Surface-modified membranes as a matrix for protein purification.

Recently introduced membrane-based chromatographic supports for protein separation are available either with a coupled ligand, e.g., protein A, protein G or ion-exchange groups, or as activated matrices for coupling a desired ligand. The coupling conditions for protein A and immunoglobulin G to an epoxy-activated membrane were determined. The performance of the prepared affinity membranes was investigated using pure rabbit immunoglobulin G and protein A as a model system. For practical application monoclonal antibodies from cell culture supernatant were purified with a prepared protein A membrane and for comparison with a sulphonic acid ion exchange membrane.     

Protein G-liposomal nanovesicles as universal reagents for immunoassays

To improve the antigen-binding activity of liposome-coupled antibodies and to develop universal liposomal nanovesicles for immunoassays, protein G was conjugated to dye-loaded liposomal nanovesicles for the preparation of immunoliposomes. Sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC), a heterobifunctional cross-linker, was used to modify protein G for conjugation to the liposomal nanovesicles. Liposome immunosorbent assays were used to evaluate the binding ability of protein G after sulfo-SMCC modification, to optimize the protein G density on the liposome surface and to determine the amount of IgG binding to the protein G-liposomal nanovesicles. Test strips coated with a narrow zone of antibodies were used to show the successful conjugation. Immunomagnetic beads were used to demonstrate the feasibility of protein G-tagged universal liposomal nanovesicles for immunoassays. Results indicate that the Fc-binding capacity of protein G decreased by only 5.3% after sulfo-SMCC modification. Antibodies were easily conjugated to universal protein G-liposomal nanovesicles in 30 min. The conjugates (protein G-immunoliposomes) were successfully used in immunomagnetic bead assays for the detection of Escherichia coli O157:H7 with a detection limit of approximately 100 CFU/ml. This work demonstrated that protein G-liposomal nanovesicles are a successful universal reagent for easily coupling antibodies in an active orientation on the liposome surface for use in immunoassays.     

AFM imaging of ALYGNSA polymer–protein surfaces: evidence of antibody orientation

Previous investigations found the combination of recombinant bacterial protein G (rProG) and poly(methyl methacrylate) (PMMA) to produce a greater proportion of oriented antibodies. PMMA–rProG yielded a sixfold greater availability of antibody Fab regions compared with other bacterial affinity linker protein and polymer pairings, including commercially available polystyrene (PS) high-binding 96-well microplates. Given the name ALYGNSA, the PMMA–rProG combination was developed into a fluorescence assay and evaluated in conjunction with commercially available cancer biomarker enzyme-linked immunosorbent assays (ELISAs). In each study, a lower limit of detection was seen with the ALYGNSA assay. The purpose of this investigation was to examine the ALYGNSA substrate in contrast with a commonly used ELISA substrate and analyze the affinity-immobilized antibodies for additional evidence of orientation. Non-contact atomic force microscopy is a logical method as it operates in ambient conditions, can be used directly on biological samples without modification, and offers the resolution necessary to identify the position of the antibody on the surface. Dynamic contact angle studies were employed to examine untreated PMMA and PS samples and revealed important differences in their surface characters. Comparative height threshold grain analysis of the prepared ALYGNSA surface, a similarly treated mica surface, and a gold colloid sizing standard evaluated and confirmed the antibody orientation of the ALYGNSA system.     

Effect of the conserved oligosaccharides of recombinant monoclonal antibodies on the separation by protein A and protein G chromatography

Glycosylation of the conserved asparagine residue in CH2 domains of IgG molecules is an important post-translational modification. The presence of oligosaccharides is critical for structure, stability and biological function of IgG antibodies. Effect of the glycosylation states of recombinant monoclonal antibodies on protein A and protein G chromatography was evaluated. Antibodies lacking oligosaccharides eluted later from protein A and earlier from protein G columns than antibodies with oligosaccharides using a gradient of decreasing pH. Interestingly, different types of oligosaccharides also affected the elution of the antibodies. Antibodies with high mannose type oligosaccharides were enriched in later eluting fractions from protein A and earlier eluting fractions from protein G. While antibodies with more mature oligosaccharides, such as core fucosylated biantennary complex oligosaccharides with zero (Gal 0), one (Gal 1) or two (Gal 2) terminal galactoses, were enriched in earlier eluting fractions from protein A and in the later eluting fractions from protein G. However, analysis by enzyme-linked immunosorbent assay (ELISA) revealed that antibody binding affinity to protein A and protein G was not affected by the absence or presence of oligosaccharides. It was thus concluded that the elution difference of antibodies with or without oligosaccharides and antibodies with different types of oligosaccharides were due to differential structural changes around the CH2–CH3 domain interface under the low pH conditions used for protein A and protein G chromatography.     

Comparative Assessment of Oriented Antibody Immobilization on Surface Plasmon Resonance Biosensing

Protein A and protein G are extremely useful molecules for the immobilization of antibodies. However, there are limited comparative reports available to evaluate their immobilization performance for use as biosensors. In this study, a comparative analysis was made of approaches that use protein A and protein G for avian leukosis virus detection. The antibody‐protein binding affinities were determined using surface plasmon resonance (SPR) analysis. The immobilization efficiency was obtained by calculating the number of the protein molecular binding sites. The positive influence of sensor response on antigen detection indicates that the amount of immobilized antibody plays a major role in the extent of immobilization. Moreover, the biosensors constructed using both proteins were found to be regenerative. The SPR results from this study suggest that the surfaces of protein G provide a better equilibrium constant and binding efficacy for immobilized antibodies, resulting in enhanced antigen detection.     

Construction of a functional IgG-binding luciferase fusion protein for the rapid detection of specific bacterial strains

A fusion protein consisting of two IgG-binding domains of streptococcal protein G and firefly luciferase was constructed, and a simple and specific bioluminescent immunodetection system for bacterial strains was developed.     

Genetic expression of an amyloid peptide fragment and analysis of formylated products

The model amyloid peptide AAKLVFF was expressed as a His-tagged fusion protein with the immunoglobulin-binding domain B1 of streptococcal protein G (GB1), a small (56 residues), stable, single-domain protein. It is shown that expression of this model amyloid peptide is possible and is not hindered by aggregation. Formylation side reactions during the CNBr cleavage are investigated via synthesis of selectively formylated peptides.     

Surface‐Independent and Oriented Immobilization of Antibody via One‐Step Polydopamine/Protein G Coating: Application to Influenza Virus Immunoassay

For the construction of high‐performance biosensor, it is important to interface bioreceptors with the sensor surface densely and in the optimal orientation. Herein, a simple surface modification method that can optimally immobilize antibodies onto various kinds of surfaces is reported. For the surface modification, a mixture of polydopamine (PDA) and protein G was employed. PDA is a representative mussel‐inspired polymer, and protein G is an immunoglobulin‐binding protein that enables an antibody to have an optimal orientation. The surface characteristics of PDA/Protein G mixture‐coated substrates are analyzed and the PDA/protein G ratio is optimized to maximize the antibody binding efficiency. Moreover, the antibody‐immobilized substrates are applied to the detection of influenza viruses with the naked eye, providing a detection limit of 2.9 × 10³ pfu mL^‐1. Importantly, the several substrates (glass, SiO₂, Si, Al₂O₃, polyethylene terephthalate, polyethylene, polypropylene, and paper) can be modified by simple incubation with the mixture of PDA/protein G, and then the anti‐influenza A H1N1 antibodies can be immobilized on the substrates successfully. Regardless of the substrate, the influenza viruses are detectable after the sandwich immunoreaction and silver enhancement procedure. It is anticipated that the developed PDA/protein G coating method will extend the range of applicable materials for biosensing.     

特异性吗啡抗原和单克隆抗体的制备

吗啡分子的不同部位与蛋白偶联诱导出的抗体的专一性差异很大。为减少交叉反应,提高抗体分子对游离吗啡的特异性识别,选择吗啡分子N位进行修饰,合成了半抗原降吗啡,并设计和合成不同的连接臂,将半抗原用不同的连接臂与不同的载体蛋白共价结合分别制备了免疫抗原和筛选抗原,经细胞融合和筛选,成功地获得了5株可分汔抗吗啡单克隆抗体的细胞株：28H10,29D5,36G3,42D5,43C4。     

基于自动磁珠转运的转基因蛋白Cry1Ab检测

基于自动磁珠转运,建立了转基因蛋白Cry1Ab免疫检测的新方法.利用水热法制备了粒径约400 nm的纳米磁球,并进行电镜表征,通过溶胶法对磁球表面进行氨基修饰,采用戊二醛偶联对磁珠实现抗体包被,在核酸提取仪中进行酶联免疫反应,采用分光光度法进行检测.本方法对转基因蛋白Cry1Ab的检出限低于1 μg/L,与商品化酶联免...     

The potential of magnetic nanocluster and dual-functional protein-based strategy for noninvasive detection of HBV surface antibodies

Magnetic nanoclusters (MNCs) were synthesized in a one-pot process, carboxylic MNCs and dual-functional protein were prepared and used to capture hepatitis B virus surface antibodies (anti-HBs) in simulated diseased oral mucosal transudate (OMT) samples. The specific substrate of dual-functional protein, dual-labeled double-chained DNA molecules, based on Fluorescence Resonance Energy Transfer (FRET), was used to amplify the detection signal and the detection limit of 0.1 ng mL(-1) of anti-HBs monoclonal antibodies was achieved. Combination MNCs with dual-functional protein enables the noninvasive detection of hepatitis B virus (HBV) surface antibodies in OMT samples, showing promise as a diagnostic tool for the OMT diagnosis of infectious diseases with sensitive, specific and facile capabilities.     

Site-specific dense immobilization of antibody fragments on polymer brushes supported by silicone nanofilaments

For site-specific dense immobilization of antibodies on a solid support, we prepared phosphorylcholine copolymer brushes on silicone nanofilaments. The nanofilaments were prepared on a silicon wafer by treatment with trichloromethylsilane (MeSiCl 3). To generate Si-OH groups on the nanofilaments, O 2 plasma was irradiated on the surface. Initiators for atom transfer radical polymerization (ATRP) were then coupled on the filaments. Phosphorylcholine copolymer brushes were prepared by a "grafting from" process, and pyridyl disulfide groups were introduced into the polymer chains. F(ab') fragments were then specifically immobilized onto these surfaces via a thiol-disulfide interchange reaction. The amount of antibodies immobilized on the nanofilament-supported copolymer brushes was approximately 65 times greater than that on smooth wafer-supported copolymer brushes.     

Atomistic mechanism of protein denaturation by urea

Effects of urea on protein stability have been studied from all-atom molecular dynamics simulations of ubiquitin, G311 protein, and immunoglobulin binding domain (B1) of streptococcal protein G (GB1) in water and 8 M aqueous urea solution. The mechanism of the change in the solvent environment and the early events in protein unfolding by urea have been identified with emphasis on the change in the interactions of hydrophilic and hydrophobic parts of the protein by calculating the potential of mean force (PMF). Urea replaces the protein-protein and protein-water contacts by forming stronger contacts with the protein, which is indicated by the longer survival times of the protein-urea hydrogen bonds.     

Bioreactive surfaces prepared via the self-assembly of dendron thiols and subsequent dendrimer bridging reactions

Here, we report a novel route to prepare bioreactive surfaces on gold by the self-assembly of generation-three hydroxyl-terminated dendron thiols (G3-OH) and subsequent bridging reactions using generation-two amine-terminated dendrimers (G2-NH(2)). It has been shown that G3-OH dendron thiols form a stable and uniform self-assembled monolayer on gold, which can be activated by the homobifunctional cross-linker N,N-disuccinimidyl carbonate (DSC). Subsequent derivatization of the activated monolayer via dendrimer bridging reactions with G2-NH(2) enhances the stability, reactivity, and versatility of the prepared surface. Each step of the surface formation reaction has been monitored, and the resulting surface has been characterized by wetting, electrochemistry, scanning tunneling microscopy (STM), and infrared (IR) spectroscopy measurements. The reactivity of this surface was demonstrated by a Schiff base coupling reaction with 4-cyanobenzaldehyde, by immobilizing biotin molecules onto the peripheral amine groups using one of the conjugation methods, and by further binding avidin onto the biotinylated surface. We believe that the prepared bioreactive surface with a high density of amine groups will be useful for the immobilization of biological macromolecules for various biosensor applications, such as the fabrication of DNA microarrays and protein chips.     

A preliminary study for isolation of catalytic antibodies by histidine ligand affinity chromatography as an alternative to conventional protein A/G methods

Catalytic autoimmune antibodies from the sera of lupus patients were purified using histidyl-aminohexyl-Sepharose gel and compared with the antibodies purified with protein A and protein G affinity chromatography. The IgG preparations from the histidine affinity column had a much higher catalytic activity in hydrolyzing the peptide substrate Pro-Phe-Arg-methyl-coumarinamide compared to the antibodies obtained by the conventional protein A/G method. This preservation of catalytic activity is attributed to the gentle buffer conditions used in the histidine ligand method that allowed the integrity of three-dimensional structure of purified catalytic antibodies. Thus, histidine affinity offer a superior method for isolating autoimmune catalytic antibodies.     

Micro-Scale Cell Patterning on Nonfouling Plasma Polymerized Tetraglyme Coatings by Protein Microcontact Printing

Nonfouling thin films were prepared by the plasma deposition of tetraethylene glycol dimethyl ether (pp4G) on fluorinated ethylene propylene polymer (FEP) and glass substrates. Ordered cell patterns were created on these surfaces by microcontact printing of proteins. Pp4G was found to be stable in aqueous environments and resistant to an ethanol sterilization procedure, as verified by surface analysis. Pp4G also reduced nonspecific protein adsorption by more than 65-fold before and after sterilization. Despite the low adsorption of proteins to pp4G in solution, protein microcontact printing was achieved and we were able to print laminin, an adhesive extracellular matrix protein, from an elastomeric stamp onto pp4G. The printed laminin supported the attachment and spreading of cardiomyocytes and the nonprinted pp4G regions remained cell repulsive in culture conditions. Microscale patterns of cardiomyocytes were maintained on printed pp4G for more than 7 days. This cell patterning process should be viable for other cell types. The potential applications include tissue engineering and microdevices for biosensor, diagnostic, and pharmacological applications.     

用于识别不同细胞蛋白质组的噬菌体抗体芯片

将4个鼠源噬菌体抗体克隆和1个人源噬菌体抗体克隆偶联到羧基终止的硅片表面,制成分析型模型芯片.挑选健康人体淋巴细胞为正常细胞的代表, HeLa细胞为肿瘤细胞的代表,提取细胞的全部蛋白质并用荧光染料Cy3标记,与制成的分析芯片反应,得到了不同的结合图谱.实验结果表明,以噬菌体抗体为分子感受器的分析芯片可用于识别不同细胞的蛋白质组.     

Factors affecting the specific activity of immobilized antibodies and their biologically active fragments.

Factors affecting the specific activity of immobilized antibodies and their biologically active fragments were studied with goat anti-mouse and goat anti-human immunoglobulin G. Antibodies were immobilized on HW 65 polymeric support matrix activated with carbonyldiimidazole, hydrazide and iodoacetic acid. The most significant factors influencing the specific activity of stochastic coupling of antibodies are multi-site attachment, multiple orientations and steric hindrance imposed by crowding of antibody and the size of the antigen. In oriented immobilization the specific activity is affected only by steric hindrance. The specific activity of immunosorbents prepared by immobilization of F(ab') fragments can be improved to almost 100% by limiting the amount of protein immobilization and the size of the antigen. The present study shows the protocols for optimizing immobilized antibody performance.     

Site-specific covalent attachment of an engineered Z-domain onto a solid matrix: An efficient platform for 3D IgG immobilization

Immobilized antibodies with oriented and homogeneous patterns are crucial to solid-phase molecular recognition assay. Antibody binding protein-based immobilization can effectively present the desired antibodies. However, steadily installing the stromatoid protein with site-specific attachment manner onto a matrix surface remains to be elucidated. In this study, we present an optimal protocol to tightly attach an immunoglobulin G (IgG)-binding protein (Z-domain) through covalent incorporation of Cys-tag and maleimide group onto polystyrene surface to guarantee site-specific, oriented, and irreversible attachment, resulting in a highly efficient platform for three-dimensional IgG immobilization. The actual IgG-binding characteristic of immobilized Z-Cys was investigated by employing affinity chromatography and size exclusion chromatography. And the efficacy and potential of this platform was demonstrated by applying it to the analysis of interaction between rabbit anti-HRP IgG and its binding partner HRP. The proposed approach may be an attractive strategy to construct high performance antibody arrays and biosensors given that the antibody is compatible with the Z-domain.     

Immune complexes in blood: a new strategy for the analysis of their antigen portion.

A method is described for the characterization of protein antigens from circulating immune complexes from plasma. Free immunoglobulins G were separated from larger immune complexes by gel filtration with a fast protein liquid chromatographic system. The collected immune complexes were dissociated with 4M urea into antigens and antibodies. With a second column run with 4M urea, antigens smaller than 120 kDa were separated from unloaded antibody fractions. After concentration, they were analyzed by two-dimensional gel electrophoresis.