A novel magnetic adsorbent for immunoglobulin-g purification in a magnetically stabilized fluidized bed

A novel magnetic poly(ethylene glycol dimethacrylate-N-methacryloly-L-histidinemethylester) [m-poly(EGDMA-(MAH)] support was prepared for purification of immunoglobulin G (IgG) in a magnetically stabilized fluidized bed by suspension polymerization. Elemental analysis of the magnetic beads for nitrogen was estimated as 70 micromol MAH/g polymer. Magnetic poly(EGDMA-MAH) beads were used in the separation of immunoglobulin-G (IgG) from aqueous solutions and/or human plasma in a magnetically stabilized fluidized bed system. IgG adsorption capacity of the beads decreased with an increase in the flow rate. The maximum IgG adsorption was observed at pH 6.0 for MES buffer. IgG adsorption onto the m-poly(EGDMA) was negligible. Higher adsorption values (up to 262 mg/g) were obtained in which the m-poly(EGDMA-MAH) sorbents were used from aqueous solutions. Higher amounts of IgG were adsorbed from human plasma (up to 320 mg/g) with a purity of 87%. IgG molecules could be repeatedly adsorbed and desorbed with these sorbents without noticeable loss in their IgG adsorption capacity.     

Amphiphilic core-shell nanoparticles with poly(ethylenimine) shells as potential gene delivery carriers

Spherical, well-defined core-shell nanoparticles that consist of poly(methyl methacrylate) (PMMA) cores and branched poly(ethylenimine) shells (PEI) were synthesized via a graft copolymerization of methyl methacrylate from branched PEI induced by a small amount of tert-butyl hydroperoxide. The PMMA-PEI core-shell nanoparticles were between 130 to170 nm in diameter and displayed zeta-potentials near +40 mV at pH 7 in 1 mM aqueous NaCl. Plasmid DNA (pDNA) was mixed with nanoparticles and formed complexes of approximately 120 nm in diameter and was highly monodispersed. The complexes were characterized with respect to their particle size, zeta-potential, surface morphology, and DNA integrity. The complexing ability of the nanoparticles was strongly dependent on the molecular weight of the PEI and the thickness of the PEI shells. The stability of the complexes was influenced by the loading ratio of the pDNA and the nanoparticles. The condensed pDNA in the complexes was significantly protected from enzymatic degradation by DNase I. Cytotoxity studies using MTT colorimetric assays suggested that the PMMA-PEI (25 kDa) core-shell nanoparticles were three times less toxic than the branched PEI (25 kDa). Their transfection efficiencies were also significantly higher. Thus, the PEI-based core-shell nanoparticles show considerable potential as carriers for gene delivery.     

Diphtheria toxoid loaded poly-(epsilon-caprolactone) nanoparticles as mucosal vaccine delivery systems

Poly-(epsilon-caprolactone) (PCL), a poly(lactide-co-glycolide) (PLGA)-PCL blend and co-polymer nanoparticles encapsulating diphtheria toxoid (DT) were investigated for their potential as a mucosal vaccine delivery system. The nanoparticles, prepared using a water-in-oil-in-water (w/o/w) double emulsion solvent evaporation method, demonstrated release profiles which were dependent on the properties of the polymers. An in vitro experiment using Caco-2 cells showed significantly higher uptake of PCL nanoparticles in comparison to polymeric PLGA, the PLGA-PCL blend and co-polymer nanoparticles. The highest uptake mediated by the most hydrophobic nanoparticles using Caco-2 cells was mirrored in the in vivo studies following nasal administration. PCL nanoparticles induced DT serum specific IgG antibody responses significantly higher than PLGA. A significant positive correlation between hydrophobicity of the nanoparticles and the immune response was observed following intramuscular administration. The positive correlation between hydrophobicity of the nanoparticles and serum DT specific IgG antibody response was also observed after intranasal administration of the nanoparticles. The cytokine assays showed that the serum IgG antibody response induced is different according to the route of administration, indicated by the differential levels of IL-6 and IFN-gamma. The nanoparticles eliciting the highest IgG antibody response did not necessarily elicit the highest levels of the cytokines IL-6 and IFN-gamma.     

Affinity separation of immunoglobulin G subclasses on dye attached poly(hydroxypropyl methacrylate) beads

Poly(hydroxypropyl methacrylate) [poly(HPMA)] gel beads with an average size of 150–200 μm were prepared by suspension polymerization of hydroxypropyl methacrylate (HPMA). The poly(HPMA) gel beads were characterized by swelling studies, surface area measurements, scanning electron microscopy (SEM) and elemental analysis. Poly(HPMA) gel beads had a specific surface area of 88.6 m²/g. The dye Reactive Green HE 4BD was chemically attached to yield dye-poly(HPMA) gel beads at an average concentration of 44.3 μmol dye/g bead with a swelling ratio of 75%. These dye attached gel beads were used in the separation of immunoglobulin-G (IgG) through adsorption–elution studies. The non-specific adsorption of IgG on the poly(HPMA) gel beads was 0.5 mg/g. The attachment of Reactive Green HE 4BD significantly increased the adsorption of IgG up to 71 mg/g. The Langmuir adsorption model was found to be applicable in interpretation of data pertaining to the adsorption studies of IgG with Reactive Green HE 4BD attached to the poly(HPMA) gel beads. The adsorption of IgG was found to be optimal at pH 7.0. The adsorption of IgG was observed to decrease by about 76% as the NaCl concentration was increased from 0.001 to 0.1 M. The IgG adsorption capacity of the dye attached poly(HPMA) gel beads was determined for a commercially available IgG solution to be 4.2 mg/g for IgG₁, 64.5 mg/g for IgG₂, 7.1 mg/g for IgG₃ and 10.8 mg/g for IgG₄. The Reactive Green HE 4BD attached poly(HPMA) gel beads have a significant adsorption capacity for IgG₂. The quantity of adsorbed IgG₂ is three times higher than the quantity of the other subclasses, IgG₁, IgG₃ and IgG₄. A similar adsorption behaviour was observed when the albumin free human plasma was used. The quantity of adsorbed IgG₂ is higher than the quantity of the other subclasses, IgG₁, IgG₃ and IgG₄. Adsorption capacities for albumin free human plasma were obtained as 6.4 mg/g for IgG₁, 67.8 mg/g for IgG₂, 5.2 mg/g for IgG₃ and 8.6 mg/g for IgG₄. Significant amount of the adsorbed IgG (up to 95%) was eluted in 1 h in the elution medium containing 2.0 M NaCl. Repeated adsorption/elution processes showed that these dye attached gel beads are suitable for IgG adsorption.     

Antibody immobilization onto glow discharge treated polymers.

Previous studies have shown that certain glow discharge treated polymers strongly retain adsorbed albumin and fibrinogen. On the basis of this phenomenon, we have investigated the possibility of immobilizing antibodies on glow discharge treated surfaces for diagnostic immunoassay applications. As a model for antibody immobilization, bovine IgG was immobilized on the following polymers: polyethylene (PE), tetrafluoroethylene glow discharge treated PE (TFE/PE), poly(ethylene terephthalate) (PET), TFE/PET, poly(tetrafluoroethylene) (PTFE), ethylene glow discharge treated PET (E/PET) and hexamethyldisiloxane glow discharge treated PET (HMDS/PET). IgG was radiolabeled with 125I and immobilized by either of the following two methods: (a) physical adsorption of IgG on untreated and glow discharge treated polymers or (b) physical adsorption of albumin followed by chemical coupling of IgG to albumin by glutaraldehyde. IgG concentration as well as adsorption times were varied in order both to optimize the immobilization conditions and to investigate the adsorption and retention mechanisms. To evaluate the efficiency of the immobilization techniques, blood plasma, Tween-20, and sodium dodecyl sulfate (SDS) were used to elute the adsorbed IgG layer. We found that IgG was successfully immobilized on the fluorocarbon glow discharge treated surfaces by using either the physical adsorption or the glutaraldehyde coupling method, although the former is more efficient than the latter method.     

Translocation of poly(ethylene glycol-co-hexadecyl)cyanoacrylate nanoparticles into rat brain endothelial cells: role of apolipoproteins in receptor-mediated endocytosis

Previous in vivo observations in rats have shown that poly(ethylene glycol) polyhexadecylcyanoacrylate (PEG-PHDCA) nanoparticles could translocate into the brain after intravenous injection, which polyhexadecylcyanoacrylate (PHDCA) nanoparticles did not. Through the detailed analysis of the plasma protein adsorption onto the surface of PEG-PHDCA nanoparticles, the present study aimed at clarifying the mechanism by which nanoparticles could penetrate into rat brain endothelial cells (RBEC). Two-dimensional polyacrylamide gel electrophoresis and Western blotting revealed that, after incubation with rat serum, apolipoprotein E (ApoE) adsorbed more onto PEG-PHDCA than on PHDCA nanoparticles. Adsorption of apolipoprotein B-100 (ApoB-100) onto PEG-PHDCA nanoparticles was demonstrated by capillary electrophoresis experiments. Moreover, only when ApoE or ApoB-100 were preadsorbed onto PEG-PHDCA nanoparticles, nanoparticles were found to be more efficient than control nanoparticles for penetrating into RBEC, suggesting the involvement of a low density lipoprotein receptor in this process. Thus, these data clearly demonstrate the involvement of apolipoproteins in the brain transport of PEG-PHDCA nanoparticles, which may open interesting prospects for brain drug delivery.     

Complex catalytic colloids on the basis of firefly luciferase as optical nanosensor platform

In the present work the layer-by-layer nano-assembly technique was used for the development of complex catalytic microparticles on the basis of firefly luciferase (FL). FL films containing 1, 2, or 3 monolayers were assembled on silver electrode QCM-resonators and on 520-nm diameter sulfonated polystyrene latex by alternate adsorption of FL and polycations using electrostatic interactions for the interlayer interaction. The assembly process was studied with quartz crystal microbalance, UV-vis spectroscopy, and microelectrophoresis (surface potential). Structural studies of the resulting multilayers confirmed stepwise deposition of FL and cationic poly(dimethyldiallyl ammonium chloride) with a bilayer thickness of 14 nm; a systematic shift of the surface potential from +28 mV for poly(dimethyldiallyl ammonium chloride) to -14 mV for luciferase outermost layer was established. The functionality and stability of the biocolloids were demonstrated by monitoring the intensity of the light emission. Factors influencing the light emitted upon catalytic activity of FL such as the number of luciferase layers in the film and polyion layer at the outermost layer were studied.     

The platelet open canalicular system: a final common pathway.

Channels of the surface-connected, open canalicular system (OCS) of human platelets serve as the pathway for transport of substances into the cells and as conduits for the discharge of alpha granule products secreted during the platelet release reaction. The purpose of the present study was to determine if both functions of the OCS can take place simultaneously. Suspensions of washed platelets were exposed to thrombin at 1 U/ml for 5, 60, or 180 seconds in the presence of fibrinogen molecules coupled to particles of colloidal gold (Fgn/Au). The samples were fixed in a low concentration of glutaraldehyde and embedded in L.R. White resin to preserve antigenicity. Thin sections were exposed to a rabbit polyclonal antibody to human fibrinogen followed by an anti-rabbit IgG coupled to 5-nm gold beads. Thrombin caused Fgn/Au particles to bind to platelets and enter channels of the surface-connected OCS. Endogenous fibrinogen detected by immunogold 5-nm beads were localized to alpha granules in resting platelets and 5 seconds after thrombin stimulation. At 60 seconds and 3 minutes Fgn/Au particles were present in swollen alpha granules, as well as OCS channels. Fibrinogen gold beads were evident in alpha granules and OCS channels connected to the platelet surface. The 18- to 20-nm Fgn/Au particles were in the same channels of the OCS as fibrinogen gold beads. The OCS is a final common pathway for uptake of particulates and discharge of secretory products in thrombin-activated human platelets.     

Chemical camouflage of nanospheres with a poorly reactive surface: towards development of stealth and target-specific nanocarriers

A two-step approach is described to chemically camouflage the inert surface of model polystyrene nanospheres of 60 nm in diameter against recognition by the body's defenses. The first step was based on the strong protein adsorbing potency of polystyrene, and the second step utilized the chemical reactivity of the adsorbed proteins for conjugation with cyanuric chloride-activated methoxypoly(ethyleneglycol)5000, mPEG5000. Bovine serum albumin (BSA) and rat IgG were used as models of non-immune and immune proteins, respectively. The maximum adsorbance values for both proteins were near expectation for a close-packed monolayer. Adsorption isotherms studies and analysis of the hydrodynamic thickness of the adsorbed protein layer confirmed the close-packed side-on mode of adsorption for BSA and the end-on mode of adsorption for IgG, respectively. Nucleophiles on the adsorbed proteins were then reacted with cyanuric chloride activated mPEG5000. The average poly(ethyleneglycol) (PEG) content for a 60-nm nanospheres was found to be 13.7+/-0.4 micromol PEG/micromol BSA and 3.6+/-0.3 micromol PEG/micromol IgG. The interaction of both PEG-bearing nanospheres with the hydrophobic column material octyl-agarose indicated surface heterogeneity among the nanospheres. Only nanospheres with the most hydrophilic phenotype (approximately 70% of the total population) exhibited stealth properties after intravenous injection to rats. In contrast to the described approach, incubation of uncoated nanospheres with preformed BSA-mPEG5000 conjugates failed to produce long circulating entities. For design of splenotropic particles cyanuric chloride-activated mPEG5000 was conjugated to BSA-coated polystyrene beads of 225 nm in diameter. Despite their stealth property to hepatic Kupffer cell recognition, these nanospheres were cleared by the splenic red pulp macrophages.     

Immunoglobulin G depletion from human serum with metal-chelated beads under magnetic field

Magnetic poly(ethylene glycol dimethacrylate-N-methacryloyl-(L)-histidine methyl ester) [mag-poly(EGDMA-MAH) beads, 50-100 microm in diameter, were produced by suspension polymerization for affinity depletion of immunoglobulin G (IgG) from human serum. Cu2+ ions were complexed directly via MAH groups (Cu2+ loading: 4.1 micromol/g). IgG depletion studies were performed by magnetically stabilized fluidized bed column. Acetate, Tris-HCl, MES and phosphate buffers all allow adsorption of similar quantities of IgG (27.3-45.6 mg/g). MOPS and HEPES allow higher adsorption quantities (79.6 mg/g and 74.1mg/g, respectively). Maximum adsorption capacities in MOPS buffer were 46.8 mg/g for mag-poly(EGDMA-MAH) and 102.1mg/g for Cu2+ chelated mag-poly(EGDMA-MAH) beads. The adsorption capacity decreased drastically from 102.1mg/g to 30.7 mg/g with the increase of the flow rate from 0.2 ml/min to 3.5 ml/min. The elution studies were performed by 1.0M NaCl. The elution results demonstrated that the adsorption of IgG to the adsorbent was reversible. To test the efficiency of IgG depletion from human serum, proteins in the serum and eluted portion were analyzed by two-dimensional gel electrophoresis. The depletion efficiency for IgG was above 99.4%. Eluted proteins include mainly IgG, and a small number of non-albumin proteins such as apo-lipoprotein A1, sero-transferrin, haptoglobulin and alpha1-antitrypsin. When anti-HSA-sepharose adsorbent is used together with our metal-chelated mag-beads, IgG and HSA can be depleted in a single step.     

Multiple Protein-immobilized Phospholipid Polymer Nanoparticles: Effect of Spacer Length on Residual Enzymatic Activity and Molecular Diagnosis

We report the development of phosphorylcholine (PC) group-covered nanoparticles for multiple immobilization reactions; the surface of these nanoparticles facilitates bioreactions such as enzymatic reactions and molecular diagnoses. The nanoparticles were covered with a bioconjugate PC group containing a polymer backbone, and their surface properties were as follows: (1) suppression of nonspecific protein adsorption and (2) stabilization of immobilized biomolecules. In this study, biomolecules were immobilized on PC-covered nanoparticles by using different spacer lengths between the polymer backbone and biomolecules. The stability of the immobilized biomolecules was evaluated using horseradish peroxidase-labeled IgG, and the bioconjugate nanoparticles were stored at 4, 25, and 40 °C. The residual enzymatic activity of the peroxidase was monitored at a particular time. On the other hand, to test the role of these nanoparticles in molecular diagnosis, we used IgG-conjugated nanoparticles and the fluorescence resonance energy transfer (FRET) phenomenon. The IgG molecules were labeled with either donor or acceptor molecules, and each labeled IgG was simultaneously immobilized on the PC-covered nanoparticles. These labeled IgG molecules induce the FRET phenomenon upon capture of the target antigen provided they are in close proximity. The resulting fluorescence was readable via the FRET phenomenon. In the present study, C-reactive protein (CRP) was used as the target antigen, and the effect of the spacer length is discussed. The bioconjugated nanoparticles covered with PC groups are promising tools for tuning bioreactions.     

Fluorescent biorecognition of gold nanoparticle-IgG conjugates self-assembled on E-beam patterns

A new concept for line patterning of immunoglobulin G (IgG) in nanometer scale using gold nanoparticles (AuNPs) self-assembled in a nanochannel written with an electron beam is proposed and demonstrated. AuNPs are synthesized by reducing KAuCl4 with NaBH4, producing AuNPs 40-70 nm in size, where Cl- ions are capping AuNPs thus making them negatively charged and subsequently stabilized. IgG is conjugated to these AuNPs by simple adsorption. Single or multiple nanochannels are written with an electron beam using a scanning electron microscope (SEM) in a layer of poly(methyl methacrylate) (PMMA), which is spin-coated on a p-doped Si wafer. AuNPs bind into the etched nanochannel where the Si surface is exposed, while the relatively hydrophobic PMMA area repels the particles. The particles with a diameter larger than the channel width are not able to go inside of it. Anti-IgG, conjugated with fluorescein isothiocyanate (FITC), is then exposed to the patterned surface, binding specifically to the IgG-AuNP conjugates within the line patterns. These antibody-antigen bindings can be visualized with a fluorescent microscope, showing the fluorescent signal only along with the nanometer line pattern. These initial steps will lead to the formation of complex protein nanoarrays, based on the size-dependent self-assembly of AuNPs within variously sized nanopatterns.     

Mercaptoheterocyclic ligands grafted on a poly(ethylene vinyl alcohol) membrane for the purification of immunoglobulin G in a salt independent thiophilic chromatography

In this study, we attempted a limited combinatorial approach for designing affinity ligands based on mercaptoheterocyclic components. The template, divinyl sulfone structure (DVS), which was grafted on poly(ethylene vinyl alcohol) (PEVA) hollow fiber membrane, has served for the tethering of different heterocyclic compounds as pyridine, imidazole, purine and pyrimidine rings. Their ability to adsorb specifically IgG in a salt independent manner out of pure IgG solution, mixture of IgG/albumin and human plasma was demonstrated. Mercapto methyl imidazole (MMI) has shown the best adsorption of IgG in terms of binding capacity. No subclass discrimination was observed on all tested ligands except for mercapto methyl pyrimidine where the major IgG subclass adsorbed was IgG3. MMI gave an IgG binding capacity of 100 microg/cm2 of hollow fiber membrane surface area.     

Silane-modified magnetic beads: application to immunoglobulin G separation

The magnetic poly(2-hydroxyethyl methacrylate ethylene glycol dimethacrylate) [m-poly(HEMA-EGDMA)] beads (150-250-microm diameter in spherical form) were prepared by a radical suspension polymerization technique. The pseudo-specific ligand, reactive imidazole containing 3-(2-imidazoline-1-yl)propyl (triethoxysilane) (IMEO) was selected as a silanization agent. IMEO was covalently immobilized onto the magnetic beads. IMEO-immobilized m-poly(HEMA-EGDMA) beads were used for the affinity adsorption of immunoglobulin-G (IgG) from aqueous solutions and human plasma. To evaluate the degree of IMEO attachment, the m-poly(HEMA-EGDMA) beads were subjected to Si analysis by using flame atomizer atomic absorption spectrometer, and it was estimated as 36.6 mg IMEO/g of polymer. The nonspecific IgG adsorption onto the plain m-poly(HEMA-EGDMA) beads was very low (about 0.4 mg/g). Higher adsorption values (up to 55 mg/g) were obtained when the m-poly(HEMA-EGDMA)/IMEO beads were used from both aqueous solutions and human plasma. The maximum IgG adsorption on the m-poly(HEMA-EGDMA)-IMEO beads was observed at pH 7.0. The IgG molecules could be repeatedly adsorbed and desorbed with m-poly(HEMA-EGDMA)-IMEO beads without noticeable loss in the IgG adsorption capacity. The adsorption capacity from human plasma in magnetically stabilized fluidized bed decreased drastically from 78.9 to 19.6 mg/g with the increase of the flow rate from 0.2 to 3.5 mL/min.     

UV-induced graft copolymerization of monoacrylate-poly(ethylene glycol) onto poly(3-hydroxyoctanoate) to reduce protein adsorption and platelet adhesion

Homogeneous solutions of poly(3-hydroxyoctanoate) (PHO) and the monoacrylate-poly(ethylene glycol) (PEGMA) monomer in chloroform were irradiated with UV light to obtain PEGMA-grafted PHO (PEGMA-g-PHO) copolymers. Variables affecting the degree of grafting (DG), such as the time of UV irradiation and the concentrations of the PEGMA monomer and initiator, were investigated. The PEGMA-g-PHO copolymers were characterized by measuring the water contact angle, molecular weight, thermal transition temperatures and mechanical properties, as well as by nuclear magnetic resonance spectroscopy. The results from all of these measurements indicate that PEGMA groups were present on the PHO polymer. The protein adsorption and platelet adhesion on the PEGMA-g-PHO surfaces were examined using poly(L-lactide) (PLLA) surfaces as the control. The proteins and platelets had a significantly lower tendency to adhere to the PEGMA-g-PHO copolymers than to PLLA. The graft copolymer with a high DG of PEGMA was very effective in reducing the protein adsorption and platelet adhesion and did not activate the platelets. The results obtained in this study suggest that PEGMA-g-PHO copolymers have the potential to be used as blood-contacting devices in a broad range of biomedical applications.     

Modulation of immunogenicity of poly(sarcosine) displayed on various nanoparticle surfaces due to different physical properties

Poly(sarcosine) displayed on polymeric micelle is reported to trigger a T cell‐independent type2 reaction with B1a cells in the mice to produce IgM and IgG3 antibodies. In addition to polymeric micelle, three kinds of vesicles displaying poly(sarcosine) on surface were prepared here to evaluate the amounts and avidities of IgM and IgG3, which were produced in mice, to correlate them with physical properties of the molecular assemblies. The largest amount of IgM was produced after twice administrations of a polymeric micelle of 35 nm diameter ( G1 ). On the other hand, the production amount of IgG3 became the largest after twice administrations of G3 (vesicle of 229 nm diameter) or G4 (vesicle of 85 nm diameter). The augmented avidity of IgG3 after the twice administrations compared with that at the single administration was the highest with G3 . These differences in immune responses are discussed in terms of surface density of poly(sarcosine) chains, nanoparticle size, hydrophobic component of poly(L‐lactic acid) or (Leu‐ or Val‐Aib)_n, and membrane elasticity of the nanoparticles. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Behavior of human immunoglobulin G adsorption onto immobilized Cu(II) affinity hollow‐fiber membranes

Iminodiacetic acid (IDA) and tris(2‐aminoethyl)amine (TREN) chelating ligands were immobilized on poly(ethylene vinyl alcohol) (PEVA) hollow‐fiber membranes after activation with epichlorohydrin or butanediol diglycidyl ether (bisoxirane). The affinity membranes complexed with Cu(II) were evaluated for adsorption of human immunoglobulin G (IgG). The effects of matrix activation and buffer system on adsorption of IgG were studied. Isotherms of batch IgG adsorption onto finely cut membranes showed that neither of the chelates, IDA‐Cu(II) or TREN‐Cu(II), had a Langmuirean behavior with negative cooperativity for IgG binding. A comparison of equilibrium and dynamic maximum capacities showed that the dynamic capacity for a mini‐cartridge in a cross‐flow filtration mode (52.5 and 298.4 mg g^?1 dry weight for PEVA‐TREN‐Cu(II) and PEVA‐IDA‐Cu(II), respectively) was somewhat higher than the equilibrium capacity (9.2 and 73.3 mg g^?1 dry weight for PEVA‐TREN‐Cu(II) and PEVA‐IDA‐Cu(II), respectively). When mini‐cartridges were used, the dynamic adsorption capacity of IDA‐Cu(II) was the same for both mini‐cartridge and agarose gel. Copyright © 2013 John Wiley & Sons, Ltd.     

C-reactive protein and C1q regulate platelet adhesion and activation on adsorbed immunoglobulin G and albumin

Blood platelets and C-reactive protein (CRP) are both used clinically as markers of ongoing inflammation, and both participate actively in inflammatory responses, although the biological effects are still incompletely understood. Rapidly adhering platelets express receptors for complement factor 1q (C1q) and the Fc part of immunoglobulin G (IgG), and CRP is known to activate/regulate complement via C1q binding, and to ligate FcgammaRs. In the present study, we used normal human IgG pre-adsorbed to a well-characterized methylated surface as a model solid-phase immune complex when investigating the effects of CRP and C1q on platelet adhesion and activation. Protein adsorption was characterized using ellipsometry and polyclonal antibodies, and human serum albumin (HSA) and non-coated surfaces were used as reference surfaces. Platelet adhesion to IgG and HSA was inhibited by both C1q and CRP. Furthermore, CRP (moderately) and C1q (markedly) decreased the spreading of adhering platelets. The combination of C1q and CRP was slightly more potent in reducing cell adhesion to IgG, and also impaired the adhesion to HSA and non-coated surfaces. Platelet production of thromboxane B2 (TXB(2)) was also reduced by C1q both in the presence and absence of CRP, whereas CRP alone had no effect on TXB(2) production. We conclude that CRP and C1q regulate the behaviour of platelets, and that this may be an important immunoregulatory mechanism during inflammatory conditions.     

Interaction of human immunoglobulin G with l-histidine immobilized onto poly(ethylene vinyl alcohol) hollow-fiber membranes

l-Histidine as pseudobiospecific ligand was immobilized onto poly(ethylene vinyl alcohol) hollow-fiber membranes to obtain an affinity support for immunoglobulin G (IgG) purification. The interaction of human IgG with the affinity membranes was studied by chromatography and equilibrium binding analysis. Adsorption was possible over a broad pH range and was found to depend strongly on the nature of the buffer ions rather than on ionic strength. With zwitterionic buffers like morpholinopropanesulfonic acid (Mops) and hydroxyethylpiperazineethanesulfonic acid (Hepes), much higher adsorption capacities were obtained than with other buffers like Tris-HCl and phosphate buffers. An inhibition analysis revealed that non-zwitterionic buffers competitively inhibit IgG binding, whereas Mops and Hepes in their zwitterionic form do not. By choosing the appropriate buffer system, it was possible to adsorb specifically different IgG subsets. The IgG molecules were found to adsorb on membrane immobilized histidine via their F_ab part. Determination of dissociation constants at different temperatures allowed calculation of thermodynamic adsorption parameters. Decrease in K_D with increasing temperature and a positive entropy value between 20 and 35°C (in Mops buffer) indicated that adsorption is partially governed by hydrophobic forces in that temperature range, whereas at lower temperatures, electrostatic forces are more important for adsorption.     

Studies of the mechanism of the human platelet release reaction induced by immunologic stimuli. III. Relationship between the binding of soluble IgG aggregates to the Fc receptor and cell response in the presence and absence of plasma

Aggregated IgG coupled covalently with bis-diazobenzidine (BDB-IgG) and labeled with 3H-diazobenzene (3H-BDB-IgG) has been used to study the binding of soluble IgG aggregates to human platelets in relationship to the release of the contents of intracellular granules (e.g., serotonin). In washed cell suspensions a minimum of 0.14 to 0.2 mug 3H-BDB-IgG per 5 X 10(8) platelets (40 to 70 aggregates per cell) was required for the triggering of the release reaction and cell aggregation. Binding was independent of divalent cations. The Arrhenius plot gave a straight line between 0 to 37 degrees C and a Q10 of 1.6. Neither inhibitors of the release reaction nor energy metabolism, nor formaldehyde fixation of the platelets affected binding. Bound 3H-BDB-IgG was not significantly eluted by IgG, bovine albumin (BSA), buffer, or plasma. Binding to washed platelets was more strongly inhibited by human IgG than by F(ab')2, bovine IgG, human albumin (HSA), or BSA. Plasma was an even more effective inhibitor of both binding and release. Plasma deficient in IgG or depleted of complement retained its inhibitory capacity. In the presence of plasma, at physiologic ratios of plasma and platelets, no release of serotonin was observed. Binding, although inhibited in rate, nevertheless occurred. It was enhanced by divalent cation chelation and had a Q10 of 2.5. The release reaction of washed platelets to which 3H-BDB-IgG had been bound in the presence of HSA or BSA was also inhibited by the subsequent addition of plasma or plasma proteins (human IgG being more effective than bovine IgG, F(ab')2, HSA, or BSA). 3H-BDB-IgG bound in the presence of either plasma or human IgG did not induce release when the platelets were subsequently suspended in media lacking these proteins. Thus, it appears that the platelet Fc receptor binds 3H-BDB-IgG by a process which is effectively inhibited by plasma, or by free IgG with an intact Fc, and to some extent by high concentrations of other proteins. The effects of bound IgG aggregates are dependent on the other proteins present both during binding and subsequently added. The mechanism of such receptor modulation and its implications in vivo are discussed.