Ferritin as a label for high-gradient magnetic separation.

In three model systems, particles the size of cells or smaller have been surface labeled with ferritin to make them slightly paramagnetic, by virtue of the iron in the ferritin. In each case it was possible to show that labeled particles could be magnetically removed from a flowing suspension by the high-gradient magnetic separation (HGMS) technique. The first system of particles consisted of small (1 micron) carboxylate-modified latex spheres to which ferritin was covalently bound to create stable paramagnetic particles analogous to a ferritin-labeled subcellular membrane preparation. In the second system polyacrylamide beads that more closely approximated whole cells in size (5-50 microns) were labeled with immunoferritin. The third system was a biomembrane preparation: erythrocyte ghosts labeled with a ferritin-lectin conjugate. A field of 7 T (tesla) (70 kG) was used in each case, along with buffer flow rates through the HGMS column in the range 0.1-1.0 ml/min.     

Protein loading,elution, and resolution behavior in a novel device that integrates ultrafiltration and chromatographic separation

Hollow fiber membranes and chromatographic resin beads are commonly employed in a variety of bioseparation processes. A new class of integrated separation devices is being studied in which the shell side of a hollow fiber device is filled with adsorbents/chromatographic resin beads. Such devices and the corresponding separation methods integrate feed broth clarification by the microfiltration/ultrafiltration membrane with bioproduct purification by the shell-side resin beads either as an adsorbent or as beads in elution chromatography. A mathematical model has been developed for the prediction of the chromatographic behavior of such an integrated device. Simulations have been done to study the effects of axial dispersion, feed flow rate, water permeation rate, fiber packing density, and void fraction. Numerical solutions were obtained by solving the governing equations. This model can reasonably describe the concentration profiles as well as the breakthrough and elution behaviors in the integrated device.     

An on-chip magnetic bead separator using spiral electromagnets with semi-encapsulated permalloy.

A new planar bio-magnetic bead separator on a glass chip has been designed, fabricated and tested. The separator is composed of micromachined semi-encapsulated spiral electromagnets and fluid channels, which have been separately fabricated and then bonded. The device was tested with super-paramagnetic beads of mean diameter 1 microm which were suspended in a buffered solution. When a DC current of 300 mA was applied to the inductor, the bio-magnetic beads were successfully separated on the electromagnets, showing a functional capability as a magnetic bead separator. To evaluate separation rate and capability, the inductance measurement method has been introduced and the inductance variation according to the separation rate has been characterized. Using this separator, cells or cell fragments and magnetic beads bonded with protein or enzyme suspended in bio-buffer solutions can be successfully separated from their suspensions, envisaging a filterless bio-separator.     

Depletion effect and biomembrane budding

Depletion effects are well known to lead to phase separation in microsystems consisting of large and small particles with short-range repulsive interactions that act over macromolecular length scales. The equilibrium mechanics between an enveloped colloidal particle and a biomembrane caused by entropy is investigated by using a continuum model. We show that the favorable contact energy stems from entropy, which is sufficient to drive engulfment of the colloidal particle, and deformation of the biomembrane determines the resistance to the engulfment of the colloidal particle. The engulfment process depends on the ratio of the radii of the larger particle and smaller particles and the bending rigidity. The results show insights into the effects of depletion on biomembrane budding and nanoparticle transportation by a vesicle.     

Monosized, magnetic polymer particles: their use in separation of cells and subcellular components, and in the study of lymphocyte function in vitro

By employing the principles of "activated swelling", monosized, superparamagnetic polymer particles have been prepared ranging in size from 1-100 microns. Both during and after the swelling process, the particles can be modified to meet a series of specific demands making them potentially very interesting for many separation and assay purposes. Using monoclonal antibodies to direct the magnetic beads to their targets, immunomagnetic separation has turned out to be one of the most specific, reliable and, above all, the fastest technique available today to isolate particulate material for further studies. So far, most efforts have been concentrated on methodology for fractionation of cells in suspension, such as removal of tumour cells from bone marrow or isolation of lymphoid cells from peripheral blood. These studies have both established the parameters necessary for optimal performance and at the same time laid the groundwork for future developments making immunomagnetic separation an exciting new tool in many research areas. High speed and specificity are the most conspicuous features of immunomagnetic cell separation. These properties have been exploited in the successful development of a new technique for tissue typing of cells directly from peripheral blood specimens. Both higher sensitivity and specificity have been obtained. The same principles can be used for fast and safe quantification of cell populations and subpopulations in blood and cell suspensions. The functions of, and interactions between, peripheral blood cell populations or subpopulations in the immune response have also been studied with high precision. The significance of direct cell contact on the one hand, and soluble factors on the other, can now be established in detail. Immunomagnetic beads have also been used to study the interaction between various T lymphocyte membrane molecules in the early phases of the activation process. Finally, the usefulness of specially developed particles for the fractionation of subcellular components is described.     

Separation and enrichment of neural stem cells using segregation in an expanded bed

An expanded bed system has been developed for a novel application in which the separation and enrichment of neural stem cells from a sample containing a mixture of stem and progenitor cells is achieved based on the difference in the sizes of the aggregates of these types of cells. Inert Sephadex beads and flocculated yeast cells were used as experimental controls and references. The characteristics of the separation of neural stem cell aggregates based on size are similar to those achieved with flocculated yeast where cell-to-cell aggregation controls the pattern of size separation different from those of inert Sephadex beads.     

Influence of different magnetites on properties of magnetic Pseudomonas aeruginosa immobilizates used for biosurfactant production

During the last decades, whole‐cell immobilization has been used successfully in many bioprocesses. In particular, it is aimed at implementing continuous production processes, reaching higher production rates, and reusing the biocatalyst. In some cases, effective retention of immobilizates in the bioprocess is not feasible by membranes or sieves due to pore plugging or undesired losses of immobilizates. In the present publication, it is reported about the investigation of magnetic immobilizates of Pseudomonas aeruginosa for application in continuous biosurfactant production of rhamnolipids by foam fractionation and retention of entrained immobilizates by high‐gradient magnetic separation from foam. Different materials and methods were tested with respect to important parameters, such as stability, diffusion properties or magnetic separation. Good magnetic separation of immobilizates was achieved at 5% (w/w) magnetite loading. Best results in terms of homogeneous embedding, good diffusion properties, and stability enhancement vis‐à‐vis pure alginate beads was achieved with alginate beads with embedded Bayoxide® magnetite or MagPrep® silica particles. Although polyurethane immobilizates showed higher stabilities compared with alginate beads, rhamnolipid diffusion in immobilizates was superior in magnetic alginate beads. Regarding bead production, smaller immobilizates were achieved with suspension polymerization compared to droplet extrusion by the JetCutting® technology. In total, magnetic immobilizates are a promising tool for an easier handling of biocatalysts in a continuous biological production process, but they have to be adapted to the current production task.© 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Lectin receptor sites at the cell surface employed for affinity separation of tissue culture cells : Basic requirements as realized by lens culinaris lectin (LCL) immobilized on 2B-Sepharose

Lens culinaris lectin (LCL) immobilized on large 2B-Sepharose beads has been used to investigate the lectin-binding capacity of cell surfaces for the separation of cells by affinity chromatography. Immobilized LCL induced the tissue culture cells studied (HeLa, SV3T3) to bind to the agarose beads. This binding could be prevented by the addition of hapten sugars such as methyl-α -mannopyranoside (MαMP) and methyl-α -glucopyranoside (MαGP) according to the affinity of these sugars to LCL in a concentration-dependent manner. The cell-bead linkages were sufficiently strong to resist any appreciable mechanical breakage. The binding of the cells occurred so fast that any release could be started before the cells interacted unspecifically with the beads. Immobilized LCL released most of the cells upon addition of the competing sugar MαMP in a concentration-dependent manner under physiological conditions. The cells retained viability during the separation procedure as demonstrated by subsequent growth in culture. The difficulties so far observed with procedures involving columns have been overcome by a batch technique for controlled cell binding and release. For rapid separation of free from bound cells a gauze of defined pore size is introduced. Methodological problems such as agarose concentration, cell stickiness, lectin amount, and mechanical stability of the bead-cell complex are discussed.     

Capture of rare cells in suspension with antibody-coated polystyrene beads

A method for the separation of one cell type present in small number from a predominant mixture of cell types using macroscopic polystyrene beads is demonstrated. An antibody specific to murine leukocytes (CD45) was adsorbed to the surface of the beads. Beads and murine hybridoma B cells were placed in test tubes and periodically inverted at fixed time intervals, causing the beads to settle through the suspension under creeping flow conditions. Capture was dependent upon interception: the captured cells must have traveled along streamlines that brought them to within a cell radius of the bead surface. B cells attached to 99-micrometer beads (maximum shear rate 8.1 s-1) were captured with greater efficiency but in lesser quantity than those attached to 170-micrometer beads (maximum shear rate 13.9 s-1). Cell capture unexpectedly reached a plateau in less than 2 h, a phenomenon that appears to involve changes in both the cells and the beads. Capture of cells was effective out to dilutions of 1:10 000 with purity in the captured population of better than 74%. This method allows for the study of physical parameters important for cell attachment and capture as well as for practical separation of rare cells.     

Novel plasma-separation dilayer gellan-gellan-sulfate adsorber for direct removal of extra domain A containing fibronectin from the blood of rheumatoid arthritis patients

Rheumatoid arthritis (RA) patients, in whom cryogelation occurs in the presence of heparin, exhibit abnormally high concentrations of extra domain A containing fibronectin [EDA(+)FN] in their plasma. The selective removal of EDA(+)FN from patient blood is therefore of potential therapeutic benefit. Gellan-sulfate is a candidate ligand for the removal of EDA(+)FN due to its high affinity for FN. In this study, we prepare a novel adsorber for the direct removal of EDA(+)FN from patient blood. The adsorber has both a plasma separation function and EDA(+)FN trapping zones, and is prepared by cross-linking gellan-sulfate with epichlorohydrine. The ratio of gellan-sulfate to gellan in the adsorber is 48%. The surface and internal structure of gellan beads were observed by a range of microscopic techniques, and the beads were found to have a dilayer structure, consisting of a porous outer layer and an underlying gellan-sulfate phase as the adsorber. The affinity constants of the gellan-sulfate beads for EDA(+)FN were almost the same in blood as in buffer because the porous gellan coating acts to separate plasma from the cellular fraction of the blood. The removal rate of plasma proteins and blood cells from mock RA blood was measured for coated and uncoated gellan-sulfate beads. Removal rates were 30-32% for EDA(+)FN, 6-10% for fibrinogen, 10-14% for antithrombin III, 8% for C3, 4-7% for C4, and 0% for albumin. The removal rates of uncoated beads were 11% for white blood cells, 0% for red blood cells and 33% for platelets, whereas removal rates of 0% for white blood cells, 0% for red blood cells and 20% for platelets were achieved for coated beads. The coating effectively inhibits the adsorption of white blood cells and platelets. Existing problems with direct adsorbers, including selectivity and plasma separation, have been solved by this material.     

Isoelectric beads for proteome pre-fractionation. II: experimental evaluation in a multicompartment electrolyzer

Proteome pre-fractionation in multicompartment electrolyzers is proposed here, with substantial modifications as compared to the standard technique. First of all, the classical isoelectric, buffering membranes, delimiting each compartment and acting, in pairs, as isoelectric traps, have been replaced by isoelectric buffering beads, operating on the same principle, but allowing unhindered migration of proteins (lack of sieving properties, contrary to typical continuous membrane barriers). Secondly, the isoelectric beads are not made in the conventional manner, with ionic acrylamide derivative monomers throughout their central core, but are composed of a hard, ceramic core, coated with an amphoteric buffering polymer. This minimizes mass transfer resistance of proteins that are transiently adsorbed onto the beads. As a result, significantly reduced separation times, of the order of ca. 3 h, are required for developing steady-state patterns, as compared to the lengthy times (overnight and much longer) in conventional multicompartment electrolyzers operating with isoelectric membranes. Examples of separation of standard marker proteins, as well as entire Escherichia coli lysates and human serum proteins, are given. The obtained fractions are analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, two-dimensional gel electrophoresis and by surface enhanced laser desorption/ionization mass spectrometry.     

Peptides, antibodies, and FRET on beads in flow cytometry: A model system using fluoresceinated and biotinylated beta-endorphin.

BACKGROUND: Particulate surfaces such as beads are routinely used as platforms for molecular assembly for fundamental and practical applications in flow cytometry. Molecular assembly is transduced as the direct analysis of fluorescence, or as a result of fluorescence resonance energy transfer. Binding of fluorescent ligands to beads sometimes alters their emission yield relative to the unbound ligands. Characterizing the physical basis of factors that regulate the fluorescence yield of bound fluorophores (on beads) is a necessary step toward their rational use as mediators of numerous fluorescence based applications. METHODS: We have examined the binding between two biotinylated and fluoresceinated beta-endorphin peptides and commercial streptavidin beads using flow cytometric analysis. We have analyzed the assembly between a specific monoclonal antibody and an endorphin peptide in solution using resonance energy transfer and compared the results on beads in flow cytometry using steady-state and time-resolved fluorescence. RESULTS: We have defined conditions for binding biotinylated and fluoresceinated endorphin peptides to beads. These measurements suggest that the peptide structure can influence both the intensity of fluorescence and the mode of peptide binding on the bead surface. We have defined conditions for binding antibody to the bead using biotinylated protein A. We compared and contrasted the interactions between the fluoresceinated endorphin peptide and the rhodamine- labeled antibody. In solution we measure a K(d) of <38 nM by resonance energy transfer and on beads 22 nM. DISCUSSION: Some issues important to the modular assembly of a fluorescence resonance energy transfer (FRET) based sensing scheme have been resolved. The affinity of peptides used herein is a function of their solubility in water, and the emission intensity of the bound species depends on the separation distance between the fluorescein and the biotin moiety. This is due to the quasi-specific quenching interaction between the fluorescein and a proximal binding pocket of streptavidin. Detection of antibodies in solution and on beads either by FRET or capture of fluorescent ligands by dark antibodies subsequently enables the determination of K(d) values, which indicate agreement between solution and flow cytometric determinations.     

Preparation of trypsin free chymotrypsin

Summary Chitosan/chitosan-magnetite beads were prepared and binding of different enzymes were done on these beads. The difference in extent of binding of trypsin and chymotrypsin to chitosan-magnetite beads is used for the separation of these enzymes.     

Microsystem for the single molecule analysis of membrane transport proteins

Micro-chamber arrays enable highly sensitive and quantitative bioassays at the single-molecule level. Accordingly, they are widely used for ultra-sensitive biomedical applications, e.g., digital PCR and digital ELISA. However, the versatility of micro-chambers is generally limited to reactions in aqueous solutions, although various functions of membrane proteins are extremely important. To address this issue, microsystems using arrayed micro-sized chambers sealed with lipid bilayers, referred to here as a “biomembrane microsystems”, have been developed by many research groups for the analysis of membrane proteins. In this review, I would like to introduce recent progress on the single molecule analysis of membrane transport proteins using a biomembrane microsystem, and discuss the future prospects for its use in analytical and pharmacological applications.     

Chiral-recognition chromatography of β-blockers on continuous polymer beds with immobilized cellulase as enantioselective protein

Columns prepared by coupling cellulase as a chiral selector to silica beads are very efficient for the separation of enantiomers. In this paper we show that continuous polymer beds compete favorably with silica beads as chromatographic supports for such separations. The chiral stationary phase is prepared either by entrapment in and simultaneous covalent linkage of ally1 cellulase to the continuous beds during their preparation or by covalent immobilization of cellulase on an epoxy-activated continuous bed. Enantiomers of β-blockers were separated rapidly and with high resolution. The enantiomers of practolol were thus baseline resolved within 45 sec. The recognition center–or at least part of it—coincides with the active center of the enzyme, since the enantiomers could not be separated in the presence of the competitive enzyme inhibitors cellobiose and D-glucose and the separation was also impaired upon addition of the substrate carboxymethyl cellulose to the eluent. Similar observations have been reported for silica columns derivatized with cellulase. The capacity factor and the separation selectivity could be tuned by the pH and the concentration of the mobile phase, a phosphate buffer. No modifier was required, as is sometimes the case with silica-based supports. The continuous beds give faster enantiomer separations than do columns of silica and are more pH-stable and cost effective to prepare. © 1993 Wiley-Liss, Inc.     

Solubilization and hydrophobic immobilization assay of a cAMP binding protein from Dictyostelium discoideum plasma membranes

A cAMP binding site present on isolated plasma membranes of aggregation-competent $\text{D.}\text{discoideum}$ cells has been solubilized with the nonionic detergent Emulphogene BC-720. An assay has been developed based on the principle of hydrophobic chromatography, in which the detergent solubilized cAMP binding protein is immobilized on alkyl-agarose beads at low detergent concentration. This allows the necessary rapid separation of bound and free [³H]-cAMP by filtration of the beads. The kinetics and nucleotide specificity of the detergent solubilized cAMP binding protein are comparable to those of the cAMP chemotactic receptor on intact cells and plasma membranes. The alkyl-agarose bead assay may have general utility for the assay of detergent solubilized membrane receptors.     

Removing human immunodeficiency virus (HIV) from human blood using immobilized heparin

Heparin covalently attached to a water-insoluble resin suspended in HIV-infected aqueous buffer or whole blood captures the virus; subsequent physical separation of the immobilized heparin reduced the viral titers by over 80 and 50%, respectively. The detoxification concept has been validated by both circulating an HIV-1 solution through a column packed with the heparin–sepharose beads and successively mixing an HIV-1 solution with fresh beads.     

Incomplete dialysis of protein samples containing 3-[(3-chlolamidopropyl)dimethylammonio]-1-propanesulfonate may lead to erroneous estimation of histidine content on amino acid analysis

The zwitterionic detergents Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and its 2-hydroxyl derivative Chapso have been widely used in the biomembrane field as well as other fields for their nondenaturing character. Due to their structure containing an amide bond in the molecules center they are prone to hydrolysis with 6 N HCl into cholic acid and 3-aminopropyldimethylammonio-1-propanesulfonate (APS) and its 2-hydroxyl derivative (APSO), respectively. On amino acid analyses with the ninhydrin detection system, His, APS, and APSO were eluted at 24.42, 24.55, and 24.85 min, respectively, and APS was identified as His due to their close retention times. Moreover, the mixture of His and APS coeluted as a single peak at 24.51 min leading to an erroneous His content for Chaps-contaminated protein samples. For full separation of APS, APSO, and His the elution program was improved by an additional 5-min elution with Buffer 7, i.e., 1:1 mixture of Buffers 3 and 4, before elution with Buffer 4, enabling complete separation of the 18 amino acids, APS, and APSO in a single run.     

Development of a 3D‐printed single‐use separation chamber for use in mRNA‐based vaccine production with magnetic microparticles

Laboratory protocols using magnetic beads have gained importance in the purification of mRNA for vaccines. Here, the produced mRNA hybridizes specifically to oligo(dT)‐functionalized magnetic beads after cell lysis. The mRNA‐loaded magnetic beads can be selectively separated using a magnet. Subsequently, impurities are removed by washing steps and the mRNA is eluted. Magnetic separation is utilized in each step, using different buffers such as the lysis/binding buffer. To reduce the time required for purification of larger amounts of mRNA vaccine for clinical trials, high‐gradient magnetic separation (HGMS) is suitable. Thereby, magnetic beads are selectively retained in a flow‐through separation chamber. To meet the requirements of biopharmaceutical production, a disposable HGMS separation chamber with a certified material (United States Pharmacopeia Class VI) was developed which can be manufactured using 3D printing. Due to the special design, the filter matrix itself is not in contact with the product. The separation chamber was tested with suspensions of oligo(dT)‐functionalized Dynabeads MyOne loaded with synthetic mRNA. At a concentration of c_B = 1.6–2.1 g·L^–1 in lysis/binding buffer, these 1 μm magnetic particles are retained to more than 99.39% at volumetric flows of up to 150 mL·min^–1 with the developed SU‐HGMS separation chamber. When using the separation chamber with volumetric flow rates below 50 mL·min^–1, the retained particle mass is even more than 99.99%.     

MagSNiPer: a new single nucleotide polymorphism typing method based on single base extension, magnetic separation, and chemiluminescence

We have developed a new method for typing single nucleotide polymorphisms (SNPs), MagSNiPer, based on single base extension, magnetic separation, and chemiluminescence. Single base nucleotide extension reaction is performed with a biotinylated primer whose 3' terminus is contiguous to the SNP site with a tag-labeled ddNTP. Then the primers are captured by magnetic-coated beads with streptavidin, and unincorporated labeled ddNTP is removed by magnetic separation. The magnetic beads are incubated with anti-tag antibody conjugated with alkaline phosphatase. After the removal of excess conjugates by magnetic separation, SNP typing is performed by measuring chemiluminescence. The incorporation of labeled ddNTP is monitored by chemiluminescence induced by alkaline phosphatase. MagSNiPer is a simple and robust SNP typing method with a wide dynamic range and high sensitivity. Using MagSNiPer, we could perform SNP typing with as little as 10(-17) mol of template DNA.