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Baicalein, a nutritional phenolic bioactive small molecule present in the roots of Scutellaria baicalensis and Scutellaria lateriflora, shows promising therapeutic effects against a wide range of cancer cells. However, its potential binding with blood carrier proteins and its anticancer mechanisms have not been well investigated. In this study, the interaction between baicalein and immunoglobulin G (IgG) was analyzed by analyzing intrinsic fluorescence, ellipticity changes, as well as molecular docking. The anticancer effects of baicalein against a glioblastoma multiforme (GBM) cell line, U87MG, and one normal immortalized human astrocytes were assessed by MTT, real-time PCR, caspase assay, and western blot. It was shown that the fluorescence quenching mechanism of human IgG by baicalein was based on a static quenching, where one binding site was responsible for the formation of baicalein-IgG complex derived by the formation of hydrogen bonds and van der Waals forces. A molecular docking study indicated that tyrosine residues (Tyr 91 and Tyr 98) were mostly involved in the binding site of baicalein to IgG. The cellular assay demonstrated that baicalein induced U87MG cell line death with an IC50 concentration of 73.19 µM, whereas normal astrocytes depicted over 82.94% portion of viability at 100 µM baicalein. The Bax mRNA overexpression, Bcl-2 mRNA downexpression, as well as the elevation of caspase-9 and caspase-3 activities confirmed that baicalein could effectively trigger the apoptosis of U87MG cells. Moreover, it was explored that baicalein downregulated the expression of the Axl/STAT3 signaling pathway in the U87MG cells, which is responsible for cellular proliferation and growth. In conclusion, these data may provide useful information regarding the pharmaceutical application of baicalein.

This work reports new analytical applications of glassy carbon electrodes (GCE) modified with a nanohybrid obtained by non-covalent functionalization of multi-walled carbon nanotubes (MWCNTs) with human immunoglobulin G (IgG) (GCE/MWCNT-IgG). We report the label-free and non-amplified breast cancer 1 gen (BRCA1) biosensing based on the facilitated adsorption of the DNA probe at the nanohybrid modified GCE and the impedimetric detection of the hybridization event in the presence of the redox marker benzoquinone/hydroquinone. The resulting genosensor made the fast, highly selective, and sensitive quantification of BRCA1 gene possible, with a linear range between 1.0 fM and 10.0 nM, a sensitivity of (3.0 ± 0.1) × 102 Ω M−1 (R2 = 0.9990), a detection limit of 0.3 fM, and excellent discrimination of fully non-complementary and mismatch DNA sequences. The detection of BRCA1 in enriched samples of diluted human blood serum showed a recovery percentage of 94.6%. Another interesting analytical application of MWCNT-IgG-modified GCE based on the catalytic activity of the exfoliated MWCNTs is also reported for the simultaneous quantification of dopamine and uric acid in the presence of ascorbic acid, with detection limits at submicromolar levels for both compounds. [ABSTRACT FROM AUTHOR]

This work reports new analytical applications of glassy carbon electrodes (GCE) modified with a nanohybrid obtained by non-covalent functionalization of multi-walled carbon nanotubes (MWCNTs) with human immunoglobulin G (IgG) (GCE/MWCNT-IgG). We report the label-free and non-amplified breast cancer 1 gen (BRCA1) biosensing based on the facilitated adsorption of the DNA probe at the nanohybrid modified GCE and the impedimetric detection of the hybridization event in the presence of the redox marker benzoquinone/hydroquinone. The resulting genosensor made the fast, highly selective, and sensitive quantification of BRCA1 gene possible, with a linear range between 1.0 fM and 10.0 nM, a sensitivity of (3.0 ± 0.1) × 102 Ω M−1 (R2 = 0.9990), a detection limit of 0.3 fM, and excellent discrimination of fully non-complementary and mismatch DNA sequences. The detection of BRCA1 in enriched samples of diluted human blood serum showed a recovery percentage of 94.6%. Another interesting analytical application of MWCNT-IgG-modified GCE based on the catalytic activity of the exfoliated MWCNTs is also reported for the simultaneous quantification of dopamine and uric acid in the presence of ascorbic acid, with detection limits at submicromolar levels for both compounds.

This work aimed to evaluate the adsorption of human Immunoglobulin G in the hydrophobic interaction adsorbents: Phenyl Sepharose 6 Fast Flow High Sub (High Sub) and Phenyl Sepharose 6 Fast Flow Low Sub (Low Sub). Kinetic tests were performed at 298 K. Equilibrium was evaluated at 277 K, 288 K, 298 K and 310 K. Data modelling was performed by using isotherm models and artificial neural networks (ANN). The highest adsorption capacity was 56.30 ± 6.50 mg mL−1 for the experiment performed using 1.0 mol L−1 ammonium sulphate buffer and High Sub adsorbent at 288 K. In this condition of highest ligand density and salt concentration were found the best results regarding the behaviour of the isotherms. When comparing the results obtained in different temperatures, there was practically no difference, considering the standard deviation, between the values of maximum capacity adsorption. Also, if either salt concentration or ligands density was increased, higher values of adsorption capacity were obtained. All the dissociation constants showed to be between intermediate and strong. The best modelling result was obtained by the ANNs, which accurately represented the experimental isotherms with mean percentage error equal to 0.68%, and R2 above 0.999. It had 2 neurons in the hidden layer, and it was trained with the Levenberg–Marquardt algorithm. The ANN modelling was effective in predicting the adsorption isotherms, as it proved to be able to interpolate its behaviour in the studied temperature range. Furthermore, it outgrew possible errors arising from experimental procedures and/or from adsorption phenomenology. [ABSTRACT FROM AUTHOR]

Baicalein, a nutritional phenolic bioactive small molecule present in the roots of Scutellaria baicalensis and Scutellaria lateriflora, shows promising therapeutic effects against a wide range of cancer cells. However, its potential binding with blood carrier proteins and its anticancer mechanisms have not been well investigated. In this study, the interaction between baicalein and immunoglobulin G (IgG) was analyzed by analyzing intrinsic fluorescence, ellipticity changes, as well as molecular docking. The anticancer effects of baicalein against a glioblastoma multiforme (GBM) cell line, U87MG, and one normal immortalized human astrocytes were assessed by MTT, real-time PCR, caspase assay, and western blot. It was shown that the fluorescence quenching mechanism of human IgG by baicalein was based on a static quenching, where one binding site was responsible for the formation of baicalein-IgG complex derived by the formation of hydrogen bonds and van der Waals forces. A molecular docking study indicated that tyrosine residues (Tyr 91 and Tyr 98) were mostly involved in the binding site of baicalein to IgG. The cellular assay demonstrated that baicalein induced U87MG cell line death with an IC 50 concentration of 73.19 µM, whereas normal astrocytes depicted over 82.94% portion of viability at 100 µM baicalein. The Bax mRNA overexpression, Bcl-2 mRNA downexpression, as well as the elevation of caspase-9 and caspase-3 activities confirmed that baicalein could effectively trigger the apoptosis of U87MG cells. Moreover, it was explored that baicalein downregulated the expression of the Axl/STAT3 signaling pathway in the U87MG cells, which is responsible for cellular proliferation and growth. In conclusion, these data may provide useful information regarding the pharmaceutical application of baicalein. [ABSTRACT FROM AUTHOR]

Aims: Coronavirus disease 2019 (COVID-19) is rapidly spreading worldwide. Drug therapy is one of the major treatments, but contradictory results of clinical trials have been reported among different individuals. Furthermore, comprehensive analysis of personalized pharmacotherapy is still lacking. In this study, analyses were performed on 47 well-characterized COVID-19 drugs used in the personalized treatment of COVID-19. Methods: Clinical trials with published results of drugs use for COVID-19 treatment were collected to evaluate drug efficacy. Drug-to-Drug Interactions (DDIs) were summarized and classified. Functional variations in actionable pharmacogenes were collected and systematically analysed. "Gene Score" and "Drug Score" were defined and calculated to systematically analyse ethnicity-based genetic differences, which are important for the safer use of COVID-19 drugs. Results: Our results indicated that four antiviral agents (ritonavir, darunavir, daclatasvir and sofosbuvir) and three immune regulators (budesonide, colchicine and prednisone) as well as heparin and enalapril could generate the highest number of DDIs with common concomitantly utilized drugs. Eight drugs (ritonavir, daclatasvir, sofosbuvir, ribavirin, interferon alpha-2b, chloroquine, hydroxychloroquine (HCQ) and ceftriaxone had actionable pharmacogenomics (PGx) biomarkers among all ethnic groups. Fourteen drugs (ritonavir, daclatasvir, prednisone, dexamethasone, ribavirin, HCQ, ceftriaxone, zinc, interferon beta-1a, remdesivir, levofloxacin, lopinavir, human immunoglobulin G and losartan) showed significantly different pharmacogenomic characteristics in relation to the ethnic origin of the patient. Conclusion: We recommend that particularly for patients with comorbidities to avoid serious DDIs, the predictive, preventive, and personalized medicine (PPPM, 3 PM) strategies have to be applied for COVID-19 treatment, and genetic tests should be performed for drugs with actionable pharmacogenes, especially in some ethnic groups with a higher frequency of functional variations, as our analysis showed. We also suggest that drugs associated with higher ethnic genetic differences should be given priority in future pharmacogenetic studies for COVID-19 management. To facilitate translation of our results into clinical practice, an approach conform with PPPM/3 PM principles was suggested. In summary, the proposed PPPM/3 PM attitude should be obligatory considered for the overall COVID-19 management. [ABSTRACT FROM AUTHOR]

Abstract Background Spinal cord injury (SCI) is a condition with few effective treatment options. The blood-spinal cord barrier consists of pericytes, astrocytes, and endothelial cells, which are collectively termed the neurovascular unit. These cells support spinal cord homeostasis by expressing tight junction proteins. Physical trauma to the spinal cord disrupts the barrier, which leads to neuroinflammation by facilitating immune cell migration to the damaged site in a process involving immune cell adhesion. Immunosuppressive strategies, including methylprednisolone (MPSS), have been investigated to treat SCI. However, despite some success, MPSS has the potential to increase a patient’s susceptibility to wound infection and impaired wound healing. Hence, immunomodulation may be a more attractive approach than immunosuppression. Approved for modulating neuroinflammation in certain disorders, including Guillain-Barre syndrome, intravenous administration of human immunoglobulin G (hIgG) has shown promise in the setting of experimental SCI, though the optimal dose and mechanism of action remain undetermined. Methods Female adult Wistar rats were subjected to moderate-severe clip compression injury (35 g) at the C7-T1 level and randomized to receive a single intravenous (IV) bolus of hIgG (0.02, 0.2, 0.4, 1, 2 g/kg), MPSS (0.03 g/kg), or control buffer at 15 min post-SCI. At 24 h and 6 weeks post-SCI, molecular, histological, and neurobehavioral effects of hIgG were analyzed. Results At 24 h post-injury, human immunoglobulin G co-localized with spinal cord pericytes, astrocytes, and vessels. hIgG (2 g/kg) protected the spinal cord neurovasculature after SCI by increasing tight junction protein expression and reducing inflammatory enzyme expression. Improvements in vascular integrity were associated with changes in spinal cord inflammation. Interestingly, hIgG (2 g/kg) increased serum expression of inflammatory cytokines and co-localized (without decreasing protein expression) with spinal cord vascular cell adhesion molecule-1, a protein used by immune cells to enter into inflamed tissue. Acute molecular benefits of hIgG (2 g/kg) led to greater tissue preservation, functional blood flow, and neurobehavioral recovery at 6 weeks post-SCI. Importantly, the effects of hIgG (2 g/kg) were superior to control buffer and hIgG (0.4 g/kg), and comparable with MPSS (0.03 g/kg). Conclusions hIgG (2 g/kg) is a promising therapeutic approach to mitigate secondary pathology in SCI through antagonizing immune cell infiltration at the level of the neurovascular unit.

Background/introduction: The neuroinflammatory response plays a major role in the secondary injury cascade after traumatic spinal cord injury (SCI). To date, systemic anti-inflammatory medications such as methylprednisolone sodium succinate (MPSS) have shown promise in SCI. However, systemic immunosuppression can have detrimental side effects. Therefore, immunomodulatory approaches including the use of human immunoglobulin G (hIgG) could represent an attractive alternative. While emerging preclinical data suggests that hIgG is neuroprotective after SCI, the optimal time window of administration and the mechanism of action remain incompletely understood. These knowledge gaps were the focus of this research study. Methods: Female adult Wistar rats received a clip compression-contusion SCI at the C7/T1 level of the spinal cord. Injured rats were randomized, in a blinded manner, to receive a single intravenous bolus of hIgG (2 g/kg) or control buffer at 15 minutes (min), 1 hour (h) or 4 h post-SCI. At 24 h and 8 weeks post-SCI, molecular, histological and neurobehavioral analyses were undertaken. Results: At all 3 administration time points, hIgG (2 g/kg) resulted in significantly better short-term and long-term outcomes as compared to control buffer. No significant differences were observed when comparing outcomes between the different time points of administration. At 24 h post-injury, hIgG (2 g/kg) administration enhanced the integrity of the blood spinal cord barrier (BSCB) by increasing expression of tight junction proteins and reducing inflammatory enzyme expression. Improvements in BSCB integrity were associated with reduced immune cell infiltration, lower amounts of albumin and Evans Blue in the injured spinal cord and greater expression of anti-inflammatory cytokines. Furthermore, hIgG (2 g/kg) increased expression of neutrophil chemoattractants in the spleen and sera. After hIgG (2 g/kg) treatment, there were more neutrophils in the spleen and fewer neutrophils in the blood. hIgG also co-localized with endothelial cell ligands that mediate neutrophil extravasation into the injured spinal cord. Importantly, short-term effects of delayed hIgG (2 g/kg) administration were associated with enhanced tissue and neuron preservation, as well as neurobehavioral and sensory recovery at 8 weeks post-SCI. Discussion and conclusion: hIgG (2 g/kg) shows promise as a therapeutic approach for SCI. The anti-inflammatory effects mediated by hIgG (2 g/kg) in the injured spinal cord might be explained in twofold. First, hIgG might antagonize neutrophil infiltration into the spinal cord by co-localizing with endothelial cell ligands that mediate various steps in neutrophil extravasation. Second, hIgG could traffic neutrophils towards the spleen by increasing expression of neutrophil chemoattractants in the spleen and sera. Overall, we demonstrate that delayed administration of hIgG (2 g/kg) at 1 and 4-h post-injury enhances short-term and long-term benefits after SCI by modulating local and systemic neuroinflammatory cascades.

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 work aimed to evaluate the interaction of human IgG in non-conventional adsorbents based on chitosan and alginate in the absence and presence of Reactive Green, Reactive Blue and Cibacron Blue immobilized as ligands. The adsorption was evaluated at 277, 288, 298 and 310 K using sodium phosphate buffer, pH 7.6, at 25 mmol L−1. The highest adsorption capacity was observed in the experiments performed with no immobilized dye, although all showed adsorption capacity higher than 120 mg g−1. Data modeling was done using Langmuir, Langmuir-Freundlich and Temkin classical nonlinear models, and artificial neural networks (ANN) for comparison. According to the parameters obtained, a possible adsorption in multilayers was observed due to protein-adsorbent and protein-protein interactions, concluding that IgG adsorption process is favorable and spontaneous. Using an ANN structure with 3 hidden neurons (single hidden layer), the MSE (RMSE) for training, test and validation were 13.698 (3.701), 11.206 (3.347) and 7.632 (2.763), respectively, achieving correlation coefficients of 0.999 in all steps. ANN modeling proved to be effective in predicting the adsorption isotherms in addition to overcoming the difficulties caused by experimental errors and/or arising from adsorption phenomenology. [ABSTRACT FROM AUTHOR]

The present study represents the fabrication of electrochemical impedimetric immunosensor based on host–guest interaction. The β-cyclodextrin self-assembled monolayer was first modified on the surface of Au electrode. Then, ionic liquids functionalized with both aldehyde and ferrocene groups were introduced on the electrode via host–guest interaction between β-cyclodextrin and ferrocene. Here, aldehyde groups were used to directly immobilize antibody, in which no additional chemical cross-linker is required. The electron transfer resistance before and after human immunoglobulin G attachment was monitored as a function of its concentration to perform the analysis. The proposed immunosensor had a good performance in a linear range of 0.1–50 ng mL−1 with a detection limit of 0.03 ng mL−1 (S/N = 3). The process of fabricating immunosensor was characterized using cyclic voltammetric and electrochemical impedance spectroscopy. The impedimetric immunosensor provided a simple and sensitive detection method for the specific determination of IgG in human serum. [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]

We report an ultrasensitive protocol for electrochemical sensing using the hydroxyl-rich C-dots assisted synthesis of gold nanoparticles (C-dots@AuNP) as labels with copper depositon reaction. The C-dots catalyzing copper deposition reaction was implemented for the first time. We constructed a sandwich-type immunosensor on the chitosan modified glassy carbon electrode (GCE) by glutaraldehyde (GA) crosslinking, with C-dots@AuNP as biolabels. Copper was deposited on the catalytic surfaces of second antibody-conjugated C-dots@AuNP nanoparticles through CuSO4-ascorbic acid reduction, because both C-dots and AuNPs could strongly catalyze the CuSO4 and ascorbic acid to form Cu particles, which amplified the detection signal. Then the corresponding antigen was quantified based on simultaneous chemical-dissolution/cathodic-preconcentration of copper for in-situ analysis using anodic stripping square wave voltammetry (ASSWV) directly on the modified electrode. Under optimized conditions, these electrodes were employed for sandwich-type immunoanalysis, pushing the lower limits of detection (LODs) down to the fg mL−1 level for human immunoglobulin G (IgG) and cardiac troponin I (cTnI), a cardiac biomarker. These novel sensors have good stability and acceptable accuracy and reproducibility, suggesting potential applications in clinical diagnostics. [ABSTRACT FROM AUTHOR]

ABSTRACT A biosensing platform based on the covalent attachment of biomolecules on electropolymerized carboxylated conducting polymers, poly(3-aminobenzoic acid) and poly(3-pyrrole carboxylic acid), were developed for the selective simultaneous detection of two biomolecules using electrochemical-surface plasmon resonance (EC-SPR) spectroscopy. The surface morphology of the developed biosensors was studied by scanning electron microscopy and atomic force microscopy. The EC-SPR dual biosensor was developed for the label-free, simultaneous, and selective detection of glucose and human immunoglobulin G (IgG). A change in current density was clearly observed after the injection of glucose, whereas a change in SPR reflectivity was clearly observed after the injection of human IgG. The present work demonstrates the potential of this biosensing platform for real sample analysis in the future. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018, 135, 45641. [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]

Mechanistic and conformational studies on the interaction of sulfamethoxazole (SMX) with human immunoglobulin G (HIgG) were performed by molecular modeling and multi-spectroscopic methods. The interaction mechanism was firstly predicted through molecular modeling that confirmed the interaction between SMX and HIgG. The binding parameters and thermodynamic parameters at different temperatures had been calculated according to the Stern−Volmer, Scatchard, Sips and Van 't Hoff equations, respectively. Experimental results showed that the fluorescence intensity of HIgG was quenched by the gradual addition of SMX. The binding constants of SMX with HIgG decreased with the increase of temperature, which meant that the quenching mechanism was a static quenching. Meanwhile, the results also confirmed that there was one independent class of binding site on HIgG for SMX during their interaction. The thermodynamic parameters of the reaction, namely standard enthalpy ΔH0 and entropy ΔS0, had been calculated to be −14.69 kJ·mol−1 and 22.99 J·mol−1·K−1, respectively, which suggested that the electrostatic and hydrophobic interactions were the predominant intermolecular forces in stabilizing the SMX−HIgG complex. Furthermore, experimental results obtained from three-dimensional fluorescence spectroscopy, UV−vis absorption spectroscopy and circular dichroism (CD) spectroscopy confirmed that the conformational structure of HIgG was altered in the presence of SMX. Copyright © 2015 John Wiley & Sons, Ltd. [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]

Binding interaction of sulfamethazine (SMZ) with human immunoglobulin G (HIgG) has been explored under physiological conditions. The interaction mechanism was firstly predicted through molecular modeling which showed that several hydrogen bonds participated in stabilizing the SMZ − HIgG complex. Fluorescence spectroscopy, ultraviolet-visible (UV-vis) light absorption and circular dichroism (CD) spectroscopy were used to analyze the binding site, binding constants and effects of SMZ on HIgG stability and secondary structure. The binding parameters and thermodynamic parameters at different temperatures for the reaction have been calculated according to the Scatchard, Sips and Van 't Hoff equations, respectively. Experimental results showed that the quenching mechanism was a static quenching and there was one independent class of binding site on HIgG for SMZ during their interaction. The thermodynamic parameters of the reaction, namely standard enthalpy ΔH0 and entropy ΔS0, had been calculated to be −19.12 kJ · mol−1 and 20.22 J · mol−1 · K−1, respectively, which meant that the electrostatic interaction was the predominant intermolecular force in stabilizing the SMZ − HIgG complex. Moreover, the conformational changes of HIgG in the presence of SMZ were confirmed by three-dimensional fluorescence spectroscopy, UV-vis absorption spectroscopy and CD spectroscopy. Copyright © 2014 John Wiley & Sons, Ltd. [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]