原子力显微镜研究小鼠免疫球蛋白G自组装

应用原子力显微镜观察小鼠免疫球蛋白G样品,结果不管是IgG1还是IgG1与IgG2的混合样品,在制样2-3d后,均发现有圆环状聚集区,也有部分圆面状聚集区域,一般环中心有核,核为IgG分子多聚体的再聚集,多聚体中亚基（单体）数目因环而异,在圆环和贺湎状区域外,也有零星分散的聚集体,不参与成环,另外,IgG1和IgG1 IgG2聚集情况有所不同,后者更倾向于形成圆面状聚集,另外,在聚集发生前,亚基已自组装形成各种形状的多聚体,然后多聚体进一步聚集形成各种圆环或圆面状,对免疫球蛋白G先自组装而后聚集的过程,从生物学,物理学角度作了初步解释。     

Sex effect on the correlation of immunoglobulin G glycosylation with rheumatoid arthritis disease activity

Rheumatoid arthritis (RA) is a chronic autoimmune disease which affects females more than males with a presence of autoantibodies. Immunoglobulin G (IgG) produced by adaptive arm has 2 functional domains, Fc and Fab. The Fc domain binds Fc gamma receptors and C1q proteins of the innate arm. Therefore, the IgG Fc domain serves as a bridge between the innate and adaptive arms and is regulated by an evolutionarily conserved N-glycosylation with variable structures. These glycans are classified as agalactosylated G0, monogalactosylated G1, and digalactosylated G2, which are further modified by core-fucosylation (F) and bisecting N-acetylglucosamine (B) moieties such as G0F and G0FB. Interestingly, proinflammatory G0F is shown to be regulated by estrogen in vivo. Here, it is hypothesized that the regulation of G0F by estrogen contributes to sex dichotomy in RA by setting up the level of IgG-dependent inflammation and therefore, RA disease activity (Das28-CRP3). To investigate this hypothesis, IgG glycosylation was characterized in serum samples from active RA patients (n = 232) and healthy controls (n = 232) by serum N-glycan analysis using the high performance liquid chromatography. According to the results, the IgG Fc glycan phenotype originates predominantly from the structure of G0F, and both G0F and G0FB correlate with Das28-CRP3 in females, but not in males. In conclusion, IgG G0F-dependent inflammation differs in males and females, and these differences point to the differential regulation of inflammation by sex hormone estrogen via IgG glycosylation.     

Picoplankton size distributions in marine and fresh waters: problems with filter fractionation studies

Abstract The retention of algal picoplankton by Nuclepore polycarbonate filters of 0.2, 1.0, 2.0 and 3.0 μm pore size was tested in 2 marine and 3 freshwater sites. When 1 μm Nuclepore filters were used, the percentage of the total cyanobacterial cells passing the filter varied between sites and with increasing depth within sites. As much as 99% of the Synechococcus -like cells was retained by a 1 μm filter. This could lead to an underestimation of the picoplanktonic contribution or, more seriously, an apparent distribution pattern that is an artifact of the choice of filter pore size. Filter retention was also dependent on vaccum pressure during filtration. This study emphasizes the need for direct observation of picoplankton numbers in filter fractionation studies.     

On the denaturation of enzymes in the process of foam fractionation

Experimental study on the denaturation of enzyme during the separation by foaming was conducted with trypsin and catalase in aqueous medium as model system respectively. The effects of operating pH and sparging gas composition on the denaturation of an enzyme were examined respectively. The oxidative deactivation of enzyme at the gas-liquid interface was identified, which could be reduced by applying nitrogen or carbon dioxide as sparging gas. At suitable conditions, the loss of enzyme activity can be reduced to less than 10% in case of trypsin and to zero in case of catalase. With its proven mildness and effectiveness, foam fractionation in a loop bubble column is applicable for recovery and concentration of enzymes from aqueous solutions.     

Online fluorescent dye detection method for the characterization of immunoglobulin G aggregation by size exclusion chromatography and asymmetrical flow field flow fractionation

The aim of this study was to develop an online fluorescent dye detection method suitable for high-pressure size exclusion chromatography (HP-SEC) and asymmetrical flow field flow fractionation (AF4). The noncovalent extrinsic fluorescent dye 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (Bis-ANS) was added to the mobile phase or the sample, and the fluorescence emission at 488 nm was recorded on excitation at 385 nm. By combining HP-SEC and AF4 with online dye detection, it was possible to simultaneously detect heat-induced aggregation and structural changes of monomeric and aggregated immunoglobulin G (IgG); an increase in Bis-ANS fluorescence was observed in both the aggregate and monomer fractions. These structural changes of individual fractions, which were not detectable by online UV and multiangle laser light scattering (MALLS) or by stand-alone dynamic light scattering (DLS), intrinsic IgG fluorescence, and far-UV circular dichroism (CD), resulted in progressive aggregation on storage. The developed online fluorescent dye detection for HP-SEC or AF4 with Bis-ANS is a powerful method to detect both aggregation and structural changes of both monomeric and aggregated IgG in heat-stressed formulations.     

A further fractionation of agarose

Hydrobiologia - Commercial agarose can be separated into two components by fractional precipitation using alcohols or glycols. The less soluble component has a much higher gel strength and lower...     

Electrochemical immune-biosensor for immunoglobulin G based bioelectrocatalytic reaction on micro-comb electrodes

A new amplification strategy of electrochemical signaling from antigen-antibody interactions was proposed via back-filling immobilization of horseradish peroxidase (HRP), immunoglobulin G antibodies (anti-IgG) and gold nanoparticles onto a three-dimensional sol-gel (3DSG)-functionalized biorecognition interface. The 3DSG sol-gel network was employed not only as a building block for the surface modification but also as a matrix for ligand functionalization. The signal-amplification was based on the bioelectrocatalytic reaction of the back-filling immobilization of HRP to H(2)O(2). With the non-competitive format, the formation of the antigen-antibody complex by a simple one-step immunoreaction between the immobilized anti-IgG and IgG in sample solution inhibited partly the active center of HRP, and decreased the immobilized HRP towards H(2)O(2) reduction. Under optimal conditions, the proposed immunosensor exhibited a good electrochemical behavior to IgG in a dynamic range of 1.12-162 ng/mL with a detection limit of 0.56 ng/mL (at 3delta). Moreover, the precision, reproducibility and stability of the as-prepared immunosensor were acceptable. Importantly, the proposed methodology would be valuable for diagnosis and monitoring of biomarkers and its metastasis.     

Structural changes of immunoglobulin G oligosaccharides with age in healthy human serum

Age-related changes of IgG N-linked oligosaccharides isolated from normal human serum are reported for 403 individuals (male 227 and female 176), varying in age from 0 to 85 years. The IgG N-linked oligosaccharides were released from the protein by digestion with a glycoamidase and reductively aminated with the fluorescent reagent, 2-aminopyridine. The mixture of pyridylaminated oligosaccharides was separated at high resolution by HPLC using a reverse-phase column. From the results of neutral oligosaccharide analysis, agalactosyl glycoform and bisecting GlcNAc-containing glycoform were shown to increase with increasing age. Spearman's correlation coefficients were 0.503 and 0.473, respectively. Thus, in healthy people, an increase of both types of glycoforms correlates weakly with age. In addition, differences were demonstrated between male and female groups in their twenties. The quantity of agalactosyl glycoform was found to be lower in females than in males. No significant differences, however, were observed in the quantity of bisecting GlcNAc-containing glycoforms between males and females. Abbreviations: Gal, D-galactose: GlcNAc, N-acetyl-D-glucosamine; Man, D-mannose; Fc, C-terminal half of the heavy chain dimers of IgG; HPLC, high-performance liquid chromatography; IgG, immunoglobulin G; ODS, octadecylsilyl; PA, pyridylamino This revised version was published online in November 2006 with corrections to the Cover Date.     

Purification of IgG and albumin from human plasma by aqueous two phase system fractionation

The current shortages in human plasma products at global levels justify the development of new, cost effective plasma fractionation methods. We have developed a fractionation process to obtain immunoglobulin G (IgG) and albumin‐enriched fractions based on polymer‐salt aqueous two phase system (ATPS). A small‐scale (0.02 L) ATPS composed of polyethyleneglycol 3350 (PEG), potassium phosphate and sodium chloride, at pH 6.1, was evaluated and subjected to 50‐fold scale‐up (1 L). Further purification of the fractions was performed using caprylic acid precipitation and ion exchange chromatography. Similar yield and purity were obtained at both small and large scales. IgG precipitated in the PEG rich upper phase at 83% recovery and 2.75‐fold purification factor. An 81% pure albumin fraction was obtained in the salt rich bottom phase with a 91% yield. After polishing, IgG was obtained at a recovery of 70% and a purity of 92%. Corresponding values for albumin were 91% and 90%. This IgG and albumin fractionation technology deserves further evaluation as it may represent a potential alternative to conventional plasma fractionation methods. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1005–1011, 2012     

Lectin analysis of human immunoglobulin G N-glycan sialylation

The lectins Sambucus nigraagglutinin (SNA) and Ricinus communisagglutinin (RCA), specific for 2,6 linked sialylation, and terminal galactose respectively were used to study the occurrence, linkage and distribution of human immunoglobulin G (IgG) sialylation. SNA was shown to bind N-glycan 2,6-linked sialic acid only. Sialidase analysis confirmed that this is the dominant, if not exclusive linkage. Total IgG sialylation was estimated at 1.0[emsp4 ]g SA/mg IgG (or about 0.5 mole per mole) using a biochemical sialic acid assay. SNA displayed strong binding to the IgG Fab fragment in both its native and denatured state. In contrast, SNA failed to bind the IgG Fc fragment in its native form, but displayed strong binding after the Fc was denatured. This allowed the construction of quantitative assays capable of measuring both IgG Fab and Fc 2,6-sialylation without the need for enzymatic peptide digestion.     

Immunodepletion of albumin and immunoglobulin G from bovine plasma

Current MS-based proteomics has facilitated the identification of large numbers of proteins from complex mixtures. The bovine plasma proteome has the potential to provide a wealth of information concerning the biological state of an animal. However, during MS-based experiments, higher abundance proteins such as albumin and immunoglobulin G (IgG) can hinder the identification of potentially important proteins that are present in much lower abundance. While a variety of readily available technologies exist for the depletion of multiple high-abundance proteins from human, mouse and rat samples, there are few available for bovine. In this study, we report the depletion of >97% of albumin and >92% of IgG from bovine plasma.     

Purification of animal immunoglobulin G (IgG) using peptoid affinity ligands

The availability of highly pure animal antibodies is critical in the production of diagnostic tools and biosensors. The peptoid PL16, previously isolated from an ensemble of peptoid variants of the IgG-binding peptide HWRGWV, was utilized in this work as affinity ligand on WorkBeads resin for the purification of immunoglobulin G (IgG) from a variety of mammalian sources and chicken immunoglobulin Y (IgY). The chromatographic protocol initially optimized for murine serum and ascites was subsequently employed for processing rabbit, goat and sheep, donkey, llama, and chicken sera. The PL16-WorkBeads resin proved able to recover all antibody targets with values of yield between 50 and 90%, and purity consistently above 90%. Notably, PL16 not only binds a broader spectrum of animal immunoglobulins than the reference ligands Protein A and G, but it also binds equally well with all their subclasses. Unlike the protein ligands, in fact, PL16 afforded excellent values of yield and purity of mammalian polyclonal IgG, namely murine (47 and 94%), rabbit (66.5 and 91.7%), caprine IgG (63 and 91–95%), donkey, and llama (93 and 97%), as well as chicken IgY (42 and 92%). Of notice, it is also the ability of PL16 to target monomeric IgG without binding aggregated IgG; when challenged with a mixture of monomeric and aggregated murine IgG, PL16 eluted <3% of fed aggregates, against 11–13% eluted by Protein A and G. Collectively, these results prove the potential of the proposed peptoid ligand for large-scale purification of animal immunoglobulins.     

High-throughput analysis of immunoglobulin G glycosylation

Introduction: Glycosylation of immunoglobulin G (IgG) is important for its effector functions and was shown to be related to age, sex and disease status of an individual. Adding glycomic information to genome-wide association studies (GWAS) and large clinical trials is enabling insight into the functional relevance of changes in glycosylation, as well as molecular mechanisms behind these changes. Large-scale studies require sensitive, robust and affordable high-throughput methodologies for glycosylation analysis, which are currently available in only a limited number of laboratories.

Areas covered: This review focuses on currently used high-throughput approaches for N-glycosylation analysis of IgG, as well as some recent advances in the areas of deglycosylation, trypsin digestion, labeling, purification, derivatization and automation of current workflows. Relevant literature was searched using the PubMed database.

Expert commentary: Development, optimization and validation of robust, affordable and simple high-throughput glycosylation analysis methods is essential for discovery and validation of diagnostic and prognostic glycan biomarkers. Although significant advances in glycosylation analysis have been made in recent years, currently used protocols will have to be further optimized to enable subsequent analysis of glycosylation on all levels with the limited initial sample and in the minimal amount of time, which is still a challenging task.     

Increased aggregation propensity of IgG2 subclass over IgG1: Role of conformational changes and covalent character in isolated aggregates

Aggregation of human therapeutic antibodies represents a significant hurdle to product development. In a test across multiple antibodies, it was observed that IgG1 antibodies aggregated less, on average, than IgG2 antibodies under physiological pH and mildly elevated temperature. This phenomenon was also observed for IgG1 and IgG2 subclasses of anti‐streptavidin, which shared 95% sequence identity but varied in interchain disulfide connectivity. To investigate the structural and covalent changes associated with greater aggregation in IgG2 subclasses, soluble aggregates from the two forms of anti‐streptavidin were isolated and characterized. Sedimentation velocity analytical ultracentrifugation (SV‐AUC) measurements confirmed that the aggregates were present in solution, and revealed that the IgG1 aggregate was composed of a predominant species, whereas the IgG2 aggregate was heterogeneous. Tertiary structural changes accompanied antibody aggregation as evidenced by greater ANS (8‐Anilino‐1‐naphthalene sulfonic acid) binding to the aggregates over monomer, and differences in disulfide character and tryptophan environments between monomer, oligomer and aggregate species, as observed by near‐UV circular dichroism (CD). Differences between subclasses were observed in the secondary structural changes that accompanied aggregation, particularly in the intermolecular β‐sheet and turn structures between the monomer and aggregate species. Free thiol determination showed ～2.4‐fold lower quantity of free cysteines in the IgG1 subclass, consistent with the 2.4‐fold reduction in aggregation of the IgG1 form when compared with IgG2 under these conditions. These observations suggested an important role for disulfide bond formation, as well as secondary and tertiary structural transitions, during antibody aggregation. Such degradations may be minimized using appropriate formulation conditions.     

Miniaturization of asymmetrical flow field-flow fractionation and application to studies on lipoprotein aggregation and fusion

Asymmetrical flow field-flow fractionation (AsFlFFF), a technique that provides direct measurement of particle size and diffusion coefficient, is converted into miniaturized scale. In comparison with conventional AsFlFFF, the separation of proteins in miniaturized AsFlFFF is achieved within shorter time periods, with smaller sample amounts, and with lower mobile phase consumption. Minimization of the overloading and optimization of the separation efficiency are prerequisites to good results. Miniaturized AsFlFFF is applied to the measurement of particle sizes of high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL). The average hydrodynamic diameters at pH 7.4 in 8.5mM phosphate buffer containing 1mM EDTA and 150 mM NaCl are 8.6+/-0.5, 11.2+/-0.2, 22.1+/-0.7, and 48.9+/-7.5 nm for subgroups HDL3, HDL2, LDL, and VLDL, respectively. In addition, the effect of different factors on the aggregation and fusion of LDL particles is studied. LDL particle sizes are unaffected by the addition of up to 300 mM NaCl and by an increase of the carrier solution pH from 3.2 to 7.4, but treatment of LDL with alpha-chymotrypsin, sphingomyelinase, or copper sulfate leads to the formation of aggregated and fused LDL particles.     

Oligosaccharide structures of immunoglobulin G from two patients with carbohydrate-deficient glycoprotein syndrome

The carbohydrate moiety of immunoglobulin G (IgG) from patients with carbohydrate-deficient glycoprotein (CDG) syndrome was analyzed. Galactosyl species were reduced in the reversed-phase chromatogram of pyridylaminated oligosaccharides as compared with child controls, and the hypogalactosylation was remarkable in a patient with typical manifestations. The abnormality was verified by composition analysis of the hydrolyzed monosaccharides from this patient, but the contents of mannose andN-acetylglucosamine were not reduced. Hypogalactosylation is the characteristic feature of IgG molecules in CDG syndrome, in contrast to the oligosaccharide deficiency of transferrin from the same patients. These findings suggest that the molecular phenotypes of different glycoproteins from patients with CDG syndrome are diverse.     

Separation of low molecular weight humic acids with Sephadex G25

Summary A method is pressented using Sephadex G25 to separate the smallest molecules from a humic acid sample. This procedure can also be used to select a certain molecular range.     

Temperature stability of proteins: Analysis of irreversible denaturation using isothermal calorimetry

The structural stability of proteins has been traditionally studied under conditions in which the folding/unfolding reaction is reversible, since thermodynamic parameters can only be determined under these conditions. Achieving reversibility conditions in temperature stability experiments has often required performing the experiments at acidic pH or other nonphysiological solvent conditions. With the rapid development of protein drugs, the fastest growing segment in the pharmaceutical industry, the need to evaluate protein stability under formulation conditions has acquired renewed urgency. Under formulation conditions and the required high protein concentration (～100 mg/mL), protein denaturation is irreversible and frequently coupled to aggregation and precipitation. In this article, we examine the thermal denaturation of hen egg white lysozyme (HEWL) under irreversible conditions and concentrations up to 100 mg/mL using several techniques, especially isothermal calorimetry which has been used to measure the enthalpy and kinetics of the unfolding and aggregation/precipitation at 12°C below the transition temperature measured by DSC. At those temperatures the rate of irreversible protein denaturation and aggregation of HEWL is measured to be on the order of 1 day^?1. Isothermal calorimetry appears a suitable technique to identify buffer formulation conditions that maximize the long term stability of protein drugs.     

Quantitative analysis of virus-like particle size and distribution by field-flow fractionation

Asymmetric flow field-flow fractionation (AFFFF) coupled with multiple-angle light scattering (MALS) is a powerful technique showing potential for the analysis of pharmaceutically-relevant virus-like particles (VLPs). A lack of published methods, and concerns that membrane adsorption during sample fractionation may cause sample aggregation, have limited widespread acceptance. Here we report a reliable optimized method for VLP analysis using AFFFF-MALS, and benchmark it against dynamic light scattering (DLS) and transmission electron microscopy (TEM). By comparing chemically identical VLPs having very different quaternary structure, sourced from both bacteria and insect cells, we show that optimized AFFFF analysis does not cause significant aggregation, and that accurate size and distribution information can be obtained for heterogeneous samples in a way not possible with TEM and DLS. Optimized AFFFF thus provides a quantitative way to monitor batch consistency for new vaccine products, and rapidly provides unique information on the whole population of particles within a sample.     

Detection of conformational changes in immunoglobulin G using isothermal titration calorimetry with low-molecular-weight probes

Proteins for therapeutic use may contain small amounts of partially misfolded monomeric precursors to postproduction aggregation. To detect these misfolded proteins in the presence of an excess of properly folded protein, fluorescent probes such as 8-anilino-1-naphthalene sulfonate (ANS) are commonly used. We investigated the possibility of using isothermal titration calorimetry (ITC) to improve the detection of this type of conformational change using hydrophobic probes. As a case study, conformational changes in human polyclonal immunoglobulin G (IgG) were monitored by measuring the enthalpies of binding of ANS using ITC. Results were compared with those using fluorescence spectroscopy. IgG heated at 63 °C was used as a model system for “damaged” IgG. Heat-treated IgG can be detected already at levels below 5% with both ITC and fluorescence. However, ITC allows a much wider molar probe-to-protein ratio to be sampled. In particular, using reverse titration experiments (allowing high probe-to-protein ratios not available to fluorescence spectroscopy), an increase in the number of binding sites with a K_d > 10 mM was observed for heat-treated IgG, reflecting subtle changes in structure. Both ITC and fluorescence spectroscopy showed low background signals for native IgG. The nature of the background signals was not clear from the fluorescence measurements. However, further analysis of the ITC background signals shows that a fraction (8%) binds ANS with a dissociation constant of approximately 0.2 mM. Measurements were also carried out at pH 4.5. Precipitation of IgG was induced by ANS at concentrations above 0.5 mM, interfering with the ITC measurements. Instead, with the nonfluorescent probes 4-amino-1-naphthalene sulfonate and 1-naphthalene sulfonate, no precipitation is observed. These probes yield differences in the enthalpies of binding to heated and nonheated IgG similar to ANS. The data illustrate that ITC with low-molecular-weight probes is a versatile tool to monitor conformational changes in proteins with a wider application potential than fluorescence measurements.