Carboxylate-modified Latex Research Articles

Transport Behavior of Selected Nanoparticles with different Surface Coatings in Granular Porous Media coated with Pseudomonas aeruginosa Biofilm

Well-controlled laboratory column experiments were conducted to understand the influence of Pseudomonas aeruginosa (P. aeruginosa) biofilms on the transport of selected engineered nanoparticles (ENPs) in granular porous media representative of groundwater aquifers or riverbank filtration settings. To understand the importance of particle size on retention in the biofilm-coated granular (quartz sand) matrix, column experiments were carried out using nanosized (20 nm) and micrometer-sized (1 μm) sulfate-functionalized polystyrene latex particles (designated as 20 nSL and 1 mSL, respectively). Additional experiments conducted with nanosized (20 nm) carboxyl-modified latex particles (20nCL) and carboxyl-modified CdSe/ZnS quantum dots (QDs) provide information on the influence of particle surface chemistry on retention. Biofilm grown on the surface of the sand was characterized by total biomass quantification, confocal laser scanning microscopy (CLSM), and electrokinetic analysis. All four particles exhibit increased retention in the biofilm-coated packed bed: e.g., the attachment efficiency (α) of the 1 mSL particle increases from 0.40 to 1.7, whereas α for the 20 nSL particle increases from 0.04 to 0.10 in the biofilm-coated system. Particle surface chemistry can also influence the affinity of the ENPs for the biofilm coating as revealed by the greater attachment of the 20 nSL particle onto the biofilm-coated sand (α = 0.10) than its carboxylated counterpart (α = 0.04). Column experiments conducted using sand coated with growth medium (LB) or extracellular polymeric substances (EPS) extracted from P. aeruginosa biofilms further reveal that particle surface chemistry influences the interaction between the different ENPs and these coated sand surfaces. Namely, coating of sand surfaces with LB medium or bacterial EPS does not affect the transport of the sulfonated nanoparticle, but the LB coating leads to decreased retention of the carboxylated latex nanoparticle. Furthermore, our results show that EPS coatings are not necessarily good surrogates for biofilm-coated sand. Electrokinetic characterization of the clean and coated sand surfaces also reveals that the extent of particle retention is not controlled by electrical double layer interactions. Future studies should thus be aimed at improving our understanding of the fundamental mechanisms (both colloidal and noncolloidal) governing nanoparticle transport and fate in biofilm-laden granular aquatic environments.

Surface Properties and Reduced Biofouling of Graft-Copolymers That Possess Oppositely Charged Groups

Microbial biofilms and their components present a major obstacle for ensuring the long-term effectiveness of membrane processes. Graft polymerization on membrane surfaces, in general, and grafting with oppositely charged monomers, have been shown to reduce biofouling significantly. In this study, surface forces and macromolecular properties of graft copolymers that possess oppositely charged groups were related to their potent antibiofouling behavior. Graft polymerization was performed using the negatively charged 3-sulphopropyl methacrylate (SPM) and positively charged [2-(methacryloyloxy)ethyl]-trimethylammonium (MOETMA) monomers to yield a copolymer layer on polyvinylidene fluoride (PVDF) surface. Quartz crystal microbalance with dissipation monitoring (QCM-D) technology was used to monitor the reduced adsorption of extracellular polymeric substances (EPS) extracted from a membrane bioreactor (MBR) wastewater treatment facility. Complemented measurements of attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy provided evaluation of the antifouling properties of the surface. Increase in water content in grafted layer exposed to 100 mM aqueous NaCl solution was observed by QCM-D. Therefore, the grafted copolymer layer is swelled in the presence of 100 mM NaCl because of reversing of polymer self-association by counterions. Force measurements by atomic force microscopy (AFM) showed an increased repulsion between a carboxylate-modified latex (CML) particle probe and a modified PVDF surface, especially in the presence of 100 mM NaCl. The hydration and swelling of the grafted polymer layer are shown to repel EPS and reduce their adsorption. Delineating the surface properties of antifouling grafted layers may lead to the design of novel antifouling surfaces.

Identification of Interacting Hot Spots in the β3 Integrin Stalk Using Comprehensive Interface Design

Protein-protein interfaces are usually large and complementary surfaces, but specific side chains, representing energetic "hot spots," often contribute disproportionately to binding free energy. We used a computational method, comprehensive interface design, to identify hot spots in the interface between the stalk regions of the β3 and the complementary αIIb and αv integrin subunits. Using the Rosetta alanine-scanning and design algorithms to predict destabilizing, stabilizing, and neutral mutations in the β3 region extending from residues Lys(532) through Gly(690), we predicted eight alanine mutations that would destabilize the αIIbβ3 interface as well as nine predicted to destabilize the αvβ3 interface, by at least 0.3 kcal/mol. The mutations were widely and unevenly distributed, with four between residues 552 and 563 and five between 590 and 610, but none between 565 and 589, and 611 and 655. Further, mutations destabilizing the αvβ3 and αIIbβ3 interfaces were not identical. The predictions were then tested by introducing selected mutations into the full-length integrins expressed in Chinese hamster ovary cells. Five mutations predicted to destabilize αIIb and β3 caused fibrinogen binding to αIIbβ3, whereas three of four predicted to be neutral or stabilizing did not. Conversely, a mutation predicted to destabilize αvβ3, but not αIIbβ3 (D552A), caused osteopontin binding to αvβ3, but not fibrinogen binding to αIIbβ3. These results indicate that stability of the distal stalk interface is involved in constraining integrins in stable, inactive conformations. Further, they demonstrate the ability of comprehensive interface design to identify functionally significant integrin mutations.

Effect of Particle Shape on Colloid Retention and Release in Saturated Porous Media

Colloidal particles of environmental concern often have nonspherical shapes. However, theories and models such as the classical filtration theory have been developed based on the behavior of spherical particles. This study examined the effect of particle shape on colloid retention (e.g., attachment and straining) and release in saturated porous media. Two- and three-step transport experiments were conducted in water-saturated glass bead columns using colloids dispersed in deionized water and an electrolyte solution. The particles used in the experiments were carboxylate-modified latex colloids of spherical (500 nm diam.) and rod (aspect ratio, 7.0) shapes. The rod-like particles were prepared by stretching the spherical particles. Analysis of the colloid breakthrough curves indicates that particle shape affected transport behavior, but retention did not increase with increasing aspect ratio. Retention of the spherical particles occurred mainly in the secondary energy minimum, whereas retention of rod-like particles occurred in primary and secondary energy minima. There was less straining of rod-like particles compared with spherical ones, indicating that the minor axis was the critical dimension controlling the process. Release of spherical particles on elution was instantaneous, whereas release of rod-like particles was rate limited, giving rise to long tails, implying an orientation effect for rod-like colloids. The results suggest that the differences in electrostatic properties and shape contributed to the observed different retention and release behaviors of the two colloids.

Niemann-Pick C1 Functions Independently of Niemann-Pick C2 in the Initial Stage of Retrograde Transport of Membrane-impermeable Lysosomal Cargo

The rare neurodegenerative disease Niemann-Pick Type C (NPC) results from mutations in either NPC1 or NPC2, which are membrane-bound and soluble lysosomal proteins, respectively. Previous studies have shown that mutations in either protein result in biochemically indistinguishable phenotypes, most notably the hyper-accumulation of cholesterol and other cargo in lysosomes. We comparatively evaluated the kinetics of [(3)H]dextran release from lysosomes of wild type, NPC1, NPC2, and NPC1/NPC2 pseudo-double mutant cells and found significant differences between all cell types examined. Specifically, NPC1 or NPC2 mutant fibroblasts treated with NPC1 or NPC2 siRNA (to create NPC1/NPC2 pseudo-double mutants) secreted dextran less efficiently than did either NPC1 or NPC2 single mutant cell lines, suggesting that the two proteins may work independently of one another in the egress of membrane-impermeable lysosomal cargo. To investigate the basis for these differences, we examined the role of NPC1 and NPC2 in the retrograde fusion of lysosomes with late endosomes to create so-called hybrid organelles, which is believed to be the initial step in the egress of cargo from lysosomes. We show here that cells with mutated NPC1 have significantly reduced rates of late endosome/lysosome fusion relative to wild type cells, whereas cells with mutations in NPC2 have rates that are similar to those observed in wild type cells. Instead of being involved in hybrid organelle formation, we show that NPC2 is required for efficient membrane fission events from nascent hybrid organelles, which is thought to be required for the reformation of lysosomes and the release of lysosomal cargo-containing membrane vesicles. Collectively, these results suggest that NPC1 and NPC2 can function independently of one another in the egress of certain membrane-impermeable lysosomal cargo.

Cellular Internalization of Exosomes Occurs Through Phagocytosis

Exosomes play important roles in many physiological and pathological processes. However, the exosome-cell interaction mode and the intracellular trafficking pathway of exosomes in their recipient cells remain unclear. Here, we report that exosomes derived from K562 or MT4 cells are internalized more efficiently by phagocytes than by non-phagocytic cells. Most exosomes were observed attached to the plasma membrane of non-phagocytic cells, while in phagocytic cells these exosomes were found to enter via phagocytosis. Specifically, they moved to phagosomes together with phagocytic polystyrene carboxylate-modified latex beads (biospheres) and were further sorted into phagolysosomes. Moreover, exosome internalization was dependent on the actin cytoskeleton and phosphatidylinositol 3-kinase, and could be inhibited by the knockdown of dynamin2 or overexpression of a dominant-negative form of dynamin2. Further, antibody pretreatment assays demonstrated that tim4 but not tim1 was involved in exosomes uptake. We also found that exosomes did not enter the internalization pathway involving caveolae, macropinocytosis and clathrin-coated vesicles. Our observation that the cellular uptake of exosomes occurs through phagocytosis has important implications for exosome-cell interactions and the exosome intracellular trafficking pathway.

Coupled Factors Influencing Concentration-Dependent Colloid Transport and Retention in Saturated Porous Media

The coupled influence of input suspension concentration (Ci), ionic strength (IS), and hydrodynamics on the transport and retention of 1.1 microm carboxyl-modified latex colloids in saturated quartz sand (150 microm) under unfavorable attachment conditions (pH 10) was investigated. The percentage of retained colloids in column experiments decreased with Ci at intermediate IS conditions (31 or 56 mM) when colloids were weakly associated with the solid phase by a shallow secondary energy minima. In contrast, the effects of Ci on colloid retention were absent when IS was too low (6 mM) or too high (106 mM). The concentration effects under intermediate IS conditions were dependent on the system hydrodynamics, magnitude of Ci, and injection order of Ci, but they were largely independent of the input colloid mass. These observations were explained in part by time- and concentration-dependent filling of retention sites. Only a small fraction of the solid surface area was found to contribute to retention when IS was 31 mM, and micromodel observations indicated that colloid retention was enhanced in lower velocity regions of the pore space that occurred near grain-grain contacts. Consequently, retention profiles for IS = 31 mM conditions were increasingly nonexponential at lower values of Ci (during filling), whereas the observed concentration effect was largely eliminated as retention locations became filled. In addition, micromodel observations indicated that liquid and solid phase mass transfer of colloids to retention locations was influenced by Ci under intermediate IS conditions. Higher values of Ci are expected to produce less relative mass transfer to retention locations due to increased numbers of collisions that knock weakly associated colloids off the solid phase. Hence, the concentration effects were found to be largely independent of input colloid mass during filling of retention sites.

Label-free and quantitative analysis of C-reactive protein in human sera by tagged-internal standard assay on antibody arrays

We have developed a new, high-throughput, competition-based tagged-internal standard (TIS) assay to measure the levels of blood proteins in human serum. In this assay, target proteins in the sample serum compete with tagged-internal standard proteins for binding to an antibody array. Antibody arrays are fabricated by immobilizing a target protein-specific antibody on the carboxylate-modified latex bead surface of well-type arrays. A solution of Alexa 546-conjugated target protein is added to a sample of human serum and applied to the well-type antibody array. The array is then analyzed with a fluorescence scanner and the level of unlabeled target protein in the human sera is inferred from the amount of tagged protein bound to the array. We successfully applied this assay to measure the level of C-reactive protein (CRP) in 92 unlabeled human sera. The TIS assay was found to be specific and reproducible for the quantitative analysis of CRP. The antibody array data from the TIS assay correlate well with clinical laboratory data obtained using the commercialized latex-enhanced turbidimetry immunoassay ( n = 3, r = 0.967, CV = 0.32%). Thus, the antibody array-based TIS assay system is high-throughput, quantitative, and label-free and may be useful in the rapid serodiagnosis of human disease.

High-Resolution Measurement of Pore Saturation and Colloid Removal Efficiency in Quartz Sand Using Fluorescence Imaging

Colloid deposition in unsaturated, nonuniform porous media is poorly explained by current models and difficult to measure using breakthrough curves and retained mass profiles. We present new methods which enable time-lapse fluorescence imaging to quantify variations in pore saturation, theta, and colloid deposition in 2D, nonuniform unsaturated flow fields. Calibration experiments revealed direct proportionality between fluorescence F and theta in 20/30 mesh quartz sand. Analysis of breakthrough data in fluorescence images allows quantification of the mean mobile concentration, mean deposition rate, and hence the colloid removal efficiency eta directly from data at the pixel-scale throughoutthe flow field. We imaged carboxylate-modified latex microspheres from a point source in saturated flow and unsaturated flow across a capillary fringe at 10(-3), 10(-2), and 10(-1) M NaCl. Total numbers of colloids deposited and values of eta increased with ionic strength. We modeled the observed variations in eta with theta to estimate the partitioning of colloid deposition between air-water and solid-water interfaces. In the broad saturation range 0.2 < theta < 1, our results suggest that only at the lowest ionic strength, where deposition at solid-water interfaces was strongly unfavorable, did colloid deposition associated with air-water interfaces significantly influence the total colloid removal.

Observed and Simulated Fluid Drag Effects on Colloid Deposition in the Presence of an Energy Barrier in an Impinging Jet System

The deposition and re-entrainment behaviors of carboxylate-modified latex microspheres (0.2-, 0.5-, 1.0-, and 2.0-microm diameter) were examined in an impinging jet system on both glass and quartz substrata under a variety of environmentally relevant fluid velocities (2.12 x 10(-4) to 1.06 x 10(-3) m sec(-1)) in both the absence and the presence of an energy barrier to deposition. In the absence of an energy barrierto deposition, deposition fluxes onto glass and quartz substrata increased with increasing fluid velocity for all four microsphere sizes, in accordance with expectations from theory. In contrast, in the presence of an energy barrier to deposition, deposition efficiencies onto glass and quartz substrata decreased with increasing fluid velocity for all four microsphere sizes. Lack of re-entrainment and observed strong attachment were consistent with the expectation that deposition occurs via the primary energy minima where nanoscale surface heterogeneity locally reduces or eliminates the energy barrier to deposition. Colloid deposition onto an overall opposite-charged surface was simulated using a particle transport model with randomly distributed hetero domains that were like-charged relative to the colloid. Varying the size and number of the hetero domains showed that simulated colloid deposition efficiencies decreased with increasing fluid velocity when the hetero domains were small relative to the colloid. The simulations thereby demonstrate that observed decreases in colloid deposition efficiencies with increasing fluid velocity are consistent with the hypothesis that colloid deposition onto overall like-charged surfaces occurs at nanoscale hetero domains where repulsion is locally reduced or eliminated.

Direct force measurements between carboxylate-modified latex microspheres and glass using atomic force microscopy

Depths of colloid-surface interaction energy minima have been sometimes utilized for estimation of the force holding a colloid to the surface upon contact. Since this approach assumes that non-contact forces prevail following attachment, a comparison of this approach to direct measurement via atomic force microscopy is warranted. Interaction and adhesion forces between 1.0-μm diameter carboxylate-modified polystyrene latex microspheres and a glass surface were measured directly with an atomic force microscope using the colloidal probe technique. Measurements were conducted as a function of ionic strength in NaCl with and without added MOPS (3-( N-morpholino)-propanesulfonic acid) buffer, at pH 6.8–6.9. Theoretical DLVO force curves were fit to the AFM approach curves by varying the surface potential of the microspheres. The depths of the primary minima of the theoretical DLVO curves fitted to AFM approach curves, were used to estimate adhesion forces according to previously published approaches, and were compared to the pull-off forces measured by AFM. Pull-off forces measured by AFM in both electrolytes were consistently a factor of about 20–30 lower than the pull-off forces estimated from theoretical adhesion forces obtained from DLVO curves. AFM-measured pull-off forces decreased with increasing the ionic strength in both electrolytes, whereas the adhesion forces calculated from DLVO showed either no change or a slight increase with increasing the ionic strength. Possible reasons for these discrepancies include roughness on one or both surfaces, which would presumably reduce the adhesion force via reduced contact area and presence of hydration forces that could reduce adhesion via strong short-range repulsion in the neighborhood of the contact points. These observations demonstrate that DLVO-based approach for determining adhesion force overestimates actual adhesion force, likely because a DLVO-based approach neglects interactions that manifest at very close separation distances and upon contact.

Moving-Wall-Driven Flows in Nanofluidic Systems

We describe fluidic control in lipid nanotubes 50−150 nm in radius, conjugated with surface-immobilized unilamellar lipid bilayer vesicles (∼5−25 μm in diameter). Transport in nanotubes was induced by continuously increasing the surface tension of one of the conjugated vesicles, for example, by ellipsoidal shape deformation using a pair of carbon microfibers controlled by micromanipulators as tweezers. The shape deformation resulted in a flow of membrane lipids toward the vesicle with the higher membrane tension; this lipid flow in turn moved the liquid column inside the nanotube through viscous coupling. Thus, micrometer-sized vesicles are used as a handle for controlling fluid flow inside nanometer-sized channels. We show transport and trapping of a single 30-nm-diameter carboxylate-modified latex particle inside a ∼100-nm-radius nanotube. Fluidic control in nanometer-sized channels using a moving wall provides pluglike liquid flows, offers a means for efficient routing and trapping of small molecules, ...

Factors affecting membrane fouling reduction by surface modification and backpulsing

Several factors affecting microfiltration membrane fouling and cleaning, including backpulsing, crossflushing, backwashing, particle size, membrane surface chemistry, and ionic strength, were investigated with suspensions of latex beads. Approximately two-fold permeate volume enhancements over 1 h of filtration were obtained by using water or gas backpulsing, and 50% enhancement was obtained with crossflushing, for filtration of 1.0 μm diameter carboxylate modified latex (CML) particles using unmodified polypropylene (PP) membranes of 0.3 μm nominal pore diameter. When 0.2 μm diameter CML particles or mixtures of 1.0 and 0.2 μm CML particles were used, however, the average flux decreased 60% compared with using 1.0 μm CML particles for experiments with or without backpulsing. PP membranes were rendered hydrophilic with neutral or positively on negatively charged surfaces by grafting monomers of poly(ethylene glycol 200) monomethacrylate (PEG200MA), dimethyl aminoethyl methacrylate (DMAEMA), or acrylic acid (AA), respectively, to the base PP membranes. Filtration experiments show that fouling is not strongly dependent on membrane surface chemistry for filtration of 1.0 μm CML particles without backpulsing. With backpulsing, however, a 10% increase and a 20% decrease of permeate volumes collected in 1 h were observed when the CML particles and the membranes had like charges and opposite charges, respectively, compared to the permeate collected with the unmodified membrane. Using the PP membranes modified with AA, permeate volumes with backpulsing decreased 30 and 40% when NaCl concentrations of 0.01 and 0.1 M, respectively, were added to the feed. However, the permeate volumes did not vary significantly with changing ionic strength for filtration without backpulsing.

The modeling of excimer laser particle removal from hydrophilic silicon surfaces

We summarize experimental results on the successful removal of submicron-sized polystyrene latex, carboxylate-modified latex, SiO2, and Al2O3 particles from hydrophilic silicon surfaces by excimer laser, using both dry and steam cleaning; the cleaning and damage thresholds have also been determined for these particles. Adhesion and removal models for an ideal sphere particle, that include van der Waals forces, hydrogen bonding, and thermoelastic effects, theoretically explain the laser cleaning results. Two models of the removal force due to the explosive evaporation of liquid film have been calculated and compared. The effects of both asperities on the particle surface and particle aggregation have also been considered. The results of the calculations show that even those surface asperities which are small compared with the particle dimension can cause a large reduction in both adhesion and thermoelastic removal forces. The theoretical predictions are consistent with the experimental observations.

Transport and Attenuation of Carboxylate‐Modified Latex Microspheres in Fractured Rock Laboratory and Field Tracer Tests

AbstractUnderstanding colloid transport in ground water is essential to assessing the migration of colloid‐size contaminants, the facilitation of dissolved contaminant transport by colloids, in situ bioremediation, and the health risks of pathogen contamination in drinking water wells. Much has been learned through laboratory and field‐scale colloid tracer tests, but progress has been hampered by a lack of consistent tracer testing methodology at different scales and fluid velocities. This paper presents laboratory and field tracer tests in fractured rock that use the same type of colloid tracer over an almost three orders‐of‐magnitude range in scale and fluid velocity. Fluorescently‐dyed carboxylate‐modified latex (CML) microspheres (0.19 to 0.98 μm diameter) were used as tracers in (1) a naturally fractured tuff sample, (2) a large block of naturally fractured granite, (3) a fractured granite field site, and (4) another fractured granite/schist field site. In all cases, the mean transport time of the microspheres was shorter than the solutes, regardless of detection limit. In all but the smallest scale test, only a fraction of the injected microsphere mass was recovered, with the smaller microspheres being recovered to a greater extent than the larger microspheres. Using existing theory, we hypothesize that the observed microsphere early arrival was due to volume exclusion and attenuation was due to aggregation and/or settling during transport. In most tests, microspheres were detected using flow cytometry, which proved to be an excellent method of analysis. CML microspheres appear to be useful tracers for fractured rock in forced gradient and short‐term natural gradient tests, but longer residence times may result in small microsphere recoveries.

Impact of Ionic Strength on Colloid Mobility in Saturated And Unsaturated Porous Media

AbstractA model system consisting of well-characterized quartz sand, negatively charged carboxyl-modified latex microspheres, and synthetic J- 13 groundwater was used as a first-step in defining limiting conditions for colloid transport at the proposed Yucca Mountain Repository for radioactive waste. Colloid (280-nm diameter) and tracer (bromide) transport was determined using column methods under steady-state flow conditions for both saturated and unsaturated moisture conditions. Experiments were conducted at three water contents (13%, 70%, and 100% saturated) and four ionic strengths (deionized water, and 0.1 x, 1x, and 10x the ionic strength of J-13 groundwater, which has an ionic strength of 0.0116). Colloid mobility in the 13% saturated columns was appreciably less than in the 70% and 100% saturated columns, whereas colloid mobility in the 70% and 100% saturated columns were quite similar. Ionic strength had a significant impact on colloid retention, becoming more pronounced in the drier systems. Colloids were mobile in deionized water and the 0.1x J-13 groundwater. Retention was approximately 20% in lx J-13 groundwater and 100% in the 10x J-13 groundwater. Compared with the 70% and 100% saturated conditions, colloid mobility decreased for the 10% saturated condition, with greater than 50% retention for the 1x J-13 groundwater system. As observed for the 70% and 100% saturated conditions, colloids in the 10x J-13 solution were essentially immobile. Colloid mobility is greatly affected by the ionic strength of the soil solution and this effect is moisture saturation dependent.

Kinetics and locus of failure of receptor-ligand-mediated adhesion between latex spheres. II. Protein-protein bond

In an extension of the previous paper, we describe the force dependence of break-up of doublets of latex spheres cross-linked by protein G-IgG bonds via the Fc region of the antibody. The receptor, the monoclonal Bear-1 antibody, was either covalently linked to 4.75-microns aldehyde/sulfate (A/S) latex spheres in a one-step reaction, or physically adsorbed to the 4.63-microns carboxyl-modified latex spheres used in Part I of this paper. The spheres were suspended in 19% buffered Dextran 40 containing the ligand, the bivalent recombinant protein G (Gamma-Bind G), and observed in the counter-rotating cone and plate Rheoscope. Break-up of doublets, tracked individually under the microscope, as well as in populations of 50-150 particles, was studied over a range of normal force from 20 to 260 pN. In individual particle studies, the fraction of doublets of spheres with covalently linked IgG breaking up in the first 10 rotations, increased from 16% in the low-force to 63% in the high-force range. In population studies, the fraction broken up increased with duration and magnitude of the applied force, and decreased with increasing ligand concentration. Moreover, doublets of physically adsorbed IgG spheres required significantly lower force than doublets of covalently linked IgG spheres for the same degree of break-up, possibly because of surface detachment of IgG molecules rather than rupture of receptor-ligand bonds. Computer simulation, using the Bell stochastic model of break-up and a Poisson distribution for the number of bonds, described in Part I, showed that the parameters of the protein-protein bond differed significantly from those of the carbohydrate-protein bond studied in Part I of this paper, the former being much more responsive to force than the latter.

Preparation, purification and analysis of BSA-coated latex particles for in vivo studies: Non-ionic adsorbance of unbound protein ligand

A procedure for the preparation, purification and analysis of small, BSA-coated carboxylate-modified latex (CML) particles, suitable for use in in vivo studies has been developed. Following conjugation, uptake of unbound BSA by Amberlite XAD-8 non-ionic adsorbent beads has been shown to be an effective method by which unbound protein ligands may be removed from coated latex preparations.

Uptake and disposal of BSA-coated latex particles by the rat mesangium: reaction with subsequently administered heterologous antiserum.

Mesangial uptake and disposal of antigen-coated latex particles and the ability of subsequently injected antibody to maintain complexed antigen in the rat mesangium has been investigated. Carboxylate-modified latex particles, coated with bovine albumin (BSA) were injected i.v. to 36 Wistar rats. Twenty-two rats (group 1) were not treated further. Fourteen rats (group 2) received rabbit anti-BSA antiserum i.v. and i.p. 24 h later. Control groups were injected with uncoated, unmodified latex particles or soluble BSA with and without subsequent antibody administration. Latex was present in the mesangial matrix of rats in group 1 at 1 h in association with a diffuse mesangial distribution of BSA. At 24 h, BSA staining was markedly reduced and extracellular latex was no longer observed. Intracellular latex aggregates were present in experimental and control groups at 24 h-14 days in cytoplasmic vacuoles of hypertrophic mesangium which showed minor infiltration by macrophage-like cells. Progressive removal of latex aggregates coincided with declining mesangial reactivity. Rapid disappearance of antigen apparently results from local degradation of tracer in the mesangium. Antibody administration preserves BSA in the mesangium due to immune complex formation and is associated with retention of ingested latex by mesangial cells. However, efficient disposal of glomerular immune deposits by the mesangium appears to minimize infiltration by monocytes and prevents aggravation of glomerular inflammation.

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.