壳聚糖-纳米金复合膜修饰电化学发光免疫传感器检测人免疫球蛋白G

利用壳聚糖与纳米金良好的生物相容性及蛋白固定能力,制备了兼具导电性和透光性的人免疫球蛋白G(IgG)修饰膜,用于修饰玻碳电极,研制了新型电化学发光免疫传感器,并通过扫描电镜(SEM)及交流阻抗技术(EIS)考查了传感器表面性质.基于竞争免疫分析模式,以Ru(bpy)32+标记的羊抗人IgG为发光示踪物,采用新型共反应剂二丁基乙醇胺(DBAE)对光信号进行放大,建立了人IgG的检测方法,线性范围20ngmL-1～1.0μgmL-1,检测限6.5ngmL-1.将该电化学发光传感器应用于人血中IgG的检测,结果令人满意.     

Sensitive amplification immunoassay for human IgG and anti-human IgG by using an enzyme cascade system

A sensitive heterogeneous immunoassay for human IgG and anti-human IgG was developed using an enzyme cascade system in limulus amoebocyte as a signal amplification system. Lipopolysaccharide (LPS) was conjugated to human IgG and anti-human IgG was adsorbed on polystyrene beads. The LPS-labelled human IgG mixed with unlabelled human IgG was allowed to react in a competitive manner with the immobilized anti-IgG on the polystyrene bead surface. After B/F separation, the LPS activity in the supernatant (free) and LPS activity on the bead (bound) were measured by using the chromogenic limulus test. IgG could be measured in the range 10^?7-10^?11 g ml^?1. LPS-labelled anti-IgG and IgG absorbed on polystyrene beads were prepared, and LPS-labeled anti-IgG mixed with unlabelled anti-IgG was allowed to react again in a competitive manner with solid-phase IgG. The LPS activity specifically bound to the bead was then measured. Anti-IgG could be measured in the range 10^?7-10^?11 g ml^?1.     

Availability and development of an enzyme immunomicrosensor based on an ISFET for human immunoglobulins

A critical evaluation of the potentiometric response of an enzyme immuno-ISFET sensor has demonstrated that it is an effective, simple sensor for human immunoglobulin (IgG). The sensor was constructed using an immobilized human IgG membrane and an ISFET. The assay procedure involves the competitive immunochemical reaction of ureuse-labelled anti-human IgG with human IgG in samples and membrane-bound IgG and the electrochemical determination of membrane-bound urease activity. A linear relationship was obtained between the initial rate of response and the logarithm of IgG concentration from 0.1 to 2.0 mg ml^?1.     

Identification of kappa and lambda chains of the major immunoglobulin G subclasses by capillary zone electrophoresis

Immunoglobulins are present in most tissues and plasma and play crucial role in immune system. Alteration of the levels of the immunoglobulin G (IgG) subclasses (IgG1, IgG2, IgG3 and IgG4) is an indication of a disturbed immunological response. The aim of the present study was the development of a capillary electrophoresis (CE) method for the analysis of IgG subclasses in respect to their variable kappa and lambda chains. Various analytical conditions and CE modes, including capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF) and micellar electrokinetic capillary chromatography (MECC) have been thoroughly studied. CZE was found to be the most convenient way to separate IgG subclasses. Three of the human IgG subclasses were resolved using uncoated fused-silica and 50 mM phosphate, pH = 9.3, as operating buffer at 20 kV and detection at 214 nm. IgG1kappa was completely separated from IgG2kappa and IgG3kappa, whereas IgG2kappa co-migrated with IgG4kappa, which is the minor IgG subclass. Under the same conditions IgG4lambda was completely separated from IgG1lambda, IgG2lambda and IgG3lambda, enabling the identification of the various lambda chains. The developed CE method is rapid and can be applied to the identification of the major immunoglobulin G subclasses in respect to their variable kappa and lambda chains.     

Isolation of immunoglobulin G from bovine milk whey by poly(hydroxyethyl methacrylate)‐based anion‐exchange cryogel

Bovine milk whey contains several bioactive proteins such as α‐lactalbumin, β‐lactoglobulin, and immunoglobulin G (IgG). Chromatographic separation of these proteins has received much attention in the past few years. In this work, we provide a chromatographic method for the efficient isolation of IgG from bovine milk whey using a poly(2‐hydroxyethyl methacrylate)‐based anion‐exchange cryogel. The monolithic cryogel was prepared by grafting 2‐(dimethylamino) ethyl methacrylate onto the poly(2‐hydroxyethyl methacrylate)‐based cryogel matrix and then employed to separate IgG under various buffer pH and salt elution conditions. The results showed that the buffer pH and the salt concentration in the step elution have remarkable influences on the purity of IgG, while the IgG recovery depended mainly on the loading volume of whey for a given cryogel bed. High purity IgG (more than 95%) was obtained using the phosphate buffer with pH of 5.8 as the running buffer and the salt solution in as the elution liquid. With suitable loading volume of whey, the maximum IgG recovery of about 94% was observed. The present separation method is thus a potential choice for the isolation of high‐purity IgG from bovine milk whey.     

Surfactant-aided size-exclusion chromatography for the purification of immunoglobulin G

In the production of monoclonal antibodies, separate chains of the antibody are often present in the product mixture as well as other contaminating proteins. These fragments should be removed from the whole antibodies. This paper shows the purification of monoclonal immunoglobulin G (IgG) from its heavy chain contaminant. The heavy chain fragment is simulated experimentally using bovine serum albumin (BSA), which has approximately the same molecular weight. The purification is performed using traditional size-exclusion chromatography (SEC) and using surfactant-aided SEC (SASEC), testing two different surfactants (C(12)E(23) and Tween20) and two different gels (Sephacryl S200HR and Sephacryl S300 HR). Pulse experiments show that with SASEC both BSA and IgG are more distributed towards the solid phase than compared to using SEC. This effect is larger on IgG, the largest component than on BSA. As a consequence, azeotropes will be formed at a specific surfactant concentration. Above this concentration the selectivity is reversed and increased to values higher than obtained with conventional SEC. At 7.5% (w/w) of C(12)E(23), BSA actually elutes before IgG. These experiments further show that when using SASEC larger productivity, higher yields and lower solvent consumption can be achieved without loss of purity of IgG when compared to conventional SEC. Mathematical simulation of the separation of BSA and IgG using simulated moving bed (SMB) chromatography indicates a large increase in productivity when applying a surfactant gradient in SASEC SMB compared to conventional isocratic SEC-SMB. Furthermore, solvent consumption reductions with a factor 15 prove possible as well as concentrating the IgG by a factor 2.     

Scanning force microscopy and fluorescence microscopy of microcontact printed antibodies and antibody fragments

Unlabeled primary immunoglobulin G (IgG) antibodies and its F(ab')2 and Fc fragments were attached to oxygen-plasma-cleaned glass substrates using either microcontact printing (MCP) or physical adsorption during bath application from dilute solutions. Fluorescently labeled secondary IgGs were then bound to surface-immobilized IgG, and the relative surface coverage was determined by measuring the fluorescence intensity. Results indicated that the surface coverage of IgG increased with increasing protein solution concentration for both MCP and bath-applied IgG and that a greater concentration of IgG was transferred to a glass substrate using MCP than during physisorption during bath applications. Scanning force microscopy (SFM) showed that patterned MCP IgG monolayers were 5 nm in height, indicating that IgG molecules lie flat on the substrate. After incubation with a secondary IgG, the overall line thickness increased to around 15 nm, indicating that the secondary IgG was in a more vertical orientation with respect to the substrate. The surface roughness of these MCP patterned IgG bilayers as measured by SFM was observed to increase with increasing surface coverage. Physisorption of IgG to both unmodified patterned polydimethylsiloxane (PDMS) stamps and plasma-cleaned glass substrates was modeled by Langmuir adsorption kinetics yielding IgG binding constants of K(MCP) = 1.7(2) x 10(7) M(-1) and K(bath) = 7.8(7) x 10(5) M(-1), respectively. MCP experiments involving primary F(ab')2 and Fc fragments incubated in fluorescently labeled fragment-specific secondary IgGs were carried out to test for the function and orientation of IgG. Finally, possible origins of MCP stamping defects such as pits, pull outs, droplets, and reverse protein transfer are discussed.     

Depletion of high-abundance proteins from human plasma using a combination of an affinity and pseudo-affinity column

Human serum albumin (HSA) and immunoglobulin G (IgG) represent over 75% of all proteins present in human plasma. These high-abundance proteins prevent the detection of low-abundance proteins which are potential markers for various diseases. The depletion of HSA and IgG is therefore essential for further proteome analysis. In this paper we describe the optimization of conditions for selective depletion of HSA and IgG using affinity and pseudo-affinity chromatography. A BIA Separations CIM (convective interaction media) Protein G disk was applied for the removal of IgG and the Mimetic Blue SA A6XL stationary phase for the removal of HSA. The binding and the elution buffer for CIM Protein G disk were chosen on the basis of the peak shape. The dynamic binding capacity was determined. It was shown to be dependent on the buffer system used and independent of the flow rate and of the concentration of IgG. Beside the binding capacity for the IgG standard, the binding capacity was also determined for IgG in human plasma. The Mimetic Blue SA A6XL column was characterized using human plasma. The selectivity of the depletion was dependent on the amount of human plasma that was loaded on the column. After the conditions on both supports had been optimized, the Mimetic Blue SA A6XL stationary phase was combined with the CIM Protein G disk in order to simultaneously deplete samples of human plasma. A centrifuge spin column that enables the removal of IgG and HSA from 20 μL of human plasma was designed. The results of the depletion were examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis.     

Affinity partitioning of human antibodies in aqueous two-phase systems

The partitioning of human immunoglobulin (IgG) in a polymer-polymer and polymer-salt aqueous two-phase system (ATPS) in the presence of several functionalised polyethylene glycols (PEGs) was studied. As a first approach, the partition studies were performed with pure IgG using systems in which the target protein remained in the bottom phase when the non-functionalised systems were tested. The effect of increasing functionalised PEG concentration and the type of ligand were studied. Afterwards, selectivity studies were performed with the most successful ligands first by using systems containing pure proteins and an artificial mixture of proteins and, subsequently, with systems containing a Chinese hamster ovary (CHO) cells supernatant. The PEG/phosphate ATPS was not suitable for the affinity partitioning of IgG. In the PEG/dextran ATPS, the diglutaric acid functionalised PEGs (PEG-COOH) displayed great affinity to IgG, and all IgG could be recovered in the top phase when 20% (w/w) of PEG 150-COOH and 40% (w/w) PEG 3350-COOH were used. The selectivity of these functionalised PEGs was evaluated using an artificial mixture of proteins, and PEG 3350-COOH did not show affinity to IgG in the presence of typical serum proteins such as human serum albumin and myoglobin, while in systems with PEG 150-COOH, IgG could be recovered with a yield of 91%. The best purification of IgG from the CHO cells supernatant was then achieved in a PEG/dextran ATPS in the presence of PEG 150-COOH with a recovery yield of 93%, a purification factor of 1.9 and a selectivity to IgG of 11. When this functionalised PEG was added to the ATPS, a 60-fold increase in selectivity was observed when compared to the non-functionalised systems.     

Study of the separation of mouse monoclonal antibodies by pseudobioaffinity chromatography using matrix-linked histidine and histamine.

The selective retention of proteins on matrix-linked histidine has been shown to depend on chromatographic conditions: pH, temperature and ionic strength. An extension of this study to separate mouse monoclonal antibodies on histidyl-Sepharose is presented here; the roles of different functional groups such as imidazole, primary amine and carboxyl groups are elucidated by using histamine-Sepharose and histidine linked via the carboxyl group of the alpha-amino acid. We separated two monoclonal antibodies, immunoglobulin G1 (IgG1) from a culture supernatant and IgG2b from ascites fluid precipitated with 50% ammonium sulphate. The pseudoselective retention of monoclonal IgG1 on the three different matrices and IgG2b on histidyl-aminohexyl-Sepharose was achieved at pH 7.4. The purity of the final monoclonal antibody preparation determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing conditions proved the separation of the monoclonal antibodies (IgG1, IgG2b) from other contaminating proteins such as albumin and transferrin. Quantitation of the mouse monoclonal antibodies was carried out using enzyme-linked immunosorbent assay.     

A simple method for determination of the concentration of anti-dextran IgG in antiserum by means of affinity electrophoresis

A simple technique for determination of the concentration of anti-dextran IgG in antiserum using affinity electrophoresis is described. In the presence of an excess of intermediary molecular size dextran in the polyacrylamide gel, polyclonal anti-dextran IgG migrated in a single sharp band, separated from the nonspecific IgG fraction and other serum protein fractions. With this technique, 1-10 micrograms anti-dextran IgG in antisera can be determined within 3 h. We call the procedure ligand saturating affinity electrophoresis.     

SERS detection of protein biochip fabricated by etching polystyrene template

In this study, a nanoscale protein chip is prepared by using an etched polystyrene (PS) template. This protein chip can be directly used for immunoassay, with the help of Surface Enhanced Raman Scattering (SERS) spectra. Some glass slides submerged in aldehyde is initially prepared, modified with antibodies, human immunoglobulin G (IgG). Then PS arrays are self-assembled on these slides with the Langmuir–Blodgett method. The PS template pattern is transferred to the human IgG substrate using an etching process—slides are exposed to O₂ plasma for 90 s. The PS nanoparticles are then washed away using phosphate buffered saline solution. Next, the slides are dipped into bovine serum albumin solution to ensure that the anti IgG would bond only to the human IgG. At this moment, a patterned protein chip is obtained. When used for protein detection, the protein chip could be immersed into labeled specificity antigen solution. Here we chose fluorescein isothiocyanate anti-human IgG. After washing, only bonded antigens remain. Fluorescence microscopy and SERS is used to characterize the samples. The SERS spectra intensity shows liner correlation with the concentration of anti-human IgG. All the experiments are conducted in a phosphate buffered saline solution at 37 °C for 2 h.     

Purification of human immunoglobulin G by thermoseparating aqueous two-phase systems

The purification of human immunoglobulin G (IgG) from a Chinese hamster ovary (CHO) cells supernatant was studied using an aqueous two-phase system (ATPS) composed of ethylene oxide/propylene oxide (UCON) and dextran. In UCON/dextran systems IgG partitions preferentially to the less hydrophobic dextran-rich phase (Kp<1). The addition of triethylene glycol-diglutaric acid (TEG-COOH) shifted the IgG partition into the upper phase showing significant improvements in both the recovery yields and purity. The purification of IgG from a CHO cell supernatant with UCON 2000/dextran/TEG-COOH system was optimised using a central composite design. Using an ATPS composed of 8% UCON, 6% dextran and 20% TEG-COOH, IgG was purified, in two steps, with a global yield of 85% and 88% purity. Statistical valid models were obtained to predict the effect of the experimental conditions on the IgG yield and purity, for both extraction and back-extraction steps. A system composed of 10% UCON, 5.5% dextran and 20% TEG-COOH was identified as the best compromise between final purity and yield.     

Application of central composite design to the optimisation of aqueous two-phase extraction of human antibodies

The partition of human antibodies in aqueous two-phase systems (ATPSs) of polyethylene glycol (PEG) and phosphate was systematically studied using first pure proteins systems and then an artificial mixture of proteins containing 1mg/ml human immunoglobulin G (IgG), 10mg/ml serum albumin and 2mg/ml myoglobin. Preliminary results obtained using pure proteins systems indicated that the PEG molecular weight and concentration, the pH value and the salts concentration had a pronounced effect on the partitioning behaviour of all proteins. For high ionic strengths and pH values higher than the isoelectric point (pI) of the contaminant proteins, IgG could be selectively recovered on the top phase. According to these results, a face centred composite design was performed in order to optimise the purification of IgG from the mixture of proteins. The optimal conditions for the isolation of IgG were observed for high concentrations of NaCl and low concentrations of both phase forming components. The best purification was achieved using an ATPS containing 8% (w/w) PEG 3350, 10% (w/w) phosphate pH 6 and 15% (w/w) NaCl. A recovery yield of 101+/-7%, a purity of 99+/-0% and a yield of native IgG of 97+/-4% were obtained. Back extraction studies of IgG to a new phosphate phase were performed and higher yields were obtained using 10% phosphate buffer at pH 6. The total extraction yield was 76% and the purity 100%.     

A synthetic trivalent hapten that aggregates anti-2,4-DNP IgG into bicyclic trimers

This paper describes the synthesis of the trivalent hapten molecule 1, containing three 2,4-dinitrophenyl (2,4-DNP) groups, and the use of this molecule to aggregate three molecules of anti-2,4-DNP IgG into a complex with 3:2 stoichiometry (IgG312). The equilibrium product IgG312 was generated in approximately 90% yield upon mixing IgG and 1; during incubation, thermodynamically unstable, high-molecular-weight aggregates (>104 nm in diameter) form first and convert subsequently to IgG312. The thermodynamics and the kinetics of the formation of aggregates were studied using size-exclusion high-performance liquid chromatography (SE-HPLC), dynamic light scattering (DLS), and analytical ultracentrifugation (AUC). An analytical model based on multiple species in equilibrium was developed and used to interpret the SE-HPLC data. The aggregate IgG312 was more stable thermodynamically and kinetically than monomeric aggregates of this IgG with monomeric derivatives of 2,4-DNP; this stability suggests potential applications of these aggregates in biotechnology.     

聚乙二醇对人血清IgG的化学修饰及修饰后性质的研究

本文以三聚氯氰为活化剂，单甲氧基聚乙二醇（ＰＥＧ）５０００为载体，对人血清免疫球蛋白Ｇ（ＩｇＧ）进行化学修饰，修饰ＩｇＧ与未修饰ＩｇＧ比较，随修饰度的增高，分子量增加，保留活性降低，对温度及ｐＨ的耐受性增加，抗原性及免疫原性明显下降，ＩｇＧ的骨架结构在修饰前后无明显变化。     

Absolute Quantitation of Glycoforms of Two Human IgG Subclasses Using Synthetic Fc Peptides and Glycopeptides

Immunoglobulins, such as immunoglobulin G (IgG), are of prime importance in the immune system. Polyclonal human IgG comprises four subclasses, of which IgG1 and IgG2 are the most abundant in healthy individuals. In an effort to develop an absolute MALDI-ToF-MS quantitative method for these subclasses and their Fc N-glycoforms, (glyco)peptides were synthesized using a solid-phase approach and used as internal standards. Tryptic digest glycopeptides from monoclonal IgG1 and IgG2 samples were first quantified using EEQYN(GlcNAc)STYR and EEQFN(GlcNAc)STFR standards, respectively. For IgG1, a similar glycopeptide where tyrosine (Y) was isotopically labelled was used to quantify monoclonal IgG1 that had been treated with the enzyme Endo-F2, i.e., yielding tryptic glycopeptide EEQYN(GlcNAc)STYR. The next step was to quantify single subclasses within polyclonal human IgG samples. Although ion abundances in the MALDI spectra often showed higher signals for IgG2 than IgG1, depending on the spotting solvent used, determination of amounts using the newly developed quantitative method allowed to obtain accurate concentrations where IgG1 species were predominant. It was observed that simultaneous analysis of IgG1 and IgG2 yielded non-quantitative results and that more success was obtained when subclasses were quantified one by one. More experiments served to assess the respective extraction and ionization efficiencies of EEQYNSTYR/EEQFNSTFR and EEQYN(GlcNAc)STYR/EEQFN(GlcNAc)STFR mixtures under different solvent and concentration conditions.

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A method of printing uniform protein lines by using flat PDMS stamps

In this paper, we report a method of printing uniform protein lines on glass slides by using UV-treated flat PDMS stamps. Unlike traditional microcontact printing (μCP) which requires microstructured PDMS stamps, this μCP method only requires a flat PDMS stamp, an UV lamp and a number of straight needles. Our results show that lines of bovine serum albumin (BSA), immunoglobin (IgG), anti-biotin, anti-human IgG and anti-mouse IgG can be printed evenly on glass slides by using this μCP method. We also demonstrate that the printed protein lines are suitable for applications such as microfluidic immunoassays.     

Bead injection for biomolecular assays: Affinity chromatography enhanced by bead injection spectroscopy

Selective capture of target biomolecules by ligands immobilized on a solid support is a cornerstone of two seemingly unrelated techniques: micro-Affinity Chromatography (microAC) and micro-Bead Injection Spectroscopy (microBIS). This work shows, for the first time, how these techniques can be carried out using the same instrument and how the data obtained this way complement each other, yielding complete information on retention and elution of target biomolecules. Biomolecular association and dissociation were investigated by microAC and microBIS, using computer-controlled programmable flow and the same instrument for automated bead transport, packing of a micro-column, assay of the analyte, and bead disposal. The absorbance of the analyte was monitored within the fiber optic flow cell configured either for monitoring directly on the beads or post-column after elution. The separation, binding, and elution of immunoglobulins (human IgG, rabbit IgG, and horse IgG) on protein G-coated Sepharose beads were studied as model systems. The limit of detection of the microAC technique was determined to be 5 ng microL(-1) IgG, and that of the microBIS technique was 50 ng microL(-1) IgG.     

Performance of hexamer peptide ligands for affinity purification of immunoglobulin G from commercial cell culture media

Previous work has reported on the identification and characterization of the hexapeptide ligands HWRGWV, HYFKFD, and HFRRHL for the affinity capture of IgG through specific binding to its Fc fragment. This paper addresses issues related to the successful application of these ligands, on a commercial methacrylate chromatographic resin, for the purification of IgG from mammalian cell culture fluids. The concentrations of sodium chloride and sodium caprylate in the binding buffer were optimized to maximize the purity and yield of IgG upon elution. Screening of several regeneration conditions found that either 2M guanidine-HCl or a combination of 0.85% phosphoric acid followed by 2M urea resulted in complete recovery of the IgG adsorption capacity and that the column could be reused over many cycles. The hexapeptide ligands were used for the purification of humanized and chimeric monoclonal antibodies from two commercial CHO cell culture fluids. The chimeric MAb of IgG1 subclass was purified using the HWRGWV resin whereas the humanized MAb of IgG4 subclass was purified using the HWRGWV, HYFKFD and HFRRHL resins. The purities and yields obtained for both the MAbs were found to be higher than 94% and 85% respectively. These results compare well with the yields and purities obtained using Protein G columns. The residual DNA and host cell protein reduction obtained by the HWRGWV resin was in the range of 4 log reduction value (LRV) and 2 LRV respectively, comparable to those reported for Protein A resins. The dynamic binding capacity of all three peptide resins for the humanized monoclonal antibody was in the range of 20mg/mL.