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Magnetic Beads (magnetic + bead)

Distribution by Scientific Domains

Chemistry	37%
Life Sciences	34%
Medical Sciences	20%

Selected Abstracts

Promising Diagnostic Biomarkers for Primary Biliary Cirrhosis Identified With Magnetic Beads and MALDI-TOF-MS

THE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 3 2009
Yong-Zhe Li
Abstract (PBC) is not a rare disease worldwide. Most patients are diagnosed at the advanced stage, primarily because there are not yet any valid biomarkers available for early diagnosis. Useful biomarkers are absolutely necessary for early detection of PBC. Fortunately, the use of MALDI-TOF-MS and pattern recognition software has been successful in finding specific markers for the early detection of the disease. To screen for potential protein biomarkers in the serum for diagnosing PBC, MALDI-TOF-MS combined with magnetic beads and pattern recognition software was used to investigate 119 serum samples from 44 patients with PBC, 32 controls with other hepatic disease, and 43 healthy controls. A total of 69 discriminant m/z peaks were identified as being associated with PBC. Of them, the m/z peaks at 3445, 4260, 8133, and 16,290 were used to construct a model for the diagnosis of PBC. This diagnostic model can distinguish PBC from non-PBC controls with a sensitivity of 93.3% and a specificity of 95.1%. In our blind test, it demonstrated good sensitivity and specificity: 92.9% and 82.4%, respectively. These results indicate that useful serum biomarkers for PBC can be discovered by MALDI-TOF-MS combined with the use of magnetic beads and pattern recognition software. The pattern of multiple markers provides a powerful and reliable diagnostic method for PBC with high sensitivity and specificity. Anat Rec, 292:455,460, 2009. © 2009 Wiley-Liss, Inc. [source]

A Method for the Rapid Discovery of Naturally Occurring Products by Proteins Immobilized on Magnetic Beads and Reverse Affinity Chromatography

CHEMISTRY - AN ASIAN JOURNAL, Issue 12 2009
Midori
Abstract A highly efficient screening method for naturally occurring products that bind to a specific target protein was demonstrated by using hVDR magnetic beads. The native ligand 1,,25(OH)2 VD3 (1) was selectively bound by hVDR magnetic beads when present in a mixture of natural compounds. Furthermore, this method was shown to be applicable to the identification of natural products that interact with a specific protein immobilized on the beads from an extract of a natural resource. Two new natural compounds were isolated by this method. This approach will be helpful for the discovery of novel, naturally occurring products that bind to specific target proteins. This method has the further advantages that it can identify the HPLC peak corresponding to the target compound for isolation, as well as provide important UV, CD, or MS profile information. [source]

DNA aptamers developed against a soman derivative cross-react with the methylphosphonic acid core but not with flanking hydrophobic groups

JOURNAL OF MOLECULAR RECOGNITION, Issue 3 2009
John G. Bruno
Abstract Twelve rounds of systematic evolution of ligands by exponential enrichment (SELEX) were conducted against a magnetic bead conjugate of the para -aminophenylpinacolylmethylphosphonate (PAPMP) derivative of the organophosphorus (OP) nerve agent soman (GD). The goal was to develop DNA aptamers that could scavenge GD in vivo, thereby reducing or eliminating the toxic effects of this dangerous compound. Aptamers were sequenced and screened in peroxidase-based colorimetric plate assays after rounds 8 and 12 of SELEX. The aptamer candidate sequences exhibiting the highest affinity for the GD derivative from round 8 also reappeared in several clones from round 12. Each of the highest affinity PAPMP-binding aptamers also bound methylphosphonic acid (MPA). In addition, the aptamer with the highest overall affinity for PAPMP carried a sequence motif (TTTAGT) thought to bind MPA based on previously published data (J. Fluoresc 18: 867,876, 2008). This sequence motif was found in several other relatively high affinity PAPMP aptamer candidates as well. In studies with the nerve agent GD, pre-incubation of a large molar excess of aptamer candidates failed to protect human butyrylcholinesterase (BuChE) from inhibition. With the aid of three-dimensional molecular modeling of the GD derivative it appears that a hydrophilic cleft sandwiched between the pinacolyl group and the p -aminophenyl ring might channel nucleotide interactions to the phosphonate portion of the immobilized GD derivative. However, bona fide GD free in solution may be repulsed by the negative phosphate backbone of aptamers and rotate its phosphonate and fluorine moieties away from the aptamer to avoid being bound. Future attempts to develop aptamers to GD might benefit from immobilizing the pinacolyl group of bona fide GD to enhance exposure of the phosphonate and fluorine to the random DNA library. Copyright © 2008 John Wiley & Sons, Ltd. [source]

Highly sensitive spin-valve devices for chip-cytometers

PHYSICA STATUS SOLIDI (A) APPLICATIONS AND MATERIALS SCIENCE, Issue 7 2009
Jong Wook Roh
Abstract The highly sensitive spin-valve devices integrated with a microfluidic channel have been studied for cell counting in a chip-cytometer. The presence or absence of a single magnetic bead was successfully detected by direct measurement of fringe fields emanating from magnetic beads using spin-valve devices. The real-time detection was also successfully confirmed by the direct measurement of magnetic fields generated from the magnetic beads passing an active sensing area of a spin-valve device integrated with a microfluidic channel. Our results show the possibility of implementing a chip-cytometer for biological applications using the highly sensitive spin-valve devices integrated with a microfluidic device. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source]

Detection of C Reactive Protein (CRP) in Serum by an Electrochemical Aptamer-Based Sandwich Assay

ELECTROANALYSIS, Issue 11 2009
Sonia Centi
Abstract A disposable electrochemical assay involving magnetic particles and carbon-based screen-printed electrodes (SPCEs) was developed for the detection of C Reactive Protein (CRP). CRP is a plasma protein and is among the most expressed proteins in acute phase inflammation cases, being a known biomarker for inflammatory states. The assay was based on a sandwich format in which a RNA aptamer was coupled to a monoclonal antibody and alkaline phosphatase (AP) was used as enzymatic label. After the sandwich assay, the modified magnetic beads were captured by a magnet on the surface of a graphite working electrode and the electrochemical detection was thus achieved through the addition of the AP substrate (,-naphthyl-phosphate) and ,-naphthol produced during the enzymatic reaction was detected using differential pulse voltammetry (DPV). The parameters influencing the different steps of the assay were optimized in order to reach the best sensitivity and specificity. With the optimized conditions, the assay was applied to the analysis of CRP free serum and serum samples. [source]

A micropillar-integrated smart microfluidic device for specific capture and sorting of cells

ELECTROPHORESIS, Issue 24 2007
Yan-Jun Liu
Abstract An integrated smart microfluidic device consisting of nickel micropillars, microvalves, and microchannels was developed for specific capture and sorting of cells. A regular hexagonal array of nickel micropillars was integrated on the bottom of a microchannel by standard photolithography, which can generate strong induced magnetic field gradients under an external magnetic field to efficiently trap superparamagnetic beads (SPMBs) in a flowing stream, forming a bed with sufficient magnetic beads as a capture zone. Fluids could be manipulated by programmed controlling the integrated air-pressure-actuated microvalves, based on which in situ bio-functionalization of SPMBs trapped in the capture zone was realized by covalent attachment of specific proteins directly to their surface on the integrated microfluidic device. In this case, only small volumes of protein solutions (62.5,nL in the capture zone; 375,nL in total volume needed to fill the device from inlet A to the intersection of outlet channels F and G) can meet the need for protein! The newly designed microfluidic device reduced greatly chemical and biological reagent consumption and simplified drastically tedious manual handling. Based on the specific interaction between wheat germ agglutinin (WGA) and N -acetylglucosamine on the cell membrane, A549 cancer cells were effectively captured and sorted on the microfluidic device. Capture efficiency ranged from 62 to 74%. The integrated microfluidic device provides a reliable technique for cell sorting. [source]

A Rapid Method for the Pre-Enrichment and Detection of Salmonella Typhimurium by Immunomagnetic Separation and Subsequent Fluorescence Microscopical Techniques

ENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 3 2005
J. Steingroewer
Abstract Detection of food-borne pathogens is of great importance in order to minimize the risk of infection for customers. These analyses should be as fast as possible. Any detection method requires enrichment and quantitative analysis of the enriched microbes. Conventional enrichment methods, which take several days, need to be replaced by faster techniques such as immunomagnetic separation (IMS). This technique is based on the use of paramagnetic microspheres coated with antibodies as ligands that have specific affinity to the microbes that have to be detected. In the studies reported here, a rapid method for the detection of Salmonella enterica serovar Typhimurium (Salmonella Typhimurium), combining IMS and Direct Epifluorescence Filter Technique (DEFT), was developed. It was focused on releasing the target cells from the magnetic beads after IMS, because this is a premise for combining IMS, as an alternative pre-enrichment, with DEFT. Otherwise, the high number of beads form a layer on the filter membrane that makes the following microscopic analysis for the detection of the contaminants impossible. The CELLectionTM Dynabeads® used in this study, are coated with recombinant streptavidin (rSA) via a DNA linker. The rSA binds biotinylated antibodies that are able to capture target cells. The DNA linker provides the cleavable site, so that the beads can be removed from the captured cells after isolation. In this study a releasing procedure was developed. This procedure allows for an average 74,% ± 4,% of the bead-bound Salmonella Typhimurium cells to be released from the beads after IMS, so that the detection of the separated cells by DEFT will be possible. [source]

Comparison of ATP and in vivo bioluminescence for assessing the efficiency of immunomagnetic sorbents for live Escherichia coli O157:H7 cells

JOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2002
W. Sun
Aims:,To develop methods to assess the efficiency of immunomagnetic separation (IMS). Methods and Results:,The capturing efficiency of biosorbents for Escherichia coli O157:H7, constructed using streptavidin-coated magnetic beads and biotinylated antibodies, was tested using both in vivo and ATP bioluminescence. Both methods were suitable for the enumeration of bacteria captured by the biosorbents. The level of both ATP and in vivo bioluminescence depended on the media used, but was unaffected by the magnetic beads. The capture efficiency depended on time and sample volume, but did not depend on the length of spacer arm of the biotinylation agent. For cell concentrations of , 105 cfu ml,1, in a 1-ml sample volume, nearly 80,85% recovery of the pathogen was observed after 0·5 h of incubation. For an 11-ml sample containing 104 cfu ml,1, maximum recovery (50% of cells) was achieved only after 2 h incubation. Conclusions:,The detection limit of an ATP-based bioluminescent assay for E. coli O157:H7 was reduced by 1 log cycle after optimization of IMS. The bioluminescent methods could be used for screening and testing the affinity of antibodies or other affinity elements of biosorbents towards live bacterial cells. Significance and Impact of the Study:,Bioluminescent assays provide an easy way to optimize conditions for the capture of bacteria by biosorbents in real time. [source]

Isolation and Purification of Osteocytes

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 4 2005
A van der Plas
An isolation method for osteocytes is described. After removal of the periostea, bone cells were isolated from calvariae of 18-day-old chicken embryos by alternating treatments with collagenase and EDTA. Osteocytes were purified from the heterogeneous bone cell population with the help of the osteocyte-specific MAb OB 7.3 bound to protein G-conjugated magnetic beads. The purity of the osteocyte population ultimately obtained was more than 95%. Osteocytes were found to adhere rapidly to glass or plastic substrates. They showed numerous processes of various types. These processes could branch and make contact with those of other osteocytes. After 1,2 days of culture, the isolated osteocytes formed a network of apparently interconnected cell processes very similar to the osteocyte network in bone. [source]

The Use of Hemastix® and the Subsequent Lack of DNA Recovery Using the Promega DNA IQTM System

JOURNAL OF FORENSIC SCIENCES, Issue 6 2009
Hiron Poon M.Sc.
Abstract:, Following implementation of our automated process incorporating the Promega DNA IQTM system as a DNA extraction method, a large number of blood-containing exhibits failed to produce DNA. These exhibits had been tested with the Hemastix® reagent strip, commonly used by police investigators and forensic laboratories as a screening test for blood. Some exhibits were even tainted green following transfer of the presumptive test reagents onto the samples. A series of experiments were carried out to examine the effect of the Hemastix® chemistries on the DNA IQTM system. Our results indicate that one or more chemicals imbedded in the Hemastix® reagent strip severely reduce the ability to recover DNA from any suspected stain using the DNA IQTM magnetic bead technology. The 3,3,,5,5,-tetramethylbenzidine (TMB) used as the reporting dye appears to interact with the magnetic beads to prevent DNA recovery. Hydrogen peroxide does not seem to be involved. The Hemastix® chemistries do not interfere in any way with DNA extraction performed using phenol-chloroform. The incompatibility of the Hemastix® chemistries on the DNA IQTM system forced us to adopt an indirect approach using filter paper to carry out the presumptive test. [source]

Development of real-time detection direct test for hepatitis B virus and comparison with two commercial tests using the WHO international standard

JOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 11 2003
MOTOKAZU MUKAIDE
Abstract Aims:, A highly reproducible and sensitive hepatitis B virus real-time detection direct (HBV RTD-direct) test using DNA extraction by magnetic beads coated with polyclonal anti-HBsAg, followed by the real-time detection polymerase chain reaction (PCR) method, was developed for the detection of HBV DNA. Methods:, The HBV DNA could be extracted from the HBsAg positive viral particles without resulting in viral DNA fragmentation. The HBV RTD-direct test was validated using a serial dilution panel of the WHO standard HBV DNA 97/746 I. Results:, The test had a dynamic range of 0.7,8.0 log10 international units (IU) per mL and the results were shown to be comparable to those obtained with two commercially available tests: the HBV DNA transcription-mediated amplification-hybridization protection assay and the Amplicor HBV Monitor test. In addition, the HBV RTD-direct test, based on magnetic extraction, successfully eliminated PCR inhibitors in clinical specimens. Conclusion:, We conclude that the HBV RTD-direct test is an excellent alternative for monitoring patients undergoing antiviral treatment or for screening various clinical specimens. [source]

MYELIN PROTEIN P-0-SPECIFIC IGM PRODUCING MONOCLONAL B CELL LINES WERE ESTABLISHED FROM POLYNEUROPATHY PATIENTS WITH MONOCLONAL GAMMOPATHY OF UNDETERMINED SIGNIFICANCE (MGUS)

JOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 4 2002
M Kvarnstrom
Monoclonal expansion of B cells and plasma cells, producing antibodies against ,self' molecules, can be found not only in different autoimmune diseases, such as peripheral neuropathy (PN), but also in malignancies, such as Waldenstroms'macroglobulinaemia and B-type of chronic lymphocytic leukaemia (B-CLL), as well as in precancerous conditions including monoclonal gammopathy of undetermined significance (MGUS). About 50% of patients with PN-MGUS have serum antibodies against peripheral nerve myelin, but the specific role of these antibodies remains uncertain. The aims of the study were to establish, and characterize, myelin-specific B cell clones from peripheral blood of patients with PN-MGUS, by selection of cells bearing specific membrane Ig-receptors for myelin protein P-0, using beads coated with P-0. P-0-coated magnetic beads were used for selection of cells, which subsequently were transformed by Epstein-Barr virus. The specificity of secreted antibodies was tested by ELISA. Two of the clones producing anti-P-0 antibodies were selected and expanded. The magnetic selection procedure was repeated and new clones established. The cells were CD5(+) positive, although the expression declined in vitro over time. The anti-P-0 antibodies were of IgM-lambda type. The antibodies belonged to the V(H)3 gene family with presence of somatic mutations. The IgM reacted with P-0 and myelin-associated glycoprotein (MAG), and showed no evidence for polyreactivity, in contrast to other IgM CD5(+) clones included in the study as controls. The expanded clones expressed CD80 and HLA-DR, which is compatible with properties of antigen-presenting cells. The immunomagnetic selection technique was successfully used for isolation of antimyelin protein P-0-specific clones. The cell lines may provide useful tools in studies of monoclonal gammopathies, leukaemia, and autoimmune diseases, including aspects of antigen-presentation by these cells followed by T cell activation. [source]

The use of biomagnetic separation to recover DNA suitable for PCR from Claviceps species ,

LETTERS IN APPLIED MICROBIOLOGY, Issue 2 2000
D.L. Scott Jr
DNA analysis of agriculturally important fungi using polymerase chain reaction (PCR)-based methods is becoming routine in research and for diagnostic purposes. Rapid, small-scale DNA isolation methods that take advantage of the sensitivity, speed and automation potential of PCR technology are needed for timely analysis of important plant pathogens. DNA isolated from Claviceps africana (causal agent of ergot of sorghum) using several standard DNA extraction protocols was found to be unamplifiable using PCR. The standard methods apparently failed to separate DNA from substances inhibitory to the Taq polymerase enzyme. We obtained DNA amenable to PCR analysis using a novel method involving magnetic beads and high salt extraction buffer. The biomagnetic purification method allowed us to obtain reliable PCR amplification of the internal transcribed spacer (ITS) regions of rDNA of Claviceps africana, making genetic comparisons possible. [source]

Techniques for phosphopeptide enrichment prior to analysis by mass spectrometry

MASS SPECTROMETRY REVIEWS, Issue 1 2010
Jamie D. Dunn
Abstract Mass spectrometry is the tool of choice to investigate protein phosphorylation, which plays a vital role in cell regulation and diseases such as cancer. However, low abundances of phosphopeptides and low degrees of phosphorylation typically necessitate isolation and concentration of phosphopeptides prior to MS analysis. This review discusses the enrichment of phosphopeptides with immobilized metal affinity chromatography, reversible covalent binding, and metal oxide affinity chromatography. Capture of phosphopeptides on TiO2 seems especially promising in terms of selectivity and recovery, but the success of all methods depends on careful selection of binding, washing, and elution solutions. Enrichment techniques are complementary, such that a combination of methods greatly enhances the number of phosphopeptides isolated from complex samples. Development of a standard series of phosphopeptides in a highly complex mixture of digested proteins would greatly aid the comparison of different enrichment methods. Phosphopeptide binding to magnetic beads and on-plate isolation prior to MALDI-MS are emerging as convenient methods for purification of small (µL) samples. On-plate enrichment can yield >70% recoveries of phosphopeptides in mixtures of a few digested proteins and can avoid sample-handling steps, but this technique is likely limited to relatively simple samples such as immunoprecipitates. With recent advances in enrichment techniques in hand, MS analysis should provide important insights into phosphorylation pathways. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 29:29,54, 2010 [source]

Use of magnetic beads to extract fungal DNA

MYCOSES, Issue 1 2005
E. Faggi
Summary Authors compare two methods of extracting DNA from different fungi: the classic method with phenol/chloroform (P/C) and that with magnetic beads. Both were tested on Candida albicans and Cryptococcus neoformans var. neoformans, belonging to the yeast group and Microsporum canis, M. gypseum, Trichophyton rubrum, T. interdigitale, T. ajelloi, Epidermophyton floccosum, belonging to the dermatophytes group. Extraction products underwent polymerase chain reaction (PCR) fingerprinting with the appropriate primers to point out any disagreement in the genomic profiles. After having determined that the genomic profiles obtained from the DNA extracted from the same strain with the two methods correspond perfectly, the authors concluded that the extraction method with magnetic beads from fungal cells is simpler and quicker than with P/C extraction, greatly facilitating the obtainment of fungal DNA. [source]

Highly sensitive spin-valve devices for chip-cytometers

Affinity capture using chimeric membrane proteins bound to magnetic beads for rapid ligand screening by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 6 2009
Christian Legros
The rapid and specific detection of therapeutically important ligands in complex mixtures, that may bind to membrane proteins, remains challenging for many research laboratories and pharmaceutical industries. Through its use in the development of screening assays, mass spectrometry (MS) is currently experiencing a period of tremendous expansion. In the study presented here, we took advantage of the remarkable stability properties of a bacterial membrane protein, the KcsA K+ channel, produced in E. coli and purified as a tetrameric protein in the presence of a detergent. This membrane protein can subserve as a molecular template to display the pore-forming region of human K+ channels, which are considered as targets in the search for inhibitory ligands. The engineered chimeric proteins were linked to metal-bound magnetic beads, for the screening of complex peptide mixtures, such as that of scorpion venoms. The affinity-captured scorpion toxins were eluted prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), and to nano-electrospray ionization tandem mass QqTOF mass spectrometry (MS/MS) analysis. The de novo sequence of the toxins was deduced by combining the MS/MS fragmentation of the reduced form (up to the 33 first residues) and the trypsin digest peptides of the native toxins. This affinity-capture screening assay led to the isolation and characterization of potent and specific ligands of the human K+ channel, Kv1.3. The affinity-capture procedure is fast and reproducible. When linked to magnetic beads, the chimeric membrane protein can be re-used several times without losing any of its selectivity or specificity. This assay also benefits from the fact that it requires minimal amounts of animal venoms or complex mixtures, which can be expensive or difficult to procure. Copyright © 2009 John Wiley & Sons, Ltd. [source]

Promising Diagnostic Biomarkers for Primary Biliary Cirrhosis Identified With Magnetic Beads and MALDI-TOF-MS

Na+ -H+ exchanger 3 (NHE3) is present in lipid rafts in the rabbit ileal brush border: a role for rafts in trafficking and rapid stimulation of NHE3

THE JOURNAL OF PHYSIOLOGY, Issue 2 2001
Xuhang Li
1Rabbit ileal Na+ -absorbing cell Na+ -H+ exchanger 3 (NHE3) was shown to exist in three pools in the brush border (BB), including a population in lipid rafts. Approximately 50 % of BB NHE3 was associated with Triton X-100-soluble fractions and the other ,50 % with Triton X-100-insoluble fractions; ,33 % of the detergent-insoluble NHE3 was present in cholesterol-enriched lipid microdomains (rafts). 2The raft pool of NHE3 was involved in the stimulation of BB NHE3 activity with epidermal growth factor (EGF). Both EGF and clonidine treatments were associated with a rapid increase in the total amount of BB NHE3. This EGF- and clonidine-induced increase of BB NHE3 was associated with an increase in the raft pool of NHE3 and to a smaller extent with an increase in the total detergent-insoluble fraction, but there was no change in the detergent-soluble pool. In agreement with the rapid increase in the amount of NHE3 in the BB, EGF also caused a rapid stimulation of BB Na+ -H+ exchange activity. 3Disrupting rafts by removal of cholesterol with methyl-,-cyclodextrin (M,CD) or destabilizing the actin cytoskeleton with cytochalasin D decreased the amount of NHE3 in early endosomes isolated by OptiPrep gradient fractionation. Specifically, NHE3 was shown to associate with endosomal vesicles immunoisolated by anti-EEA1 (early endosomal autoantigen 1) antibody-coated magnetic beads and the endosome-associated NHE3 was decreased by cytochalasin D and M,CD treatment. 4We conclude that: (i) a pool of ileal BB NHE3 exists in lipid rafts; (ii) EGF and clonidine increase the amount of BB NHE3; (iii) lipid rafts and to a lesser extent, the cytoskeleton, but not the detergent-soluble NHE3 pool, are involved in the EGF- and clonidine-induced acute increase in amount of BB NHE3; (iv) lipid rafts and the actin cytoskeleton play important roles in the basal endocytosis of BB NHE3. [source]

Tumor necrosis factor , blockade exacerbates murine psoriasis-like disease by enhancing Th17 function and decreasing expansion of Treg cells

ARTHRITIS & RHEUMATISM, Issue 2 2010
Hak-Ling Ma
Objective Patients with psoriasis and psoriatic arthritis respond well to tumor necrosis factor , (TNF,) blockers in general; however, there is now mounting evidence that a small cohort of patients with rheumatoid arthritis who receive TNF, blockers develop psoriasis. This study was undertaken to explore the mechanisms underlying TNF, blockade,induced exacerbation of skin inflammation in murine psoriasis-like skin disease. Methods Skin inflammation was induced in BALB/c scid/scid mice after they received CD4+CD45RBhighCD25, (naive CD4) T cells from donor mice. These mice were treated with either anti,interleukin-12 (anti,IL-12)/23p40 antibody or murine TNFRII-Fc fusion protein and were examined for signs of disease, including histologic features, various cytokine levels in the serum, and cytokine or FoxP3 transcripts in the affected skin and draining lymph node (LN) cells. In a separate study, naive CD4+ T cells were differentiated into Th1 or Th17 lineages with anti-CD3/28 magnetic beads and appropriate cytokines in the presence or absence of TNF,. Cytokine gene expression from these differentiated cells was also determined. Results Neutralization of TNF, exacerbated skin inflammation and markedly enhanced the expression of the proinflammatory cytokines IL-1,, IL-6, IL-17, IL-21, and IL-22 but suppressed FoxP3 expression in the skin and reduced the number of FoxP3-positive Treg cells in the draining LNs. TNF, also demonstrated a divergent role during priming and reactivation of naive T cells. Conclusion These results reveal a novel immunoregulatory role of TNF, on Th17 and Treg cells in some individuals, which may account for the exacerbation of skin inflammation in some patients who receive anti-TNF treatments. [source]

Antibody-immobilized column for quick cell separation based on cell rolling

BIOTECHNOLOGY PROGRESS, Issue 2 2010
Atsushi Mahara
Abstract Cell separation using methodological standards that ensure high purity is a very important step in cell transplantation for regenerative medicine and for stem cell research. A separation protocol using magnetic beads has been widely used for cell separation to isolate negative and positive cells. However, not only the surface marker pattern, e.g., negative or positive, but also the density of a cell depends on its developmental stage and differentiation ability. Rapid and label-free separation procedures based on surface marker density are the focus of our interest. In this study, we have successfully developed an antiCD34 antibody-immobilized cell-rolling column, that can separate cells depending on the CD34 density of the cell surfaces. Various conditions for the cell-rolling column were optimized including graft copolymerization, and adjustment of the column tilt angle, and medium flow rate. Using CD34-positive and -negative cell lines, the cell separation potential of the column was established. We observed a difference in the rolling velocities between CD34-positive and CD34-negative cells on antibody-immobilized microfluidic device. Cell separation was achieved by tilting the surface 20 degrees and the increasing medium flow. Surface marker characteristics of the isolated cells in each fraction were analyzed using a cell-sorting system, and it was found that populations containing high density of CD34 were eluted in the delayed fractions. These results demonstrate that cells with a given surface marker density can be continuously separated using the cell rolling column. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source]

Highly avid magnetic bead capture: An efficient selection method for de novo protein engineering utilizing yeast surface display

BIOTECHNOLOGY PROGRESS, Issue 3 2009
Margaret Ackerman
Abstract Protein engineering relies on the selective capture of members of a protein library with desired properties. Yeast surface display technology routinely enables as much as million-fold improvements in binding affinity by alternating rounds of diversification and flow cytometry-based selection. However, flow cytometry is not well suited for isolating de novo binding clones from naïve libraries due to limitations in the size of the population that can be analyzed, the minimum binding affinity of clones that can be reliably captured, the amount of target antigen required, and the likelihood of capturing artifactual binders to the reagents. Here, we demonstrate a method for capturing rare clones that maintains the advantages of yeast as the expression host, while avoiding the disadvantages of FACS in isolating de novo binders from naïve libraries. The multivalency of yeast surface display is intentionally coupled with multivalent target presentation on magnetic beads,allowing isolation of extremely weak binders from billions of non-binding clones, and requiring far less target antigen for each selection, while minimizing the likelihood of isolating undesirable alternative solutions to the selective pressure. Multivalent surface selection allows 30,000-fold enrichment and almost quantitative capture of micromolar binders in a single pass using less than one microgram of target antigen. We further validate the robust nature of this selection method by isolation of de novo binders against lysozyme as well as its utility in negative selections by isolating binders to streptavidin-biotin that do not cross-react to streptavidin alone. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source]

Signalization and cytoskeleton activity through myosin IB during the early steps of phagocytosis in Entamoeba histolytica: a proteomic approach

CELLULAR MICROBIOLOGY, Issue 10 2005
Sabrina Marion
Summary Phagocytosis of human cells is a crucial activity for the virulence of the human parasite Entamoeba histolytica. This protozoan invades and destroys the intestine by killing and phagocytosing epithelial cells, erythrocytes and cells from the immune system. In this study, we used magnetic beads covered with proteins from human serum as a model system to study the early events involved in phagocytosis by E. histolytica. We validated the system showing that the beads uptake triggered the activation of the actin-myosin cytoskeleton and involved a PI3-kinase as previously described for erythrophagocytosis. We purified early phagosomes from wild-type (WT) amoeba and from parasites that overproduced myosin IB (MyoIB+), the unique unconventional myosin of E. histolytica. The MyoIB+ cells exhibit a slower and more synchronized uptake process than the WT strain. Proteomic analysis by liquid chromatography and tandem mass spectroscopy (LC-MS/MS) of the WT and MyoIB+ phagosomes allowed us to identify, for the first time, molecular actors involved in the early step of the uptake process. These include proteins involved in cytoskeleton activity, signalling, endocytosis, lytic activity and cell surface proteins. Interestingly, the proteins that we found specifically recruited on the phagosomes from the MyoIB+ strain were previously described in other eukarytotic cells, as involved in the regulation of cortical F-actin dynamics, such as ,-actinin and formins. This proteomics approach allows a step further towards the understanding of the molecular mechanisms involved in phagocytosis in E. histolytica that revealed some interesting differences compared with phagocytosis in macrophages or Dictyostelium discoideum, and allowed to identify putative candidates for proteins linked to myosin IB activity during the phagocytic process [source]

Organic,Inorganic Hybrid Polymer-Encapsulated Magnetic Nanobead Catalysts

CHEMISTRY - A EUROPEAN JOURNAL, Issue 3 2008
Takayoshi Arai Prof.
Abstract A new strategy for the encapsulation of magnetic nanobeads was developed by using the in situ self-assembly of an organic,inorganic hybrid polymer. The hybrid polymer of {[Cu(bpy)(BF4)2(H2O)2](bpy)}n (bpy=4,4,-bipyridine) was constructed on the surface of amino-functionalized magnetic beads and the resulting hybrid-polymer-encapsulated beads were utilized as catalysts for the oxidation of silyl enolates to provide the corresponding ,-hydroxy carbonyl compounds in high yield. After the completion of the reaction, the catalyst was readily recovered by magnetic separation and the recovered catalyst could be reused several times. Because the current method did not require complicated procedures for incorporating the catalyst onto the magnetic beads, the preparation and the application of various other types of organic,inorganic hybrid-polymer-coated magnetic beads could be possible. [source]