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Here, we determined qualitative and quantitative characteristics of the chaperone and immunoglobulin-binding activities of recombinant Skp protein (rSkp) from Yersinia pseudotuberculosis using the methods of dynamic light scattering and surface plasmon resonance. Commercial human polyclonal IgG and Fc and Fab fragments of human IgG were used as substrate proteins. The activity of rSkp strongly depended on the medium pH. The most stable low-molecular-weight complexes with a hydrodynamic radius up to 10 nm were formed by rSkp and protein substrates at acidic pH values. Under these conditions, rSkp exhibited the lowest propensity to self-association and the highest affinity for human IgG and its Fc and Fab fragments, as well as prevented their aggregation most efficiently (i.e., demonstrated the maximal chaperone activity). As the medium pH increased, the affinity of rSkp for IgG and its fragments decreased; rSkp was not able to completely prevent the aggregation of protein substrates, but significantly slowed it down. The obtained information may be of practical interest, since the stability of therapeutic IgG preparations affects their safety and efficacy in medical applications. [ABSTRACT FROM AUTHOR]

This paper builds on an earlier examination of the influence of sampling size and analyte surface density on the accuracy and precision of measurements using surface-enhanced Raman scattering (SERS) to read out heterogeneous immunoassays. Quantitation using SERS typically relies on interrogating a small area on the sample surface by using a micrometer-sized laser spot for signal generation. The information obtained using such a small portion of sample is then projected as being representative of the much larger sample, which can compromise the accuracy and precision of the measurement due to undersampling. For a heterogeneous immunoassay interrogated by SERS, quantitation is, therefore, sensitive to the size of the analyzed area and the surface density of the measured analyte. To identify conditions in which sampling error poses a threat to accuracy and precision, a simulation of a SERS immunoassay was developed and compared to experimental results. The simulation randomly distributes adsorbates across the capture surface and then measures the density of adsorbates inside areas of analysis of different sizes. This approach mimics the analysis of a heterogeneous immunoassay when using a Raman microscope with different laser spot sizes. The results of the simulations, which were confirmed experimentally by comparison to an immunoassay of human immunoglobulin G (IgG) show that the accuracy and precision of the measurement improved with larger analysis areas and higher analyte concentrations due to the increased apparent homogeneity of the analyte within the area of analysis. By imposing a threshold on precision (5%), we also begin to establish a framework for the parameters necessary to achieve reliable quantitative measurements (e.g., laser spot size, analyte concentration, and sample volume). [ABSTRACT FROM AUTHOR]

This study describes the development and testing of a simple and novel enzyme‐free nanolabel for the detection and signal amplification in a sandwich immunoassay. Gold nanoparticles decorated reduced graphene oxide (rGOAu) was used as the nanolabel for the quantitative detection of human immunoglobulin G (HIgG). The rGOAu nanolabel was synthesised by one pot chemical reduction of graphene oxide and chloroauric acid using sodium borohydride. The pseudo‐peroxidase behaviour of rGOAu makes the nanolabel unique from other existing labels. The immunosensing platform was fabricated using self‐assembled monolayers of 11‐mercaptoundecanoic acid (11‐MUDA) on a gold disc electrode. The covalent immobilisation of antibody was achieved through the bonding of the carboxyl group of 11‐MUDA and the amino group of the antibody using chemical linkers [1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide] and N ‐hydroxysuccinimide. The fabricated immunosensor exhibited a linear range that included HIgG concentrations of 62.5–500 ng ml−1. The sensor was also used for the testing of HIgG in the blood sample. [ABSTRACT FROM AUTHOR]

This work focuses on fabricating poly(2-aminobenzylamine)-modified screen-printed carbon electrode as an electrochemical immunosensor for the label-free detection of human immunoglobulin G. To selectively detect immunoglobulin G, the anti-immunoglobulin G antibody with high affinity to immunoglobulin G was covalently linked with the amine group of poly(2-aminobenzylamine) film-deposited screen-printed carbon electrode. The selectivity for immunoglobulin G was subsequently assured by being challenged with redox-active interferences and adventitious adsorption did not significantly interfere the analyte signal. To obviate the use of costly secondary antibody, the [Fe(CN)6]4-/3- redox probe was instead applied to measure the number of human immunoglobulin G through the immunocomplex formation that is quantitatively related to the level of the differential pulse voltammetric current. The resulting immunosensor exhibited good sensitivity with the detection limit of 0.15 ng mL−1, limit of quantitation of 0.50 ng mL−1 and the linear range from 1.0 to 50 ng mL−1. Given those striking analytical performances and the affordability arising from using cheap screen-printed carbon electrode with label-free detection, the immunosensor serves as a promising model for the next-step development of a diagnostic tool. [ABSTRACT FROM AUTHOR]

Sequential injection (SI) system incorporated with amperometric immunosensor was developed for sensitive determination of human immunoglobulin G (HIgG). A cost effective label-free immunosensor was fabricated by immobilizing anti-HIgG on a graphene oxide (GO) modified screen-printed carbon electrode (SPCE). The developed electrode was characterized by cyclic voltammetry(CV), scanning electron microscope(SEM), and energy dispersive spectroscopy(EDS) which confirmed the selective immunointeraction of HIgG to the anti-HIgG on the electrode, thus reduced the amperometric current of [Fe(CN) 6 ] 3-/4- redox probe. The sensing electrode was placed in a designed electrochemical flow cell of SI system, where the redox probe was propelled through and the currents before and after the immunointeraction occurred were measured amperometrically by using a simple home-made amperometer. Under the optimum condition: flow rate of 2 mL min −1 , applied potential of +350 mV, [Fe(CN) 6 ] 3-/4- concentration of 10 mM and 10 min of incubation time, a linear calibration in the range of 2–100 ng mL −1 was achieved, with detection limit of 1.70 ng mL −1 . The proposed system provided good repeatability and reproducibility and the application for urine sample analysis was demonstrated. [ABSTRACT FROM AUTHOR]

An enzyme-linked immunosorbent assay (ELISA) is reported for human immunoglobulin G based on synthesized hemin–MnO2 nanocomposite as the label. Enhanced sensitivity was obtained due to the increased catalytic activity of the hemin–MnO2 nanocomposite toward 3,3′,5,5′-tetramethylbenzidine compared to hemin and MnO2 alone. The synthesized hemin–MnO2 nanocomposite was characterized by transmission electron microscopy and its catalytic activity to 3,3′,5,5′-tetramethylbenzidine was investigated by ultraviolet–visible absorption spectroscopy. After assembly of the sandwich-type immunoassay in the 96 wells of the plate the hemin–MnO2-based label catalyzed 3,3′,5,5′-tetramethylbenzidine into blue compounds that were monitored by a plate reader. The absorbance increased with the concentration of human immunoglobulin G. The immunoassay displayed high sensitivity, a long linear dynamic range, and good selectivity for human immunoglobulin G. The immunoassay was also used for the determination of human immunoglobulin G in serum with favorable results. The developed assay combines the high throughput and low cost of ELISA with the simplicity of nanocomposite labeling and is suitable for application in clinical diagnosis. [ABSTRACT FROM AUTHOR]

Highly sensitive and selective quantitation of a variety of proteins over a wide concentration range is highly desirable for increased accuracy of biomarker detection or for multidisease diagnostics. In the present contribution, using human immunoglobulin G (HIgG) as the model target protein, an electrochemical ultrasensitive immunosensing platform was developed based on the oligonucleotide self-assembled monolayer-mediated (OSAM) sensing interface. For this immunosensor, the “signal-on” signaling mechanism and enzymatic signal amplification effect were integrated into one sensing architecture. Moreover, the thiolated flexible single-stranded DNAs immobilized onto gold electrode surface not only performs the wobbling motion to facilitate the electron transfer between the electrode surface and biosensing layer but also fundamentally prohibiting the direct interaction of proteins with gold substrate. Thus, the electrochemical signal could be efficiently enhanced and the unspecific adsorption or cross-reaction might be eliminated. As a result, utilizing the newly-proposed immunosensor, the HIgG can be detected down to 0.5 ng/mL, and the high detection specificity is offered. The successful design of OSAM and the highly desirable detection capability of new immunosensor are expected to provide a perspective for fabricating new robust immunosensing platform and for promising potential of oligonucleotide probe in biological research and biomedical diagnosis. [ABSTRACT FROM AUTHOR]

We report on an ultrasensitive metal-labeled amperometric immunoassay of proteins, which is based on the selective staining of nanocrystalline cadmium sulfide (CdS) on ZnO nanocrystals and in-situ microliter-droplet anodic stripping voltammetry (ASV) detection on the immunoelectrode. Briefly, antibody 1 (Ab 1 ), bovine serum albumin (BSA), antigen and ZnO-multiwalled carbon nanotubes (MWCNTs) labeled antibody 2 (Ab 2 -ZnO-MWCNTs) were successively anchored on a β -cyclodextrin-graphene sheets (CD-GS) nanocomposite modified glassy carbon electrode (GCE), forming a sandwich-type immunoelectrode (Ab 2 -ZnO-MWCNTs/antigen/BSA/Ab 1 /CD-GS/GCE). CdS was selectively grown on the catalytic ZnO surfaces through chemical reaction of Cd(NO 3 ) 2 and thioacetamide (ZnO-label/CdS-staining), due to the presence of an activated cadmium hydroxide complex on ZnO surfaces that can decompose thioacetamide. A beforehand cathodic "potential control" in air and then injection of 7 μL of 0.1 M aqueous HNO 3 on the immunoelectrode allow dissolution of the stained CdS and simultaneous cathodic preconcentration of atomic Cd onto the electrode surface, thus the following in-situ ASV detection can be used for immunoassay with enhanced sensitivity. Under optimized conditions, human immunoglobulin G (IgG) and human heart-type fatty-acid-binding protein (FABP) are analyzed by this method with ultrahigh sensitivity, excellent selectivity and small reagent-consumption, and the limits of detection (LODs, S/N =3) are 0.4 fg mL −1 for IgG and 0.3 fg mL −1 for FABP (equivalent to 73 FABP molecules in the 6 μL sample employed). [ABSTRACT FROM AUTHOR]

The present study evaluated the phosphorylated-tyrosine (P-Tyr) based pseudobioaffinity adsorbent for the purification of human immunoglobulin G (IgG). P-Tyr was selected as a ligand to mimic the natural interactions that occur between the immunoreceptor tyrosine-based activation motif and the IgG. The ligand was coupled to bisoxirane-activated agarose gel and the effect of buffer system, pH, and conductivity was performed to elucidate the nature of IgG-P-Tyr interactions. P-Tyr-agarose was able to purify IgG from human plasma solution in HEPES buffer at pH 7.0 exhibiting a purification factor of 9.1 with IgG purity of 91% (based on ELISA analysis of albumin, transferrin, and immunoglobulins A, G, and M). The evaluation of different functional groups of P-Tyr on the adsorption of human IgG indicated the predominance of electrostatic interactions with phosphate groups, although the contributions of aromatic and carboxylic groups also play a role. The thermodynamic parameters (ΔH°, ΔS°, ΔG°) for IgG adsorption onto P-Tyr-agarose were determined from the temperature dependence. The maximum IgG binding capacity at 20 °C was 273.51 ± 12.63 mg g −1 and the dissociation constant value of the complex IgG-P-Tyr was in the order of 10 −5 mol L −1 indicating low-affinity. [ABSTRACT FROM AUTHOR]

A substantial outstanding challenge in diagnostics and disease monitoring is the ability to assay rapidly and conveniently for protein biomarkers within complex biological media. BiSe, as an important topological insulator (TI) material, was synthesized by a solvothermal method and characterized structurally. Subsequently, the composite of BiSe and ionic liquid ([BMIm]BF IL) was used as a sensing interface to cross-link goat anti-human immunoglobulin G (anti-IgG) via glutaraldehyde (GA) to fabricate an BiSe/IL/GA/anti-IgG-carbon paste electrode (CPE). The nonspecific binding sites were enclosed with bovine serum albumin (BSA) to develop a label-free IgG immunosensor. The result showed that the proposed label-free IgG immunosensor exhibited high specificity with a detection limit of 0.8 ng mL and linear range from 2 to 300, and 300 to 2200 ng mL. Besides, the immunosensor exhibited high specificity for IgG detection, acceptable reproducibility, and stability. Thus, the strategy reported here paved a simple way to design a sensitive and cost-effective sensing platform for extension to other disease biomarkers. [ABSTRACT FROM AUTHOR]

Adsorption selectivity is critical important for mixed-mode chromatography with specially-designed ligands. Human immunoglobulin G (hIgG), Fc and Fab fragments were used in the present work to evaluate adsorption behavior and binding selectivity of four mixed-mode resins with the ligands of 4-mercatoethyl-pyridine (MEP), 2-mercapto-1-methylimidazole (MMI), 5-aminobenzimidazole (ABI) and tryptophan-5-aminobenzimidazole (W-ABI), respectively. The resins showed an obvious pH-dependent adsorption behavior. High adsorption capacities were found at neutral pH for hIgG, Fc and Fab, and almost no adsorption happened under acidic conditions. An adsorption selectivity index was proposed to evaluate separation efficiency. High specificity of hIgG/Fc was found at pH 8.9 for MEP resin, and for W-ABI resin at pH 8.0 and 8.9. In addition, isothermal titration calorimetry was used to evaluate ligand-protein interactions. Finally, the separation of hIgG and Fc (1:1) was optimized with mixed-mode resins, and the best separation performance was obtained with W-ABI-based resin. Loading at pH 8.0 resulted in the flow through of Fc with purity of 90.4% and recovery of 98.8%, while elution at pH 3.6 provided hIgG with purity of 99.7% and recovery of 86.5%. [ABSTRACT FROM AUTHOR]

Hydrophobic charge-induction chromatography is a new technology for antibody purification. To improve antibody adsorption capacity of hydrophobic charge-induction resins, new poly(glycidyl methacrylate)-grafted hydrophobic charge-induction resins with 5-aminobenzimidazole as a functional ligand were prepared. Adsorption isotherms, kinetics, and dynamic binding behaviors of the poly(glycidyl methacrylate)-grafted resins prepared were investigated using human immunoglobulin G as a model protein, and the effects of ligand density were discussed. At the moderate ligand density of 330 μmol/g, the saturated adsorption capacity and equilibrium constant reached the maximum of 140 mg/g and 25 mL/mg, respectively, which were both much higher than that of non-grafted resin with same ligand. In addition, effective pore diffusivity and dynamic binding capacity of human immunoglobulin G onto the poly(glycidyl methacrylate)-grafted resins also reached the maximum at the moderate ligand density of 330 μmol/g. Dynamic binding capacity at 10% breakthrough was as high as 76.3 mg/g when the linear velocity was 300 cm/h. The results indicated that the suitable polymer grafting combined with the control of ligand density would be a powerful tool to improve protein adsorption of resins, and new poly(glycidyl methacrylate)-grafted hydrophobic charge-induction resins have a promising potential for antibody purification applications. [ABSTRACT FROM AUTHOR]

Hydrophobic charge-induction chromatography (HCIC) is a highly promising technology for antibody separation. HCIC resins ABI-B-6FF were prepared with 5-aminobenzimidazole (ABI) as the functional ligand. The effects of ligand density on the adsorption of human immunoglobulin G (hIgG) and bovine serum albumin were focused. It was found that the adsorption capacity and dynamic binding capacity (DBC) were improved with the increase of ligand density. The adsorption capacity andDBCof hIgG reached 128.07 mg/g gel and 67.63 mg/g gel. The results indicated that ABI-B-6FF resin has a promising potential for the application of antibody purification. [ABSTRACT FROM AUTHOR]

A biomarker is related to the biological status of a living organism and shows great promise for the early prediction of a related disease. Herein we presented a novel structured light-addressable potentiometric sensor (LAPS) for the determination of a model biomarker, human immunoglobulin G (hIgG). In this system, the goat anti-human immunoglobulin G antibody was used as recognition element and covalently immobilized on the surface of light-addressable potentiometric sensor chip to capture human immunoglobulin G. Due to the light addressable capability of light-addressable potentiometric sensor, human immunoglobulin G dissolved in the supporting electrolyte solution can be detected by monitoring the potential shifts of the sensor. In order to produce a stable photocurrent, the laser diode controlled by field-programmable gate array was used as the light emitter to drive the light-addressable potentiometric sensor. A linear correlation between the potential shift response and the concentration of human immunoglobulin G was achieved and the corresponding regression equation was ΔV (V) = 0.00714C hIgG (μg/mL)–0.0147 with a correlation coefficient of 0.9968 over a range 0–150 μg/mL. Moreover, the light-addressable potentiometric sensor system also showed acceptable stability and reproducibility. All the results demonstrated that the system was more applicable to detection of disease biomarkers with simple operation, multiple-sample format and might hold great promise in various environmental, food, and clinical applications. [ABSTRACT FROM AUTHOR]

We report a new protocol for ultrasensitive electrochemical sandwich-type immunosensing, on the basis of signal amplification by gold-label/copper-staining, galvanic replacement reactions (GRRs), and in situ microliter-droplet anodic stripping voltammetry (ASV) after an enhanced cathodic preconcentration of copper. First, a sandwich-type immuno-structure is appropriately assembled at a glassy carbon electrode. Second, copper is selectively stained on the catalytic surfaces of second antibody-conjugated Au nanoparticles through CuSO 4 -ascorbic acid redox reaction, and the GRRs between HAuCl 4 and the stained copper are used to amplify the quantity of copper. Finally, the corresponding antigen is determined based on simultaneous chemical-dissolution/cathodic-preconcentration of copper for in-situ ASV analysis directly at the immunoelectrode. Cyclic voltammetry, electrochemical impedance spectroscopy, quartz crystal microbalance and scanning electron microscopy are used for film characterization and/or process monitoring. Under optimized conditions, ultrasensitive analyses of human immunoglobulin G (IgG) and human carbohydrate antigen 125 (CA125) are achieved. The limits of detection are 0.3 fg mL −1 (equivalent to 7 IgG molecules in the 6 μL sample employed) for IgG ( S / N =3) and 1.3 nU mL −1 for CA125 ( S / N =3), respectively, which are amongst the best reported to date for the two proteins. The theoretical feasibility of such a single-molecule-level amperometric immunoassay is also discussed based on the immunological reaction thermodynamics. [ABSTRACT FROM AUTHOR]

We have reported earlier on the characterization of four peptides from a library of 15 peptide ligand candidates for human IgG (hIgG) obtained by a biomimetic design strategy and identified three high-affinity octapeptides. In this work, the left 11 peptides were evaluated and we found two more octapeptides (FYCHWQDE and FYCHNQDE) that showed high affinity for hIgG. The binding pH ranges for the two ligands were different, but the optimum pH values were the same for each other (pH 6.0). Both the ligands showed high specificity and bound hIgG mainly by electrostatic interactions. Ligand binding competition experiments revealed that the binding sites on hIgG for the two octapeptides were similar to those for Protein A. Finally, hIgG was purified from human serum with high purities and recovery yields with the two peptide affinity columns. Thus, among the 15 peptide candidates, a total of five octapeptides were identified as high-affinity ligands of hIgG. The five ligands were all derived from the same peptide model FYxHxxxE (where x denotes any amino acid) and contained four common hot spots F132, Y133, H137, and E143 of the affinity motif of Protein A. Analyses and evaluation of the peptide library would help deepen our understanding of the affinity binding of Protein A to IgG, and promote application of the biomimetic strategy in the design of affinity ligands for different proteins. [ABSTRACT FROM AUTHOR]

The chemical compositions, sizes and fluorescent properties of synthesized carbon nanoparticles (CNPs) were characterized. Escherichia coli ( E. coli ) cells were used as a model to study the cytotoxicity of CNPs, and the results of the cellular uptake of CNPs yielded excellent results: the CNPs demonstrated good biocompatibility and were non-toxic to the growth of the E. coli cells. Moreover, to assess the potential toxicity of CNPs to human health, the binding behavior of CNPs with human immunoglobulin G (HIgG) was examined by fluorescence quenching spectroscopy, synchronous fluorescence spectroscopy and circular dichroism spectroscopy under physiological conditions. The fluorescence quenching constants and parameters for the interaction at different temperatures had been calculated according to Scatchard. The thermodynamic parameters, such as enthalpy change (Δ H ), entropy change (Δ S ) and free energy change (Δ G ), were calculated, and the results indicated strong static quenching and showed that van der Waals forces, hydrogen bonds and hydrophobic interactions were the predominant intermolecular forces stabilizing the CNP–HIgG complex. Synchronous fluorescence and circular dichroism spectra provided information regarding the conformational alteration of HIgG in the presence of CNPs. These findings help to characterize the interactions between CNPs and HIgG, which may clarify the potential risks and undesirable health effects of CNPs, as well as the related cellular trafficking and systemic translocation. [ABSTRACT FROM AUTHOR]

An ultrasensitive sandwich-type electrochemical immunosensor based on a novel signal amplification strategy was developed for the quantitative determination of human immunoglobulin G (IgG). Pd nanocubes functionalized magnetic graphene sheet (Pd–Fe 3 O 4 -GS) was employed as the matrix to immobilize the primary antibodies (Ab 1 ). Owing to the synergetic effect between Pd nanocubes and magnetic graphene sheet (Fe 3 O 4 -GS), Pd–Fe 3 O 4 -GS can provide an obviously increasing electrochemical signal by electrochemical catalysis towards hydrogen peroxide (H 2 O 2 ). Silicon dioxide (SiO 2 ) was functionalized as the label to conjugate with the secondary antibodies (Ab 2 ). Due to the larger steric hindrance of the obtained conjugate (SiO 2 @Ab 2 ), the sensitive decrease of the electrochemical signal can be achieved after the specific recognition between antibodies and antigens. In this sense, this proposed immunosensor can achieve a high sensitivity, especially in the presence of low concentrations of IgG. Under optimum conditions, the proposed immunosensor offered an ultrasensitive and specific determination of IgG down to 3.2 fg/mL. This immunoassay method would open up a new promising platform to detect various tumor markers at ultralow levels for early diagnoses of different cancers. [ABSTRACT FROM AUTHOR]

A new polymer bearing aldehyde groups was designed and synthesized by grafting 4-pyridinecarboxaldehyde onto poly(epichlorohydrin). Antibodies can be directly immobilized on the surface of the polymer film through the covalent bonding of aldehyde groups of the film with amino groups of antibodies. In this study, human immunoglobulin G (IgG) was used as a model analyte for the fabrication of an electrochemical impedance immunosensor. Using the proposed immunosensor, IgG in the range from 0.1 to 80 ng ml −1 was detected with a detection limit of 0.07 ng ml −1 (signal/noise [S/N] = 3). In addition, the electrochemical impedance immunosensor displays good stability and reproducibility. [ABSTRACT FROM AUTHOR]

Objective Matrix metalloproteinase ( MMP)-9 is a key molecule that indicates disruption of the blood-brain barrier ( BBB), and is recognized as a candidate biomarker of disease activity in multiple sclerosis ( MS). The aim of the present study was to determine whether human immunoglobulin G ( hIgG) could reduce the production of MMP-9 in peripheral blood mononuclear cells ( PBMC) from patients with MS. Methods We investigated the effect of hIgG on the expression of MMP-9 and tissue inhibitor of metalloproteinase ( TIMP)-1 in PBMC of patients with relapsing-remitting MS ( RRMS) compared with healthy controls ( HC) in vitro. Results Patients with RRMS were not receiving any disease-modifying therapies when blood was sampled in this study. Although levels of MMP-9 and TIMP-1 in PBMC were not different between RRMS and HC groups, the MMP-9/ TIMP-1 ratio was significantly increased in patients with RRMS when compared with HC. PBMC that were stimulated with lipopolysaccharide (LPS, 1 μg/mL) expressed a higher level of MMP-9 in RRMS than the HC groups, although the level of TIMP-1 was equal between groups. hIgG reduced the level of MMP-9 in PBMC from both patients with RRMS and HC with LPS stimulation in a dose-dependent manner, but had no effect on the expression of TIMP-1. The MMP-9/ TIMP-1 ratio in both patients with RRMS and HC was also decreased by hIgG. The effect of hIgG was not through neutralization of MMP-9. hIgG alone did not induce MMP-9 mRNA, and suppressed the upregulation of mRNA in PBMC stimulated with LPS. Conclusions These results suggest that hIgG could be effective in treating patients with RRMS though the inhibition of the transmigration of immune cells into the brain parenchyma. [ABSTRACT FROM AUTHOR]

A new hydrophobic charge-induction chromatography resin was prepared with 5-aminobenzimidazol as functional ligand and polyacrylic ester beads as matrix. Adsorption isotherms and adsorption in columns were investigated using human immunoglobulin G and bovine serum albumin as model proteins, and the influence of pH and NaCl concentration was discussed. Results showed that the ligand density was 195 μmol/mL gel, and protein selectivity can be improved by controlling pH and salt addition. An optimized purification process (sample loading at pH 8.0 with 0.2 M NaCl and elution at pH 5.0) was performed to purify human immunoglobulin G from bovine serum albumin containing feedstock, which resulted in human immunoglobulin G purity of 99.7% and recovery of 94.6%. A similar process was applied for the purification of monoclonal antibody from cell culture supernatant, which showed antibody purity of 94.9% and recovery of 92.5%. The results indicated that the new resin developed had comparable performance as Protein A chromatography and would be suitable for antibody purification from complex feedstock. [ABSTRACT FROM AUTHOR]

Au@Ag nanorods (Au@AgNRs) was utilized to construct a novel sandwich-type electrochemical immunosensor for the detection of immunoglobulin G (IgG). The sensor was prepared by immoblizing capture antibodies on the amine-terminated nanocomposite of carbon nanofibers-polyamidoamine dendrimer (CNFs-PAMAM), whilst the trace tag was prepared by loading anti-human IgG on Au@AgNRs. The “built-in” Ag layer on Au nanorods was characterized by UV–vis extinction spectra, transmission electron microscopy and energy dispersive spectroscopy. The results of cyclic voltammetry indicated that modifying CNFs-PAMAM nanocomposite on glassy carbon electrode enabled 177 times of peak current increase of Ag in the bimetallic nanorods. The peak current was quantitatively related with the concentration of the target protein IgG via the formation of immunocomplex. After the parameter optimization, the oxidative peak current of silver was proportional to the concentration of IgG in a wide linear range of six orders of magnitude with a low detection limit of 0.5 fg mL −1 . Besides, this sensor showed acceptable reproducibility and stability, and thus the strategy reported here has great promise for extension to the other disease biomarkers. [ABSTRACT FROM AUTHOR]

In this work, a 3D-structured reduced graphene oxide–multiwalled carbon nanotube–palladium nanoparticles (NPs) nanocomposite (rGO–MWCNT–Pd) was prepared by a facile and green way and used as substrate material for the development of a label-free electrochemical immunoassay for human immunoglobulin G (HIgG) detection. The network structure of rGO–MWCNT nanocomposite provides more active sites for Pd nucleation. And the synergistic effect of rGO, MWCNTs and Pd NPs significantly improved the electrochemical performance of the modified electrode. Pd particles can combine HIgG antibodies and enhance the conductivity of electrode. The structure of rGO–MWCNT–Pd nanocomposite was characterized by scanning electron microscopy (SEM) and high resolution transmission electron microscopy (HRTEM). The HRTEM of Pd NPs and energy dispersive X-ray spectroscopy (EDS) of rGO–MWCNT–Pd nanocomposite confirmed the composition of the as-synthesized nanocomposite. K 3 [Fe(CN) 6 ] was used as a redox probe. HIgG antibodies are immobilized onto the nanocomposite via Pd NH 2 bonds. Cyclic voltammetry and square wave voltammetry were used to characterize the recognizability of HIgG. Under optimum experimental conditions, the proposed immunoassay exhibited a low detection limit (3.3 pg/mL), a wide linear range (from 0.01 to 25 ng/mL) as well as good stability, reproducibility and selectivity. [ABSTRACT FROM AUTHOR]

Hydrophobic charge-induction chromatography (HCIC) is a new and effective technology for antibody separation. In the present work, HCIC resin MMI-B-XL was prepared with dextran-grafted agarose gel as the matrix and 2-mercapto-1-methyl-imidazole (MMI) as the functional ligand. The preparation procedures were optimized, and the maximum ligand density could reach as high as 200 μmol/g gel. The adsorption isotherms and kinetics on new resins were investigated with human immunoglobulin G (hIgG) as the model protein, which were compared with non-grafted HCIC resin MMI-B-6FF. It was found that the saturated adsorption capacity ( Q m ) increased with the increase of ligand density for MMI-B-XL. Moreover, the effective diffusivity ( D e ) could be dramatically enhanced with the increase of ligand density for MMI-B-XL, and the D e for MMI-B-XL with the ligand density of 200 μmol/g gel was 18–40 times higher than that for MMI-B-6FF. The breakthrough experiments indicated that new resins with the ligand density of 200 μmol/g gel could be used for high superficial velocity and high dynamic adsorption could be obtained. The results indicated that dextran-grafted layer on the resin could increase the ligand density, enhance the mass transport in the pore, and improve the dynamic adsorption at high velocity, which showed a potential application for large-scale antibody purification. [ABSTRACT FROM AUTHOR]

This work reports the development of affinity systems with dual octapeptide ligands for affinity adsorption and purification of human IgG (hIgG) and monoclonal antibody (mAb). The three octapeptide ligands, FYWHCLDE (1), FYCHWALE (2), and FYCHTIDE (3), identified earlier by the biomimetic design strategy were used; any two of the three were mixed and coupled to Sepharose gel, leading to the formation of three dual-ligand affinity systems. Research emphasis was first placed on hIgG adsorption isotherms and the results were compared to the three single-ligand affinity systems. It was found that there was synergistic effect of the two peptide ligands in a dual-ligand system, so the affinity of a dual-ligand resin for hIgG was higher than those of its counterparts, single-ligand resins. Of the three dual-ligand systems, the FYWHCLDE (1)–FYCHTIDE (3) resin showed the highest affinity, so it was selected for investigating the effects of ligand density and molar ratio on hIgG adsorption equilibrium. It was found that the synergistic effect increased with increasing the total ligand density of the two peptides in the dual-ligand affinity system. Moreover, the FYWHCLDE (1)–FYCHTIDE (3) system at a molar ratio of 2:1 displayed the highest affinity for hIgG (0.69 μM at a total ligand density of 31.1 μmol/mL), indicating that the synergistic effect reached the maximum at this ratio. This dual-ligand affinity column was then used for the purification of hIgG and mAb by affinity chromatography, resulting in over 95% pure hIgG and mAb at recovery yield over 90%. Molecular docking of the two peptides to the Fc fragment simultaneously showed that FYWHCLDE (1) stood still but FYCHTIDE (3) shifted aside the CH2 CH3 inter-domain. Molecular dynamics simulation of the binding process of the two octapeptides to Fc revealed that both the peptide ligands kept stable interactions with Fc. The synergistic effect of the dual-ligand affinity system was thus elucidated by the molecular simulations. [ABSTRACT FROM AUTHOR]

The aim of this work was to evaluate the ability of Cibacron Blue F-3GA dye-ligand chitosan/alginate epoxide on human IgG adsorption under different buffer systems at 25 mmol L: Morpholinoethane sulfonic acid, morpholinopropane sulfonic acid, hydroxyethylpiperazine ethanesulfonic acid and tris-hydroxymethyl aminomethane (Tris-HCl). Batch adsorption studies were performed in order to evaluate the effect of buffer pH and nature on IgG uptake as well the equilibrium isotherms. Chromatographic experiments were performed with both human IgG (high purity) and human serum and each buffer with NaCl 1.0 M was used in the elution step. Best results (maximum equilibrium adsorption capacity of 110.9 mg g) were found at pH 7.8 with Tris-HCl buffer. The Langmuir-Freundlich model provided a good fit for the adsorption isotherm. In chromatographic experiments with high purity IgG and Tris/HCl buffer, IgG dynamic adsorption capacity onto E-Ch/Al-Cibacron was 13.4 mg g, which accounted for 60 % of IgG injected into the column. Chromatographic experiments with diluted (tenfold) human serum were conducted and it was found by electrophoresis that IgG is preferentially adsorbed with respect to other proteins using all buffers systems, especially with Tris-HCl. The amount of desorbed protein from E-Ch/Al-Cibacron was around 7.0 mg g for all buffers. The new stationary phase showed high affinity for IgG and may be a potential adsorbent for human IgG purification. [ABSTRACT FROM AUTHOR]

Abstract: Constructions of versatile electroactive labels are key issues in the development of electrochemical immunosensors. In this study, copper-doped titanium dioxide nanoparticle (Cu@TiO2) was synthesized and used as labels for fabrication of sandwich-type electrochemical immunosensors on glassy carbon electrode (GCE). Due to the presence of copper ions, Cu@TiO2 shows a strong response current when coupled to an electrode. The prepared nanocomposite also shows high electrocatalytic activity towards reduction of hydrogen peroxide (H2O2). The dual functionality of Cu@TiO2 enables the fabrication of immunosensor using different detection modes, that is, square wave voltammetry (SWV) or chronoamperometry (CA). While Cu@TiO2 was used as labels of secondary antibodies (Ab2), carboxyl functionalized graphene oxide (CFGO) was used as electrode materials to immobilize primary antibodies (Ab1). Using human immunoglobulin G (IgG) as a model analyte, the immunosensor shows high sensitivity, acceptable stability and good reproducibility for both detection modes. Under optimal conditions, a linear range from 0.1pg/mL to 100ng/mL with a detection limit of 0.052pg/mL was obtained for SWV analysis. For CA analysis, a wider linear range from 0.01pg/mL to 100ng/mL and a lower detection limit of 0.0043pg/mL were obtained. The proposed metal ion-based enzyme-free and noble metal-free immunosensor may have promising applications in clinical diagnoses and many other fields. [Copyright &y& Elsevier]

In an earlier work, we have developed a biomimetic design strategy based on the human IgG (hIgG)–Protein A interactions and identified an affinity ligand for hIgG, FYWHCLDE, which ranked top one in a pool of 14 potential candidates. Herein, two more octapeptides, FYCHWALE and FYCHTIDE, were identified, and the binding and purification of hIgG on the affinity columns packed with the three octapeptide-modified Sepharose gels were extensively studied and compared to find more effective octapeptide-based affinity ligands. It was found that all the three ligands bound hIgG and Fc fragment but barely bound Fab fragment, and the binding to hIgG and Fc was mainly by electrostatic interactions. The optimum binding pH values for the three ligands were different from each other, but kept in the range of 5.0–6.0. Ligand binding competition revealed that the binding sites on hIgG for the three octapeptides were similar to those for Protein A. Adsorption isotherms revealed that hIgG binding capacity was in the range of 64–104 mg/mL drained gel in the order of FYWHCLDE > FYCHWALE > FYCHTIDE. Then, purifications of hIgG and human monoclonal antibody from human serum and cell culture supernatant, respectively, were achieved with the three affinity columns at high purities and recovery yields. Finally, the molecular basis for the binding affinity of the peptides for the Fc fragment of hIgG was elucidated by molecular dynamics simulations. [ABSTRACT FROM AUTHOR]

Abstract: Antibodies can be directly immobilized on the surface of dialdehyde cellulose film through the covalent bonding of the aldehyde groups of the dialdehyde cellulose with amino groups of antibodies. In this work, human IgG was used as a model analyte to fabricate an electrochemical immunosensor. Using the proposed immunosensor, we detected IgG within the range from 0.5 to 45ng/mL with a detection limit of 0.3ng/mL (S/N=3). The electrochemical immunosensor had a good specificity, stability and reproducibility. This strategy may pave a simple way to fabricate an electrochemical immunosensor in other applications. [Copyright &y& Elsevier]

This work reports the development of an octapeptide-based affinity adsorbent for the purification of human IgG (hIgG) and monoclonal antibody (mAb). The octapeptide was FYWHCLDE selected earlier by the biomimetic design of affinity peptide ligands for hIgG. The ligand was coupled to Sepharose gel at four densities from 10.4 to 31.0µmol/mL, and the effect of peptide density on the adsorption of hIgG and bovine serum albumin (BSA) was first investigated. The binding capacity of hIgG increased from 104.2 to 176.4mg/mL within the ligand density range, and the binding affinity (dissociation constant) kept at 2.4-3.7µM. Batch adsorption revealed that the selectivity of FYWHCLDE-Sepharose for IgG was 30-40 times over BSA. The effective pore diffusivity of IgG decreased somewhat with increasing ligand density, but the dynamic binding capacity at 10% breakthrough, measured by using 10-fold diluted human serum as feedstock, doubled with increasing ligand density from 10.4 to 31.0µmol/mL due to the remarkable increase of static binding capacity. By using the affinity column with a ligand density of 23.9µmol/mL, hIgG and humanized mAb purifications from human serum and cell culture supernatant, respectively, were achieved at high purities and recovery yields. Finally, the robustness of the peptide gel was demonstrated by recycled use of the affinity column in 20 breakthrough cycles [ABSTRACT FROM AUTHOR]

This paper characterizes the potential of novel hexameric peptide ligands for on-line IgG detection in bioprocesses. Surface Plasmon Resonance (SPR) was used to study the binding of human IgG to the hexameric peptide ligand HWRGWV, which was covalently grafted to alkanethiol self-assembled monolayers (SAM) on gold surfaces. Peptide coupling on SAMs was verified, followed by covalent grafting of peptides with a removable Fmoc or acetylated N-termini via their C-termini to produce active peptide SPR sensors that were tested for IgG binding. The dynamics and extent of peptide–IgG binding were compared with results from a conventional system using protein A attached on a gold surface via disulfide monolayers. IgG binding to protein A on disulfide monolayers yielded equilibrium dissociation constants of 1.4×10–7 M. The corresponding dissociation constant value for the acetylated version of the peptide (Ac-HWRGWV) supported on alkanethiol SAM was 5.8×10–7 M and that for HWRGWV on the alkanethiol SAM (after de-protection of Fmoc-HWRGWVA) was 1.2×10–6 M. Maximum IgG binding capacities, Q m of 6.7, 3.8, and 4.1mgm−2 were determined for the protein A and the two forms of HWRGWV-based biosensors, respectively. Real-time data for the kinetics of adsorption were used to determine the apparent rate constants for adsorption and desorption. The results were analyzed to understand the mechanism of IgG binding to the protein and peptide ligands. It was found that the peptide–IgG binding was reaction controlled, however the protein A–IgG binding mechanism was partially mass transfer (diffusion) controlled. The adsorption rate constants, k a, for the protein A ligand increased with decreasing concentration of analyte and the peptide ligand k a values was constant at different IgG concentrations and flow rates. [ABSTRACT FROM AUTHOR]

Highlights: [•] Aqueous two-phase system was used to separate IgG from albumin containing feedstock. [•] Hydroxypropyl starch was used as the phase-forming polymer with relatively low cost. [•] NaCl addition and pH were optimized as key factors to improve the extraction of IgG. [•] High purification factor of IgG was obtained with two-step extraction process. [Copyright &y& Elsevier]

Poly(2-hydroxyethyl methacrylate)/3-(2-imidazoline-1-yl)propyl(triethoxysilane) (PHEMA/IMEO) nanoparticles were attached on surface plasmon resonance (SPR) sensor for the real-time detection of human immunoglobulin G (IgG) in human serum. The PHEMA/IMEO nanoparticles-attached SPR sensor was characterized by Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), and contact angle measurements. IgG detection studies were performed using aqueous IgG solutions at different concentrations. In order to show the selectivity and specificity of the SPR sensor, competitive kinetic analyses were performed using IgG, albumin, hemoglobin in singular and competitive manner. Finally, IgG detection in human serum was carried out. [ABSTRACT FROM AUTHOR]

Abstract: A simple systematic approach using Fourier transform infrared (FTIR) spectroscopy, size exclusion chromatography (SEC) and design of experiments (DOE) techniques was applied to the analysis of aggregation factors for protein formulations in stress and accelerated testings. FTIR and SEC were used to evaluate protein conformational and storage stabilities, respectively. DOE was used to determine the suitable formulation and to analyze both the main effect of single factors and the interaction effect of combined factors on aggregation. Our results indicated that (i) analysis at a low protein concentration is not always applicable to high concentration formulations; (ii) an investigation of interaction effects of combined factors as well as main effects of single factors is effective for improving conformational stability of proteins; (iii) with the exception of pH, the results of stress testing with regard to aggregation factors would be available for suitable formulation instead of performing time-consuming accelerated testing; (iv) a suitable pH condition should not be determined in stress testing but in accelerated testing, because of inconsistent effects of pH on conformational and storage stabilities. In summary, we propose a three-step strategy, using FTIR, SEC and DOE techniques, to effectively analyze the aggregation factors and perform a rapid screening for suitable conditions of protein formulation. [Copyright &y& Elsevier]

Abstract: A simple, reusable and ultrasensitive electrochemical clinic immunoassay is proposed via developing a versatile CTDONG (conductive three-dimensional ordered nano-gold) film-modified gold electrode, in which ferrocene derivative and human immunoglobulin G (hIgG) are used as signaling probe and model molecule, respectively. A signal-on signaling mechanism is achieved by utilizing a sandwich format of the primary antibody/hIgG/the secondary antibody labeled with Ferrocene (PAb/Ag/SAb). Owing to the combination of the advantages of CTDONG film with the versatility of ferrocene derivatives, a substantially enhanced signal accompanied by a low background peak current is achieved. By this sensing scheme, target molecule can be readily quantified in a comparatively wide dynamic range (8.1×10−13–6.2×10−10 M) with a relatively low detection limit (2.7×10−13 M). In addition to a greatly improved signal gain, this immunosensor gives a favourable reusability and good reproducibility. Moreover, the CTDONG film-based sensing interface shows excellent anti-interference ability to the coexistent proteins. Meanwhile, the recovery test and determination of target molecule in real samples have confirmed the feasibility of designed sensing system for clinic immunoassay of protein molecules, demonstrating the potential application of described CTDONG film in the development of biomolecule assay platforms. [ABSTRACT FROM AUTHOR]

A novel experimental methodology based on a Prussian blue (PB) and gold nanoparticles (AuNPs) modified carbon ionic liquid electrode (CILE) was developed for use in a label-free amperometric immunosensor for the sensitive detection of human immunoglobulin G (HIgG) as a model protein. The CILE was fabricated by using the ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate as binder. Controllable electrodeposition of PB on the surface of the CILE and coating with 3-aminopropyl triethylene silane (APS) formed a film with high electronic catalytic activity and large surface area for the assembly of AuNPs and further immobilization of HIgG antibody. The electrochemistry of the formed nanocomposite biofilm was investigated by electrochemical techniques including cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy. The HIgG concentration was measured through the decrease of amperometric responses in the corresponding specific binding of antigen and antibody. The decreased differential pulse voltammetric values were proportional to the HIgG concentration in two ranges, 0.05–1.25 ng mL−1 and 1.25–40 ng mL−1, with a detection limit of 0.001 ng mL−1 (S/N = 3). This electrochemical immunoassay combined the specificity of the immunological reaction with the sensitivity of the AuNPs, ionic liquid, and PB amplified electrochemical detection and would therefore be valuable for clinical immunoassays. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

A simple and sensitive biosensor was developed for the determination of human immunoglobulin G (IgG). Protein A was employed as molecular receptor and electrochemical impedance spectroscopy (EIS) was used as detection technique. The biosensor was obtained by self-assembling protein A on a gold electrode. The surface morphology of the self-assembled layer before and after interaction with IgG was studied by atomic force microscopy. Protein A bound specifically to the Fc portion of IgG, and this caused a change in the resistance of the interfacial electron transfer when using a ferrocyanide redox couple as a probe. The increase of the resistance of the electron transfer was linearly related to the concentration of IgG in the range from 10 ng.mL−1 to 1.0 μg.mL−1, with a detection limit of 5 ng.mL−1. The work demonstrates that protein A is a versatile matrix for the immobilization of antibodies. [ABSTRACT FROM AUTHOR]

Objectives: Examine the antitumor activity of the histone deacetylase inhibitor vorinostat’s antitumor activity against multiple myeloma (MM) using cell lines and a murine xenograft model. Methods: RPMI8226, U266, and MM1S cells were cultured for 48 h in the presence of media, vorinostat, melphalan, or bortezomib alone, or combinations of vorinostat with melphalan or bortezomib. Cell proliferation was measured using the MTS [3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfphophenyl)-2H-tetrazolium, inner salt] assay. Severe combined immunodeficient mice bearing LAGκ-1B tumors were treated with vorinostat [30, 60, or 100 mg/kg daily for five consecutive days per week (qd×5d), 100 or 300 mg/kg daily for 2 d/wk (qd×2d)], melphalan (1, 3, or 10 mg/kg qd×1d), bortezomib (0.25 or 0.5 mg/kg qd×2d), or combinations thereof for 35 d. Tumor growth was determined via measurement of human immunoglobulin G (hIgG) levels and tumor volume. Results and Conclusions: Vorinostat enhanced the anti-MM effects of melphalan and bortezomib in vitro. Synergism was observed with vorinostat and melphalan in RPMI8226 and U266 cell lines. Vorinostat 100 mg/kg in combination with melphalan 3 mg/kg resulted in significant inhibition of tumor growth in vivo, compared with control (tumor volume P = 0.0001; hIgG P = 0.0001), single-agent vorinostat (tumor volume P = 0.0025; hIgG P = 0.0137), and single-agent melphalan (tumor volume P = 0.0043; hIgG P = 0.0426). Vorinostat also enhanced the antimyeloma effects of bortezomib in vivo. Vorinostat enhances the anti-MM activity of melphalan and bortezomib in vitro and in vivo. This study provides rationale for further evaluation of vorinostat in combination with chemotherapeutic agents and bortezomib for the treatment of MM. [ABSTRACT FROM AUTHOR]

A new strategy is described to construct disposable electrochemical immunosensors for the assay of human immunoglobulin. It is based on a carbon paste electrode constructed from chitosan nanoparticles modified with colloidal gold. The stepwise assembly process of the immunosensor was characterized by means of cyclic voltammetry and electrochemical impedance spectroscopy. Assay conditions that were optimized included the amount of chitosan nanoparticles in the preparation of carbon paste electrode, antibody concentration, and the incubation time of the antibody immobilization. Using hexacyanoferrate as a mediator, the current change increased with the concentration of human immunoglobulin G. A linear relationship in the concentration range 0.3 to 120 ng mL−1 was achieved, with a detection limit of 0.1 ng mL−1 (S/ N = 3). The method combines the specificity of the immunological reaction with the sensitivity of the gold colloid amplified electrochemical detection, and it has potential application in clinical immunoassay. [ABSTRACT FROM AUTHOR]

Abstract: A novel immunoassay using the eggshell membrane as immobilization platform for determination of human IgG (HIgG) has been developed. The immunoassay was based on a sandwich immunoreaction of rabbit anti-human IgG (primary antibody), HIgG and the goat anti-human IgG labeled with FITC (secondary antibody). Due to well permeability and highly biocompatibility of eggshell membrane, a friendly microenvironment was constructed to prolong the life-times of the immobilized proteins. Under the optimal conditions, the fluorescence intensity is linear to the concentration of HIgG in a range of 20–100ngmL−1 (r =0.9918). The proposed method was successfully applied to the determination of the HIgG in the standard blood serum, and the results are satisfactory compared with that obtained by general immunonephelometric method. [Copyright &y& Elsevier]

Abstract: A novel electrochemiluminescence (ECL) sandwich-type immunosensor for human immunoglobulin G (hIgG) on a gold nanoparticle modified electrode was developed by using N-(aminobutyl)-N-ethylisoluminol (ABEI) labeling. The primary antibody, goat-anti-human IgG was first immobilized on a gold nanoparticle modified electrode, then the antigen (human IgG) and the ABEI-labeled second antibody was conjugated successively to form a sandwich-type immunocomplex. ECL was carried out with a double-step potential in carbonate buffer solution (CBS) containing 1.5mM H2O2. The ECL intensity increased linearly with the concentration of hIgG over the range 5.0–100ng/mL. The limit of detection was 1.68ng/mL (S/N=3). The relative standard deviation was 3.79% at 60ng/mL (n =9). The present immunosensor is simple and sensitive. It has been successfully applied to the detection of hIgG in human serums. [Copyright &y& Elsevier]

Abstract: Chromatographic resins of a family of linear Fc-binding hexamer peptides (HWRGWV, HYFKFD, and HFRRHL) exhibited the ability to selectively adsorb and isolate human IgG (hIgG) from complete mammalian cell culture medium (cMEM). Among them, the HWRGWV resin with a peptide density of 0.08mequiv./g of resin was able to purify hIgG from cMEM with both purity and yield as high as 95%, comparable to Protein A and A2P agarose gels. The influences of N-terminal acetylation of the HWRGWV resin, ligand density on the resin, initial hIgG concentration, and temperature on IgG isolation were also investigated. The results indicate that these small peptide ligands, especially HWRGWV, offer a potential alternative to the use of Protein A or Protein G for large scale affinity chromatography. [Copyright &y& Elsevier]

Abstract: This paper reports a simple, sensitive, and reusable piezoelectric immunosensor using magnetic hydroxyapatite (HAP)/γ-Fe2O3/Au nanocomposite. Use of porous HAP nanocrystals embedded with γ-Fe2O3 and colloidal gold nanoparticles resulted in a multifunctional HAP/γ-Fe2O3/Au nanocomposite. Under optimized conditions, the biocompatible nanocomposites were exploited for direct adsorption of large quantities of rabbit anti-human immunoglobulin G antibodies (anti-hIgG) with well-preserved immunoactivity. In a homogeneous bulk solution, the hIgG analytes were captured by the anti-hIgG-derivatized immunocomposites followed by magnetic separation/enrichment onto a bovine serum albumin (BSA)-sealed QCM probe before measuring. This QCM immunosensor can quantitatively determine concentrations of hIgG ranging from ~20 to 800 ng/ml, with a detection limit of ~15 ng/ml. Moreover, regeneration of the immunosensor can be simply realized by canceling the controllable magnetic field. With the possibility of performing the analysis automatically and considering its ease of use, high sensitivity, and good reusability, this magnetic separation-assisted QCM immunosensor may have great potential to be further tailored as a general and promising alternative for a broad range of practical applications. [Copyright &y& Elsevier]

We report the multiplexed, simultaneous analysis of antigen–antibody interactions that involve human immunoglobulin G (IgG) on a gold substrate by the surface plasmon resonance imaging method. A multichannel, microfluidic chip was fabricated from poly(dimethylsiloxane) (PDMS) to selectively functionalize the surface and deliver the analyte solutions. The sensing interface was constructed using avidin as a linker layer between the surface-bound biotinylated bovine serum albumin and biotinylated anti-human IgG antibodies. Four mouse anti-human IgG antibodies were selected for evaluation and the screening was achieved by simultaneously monitoring protein–protein interactions under identical conditions. Antibody–antigen binding affinities towards human immunoglobulin were quantitatively compared by employing Langmuir adsorption isotherms for the analysis of SPRi responses obtained under equilibrium conditions. We were able to identify two IgG samples with higher affinities towards the target, and the determined binding kinetics falls within the typical range of values reported in the literature. Direct measurement of proteins in serum samples by SPR imaging was achieved by developing methods to minimize nonspecific adsorption onto the avidin-functionalized surface, and a limit of detection (LOD) of 6.7 nM IgG was obtained for the treated serum samples. The combination of SPR imaging and multichannel PDMS chips offers convenience and flexibility for sensitive and label-free measurement of protein–protein interactions in complex conditions and enables high-throughput screening of pharmaceutically significant molecules. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The delivery of monoclonal antibodies (mAbs) as subcutaneous (sc) injections hinges on the high dose requirement of these usually low potency molecules. This necessitates their formulation as high concentration solutions or suspensions, which presents a formidable formulation challenge due to the concentration-driven protein aggregation and high solution viscosity generated at these conditions. The objective of this study was to evaluate the feasibility of spray-drying in preparing stable, high concentration formulations of mAbs. A model polyclonal antibody, human immunoglobulin G (IgG) was formulated as dry powder using Nektar's glass stabilization technology. Formulation in sugar glasses stabilized IgG during spray-drying and maintained the protein's secondary structure. Further, in contrast to the bulk material, the glass-stabilized powders successfully reconstituted at 200 mg/mL IgG without loss of the protein monomer. Spectroscopic analysis confirmed that upon high concentration reconstitution, spray-dried glass-stabilized IgG retained both its secondary and tertiary structure. Further, the spray-dried powder reconstituted within a few minutes yielding clear, low viscosity solutions that syringed easily through narrow (28 G) needles. The results of this study suggest that formulation in spray-dried, glass-stabilized powders may enable the development of products suitable for sc administration of mAbs and other low potency protein therapeutics. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96:1504–1517, 2007 [ABSTRACT FROM AUTHOR]

Abstract: Plasma-polymerized films were formed on flat glass plates using allylamine, acrylic acid, acrolein, and allylcyanide as monomers. Adsorption of 125I-labeled-proteins such as immunoglobulin G (IgG), its F(ab′)2 and Fc fragments, and human serum albumin (HSA) was measured on these plasma-polymerized (PP) films covering the glass plates and on commercially available polymer plates. The adsorption isotherm followed the Langmuir equation, from which the binding constant and amount of saturation binding were estimated. We found that, in general, a cationic surface had higher affinity for protein adsorption than an anionic surface. Among the surfaces examined, the PP-allylamine surface showed the highest binding capacity (264.2nmol/m2) for F(ab′)2 fragment: it was remarkably high. Of the surfaces examined, the PP-acrylic acid surface showed the lowest binding capacity (12.8nmol/m2) for F(ab′)2 fragment. The PP-acrylic acid surface also indicated the lowest protein binding capacity for IgG (16.5nmol/m2), Fc-IgG (32.4nmol/m2) and HSA (16.7nmol/m2), respectively. These imply that the PP-acrylic acid film is useful to fabricate as a low protein adsorption material which expected to decrease cell adhesion. Results of our investigation indicate that the plasma-polymerization technique is promising for fabrication of a smart NanoBio-interface which can control the protein adsorption on a solid-phase substrate using a suitable monomer such as allylamine for the large adsorption and acrylic acid for the small adsorption. [Copyright &y& Elsevier]

Abstract: An immunosensor interface based on mixed hydrophobic self-assembled monolayers (SAMs) of methyl and carboxylic acid terminated thiols with covalently attached human Immunoglobulin G (hIgG), is investigated. The densely packed and organised SAMs were characterised by contact angle measurements and cyclic voltammetry. The effect of the non-ionic surfactant, Tween 20, in preventing nonspecific adsorption is addressed by ellipsometry during physical and covalent hIgG immobilization on pure and mixed SAMs, respectively. It is clearly demonstrated that nonspecific adsorption due to hydrophobic interactions of hIgG on methyl ended groups is totally inhibited, whereas electrostatic/hydrogen bonding interactions with the exposed carboxylic groups prevail in the presence of surfactant. Results of ellipsometry and Atomic Force Microscopy, reveal that the surface concentration of covalently immobilized hIgG is determined by the ratio of COOH / CH3-terminated thiols in SAM forming solution. Moreover, the ellipsometric data demonstrates that the ratio of bound anti-hIgG / hIgG depends on the density of hIgG on the surface and that the highest ratio is close to three. We also report the selectivity and high sensitivity achieved by chronoamperometry in the detection of adsorbed hIgG and the reaction with its antibody. [Copyright &y& Elsevier]

Abstract: A capacitive sensing method based on self-assembling gold nanoparticles to the surface of the sol–gel modified electrode has been developed for the direct detection of the human IgG in human serum. The capacitance of the immunosensor corresponding to the concentration of human IgG is investigated by alternating current impedance. The formed mercaptopropyltriethoxysilane (MPTS) film is ultrathin; the immobilization density of antibodies is high because of high surface-volume area of the assembled gold nanoparticles and the biological macromolecules when immobilized on gold nanoparticles can retain their bioactivity. This capacitive immunosensor prepared with present method can provide high sensitivity. The linear calibration curve was obtained in the range 8.3–2128ng/ml, with a detection limit of 3.3ng/ml when plotted versus the logarithm of the antigen concentration. After each immunoassay, the regeneration of the electrode could be performed through washing in basic solution without obvious decrease in response. No cross-reactivity was observed with other protein species. The dependence of sol–gel modified electrode stability on the pH value and ion strength was studied. The insulating properties of the different layers of the immunosensor were also investigated. [Copyright &y& Elsevier]

The competitive adsorption of blood proteins is of great importance for the treatment of thrombosis using a colloidal drug delivery system. The aim of this study is to investigate competitive adsorption of albumin (BSA) and human immunoglobulin G (HIgG) against fibrinogen (Fb). The competitive adsorption of blood proteins was investigated using interfacial rheology at physiological pH. The influence of bulk concentration, temperature and pH on the interfacial adsorption of protein molecules was determined at the air/aqueous interface. As expected, the results indicated that increase in bulk concentration enhanced the interfacial adsorption. Structure and molecular weight of the protein molecules under investigation had influence on interfacial adsorption leading to a competition at the interface. HIgG is more flexible and surface active molecule than BSA. Thus, HIgG replaced BSA and Fb at the air/aqueous interface. In the presence of Fb, BSA adsorbed rapidly initially and then, was replaced by Fb at the interface. The kinetics of displacement of albumin at the interface was rather slow. In conclusion, the investigation of competitive adsorption of blood proteins may be useful biotechnologically, as it will provide useful information for the production of an antithrombogenic material, which will adsorb albumin rather than Fb. [Copyright &y& Elsevier]

A resonant mirror biosensor (IAsys) protocol is described for the comparative kinetic analysis of the ability of monoclonal antibodies (Mabs) and bispecific antibodies (Babs) to bind immobilized antigens. The protocol has been optimized and validated using the panel of affinity-purified antibodies, including two parental Mabs, one specific to human immunoglobulin G (hIgG) and another specific to horseradish peroxidase (HRP), and a Bab derived thereof by cell fusion (anti-hIgG/HRP Bab). The real-time kinetic analysis of antigen–antibody interactions using this protocol allows to demonstrate the differences in the avidity of bivalently binding Mabs and monovalent Babs. As shown in our previous study [J. Immunol. Methods 261 (2002) 103], the observed equilibrium association constants (Kass) determined by IAsys using this protocol yield figures almost overlapping with those obtained by solid-phase radioimmunoassay (RIA). The described protocol is suited for the investigation of the effects of valency on the binding properties of antibodies. It also may be applied for the selection of Mabs and Babs with desired features, for different fields of application. [Copyright &y& Elsevier]