Passive adsorption of human antirrabic immunoglobulin onto a polystyrene surface

The latex agglutination immunoassay technique uses polymer colloids as carriers of adsorbed proteins to enhance the antigen-antibody reaction. The aim of the present work is to study the adsorption of Human Antirrabic Immunoglobulin (HA-IgG) on polystyrene latex (PS). The physical adsorption of HA-IgG on PS latex was investigated as a function of pH at 2 mM ionic strength. The amount of HA-IgG adsorbed onto PS latex greatly depends on pH; its value showed a maximum in the neighborhood of the IEP of HA-IgG. The electrophoretic method was applied to characterize latex particles. The influence of the amount of HA-IgG adsorbed (J(ads)) on the electrophoretic mobility and zeta-potential values was also studied.     

Covalent grafting of polythymidylic acid onto amine-containing polystyrene latex particles

The covalent grafting of a 35-base poly(thymidylic) acid (dT35) onto aminated polystyrene latex has been studied. The oligonucleotide was activated by 1,4-phenylene diisothiocyanate (PDC) prior to grafting reaction. The influence of various experimental conditions related either to the medium (pH, ionic strength) or to the latex (nature and surface charge density, presence of adsorbed nonionic surfactant) was studied. Discrimination between covalent grafting and adsorption was achieved by desorbing the non-covalently grafted dT35 induced by consecutive washings with a suitable buffer solution. The amount of covalently grafted oligonucleotide directly correlated to the concentration and accessible functional groups at the particle surface.     

Passive adsorption of human antirrabic immunoglobulin onto a polystyrene surface

The latex agglutination immunoassay technique uses polymer colloids as carriers of adsorbed proteins to enhance the antigen–antibody reaction. The aim of the present work is to study the adsorption of Human Antirrabic Immunoglobulin (HA-IgG) on polystyrene latex (PS). The physical adsorption of HA-IgG on PS latex was investigated as a function of pH at 2 mM ionic strength. The amount of HA-IgG adsorbed onto PS latex greatly depends on pH; its value showed a maximum in the neighborhood of the IEP of HA-IgG. The electrophoretic method was applied to characterize latex particles. The influence of the amount of HA-IgG adsorbed (J _ads) on the electrophoretic mobility and ζ -potential values was also studied.     

Covalent grafting of polythymidylic acid onto amine-containing polystyrene latex particles

The covalent grafting of a 35-base poly(thymidylic) acid(dT₃₅) onto aminated polystyrene latex has been studied. The oligonucleotide was activated by 1,4-phenylene diisothiocyanate (PDC) prior to grafting reaction. The influence of various experimental conditions related either to the medium (pH, ionic strength) or to the latex (nature and surface charge density, presence of adsorbed nonionic surfactant) was studied. Discrimination between covalent grafting and adsorption was achieved by desorbing the non-covalently grafted dT₃₅ induced by consecutive washings with a suitable buffer solution. The amount of covalently grafted oligonucleotide directly correlated to the concentration and accessible functional groups at the particle surface.     

Plasma protein adsorption pattern and tissue-implant reaction of poly(vinyl alcohol)/carboxymethyl-chitosan blend films

Various poly(vinyl alcohol)/carboxymethyl-chitosan (PVA/CMCS) blend films were prepared by a mechanical blending method and characterized by SEM for their surface and cross-section morphologies. It indicated that blending high CMCS content in PVA plastic led to a rough surface and loose structure. Bovine serum albumin (BSA) and bovine fibrinogen (BFG) were chosen as representative plasma proteins to carry out adsorption tests. Equilibrium adsorption amount of proteins onto the blends decreased with the increase of CMCS content in film matrix, and BSA was more easily adsorbed onto the films than BFG in the same conditions. The blend films also exhibited different trends for BSA and BFG adsorption when pH of the media changed, but maximum adsorption approximately occurred at the isoelectric point of proteins. Moreover, increasing the ionic strength would always decrease the adsorptions of protein onto the films. In animal experiments, it was found that incorporation of CMCS and PVA gave a lower tissue reaction than pure PVA films when they were subcutaneously implanted in Wistar rats. After two weeks subcutaneous implantation, surfaces of PVA became wrinkled and cracked; however, the blend implants exhibited a alveolate porous microstructure.     

Plasma protein adsorption pattern and tissue-implant reaction of poly(vinyl alcohol)/carboxymethyl-chitosan blend films

Various poly(vinyl alcohol)/carboxymethyl-chitosan (PVA/CMCS) blend films were prepared by a mechanical blending method and characterized by SEM for their surface and cross-section morphologies. It indicated that blending high CMCS content in PVA plastic led to a rough surface and loose structure. Bovine serum albumin (BSA) and bovine fibrinogen (BFG) were chosen as representative plasma proteins to carry out adsorption tests. Equilibrium adsorption amount of proteins onto the blends decreased with the increase of CMCS content in film matrix, and BSA was more easily adsorbed onto the films than BFG in the same conditions. The blend films also exhibited different trends for BSA and BFG adsorption when pH of the media changed, but maximum adsorption approximately occurred at the isoelectric point of proteins. Moreover, increasing the ionic strength would always decrease the adsorptions of protein onto the films. In animal experiments, it was found that incorporation of CMCS and PVA gave a lower tissue reaction than pure PVA films when they were subcutaneously implanted in Wistar rats. After two weeks subcutaneous implantation, surfaces of PVA became wrinkled and cracked; however, the blend implants exhibited a alveolate porous microstructure.     

Adsorption of bovine serum albumin onto surface-modified polyhydroxyethyl methacrylate beads

The adsorption of bovine serum albumin (BSA) onto poly(2-hydroxyethyl methacrylate) (PHEMA) beads modified by using the pair of hexamethylene diisocyonate-suberic acidbis-N-hydroxy succinimide has been studied as a function of protein concentration and adsorption time. The adsorption studies were carried out in phosphate buffer solution (PBS) at pH = 7.4. The isotherm data have been analysed using the Langmuir model and the adsorption parameters Q0 and b were calculated. It is determined that the adsorbed amount of BSA increases by the increase of the adsorption time and BSA concentration until a certain value. PHEMA beads were characterized by using FTIR spectra and SEM analysis. The adsorption of BSA onto PHEMA beads were clearly observed from SEM micrographs. The swelling tests of the beads were performed at 37 degrees C in PBS.     

Comparison of albumin binding capacities of three different reactive dye-derivatized poly(ethylene glycol dimethacrylate-hydroxyethyl methacrylate) microbeads

Bovine serum albumin (BSA) adsorption onto dye-derivatized poly(ethylene glycol dimethacrylate-hydroxyethyl methacrylate) [poly(EGDMA-HEMA)] microbeads carrying three different reactive dye ligands (i.e. Congo Red, Cibacron Blue F3GA, and Alkali Blue 6B) was investigated. Swellable poly(EGDMA-HEMA) microbeads, in the size range of 150-200 μm, were produced by a modified suspension copolymerization of EGDMA and HEMA. The dyes were covalently attached to the microbeads. The maximum amounts of dye loadings were 14.5, 16.5, and 23.7 μmol g^-1 for Congo Red, Cibacron Blue F3GA, and Alkali Blue 6B, respectively. The maximum BSA adsorption on the dye-derivatized microbeads from aqueous solutions containing different amounts of BSA were 90, 60.5, and 40 mg g^-1 for the Congo Red, Cibacron Blue F3GA, and Alkali Blue 6B carrying microbeads, respectively. The maximum BSA adsorptions were observed at pH 6.0 in all cases. Desorption of albumin molecules were achieved by using 1.0 M NaSCN (pH 8.0). High desorption ratios (more than 85% of the adsorbed BSA) were observed in all cases. It was possible to reuse these novel sorbents without significant losses in the adsorption capacities.     

Protein adsorption and zeta potentials of a biphasic calcium phosphate ceramic under various conditions

An investigation on the relationship between protein adsorption and zeta potentials of a biphasic calcium phosphate (BCP) ceramic was carried out. Zeta potentials of the BCP ceramic particles were measured at various aqueous solutions. Bovine serum album (BSA) and its competitive adsorption with lysozyme (LSZ) on BCP were investigated using conventional protein adsorption and polyacrylamide gel electrophoresis (PAGE) methods. The results showed that zeta potential and the amount of adsorbed BSA were both influenced by pH, ionic strength, Ca2+ and PO4(3-) concentrations in the buffers. The variation tendencies of BSA adsorption were consistent with that of zeta potentials to some extent. The co-adsorption of BSA and LSZ on BCP was confirmed by the PAGE gel pattern. The semi-quantitative analysis for the detected protein bands proved that LSZ had higher affinity for BCP than BSA and would preferentially bind to the surface. Electrostatic interaction played an important role in protein adsorption on the surface of the BCP ceramic particles.     

椭偏光学显微成像法研究胶原在表面的吸附

我们利用椭偏光学显微成像技术研究了胶原蛋白在亲水疏水表面的吸附,考察了不同pH值、不同吸附时间、不同离子强度以及BSA的存在对胶原在亲水、疏水表面吸附的影响。结果表明,胶原在亲水表面的吸附量在较短时间内即接近于吸附饱和值,而在疏水表面吸附则持续较长时间,最终胶原在疏水表面的吸附量大于在亲水表面的吸附量。胶原在两种表面的吸附量均在pH为7.2时为最大。BSA存在时,疏水表面上BSA对胶原的竞争吸附影响比亲水表面大。     

Drug release from a porous ion-exchange membrane in vitro.

The effect of environmental ionic strength on the rate of drug release from a cation exchange membrane was evaluated. Cationic propranolol-HCl, timolol, sotalol-HCl, atenolol and dexmedetomidine-HCl and neutral diazepam were adsorbed onto a porous poly(vinylidene fluoride) (PVDF) membrane that was grafted with bioadhesive poly(acrylic acid) chains (PAA-PVDF). Despite its porosity, the PAA-PVDF membrane acted as a cation exchange membrane. The release of adsorbed drug from the PAA-PVDF membrane was investigated by using a USP rotating basket apparatus. Adsorption of cationic drugs onto the PAA-PVDF membrane tended to increase with increasing lipophilicity of the drug. A decrease in the ionic strength of the adsorption medium increased the amount of the cationic drugs adsorbed onto the membrane, but had no effect on diazepam adsorption. The release of cationic drugs from the PAA-PVDF membrane was greatly affected by the ionic strength of both the adsorption medium and the dissolution medium, while ionic strengths did not affect diazepam release. Our results suggest that the ionic strength of both the adsorption and dissolution media substantially affects the release rate of a drug that has been adsorbed onto the ion exchange membrane, primarily via electrostatic interactions, while ionic strength has no effect on the release of a drug which has been adsorbed onto the membrane via non-electrostatic forces.     

Immunological agglutination kinetics of latex particles with physically adsorbed antigens

The influence of the properties of antigens and particles on the immunological agglutination kinetics of the antigen-coated latex particles was studied. Horse cytochrome c, hen egg-white lysozyme (HEL), bovine serum albumin (BSA), and Aspergillus sp. glucose oxidase were physically adsorbed onto the surfactant free latices of styrene-methacrylic acid (MAA) copolymer (P (S/MAA)) and polystyrene (PS). The initial rates of the immunological agglutination of these protein-coated particles initiated by the addition of antibodies were quantified by the absorbance change at a wavelength of 680 nm. The initial agglutination rates of the particles covered with smaller antigens were lower. This effect of the molecular size of antigens was larger in P(S/MAA), because small antigens are probably buried in the hydrous polymethacrylic acid layer on the surface of particles. Thus, both the molecular size of antigens and the surface properties of particles affect the sensitivity of the immunological agglutination. On the other hand, the dependence of the initial rate of the immunological agglutination on the ionic strength and pH was similar irrespective of antigen-particle systems. The initial agglutination rates were largest at an ionic strength of approximately 0.05 at pH 7.0 and decreased with increasing pH. This dependence of the sensitivity on the pH and ionic strength is attributed to the electrostatic interactions of particle-particle and antibody-particle.     

Development of a slide latex agglutination test for rotavirus antigen detection

The aim of this study was to optimize the conditions for the passive adsorption of polyclonal antibody onto plain surface polystyrene latex particles and its performance in a slide latex agglutination test for rotavirus antigen detection. Cleaning of latex particles by washing through repetitive centrifuging, decanting and resuspending in distilled water was adequate in removing surfactants from the particles' surfaces to enable coating. A study of antibody concentration, incubation temperature and buffer pH revealed that optimum coating was achieved with a 3-fold excess of antibody to the calculated total particle surface capacity for the antibody in a glycine-saline buffer of pH 9.2 at 40 degrees C for 4 hours. The ionic strength and pH of the latex suspending buffer and the sample buffer were critical factors determining the sensitivity of the test and the appearance of non-specific agglutination. Ultrasonication, addition of glycerol and Tween 20, either individually or in combination, were able to suppress non-specific agglutination in some batches of latex reagents. Polyethylene glycol 6000 enhanced the quality of agglutination as well as reduced the time of its appearance, especially in reagents that produced poor agglutination.     

Adsorption of Chitosan onto Poly(D,L‐lactic acid) Particles: A Physico‐Chemical Investigation

Summary: Particles of poly(D ,L ‐lactic acid) (PLA) obtained by emulsification‐diffusion were surface‐modified by adsorption of chitosan. Thus, positively charged biodegradable carriers were obtained as potential carriers for DNA. Various physico‐chemical parameters, susceptible to impact the elaboration of such colloids, were investigated. External parameters were the ionic strength and the nature of the buffer; pH was fixed at 4.5 in order to ensure the ionisation of both counterparts. Internal parameters were the molar mass of chitosan and its degree of acetylation (DA). Kinetically, the adsorption of chitosan was spontaneous. Adsorption isotherms of chitosan were established using two different assays for chitosan, one based on the formation of a covalent bond, and the other on the formation of an ion pair. Two adsorption regimes were evidenced in ammonium acetate buffer. The amount of adsorbed chitosan increased with its molar mass up to a limit due to steric hindrance and with the DA. The latter effect could be explained by a reduction of the molecular dimensions of chitosan with DA. On storing, desorption of chitosan was dependent on the molar mass of the polysaccharide and, to a lesser extent, on the DA.

Complexation isotherm of chitosan onto PLA particles.     

Reactive latex studies, 2. Kinetics and retention of enzymatic activity in the covalent protein bonding to a polymer latex

Bovine serum albumin (BSA) was coupled to the reactive chloromethyl groups of poly(vinylbenzyl chloride) latex following the process both by the amount of BSA covalently bound and the chloride ion released. The primary bond formation is very insensitive to temperature with protein adsorption probably rate determining. Subsequent bond formation has an unusually high temperature coefficient and may depend on the deformability of the BSA molecule. On covalent bonding of ribonuclease 10% of the enzyme activity is retained.     

N-methacryloly-(L)-histidinemethylester carrying a pseudospecific affinity sorbent for immunoglobulin-G isolation from human plasma in a column system

N-methacryloly-(L)-histidinemethylester (MAH) as a pseudospecific ligand was synthesized by using methacryloyl chloride and histidine. Spherical beads with an average size of 63-75 microm were obtained by suspension polymerization of ethylene glycol dimethacrylate (EGDMA), 2-hydroxyethyl methacrylate (HEMA) and MAH conducted in an aqueous dispersion medium. The specific surface area of the beads was found to be 18.3 m2/g. Poly(EGDMA-HEMA-MAH) beads were used in the separation of immunoglobulin-G (HIgG) from aqueous solutions and/or human plasma in a packed-bed column system. HIgG adsorption capacity of the beads decreased with an increase in the flow-rate of plasma. The maximum HIgG adsorption on the poly(EGDMA-HEMA-MAH) sorbents was observed at pH 7.4. HIgG adsorption onto the poly(EGDMA-HEMA) sorbents was negligible. Higher adsorption values (up to 135 mg/g) were obtained when the poly(EGDMA-HEMA-MAH) sorbents were used from aqueous solutions. HIgG adsorption increased with decreasing temperature and the maximum adsorption achieved at 4 degrees C. MAH incorporation significantly affected HIgG adsorption capacity (135 mg/g). Higher amounts of HIgG were adsorbed from human plasma (up to 165 mg/g). Adsorption capacities of other blood proteins were obtained as 8.7 mg/g for fibrinogen and 14.6 mg/g for albumin. The total protein adsorption was determined as 191 mg/g. The pseudospecific affinity beads allowed one-step separation of HIgG from human plasma. HIgG molecules could be repeatedly adsorbed and desorbed with these sorbents without noticeable loss in their HIgG adsorption capacity.     

免疫球蛋白G在苯乙烯—苯乙烯磺酸钠胶乳微球上的吸附行为

苯乙烯-苯乙烯磺酸钠胶乳微球有着极好的表面性能,可按希望模式大量吸附免疫球蛋白G,因而可用作免疫诊断试剂。本文讨论了抗人绒膜促性腺素免疫球蛋白G(Anti-HCG-IgG)在磺酸基胶乳微球上的影响因素,及其吸附机理实验证明,免疫球蛋白在磺酸基胶乳微球上的吸附受缓冲溶液的极性、浓度、pH、离子强度的影响。微球表面的电荷密度对吸附量及吸附模型产生影响。微球对免疫球蛋白的吸附有两种:(1)疏水基团间相互作用;(2)离子间相互作用,但以第一作用为主。本文对研究为制备免疫胶乳诊断试剂打下了良好的基础。     

Thermally-modulated on/off-adsorption materials for pharmaceutical protein purification

For the development of temperature-responsive adsorption materials for pharmaceutical protein purification, poly(N-isopropylacrylamide-co-N,N-dimethylaminopropylacrylamide-co-N-tert-butylacrylamide) (P(IPAAm-co-DMAPAAm-co-tBAAm) brush grafted silica beads were prepared through a surface-initiated atom transfer radical polymerization (ATRP). The prepared silica beads as a chromatographic stationary phase were evaluated by observing their thermo-responsive elution profiles of plasma proteins including human serum albumin (HSA) and γ-globulin. Chromatograms of two proteins indicated that negatively-charged HSA was adsorbed on the cationic copolymer brush modified silica beads at higher temperatures with low concentration of phosphate buffer (PB) (pH 7.0) as a mobile phase. The HSA adsorption was attributed to (1) an enhanced electrostatic interaction with the cationic copolymer brush at low concentration of PB and (2) an increased hydrophobic interaction from the dehydrated copolymer at high temperature. Step-temperature gradient enabled HSA and γ-globulin to be separated by the modulation of HSA adsorption/desorption onto the copolymer brush grafted silica beads. These results suggested that the prepared copolymer brush grafted silica beads adsorbed negatively-charged proteins both through electrostatic and hydrophobic interactions by the modulation of column temperature and gave attractive adsorption materials for protein purification process.     

The formation of fast-sedimenting turnip yellow mosaic virus RNA: structural rearrangement inside the capsid.

Treatment of TYMV (turnip yellow mosaic virus) virions under alkaline conditions and at high ionic strength leads to the in situ formation of RNA sedimenting uniformly at 38 S. Native TYMV-RNA has a sedimentation coefficient of 28 S in the same solvent. A systematic study in which the temperature, ionic strength, and pH were varied showed that favorable conditions for 38 S RNA formation are 1.0 M KCl, pH 10.5, 30°, and a reaction time of 5–10 min. Nonetheless, the fast-sedimenting RNA can also be obtained at neutral pH and 1.0 M KCl, provided that the time of treatment is prolonged or the temperature is raised. No evidence for breakage of the RNA chain was obtained under the latter conditions, while all of the RNA is retained by the virion. These data indicate that exposure of TYMV to high ionic strength causes a drastic structural rearrangement of the viral RNA inside the capsid without concurrent fragmentation of the RNA chain.     

Quercetin adsorption with imprinted polymeric materials

In this study, molecularly imprinted polymer membranes were synthesized for the recognition and adsorption of quercetin. For this, quercetin imprinted polymeric membranes [p(HEMA-MAH)] (Poly(2-hydroxyethyl methacrylate-co-N-methacryloly-l-histidinemethylester) were synthesized by UV polymerization technique using HEMA and MAH as monomers. Synthesized polymeric membranes were characterized with SEM, FTIR and swelling test. Characterized membranes were used for the direct adsorption of quercetin in a batch system. Quercetin adsorption conditions were optimized by using the quercetin imprinted polymeric membrane by altering the pH, temperature and initial quercetin concentration of the adsorption medium. Effect of adsorption time was also studied for up to 180?min. The optimum pH and temperature was determined between 4.0 and 45?°C. Maximum adsorbed amount of quercetin onto quercetin imprinted poly(HEMA-MAH) membrane was found to be as 299.6?mg/g membrane using the initial quercetin concentration of 2.0?mg/ml. Adsorbed quercetin was desorbed from the polymeric membranes with isopropyl alcohol with desorption yield of 98.3%. and repeated usability of the quercetin imprinted polymeric membranes was fallowed for 7 adsorption/desorption cycles. At the end of the 7^th reuse, quercetin adsorption capacity of the quercetin imprinted poly(HEMA-MAH) membranes decreased only about 10%.