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1.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1995,674(1):13-21
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 Fab part. Determination of dissociation constants at different temperatures allowed calculation of thermodynamic adsorption parameters. Decrease in KD 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. 相似文献
2.
Polyclonal preparations of therapeutic immunoglobulins, namely intravenous immunoglobulins (IVIg), are essential in the treatment of immunodeficiency and are increasingly used for the treatment of autoimmune and inflammatory diseases. Currently, patients’ accessibility to IVIg depends exclusively upon volunteer blood donations followed by the fractionation of pooled human plasma obtained from thousands of individuals. Presently, there are no in vitro cell culture procedures allowing the preparation of polyclonal human antibodies. All in vitro human therapeutic antibodies that are currently generated are based on monoclonal antibodies, which are mostly issued from genetic engineering or single cell antibody technologies. Here, we describe an in vitro cell culture system, using CD40-CD154 interactions, that leads to a 1×106-fold expansion of switched memory B lymphocytes in approximately 50 days. These expanded cells secrete polyclonal IgG, which distribution into IgG1, IgG2, IgG3 and IgG4 is similar to that of normal human serum. Such in vitro generated IgG showed relatively low self-reactivity since they interacted moderately with only 24 human antigens among a total of 9484 targets. Furthermore, up to one liter of IgG secreting cells can be produced in about 40 days. This experimental model, providing large-scale expansion of human B lymphocytes, represents a critical step toward the in vitro production of polyclonal human IgG and a new method for the ex vivo expansion of B cells for therapeutic purposes. 相似文献
3.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1986,886(1):88-95
The ligand binding properties of folate chemotactic receptors on isolated membranes of Dictyostelium discoideum were analyzed. Three out of the four receptor states (BF, BS and BSS) were detected, showing rate constants and Kd values similar to those obtained for intact cells. Guanine nucleotides changed the proportioning of the receptor states as well as the rates of several conversions. (i) The transformation of BF into BS was inhibited by GDP but not by guanylyl imidodiphosphate (GuaPP[NH]P) or GTP. (ii) The number of BS sites was lowered by GTP and GuaPP[NH]P. (iii) The binding to BSS was lowered by GTP and GDP, but increased by GuaPP[NH]P. (iv) The rate of disappearance of BSS was increased by GTP, but not by GuaPP[NH]P. Effects of guanine nucleotides were not observed after treatment of the membrane preparations with 15 mg/ml bovine serum albumin. This treatment caused the detection of a binding type different from the types described previously. The affinity of this binding site was extremely high (Kd ≤ 0.2 nM for N10-methylfolic acid), while the dissociation was relatively slow (k−1 ≤ 3·10−4 s−1). It is proposed that bovine serum albumin uncouples the folate receptor from a guanine nucleotide regulatory (G) protein in an irreversible manner. A model is presented in which the four receptor states correspond to distinct interactions with a G protein and GDP or GTP. 相似文献
4.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1988,970(3):343-354
Nucleotides such as GTP and GDP appear to be involved in signal transduction via G protein modulation of adenylate cyclase activity. Studies on direct binding of [3H]GDP to membranes prepared from cultured immature rat Sertoli cells indicated that this process was reversible, approached steady state within 10 min, had a Ka of 4.5 ·106M−1 and was specific for guanine nucleotides. The non-hydrolyzable analog, guanosine 5′-O-[3-thio]triphosphate (GPPP[S]), was most effective as an inhibitor of [3H]GDP binding (ED50 = 4.8·10−8M), whereas guanosine 5′-O-[2-thio]diphosphate (Gpp[S]) was less potent (ED50 = 3.4·10−7M). Release of bound GDP was enhanced by follitropin (FSH) in the presence of Gppp[S], although not by FSH alone. Sertoli cell membranes possess guanine nucleotide hydrolase activity, where 95% of added nucleotide was rapidly degraded to guanosine. Binding kinetics were significantly influenced by nucleotide metabolism, which was prevented by controlling the Mg2+ concentration with EDTA and including App[NH]p to reduce nonspecific hydrolysis. Kinetic studies indicated that Gpp[S] inhibited (P < 0.05) Gppp[S]-stimulated adenylate cyclase activity (Ki = 1.8·10−7M), whereas basal activity remained unaffected. Addition of Gpp[S] to pre-activated enzyme (FSH plus GTP) resulted in a time-dependent decay of adenylate cyclase activity with a Koff value of 6 ± 1·min−1. Using a two-stage pre-inculbation technique, adenylate cyclase activity was demonstrated to be sensitive to the nucleotide bound. When FSH was included, catalytic activity was not altered by the order of pre-incubation with the nucleotides. This suggested that the exchange of bound Gpp[S] for Gppp[S] was enhance by FSH. Activation and attenuation of FSH-sensitive adenylate cyclase activity is dependent on a nucleotide exchange mechanism which is driven by (1) the higher affinity of G for GTP than GDP, (2) enhanced release of GD when FSH is present and (3) GTP hydrolysis coupled to rapid metabolism of guanine nucleotides. 相似文献
5.
In vitro data showed that immunoglobulin G (IgG) from patients with lupus nephritis (LN) could bind to cultured human mesangial cells (HMC). The clinical relevance of such binding was unknown. Binding of IgG and subclasses was measured in 189 serial serum samples from 23 patients with Class III/IV±V LN (48 during renal flares, 141 during low level disease activity (LLDA)). 64 patients with non-lupus glomerular diseases (NLGD) and 23 healthy individuals were used as controls. HMC-binding was measured with cellular ELISA and expressed as OD index. HMC-binding index of total IgG was 0.12±0.09, 0.36±0.25, 0.59±0.37 and 0.74±0.42 in healthy subjects, NLGD, LN patients during LLDA, and LN flares respectively (P = 0.046, LN flare vs. LLDA; P<0.001, for healthy controls or NLGD vs. LN during flare or LLDA). Binding of serum IgG1 to HMC was 0.05±0.05, 0.15±0.11, 0.41±0.38 and 0.55±0.40 for the corresponding groups respectively (P = 0.007, LN flare vs. remission; P<0.001, for healthy controls or NLGD vs. LN during flare or remission). IgG2, IgG3 and IgG4 from patients and controls did not show significant binding to HMC. Total IgG and IgG1 HMC-binding index correlated with anti-dsDNA level (r = 0.26 and 0.39 respectively, P<0.001 for both), and inversely with C3 (r = −0.17 and −0.45, P<0.05 for both). Sensitivity/specificity of total IgG or IgG1 binding to HMC in predicting renal flares were 81.3%/39.7% (ROC AUC 0.61, P = 0.03) and 83.8%/41.8% (AUC 0.63, P = 0.009) respectively. HMC-binding by IgG1, but not total IgG, correlated with mesangial immune deposition in LN renal biopsies under electron microscopy. Our results showed that binding of serum total IgG and IgG1 in LN patients correlates with disease activity. The correlation between IgG1 HMC-binding and mesangial immune deposition suggests a potential pathogenic significance. 相似文献
6.
Ch.V. Rao 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,538(2):212-225
Preincubation of membranes with various concentrations of pronase, trypsin, lipase, phospholipase A from Vipera russelli and from Crotalus durissus terrificus, phospholipase C from Bacillus cereus and from Clostridium welchii, acetic anhydride, 2,4-dinitrofluorobenzene and tetranitromethane resulted in a dose-dependent inhibition of 125I-labeled human choriogonadotropin binding. At the submaximal concentrations of enzymes and at both submaximal and maximal concentrations of protein-modifying reagents, the losses were always greater with 125I-labeled human choriogonadotropin than with 125I-labeled human lutropin. The inhibition of binding was a consequence of changes in the membranes rather than changes in the hormone caused by the agents being carried over to the final incubation. Inhibition of binding was non-competitive and irreversible.In untreated membranes, the 125I-labeled human choriogonadotropin binding was homogenous (Kd = 1.7 · 10?10 M; N = 60 fmol/mg protein). Treatment of membranes with various enzymes and protein-modifying reagents except tetranitromethane resulted in heterogeneous binding. The number of available high affinity receptors was greatly reduced in every case. However, the affinity of these sites were either unchangedd (trypsin, lipase, phospholipase A from V. russelli, dinitrofluorobenzene and the tetranitromethane) or decreased (pronase and acetic anhydride). the newly appeared second receptor site had a Kd which varied from 3.2 · 10?10 to 7.1 · 10?9d M depending on the agent used, and the receptor numbers were low in all cases except acetic anhydride.Receptor occupancy conferred the receptors with marked protection against various hydrolytic enzymes, dinitrofluorobenzene and tetranitromethane. These data suggest the inhibition of binding by the above agents was primarily a consequence of changes in the receptor molecules themselves. 相似文献
7.
Migyeong Jo Sanghwan Ko Bora Hwang Sung-Won Min Ji Yeon Ha Ji Chul Lee Se-Eun Jang Sang Taek Jung 《Biotechnology and bioengineering》2020,117(8):i-i
The immunoglobulin G (IgG) molecule has a long circulating serum half-life (~3 weeks) through pH- dependent FcRn binding-mediated recycling. To hijack the intracellular trafficking and recycling mechanism of IgG as a way to extend serum persistence of non-antibody therapeutic proteins, we have evolved the ectodomain of a low-affinity human FcγRIIa for enhanced binding to the lower hinge and upper CH2 region of IgG, which is very far from the FcRn binding site (CH2–CH3 interface). High-throughput library screening enabled isolation of an FcγRIIa variant (2A45.1) with 32-fold increased binding affinity to human IgG1 Fc (equilibrium dissociation constant: 9.04 × 10−7 M for wild type FcγRIIa and 2.82 × 10−8 M for 2A45.1) and significantly improved affinity to mouse serum IgG compared to wild type human FcγRIIa. The in vivo pharmacokinetic profile of PD-L1 fused with engineered FcγRIIa (PD-L1–2A45.1) was compared with that of PD-L1 fused with wild type FcγRIIa (PD-L1–wild type FcγRIIa) and human PD-L1 in mice. PD-L1–2A45.1 showed 11.7- and 9.7-fold prolonged circulating half-life (t1/2) compared to PD-L1 when administered intravenously and intraperitoneally, respectively. In addition, the AUCinf of PD-L1–2A45.1 was two-fold higher compared to that of PD-L1–wild type FcγRIIa. These results demonstrate that engineered FcγRIIa fusion offers a novel and successful strategy for prolonging serum half-life of therapeutic proteins. 相似文献
8.
Fc and Fab Fragments from IgG<Subscript>2</Subscript> Human Immunoglobulins Characterized 总被引:1,自引:0,他引:1
Papain digestion of 7S immunoglobulin G (IgG) produces two 3.5S Fab fragments and one 3.5S Fc fragment1–8. The Fab fragment contains one light chain and one Fd fragment and is still able to combine specifically univalently with antigen. The Fc fragment is a dimer of the carboxyl terminal half of the heavy chain. Pepsin splits 7S IgG into some small peptides derived from Fc and one 5S F(ab′)2 fragment, which contains both antigen-binding sites. Based on this information, some investigators6,7 have postulated that pepsin splits the γ chains at the C-terminal side of the inter-heavy chain disulphide bridges, whereas papain splits at the N-terminal side of the inter-heavy chain disulphide bridges. We report here evidence that this model does not apply to all IgG subclasses. In the case of human IgG2 subclass myeloma proteins, papain splits initially at the C-terminal side of inter-heavy chain disulphide bridges. We also show that the amino-acid sequence of the Fc fragment of human IgG2 subclass so far determined has approximately 95% homology with that of human IgG1 and IgG4 subclasses reported by others9–15. 相似文献
9.
Krishna Balakrishnan Frank J. Hsu Dean G. Hafeman Harden M. McConnell 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1982,721(1):30-38
The isolation and characterization of a hybridoma cell line producing a monoclonal IgG1 antibody against a spin-label nitroxide group is described. The antibody recognizes a synthetic hapten containing linked dinitrophenyl and 2,2,6,6-tetramethylpiperidinyl 1-oxy groups, having an affinity of 3.6±1.0·106 M?1 for the soluble hapten at 25°C. The antibody binds to phospholipid vesicles containing 2 mol% of spin label-derivitized lipid (lipid hapten) with an affinity of 1.5±0.2·108 M?1. This monoclonal IgG1 mediates the binding of hapten-bearing lipid vesicles to mouse macrophage RAW264 cells bearing Fc receptors. The cellular responses to this binding are similar to those observed previously using polyclonal rabbit anti-hapten IgG. As with the heterogeneous antibodies, the monoclonal IgG1 is more efficient in mediating cellular uptake when the vesicles are in the ‘fluid’ physical state (dimyristoylphosphatidylcholine at 37°C) compared to ‘solid’ (dipalmitoylphosphatidylcholine at 37°C). Despite the enhanced binding of ‘fluid’ phospholipid vesicles to cells, only the ‘solid’ vesicles triggered a significant respiratory burst in RAW264 macrophages. 相似文献
10.
Olivier P. Dancette Jean-Luc Taboureau Eric Tournier Catherine Charcosset Pierre Blond 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1999,723(1-2)
An affinity membrane grafted with protein A/G or protein A was characterized for human and mouse immunoglobulins G purification. Breakthrough curves up to ligand saturation were measured and used to study the effects of flow velocities, feed solution concentrations and protein A/G versus protein A membranes. Increased flow-rate did not decrease the amount of IgG bound to the membranes. Increased feed solution concentration allowed more IgG to bind prior to breakthrough. Kinetic parameters for immunoglobulins G sorption to immobilized protein A were measured in batch experiments. The static binding capacity was determined to be 6.6 mg ml−1 membrane volume. Finally, this affinity membrane was used to purify IgG from cell culture supernatant. The electrophoresis of the purified IgG fractions did not show any contaminant. 相似文献
11.
Wendy A. Wallner Michael J. P. Lawman Michael D. P. Boyle 《Applied microbiology and biotechnology》1987,27(2):168-173
Summary The interaction of bovine immunoglobulins with staphylococcal Protein A and a group C streptococcal bacterial Fc receptor (FcRc) were compared. The isolated group C streptococcal receptor was reactive with both bovine IgG1 and IgG2. The reactivity of the streptococcal FcRc with IgG2 was approximately 40 fold greater than that observed with IgG1. By contrast, protein A reacted only poorly with bovine IgG2 and no detectable reactivity was observed with IgG1. A two stage competitive binding assay to measure bovine IgG in serum and secretions using 125I-labeled protein A as tracer was developed. This assay was found to be sensitive and reproducible and was used to measure serum IgG levels in cattle of differing ages and breeds. 相似文献
12.
《Biochimica et Biophysica Acta (BBA)/General Subjects》1987,926(3):359-364
By isoelectric focusing, transcobalamin from human cerebrospinal fluid was separated into the phenotypes X, M, MX, SX and MS. The corresponding plasma transcobalamins were of identical phenotypes. The unsaturated cobalamin-binding capacity in the cerebrospinal fluid was 0.12–0.54 nmol·l−1, median 0.23 nmol·l−1; no difference in binding capacity was found between the individual phenotypes. The isopeptides M, X and S bound cyano[57Co]cobalamin from pH 6 to 10. The apparent affinity constant was the same for all the isopeptides (0.4·1012 l·mol−1, pH 7.4. The isopeptide-cobalamin complexes bound to acceptors and human placenta membranes with an apparent affinity constant of 11·109 l·mol−1, pH 7.4. 相似文献
13.
Antisera to the LP and SP34 strains of polyoma virus have been prepared and their reactions with purified virions studied by double diffusion in agar and direct assay of antibody binding. One or more common antigenic determinants appear on the capsids of both strains. The form of this determinant varies slightly on each of the strains tested. The SP34 strain also carries its own strain-specific antigenic determinant. Both strains of virus were able to bind 150 anti-LP IgG3 molecules per virion and 50 anti-SP IgG molecules per virion. The slow rate of dissociation of bound IgG antibody (kdissociation = 2 × 10−6 s−1), and the rapid rate of antibody binding (kdissociation = 2 × 107m−1 s−1), suggest that IgG antibody is bound to the capsid surface through two antigen-antibody bonds. 50 anti-SP IgG molecules per capsid, divalently bound, completely inhibit the binding of 150 anti-LP IgG molecules, and vice versa. Consideration of the symmetry and molecular dimensions of the IgG molecule and the polyoma virus capsid leads to a model of the divalent interaction of IgG antibody with the common antigenic determinant(s). In this model, one species of antibody binds divalently to opposed subunits of a hexamer morphological unit. The other species of antibody binds divalently to the subunits on either side of the point of tangency of any two morphological units. 相似文献
14.
Jan Terje Andersen Muluneh Bekele Daba G?ril Berntzen Terje E. Michaelsen Inger Sandlie 《The Journal of biological chemistry》2010,285(7):4826-4836
The neonatal Fc receptor (FcRn) regulates the serum half-life of both IgG and albumin through a pH-dependent mechanism that involves salvage from intracellular degradation. Therapeutics and diagnostics built on IgG, Fc, and albumin fusions are frequently evaluated in rodents regarding biodistribution and pharmacokinetics. Thus, it is important to address cross-species ligand reactivity with FcRn, because in vivo testing of such molecules is done in the presence of competing murine ligands, both in wild type (WT) and human FcRn (hFcRn) transgenic mice. Here, binding studies were performed in vitro using enzyme-linked immunosorbent assay and surface plasmon resonance with recombinant soluble forms of human (shFcRnWT) and mouse (smFcRnWT) receptors. No binding of albumin from either species was observed at physiological pH to either receptor. At acidic pH, a 100-fold difference in binding affinity was observed. Specifically, smFcRnWT bound human serum albumin with a KD of ∼90 μm, whereas shFcRnWT bound mouse serum albumin with a KD of 0.8 μm. shFcRnWT ignored mouse IgG1, and smFcRnWT bound strongly to human IgG1. The latter pair also interacted at physiological pH with calculated affinity in the micromolar range. In all cases, binding of albumin and IgG from either species to both receptors were additive. Cross-species albumin binding differences could partly be explained by non-conserved amino acids found within the α2-domain of the receptor. Such distinct cross-species FcRn binding differences must be taken into consideration when IgG- and albumin-based therapeutics and diagnostics are evaluated in rodents for their pharmacokinetics. 相似文献
15.
Nicola L. Brown Stephen P. Bottomley Michael D. Scawen Michael G. Gore 《Molecular biotechnology》1998,10(1):9-16
The nonantigenic interaction between a recombinant immunoglobulin G (IgG)-binding protein based on the B domain of Protein
A fromStaphylococcus aureus (termed SpA1) and the Fc fragment of rabbit IgG has been investigated. The contribution to binding of four putative hydrogen bond contacts
between SpA1 and IgG-Fc were examined by the individual substitution of the residues in SpA1 involved in these interactions by others unable to form hydrogen bonds. It was found that the most important of the hydrogen
bonds involved Tyr 18 which, when replaced by Phe, resulted in a twofold decrease in IgG-binding affinity. The residues of
SpA1 proposed to make close, mainly hydrophobic, contacts with Fc were replaced by residues with potential electrostatic charge
to establish the importance of the hydrophobic interaction in the complex. The IgG-binding affinities of the mutant proteins
were compared to the wild-type protein by a competitive enzyme-linked immunosorbant assay. The replacement of individual hydrophobic
residues by His generated a number of novel IgG-binding proteins with reduced binding affinity at pH 5.0 but which maintained
strong binding affinities at pH 8.0. The elution profile of human IgG1-Fc (Fc fragment of human IgG1) from a column made from an immobilized two-domain mutant protein shows that the complex dissociates at a higher pH relative
to that of the non-mutated protein thus offering favorable elution characteristics. 相似文献
16.
Staphylococcus aureus Cowan I bacteria (SpA CoI) is known to be a polyclonal B-cell activator of human lymphocytes. In this study, we investigated which of the B-cell subsets SpA CoI could stimulate and induce immunoglobulin (Ig) production. B-Cell subsets were separated from peripheral blood and tonsil lymphocytes by rosette formation with E, EAIgG, EAC, anti-Ig-conjugated ox erythrocytes (OE-anti-Ig), and protein A-conjugated OE (OE-Pro A), or on a bovine serum albumin (BSA) discontinuous density gradient. The cells responding to SpA CoI included E?, C3 receptor-positive (C3R+), Fc receptor-negative (FcR?), and surface Ig-positive (SIg+) B-cell subsets. These B-cell populations responded well to SpA CoI and produced significant amounts of IgG, IgM, and a lesser amount of IgA. Among SIg+ B cells, IgG, IgA, and IgM+ B-cell subsets responded to SpA CoI and produced large amounts of Ig belonging to each corresponding Ig class. IgD+ B cells failed to produce Ig of any class, except for minimal amounts of IgG and IgM. While both the protein A receptor-positive (Pro A · R+) and negative (Pro A · R?) cells responded well to SpA CoI, Pro A · R+ B cells produced IgG mainly and Pro A · R? B cells produced IgM. Fractionation of B cells on a BSA gradient revealed that comparatively small-sized and denser B-cell subsets responded well to SpA CoI and produced every class of Ig. 相似文献
17.
《Plant science》1986,45(1):43-50
Maize shoot plasma membranes were prepared using either polyethyleneglycol (PEG)-dextran phase partition or centrifugation through a 30% sucrose cushion. The ATPase specific activity of membranes obtained with the phase partition method (1.4 μmol Pi · min−1 · mg−1 protein) was twice that of those prepared with the sucrose cushion method. After solubilization by lysolecithin and precipitation by ammonium sulfate, ATPase activities of the order of 3.0–3.5 μmol Pi · min−1 · mg−1 were obtained. A polypeptide of Mr = 90 000 was enriched during ATPase purification.Antibodies against pure plasma membrane ATPase from Saccharomyces cerevisiae inhibited the plant ATPase activity. Immunodetection during purification of the plant enzyme strongly supported the conclusion that the polypeptide of Mr = 90 000 belongs to plant plasma membrane ATPase. 相似文献
18.
The purpose of these studies was to determine the proportion of each immunoglobulin class/subclass in blood and colostrum of the pig and sheep, which would bind to staphylococcal Protein A. The concentrations of porcine IgG, IgM, and IgA were determined for serum and colostral whey from five sows. Similar measurements were made on two fractions produced by elution of the sample through a Protein A-Sepharose column: fraction 1, immunoglobulins which did not bind to Protein A, and fraction 2, immunoglobulins which bound to Protein A. The concentrations of ovine IgG1, IgG2, IgM, and IgA were measured for serum and colostral whey from six ewes, and again similar measurements were made after elution of each ovine sample through Protein A-Sepharose. All classes/subclasses of porcine and ovine serum and colostral immunoglobulins bound to Protein A to some extent. More than 90% of IgG from both porcine colostral whey and serum bound to Protein A. Ovine IgG1 from most ewes possessed a low affinity for Protein A whereas ovine IgG2 generally possessed a high affinity; 100% of the IgG2 in ovine colostral whey samples bound to Protein A. There was remarkable variation between individuals in the binding capacity of porcine IgM and each of the ovine immunoglobulins. For the ovine samples, in particular there were distinct differences between Protein A binding capacity of serum and colostral immunoglobulins of the same class/subclass. 相似文献
19.
Mariana Borsoi‐Ribeiro Igor Tadeu Lazzarotto Bresolin Mookambeswaran Vijayalakshmi Sônia Maria Alves Bueno 《Journal of molecular recognition : JMR》2013,26(10):514-520
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. 相似文献
20.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》1989,994(3):285-288
At pH 8.0 and 25°C α1-proteinase inhibitor and α2-macroglobulin bind human pancreatic elastase with rate constants of 4.7·105 M−1·s−1 and 6.4·106 M−1·s−1, respectively. The corresponding delay times of elastase inhibition in plasma are 0.4 s and 0.2 s, respectively, indicating that both inhibitors may act as physiological antielastases. Elastin impairs the elastase inhibitory capacity of α1-proteinase inhibitor and α2-macroglobulin. In presence of human elastin, the former behaves like a slow-binding elastase inhibitor, with a rate constant of about 260 M−1·s−1. In contrast, α2-macroglobulin is a fast-binding inhibitor of elastin-bound elastase, but only one of its two sites is functioning in presence of elastin. 相似文献