simulated gastric solution, the size of the beads decreased and their hardness increased with time; however, the opposite trend was observed for pomegranate juice, indicating that there is no correlation between cell survival and the hardness of the beads.     

Plucking of lectin receptors from erythrocytes: Isolation of cell surface components without the use of detergents

A new approach is described for the isolation of lectin receptors without the use of detergents, by plucking them from the cell surface. Cells bound to lectin-coated Sepharose beads are sheared off the beads by mechanical disruption, whereupon the receptors remain attached to the beads and are released specifically by inhibitory sugars. Material plucked from neuraminidase-treated human erythrocytes by beads coated with peanut agglutinin and released by D-galactose was identified as asialoglycophorin. The same membrane glycoprotein was plucked from neuraminidase-treated erythrocytes by beads coated with soybean agglutinin, but at considerably lower yield.     

Laminin and a Plasmodium ookinete surface protein inhibit melanotic encapsulation of Sephadex beads in the hemocoel of mosquitoes

In refractory mosquitoes, melanotic encapsulation of Plasmodium ookinetes and oocysts is a commonly observed immune response. However, in susceptible mosquitoes, Plasmodium oocysts develop extracellularly in the body cavity without being recognized by the immune system. Like Plasmodium gallinaceum oocysts, negatively charged carboxymethyl (CM)-Sephadex beads implanted in the hemocoel of Aedes aegypti female mosquitoes were not usually melanized, but were coated with mosquito-derived laminin. Conversely, electrically neutral G-Sephadex beads were routinely melanized. Since mosquito laminin coated both CM-Sephadex beads and P. gallinaceum oocysts, we hypothesized that laminin prevents melanization of both. To test this hypothesis, we coated cyanogen-bromide-activated G-Sephadex beads with laminin, recombinant P. gallinaceum ookinete surface protein (PgS28) or bovine serum albumin (BSA). Beads were implanted into the abdominal body cavity of female Aedes aegypti and retrieved 4 days later. Uncoated controls as well as BSA-coated G-Sephadex beads were melanized in a normal manner. However, melanization of beads coated with mouse laminin, Drosophila L2-secreted proteins or PgS28 was markedly reduced. Fluorescent antibody labeling showed that PgS28-coated beads had adsorbed mosquito laminin on their surface. Thus, mosquito laminin interacting with Plasmodium surface proteins probably masks oocysts from the mosquito's immune system, thereby facilitating their development in the body cavity.     

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 adsorbed. 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.     

Studies of the mechanical properties and the fermentation behavior of double layer alginate–chitosan beads,using <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> entrapped cells

Double layer alginate beads coated with chitosan were constructed for the entrapment of yeast cells used in alcoholic fermentations. Several construction parameters of the beads were studied. Among these parameters were the composition of the inner and the outer layer, the initial cell loading, the concentration of chitosan in the coating solution. Improved bead behavior was noticed by the use of chitosan as a coating agent to double layer alginate beads. The mechanical strength and the stability of the beads were enhanced. The polyelectrolyte complex membrane of alginate–chitosan reduced significantly the leakage of the entrapped cells into the medium. The aim of this work was to define the optimal conditions for the construction of the double layer alginate beads coated with chitosan with the purpose of using them for the fermentation of carbohydrates. This paper is based on a presentation at the “International Congress on Bioprocesses in Food Industries – ICBF 2006” conference, Patras 2006     

Development of sensors for direct detection of organophosphates. Part I: Immobilization, characterization and stabilization of acetylcholinesterase and organophosphate hydrolase on silica supports

Biosensors for organophosphates in solution may be constructed by monitoring the activity of acetylcholinesterase (AChE) or organophosphate hydrolase (OPH) immobilized to a variety of microsensor platforms. The area available for enzyme immobilization is small (< 1 mm2) for microsensors. In order to construct microsensors with increased surface area for enzyme immobilization, we used a sol-gel process to create highly porous and stable silica matrices. Surface porosity of sol-gel coated surfaces was characterized using scanning electron microscopy; pore structure was found to be very similar to that of commercially available porous silica supports. Based upon this analysis, porous and non-porous silica beads were used as model substrates of sol-gel coated and uncoated sensor surfaces. Two different covalent chemistries were used to immobilize AChE and OPH to these porous and non-porous silica beads. The first chemistry used amine-silanization of silica followed by enzyme attachment using the homobifunctional linker glutaraldehyde. The second chemistry used sulfhydryl-silanization followed by enzyme attachment using the heterobifunctional linker N-gamma-maleimidobutyryloxy succinimide ester (GMBS). Surfaces were characterized in terms of total enzyme immobilized, total and specific enzyme activity, and long term stability of enzyme activity. Amine derivitization followed by glutaraldehyde linking yielded supports with greater amounts of immobilized enzyme and activity. Use of porous supports not only yielded greater amounts of immobilized enzyme and activity, but also significantly improved long term stability of enzyme activity. Enzyme was also immobilized to sol-gel coated glass slides. The mass of immobilized enzyme increased linearly with thickness of coating. However, immobilized enzyme activity saturated at a porous silica thickness of approximately 800 nm.     

Latex beads as probes of a neural crest pathway: effects of laminin, collagen, and surface charge on bead translocation

In the trunk region of avian embryos, neural crest cells migrate along two pathways: dorsally just under the ectoderm, and ventrally between the neural tube and the somites. Previous work from this laboratory has shown that uncoated latex beads are able to translocate along the ventral neural crest pathway after injection into young embryos; however, beads coated with fibronectin are restricted from the ventral route ( Bronner -Fraser, M.E., 1982, Dev. Biol., 91: 50-63). Here, we extend these observations to determine the effects of other macromolecules on bead distribution. The data show that laminin-coated beads, like fibronectin-coated beads, are restricted from the ventral pathway. In contrast, beads coated with type I collagen translocate ventrally after injection. Because macromolecules have characteristic charge properties, changes in surface charge caused by coating the beads may confound interpretation of the results. Electrostatic effects on bead movement were examined by coating the latex beads with polyamino acids in order to predictably alter the initial surface charge. The surface charge before injection was measured for beads coated with amino acid polymers or with various biologically important macromolecules; the subsequent translocation ability of these beads was then monitored in the embryo. Polylysine-coated beads (positively charged) were restricted from the ventral pathway as were fibronectin and laminin-coated beads, even though fibronectin and laminin beads were both negatively charged. In contrast, polytyrosine -coated beads ( neutrally charged) translocated ventrally as did negatively charged collagen-coated or uncoated beads. The results demonstrate that no correlation exists between the charge properties on the latex bead surface and their subsequent ability to translocate along the ventral pathway. Therefore, an adhesion mechanism independent of surface charge effects must explain the restriction or translocation of latex beads on a neural crest pathway.     

T lymphocyte aggregation with immobilized anti-TCR-antibodies is dependent upon energy and microfilament assembly

An assay has been developed to quantitate the binding of beads coated with anti-T cell receptor (TCR) monoclonal antibodies (MoAb) to T lymphocytes. The Ab used were a hamster MoAb, 145.2C11 (2C11), directed against the epsilon chain of the CD3 complex of the murine TCR, and a murine MoAb, F23.1, directed against the V beta 8-encoded determinant of the alpha/beta heterodimer of the TCR. Ab were adsorbed onto polystyrene beads and the beads labeled with [125I]bovine serum albumin [( 125I]BSA). The labeled, Ab-coated beads were mixed at 4 degrees C with murine, cloned T-helper (Th) cells and contact between beads and cells was promoted by centrifugation. The mixtures were incubated at 37 degrees C for 10-20 min, and unbound beads were separated from cell-bound beads by Percoll gradient centrifugation. Beads coated with anti-TCR Ab formed stable conjugates with Th cells; an average of 6-10 2C11 Ab-coated beads/cell, or 10-15 F23.1 Ab-coated beads/cell was measured under optimal conditions. Beads coated with control Ab (hamster or mouse IgG) did not appreciably bind to the cells. Conjugation with 2C11 Ab-coated beads could be prevented by coating the cells with soluble 2C11 Ab, but not with soluble F23.1 Ab. Blocking the CD3 epsilon chain with soluble 2C11 Ab also reduced conjugate formation with F23.1 Ab-coated beads, suggesting a steric hindrance phenomenon. The extent of conjugation depended on the density of immobilized Ab. Maximum conjugation was observed when 100 micrograms of 2C11 Ab was used to coat 10(6) beads; higher Ab amounts did not further increase binding. Increasing the bead to cell ratio in the mixture increased binding, reaching optimal binding at 300:1, irrespectively of the amount of Ab adsorbed onto the beads. Stable binding of anti-TCR Ab-coated beads to T cells was temperature and energy dependent. It was prevented when glucose was removed from the medium and the glycolysis inhibitor, 2-deoxy-D-glucose was added, or when cells were treated with sodium azide. Conjugate formation was prevented by pretreatment of the cells with cytochalasins, indicating that microfilament assembly was essential. Microtubules were not involved, as vinca alkaloids were without effect. This novel assay system provides a simple means of studying aspects of TCR function including its physical and metabolic regulation.