Multiplexed Antibody Glycosylation Profiling Using Dual Enzyme Digestion and Liquid Chromatography-Triple Quadrupole Mass Spectrometry Method.

Antibody glycosylation plays a crucial role in the humoral immune response by regulating effector functions and influencing the binding affinity to immune cell receptors. Previous studies have focused mainly on the immunoglobulin G (IgG) isotype owing to the analytical challenges associated with other isotypes. Thus, the development of a sensitive and accurate analytical platform is necessary to characterize antibody glycosylation across multiple isotypes. In this study, we have developed an analytical workflow using antibody-light-chain affinity beads to purify IgG, IgA, and IgM from 16 µL of human plasma. Dual enzymes, trypsin and Glu-C, were used during on-bead digestion to obtain enzymatic glycopeptides and protein-specific surrogate peptides. Ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry was used in order to determine the sensitivity and specificity. Our platform targets 95 glycopeptides across the IgG, IgA, and IgM isotypes, as well as eight surrogate peptides representing total IgG, four IgG classes, two IgA classes, and IgM. Four stable isotope-labeled internal standards were added after antibody purification to calibrate the preparation and instrumental bias during analysis. Calibration curves constructed using serially diluted plasma samples showed good curve fitting (R2 > 0.959). The intrabatch and interbatch precision for all the targets had relative standard deviation of less than 29.6%. This method was applied to 19 human plasma samples, and the glycosylation percentages were calculated, which were comparable to those reported in the literature. The developed method is sensitive and accurate for Ig glycosylation profiling. It can be used in clinical investigations, particularly for detailed humoral immune profiling.

A phase 1 study in healthy volunteers to investigate the safety, tolerability, and pharmacokinetics of VIR-2482: a monoclonal antibody for the prevention of severe influenza A illness.

The objective of this study was to evaluate the safety, tolerability, pharmacokinetics (PK), and immunogenicity of VIR-2482 in healthy adult subjects. A phase 1, first-in-human, randomized, double-blind, placebo-controlled dose-escalation study was conducted. One hundred participants were allocated to four cohorts (60 mg, 300 mg, 1,200 mg, and 1,800 mg). In each cohort, participants were randomized in a 4:1 ratio (active:placebo) to receive either VIR-2482 or volume-matched placebo by gluteal intramuscular injection. Participants remained at the investigative site under observation for 48 h, and adverse events (AEs) were collected for 56 days. PK and immunogenicity were measured up to 52 weeks post-dose. VIR-2482 was well tolerated at all doses studied. The overall incidence of AEs was comparable between VIR-2482 (68.8%) and placebo (85.0%). Nineteen VIR-2482 (23.8%) and six placebo (30.0%) recipients had Grade 1 or 2 AEs that were considered to be related to the study intervention. There were no treatment-related serious AEs. Injection-site reactions (ISRs) were reported in six (7.5%) VIR-2482 recipients, while no such reactions were reported among the placebo recipients. All ISRs were Grade 1, and there was no relationship with the dose. Median VIR-2482 serum elimination half-life ranged from 56.7 to 70.6 days across cohorts. The serum area under the curve and Cmax were dose-proportional. Nasopharyngeal VIR-2482 concentrations were approximately 2%-5% of serum levels and were less than dose-proportional. The incidence of immunogenicity across all cohorts was 1.3%. Overall, the safety, tolerability, and pharmacokinetic profile of VIR-2482 at doses up to 1,800 mg supported its further investigation as a long-acting antibody for the prevention of influenza A illness. This study has been registered at ClinicalTrials.gov under identifier NCT04033406.

Dynamics of scFv-targeted VAP2 correlating with IL-16, MIF and IL-1Ra in ANCA-associated vasculitis.

BACKGROUND AND HYPOTHESIS: Using a mouse model of MPA with microvascular lesion with a clone (VasSF) of recombinant single chain fragments of the variable region of human IgG, we previously showed that vasculitis-associated apolipoprotein A2 (VAP2) may be a therapeutic target for vasculitis. The present study estimated the target molecules for VasSF and the association between VAP2 and cytokine levels in patient sera in terms of microvascular lesion severity. METHODS: Sera and clinical information were collected from patients with microscopic polyangiitis and granulomatosis with polyangiitis (MPA/GPA) and infectious disease. Neutrophil counts, levels of C-reactive protein (CRP), creatinine, total cholesterol associated with microvascular lesion, HDL cholesterol, low-density lipoprotein cholesterol, triglycerides, glomerular filtration rate (eGFR), and cytokines were estimated. Serum VAP2 signals were determined with Western blotting. RESULTS: VasSF bound to a 24 kDa molecule in the serum of active MPA/GPA patients. Anti-AP2 antibody also bound with the same 24 kDa molecule, named VAP2, because of size difference from normal APOA2. The VAP2 signal was significantly stronger in the active-disease group but significantly weakened in remission. The signal correlated positively with eGFR but not with the Birmingham Vasculitis Activity Score, CRP, MPO-ANCA, or PR3-ANCA levels. It correlated negatively with MPO activity, IL-16, MIF, and IL-1Ra. Moreover, VasSF bound to a 17 kDa molecule in the remission phase. CONCLUSION: The 24 kDa VAP2 molecule may be associated with neutrophil functions because of its inverse correlation with MPO activity, IL-16, MIF, and IL-1Ra, suggesting that VAP2-APOA1 formation in HDL triggers microvascular injury. VasSF may reverse the injury by removing APOA1-VAP2 heterodimers from peripheral blood vessels.

Delayed administration of high dose human immunoglobulin G enhances recovery after traumatic cervical spinal cord injury by modulation of neuroinflammation and protection of the blood spinal cord barrier.

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.

Orally Administered Human Immunoglobulin Therapy for Norovirus Enteritis in Solid Organ Transplant Recipients: A Case Series at a Single Academic Transplant Center.

Norovirus enteritis can cause intractable diarrhea in solid organ transplant (SOT) recipients, for which there are no established treatments. We reviewed medical records of 9 SOT recipients at our center who received orally administered human immunoglobulin for norovirus enteritis, and it appeared to be an effective treatment modality.

SARS-CoV-2 Inflammatory Syndrome. Clinical Features and Rationale for Immunological Treatment.

The current pandemic coronavirus, SARS-CoV-2, is a global health emergency because of its highly contagious nature, the great number of patients requiring intensive care therapy, and the high fatality rate. In the absence of specific antiviral drugs, passive prophylaxis, or a vaccine, the treatment aim in these patients is to prevent the potent virus-induced inflammatory stimuli from leading to the acute respiratory distress syndrome (ARDS), which has a severe prognosis. Here, the mechanism of action and the rationale for employing immunological strategies, which range from traditional chemically synthesized drugs, anti-cytokine antibodies, human immunoglobulin for intravenous use, to vaccines, are reviewed.

The effects of human immunoglobulin G on enhancing tissue protection and neurobehavioral recovery after traumatic cervical spinal cord injury are mediated through the neurovascular unit.

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.

Intravenous human immunoglobulin and/or methylprednisolone pulse therapies as a possible treat-to-target strategy in immune-mediated necrotizing myopathies.

To evaluate the relevance of immunoglobulin (IVIg) and/or methylprednisolone pulse therapies in immune-mediated necrotizing myopathy (IMNM). Secondarily, to analyze the muscle damage measured by late magnetic resonance images (MRI). This retrospective study included 13 patients with defined IMNM (nine patients positive for the anti-signal recognition particle and four patients positive for hydroxyl-methyl-glutaryl coenzyme A reductase) who were followed from 2012 to 2018. International Myositis Assessment and Clinical Studies Group (IMACS) scoring assessed the response to a standardized treat-to-target protocol with disease activity core-set measures and late magnetic resonance imaging (MRI). The patients had a mean age of 53.5 years and were predominantly female and of white ethnicity. Median symptom and mean follow-up durations were 4 and 39 months, respectively. All patients received IVIg and/or methylprednisolone pulse therapies. All IMACS core-set measurements improved significantly after initial treatment. Nine patients achieved complete clinical response and among them 2 had complete remission. Eleven patients had discontinued glucocorticoid use by the end of the study. Only 2 patients had moderate muscle atrophy or fat replacement observed by MRI, with the remainder presenting normal or mild findings. Our patients with IMNM treated with an aggressive immunosuppressant therapy had a marked improvement in all IMACS core-set domains. Moreover, the MRI findings suggest that an early treat-to-target approach could reduce the odds of long-term muscle disability. Methylprednisolone and/or IVIg pulse therapies aiming at a target of complete clinical response are potential treatment strategies for IMNM that should be studied in future prospective studies.

Immunoglobulin Attenuates Streptokinase-Mediated Virulence in Streptococcus dysgalactiae Subspecies equisimilis Necrotizing Fasciitis.

Background: Necrotizing fasciitis (NF) retains a very high mortality rate despite prompt and adequate antibiotic treatment and surgical debridement. Necrotizing fasciitis has recently been associated with Streptococcus dysgalactiae subspecies equisimilis (SDSE). Methods: We investigated the causes of a very severe clinical manifestation of SDSE-NF by assessing both host and pathogen factors. Results: We found a lack of streptokinase-function blocking antibodies in the patient resulting in increased streptokinase-mediated fibrinolysis and bacterial spread. At the same time, the clinical SDSE isolate produced very high levels of streptokinase. Exogenous immunoglobulin Gs (ex-IgGs) efficiently blocked streptokinase-mediated fibrinolysis in vitro, indicating a protective role against the action of streptokinase. In vivo, SDSE infection severity was also attenuated by ex-IgGs in a NF mouse model. Conclusions: These findings illustrate for the first time that the lack of specific antibodies against streptococcal virulence factors, such as streptokinase, may contribute to NF disease severity. This can be counteracted by ex-IgGs.

[Efficacy of the combination of glucocorticoids, mycophenolate-mofetil and human immunoglobulin for the therapy of immune mediated haemolytic anaemia in dogs]. / Wirksamkeitsstudie zur Kombinationstherapie von Glukokortikoid, Mycophenolat- Mofetil und humanem Immunglobulin zur Therapie von primärer immun vermittelter hämolytischer Anämie beim Hund.

INTRODUCTION: Idiopathic immune-mediated haemolytic anaemia (IMHA) is one of the most common immune-mediated diseases in dogs with a high mortality rate. In this retrospective study, we examined the effect of mycophenolate- mofetil (MMF) and human intravenous immunoglobulin (hIVIG) in combination with glucocorticoids on canine IMHA patients. Six dogs were treated with prednisolone and MMF (hIVIG-) and in 15 patients hIVIG was added (hIVIG+). There was no significant difference between the groups regarding age, weight, number of blood transfusions or hematocrit on the day of diagnosis. The hIVIG+ group showed a significantly faster recovery of the hematocrit, but this difference was only short-lived. The survival in the first year was similar in both groups and was with 71.5% somewhat higher than in other published studies. The addition of MMF to prednisolone for the treatment of dogs with acute IMHA was well tolerated and seemed to positively affect the course of the disease. Randomized studies are necessary to confirm this observation. Human immunoglobulin had only minimal clinical advantages and no effect on mortality.

INTRODUCTION: Une anémie hémolytique auto-immune idiopathique (IMHA) compte chez le chien au nombre des affections auto-immunes les plus fréquentes avec un taux de mortalité élevé. Dans le cadre de la présente étude rétrospective, nous avons examiné l'effet du mycophénolate mofétil (MMF) et des globulines humaines par voie intraveineuse (hIVIG) en combinaison avec des glucocorticoïdes sur des chiens souffrant d'IMHA. Six chiens ont été traités au moyen d'une combinaison prédnisolone/ MMF (hIVIG­) et chez 15 patients on a en plus utilisé des hlVIG (hIVIG+). Les deux groupes ne se différenciaient pas de façon significative en ce qui concerne l'âge, le poids, le nombre de transfusions sanguines ou l'hématocrite lors du diagnostic. Les patients du groupe hIVIG+ ont montré une normalisation significativement plus rapide mais de courte durée de l'hématocrite. Le taux de survie à une année était similaire dans les deux groupes avec 71,5%, ce qui est un peu plus élevé que dans d'autres études. L'ajout de MMF à la prédnisolone dans le traitement de chiens atteints d'IMHA est bien supporté et semble influencer le cours de la maladie de façon positive. Des études randomisées sont nécessaires pour confirmer ces observations. Les immunoglobulines humaines n'ont eu qu'un effet limité et pas d'influence sur la mortalité.

A megaporous material harbouring a peptide ligand for affinity IgG purification.

Common limitations of Protein A affinity chromatography include high adsorbent costs, ligand instability and possible ligand leakage. In this study, a short peptide with affinity for IgG was synthesized chemically and subsequently immobilized on a megaporous support. The support was prepared utilising the cryogel technique while the peptide-ligand was covalently immobilised via thiol-epoxy click chemistry. The cryogel support was chemically grafted to increase the number of reaction sites. This adsorbent was designated as "MP-Pep". Adsorption isotherms were employed to evaluate protein binding capacity. A maximum static binding capacity within the range of 30-60 mg/mL was observed for T hIgG. This parameter compares well with other commercial and non-commercial adsorbents, as reported in the literature. As a control material, a Protein A grafted megaporous cryogel was synthesized. Dynamic binding capacity values were obtained by breakthrough analysis. The peptide cryogel showed a dynamic capacity value 9.0 mg/mL in comparison to 9.7 mg/mL in the case of the Protein A based adsorbent. The ratio of dynamic binding capacity to static binding capacity was 20%, indicating suboptimal product capture. However, the advantage of MP-Pep lies in its cost-effective preparations while maintaining a reasonable binding capacity for the targeted product. The presence of cooperative effects during protein binding could also represent an advantage during the processing of a feedstock containing a product in high concentration.

Central nervous system vasculitis in systemic lupus erythematosus: a case series report in a tertiary referral centre.

Central nervous system (CNS) vasculitis (CNS) in systemic erythematosus lupus (SLE) is a rare and challenging diagnosis. We report four cases of CNS vasculitis that occurred 5 to 16 years after the diagnosis of SLE. Magnetic resonance imaging (MRI) detected different features suggestive of CNS vasculitis: enhancement and thickening of the vascular wall, vascular stenosis, ischemic brain lesions and intracerebral haemorrhage unlikely to correspond to other mimic aetiologies. Three patients received combination therapy with glucocorticoids (GC) and cyclophosphamide (CYC). Intravenous human immunoglobulin (IVIG) was administered when the patient had a past history of serious adverse event to CYC or high infectious risk. All patients showed imagiological improvement, at least partially, 5 to 23 days after starting treatment. We discuss the management of CNS in SLE including the role of magnetic resonance imaging (MRI).

Electrochemical Detection of Plasma Immunoglobulin as a Biomarker for Alzheimer's Disease.

The clinical diagnosis and treatment of Alzheimer's disease (AD) represent a challenge to clinicians due to the variability of clinical symptomatology as well as the unavailability of reliable diagnostic tests. In this study, the development of a novel electrochemical assay and its potential to detect peripheral blood biomarkers to diagnose AD using plasma immunoglobulins is investigated. The immunosensor employs a gold electrode as the immobilizing substrate, albumin depleted plasma immunoglobulin as the biomarker, and polyclonal rabbit Anti-human immunoglobulin (against IgA, IgG, IgM) as the receptor for plasma conjugation. The assay showed good response, sensitivity and reproducibility in differentiating plasma immunoglobulin from AD and control subjects down to 10-9 dilutions of plasma immunoglobulin representing plasma content concentrations in the pg mL-1 range. The newly developed assay is highly sensitive, less time consuming, easy to handle, can be easily modified to detect other dementia-related biomarkers in blood samples, and can be easily integrated into portable devices.

SMART Digest™ compared with pellet digestion for analysis of human immunoglobulin G1 in rat serum by liquid chromatography tandem mass spectrometry.

The newly developed SMART Digest™ kit was applied for the sample preparation of human immunoglobulin G1 (hIgG1) in rat serum prior to qualitative and quantitative analyses by liquid chromatography tandem mass spectrometry (LC-MS/MS). The sequence coverages obtained for the light and heavy chains of hIgG1A were 50 and 76%, respectively. The calibration curve was linear from 1.00 to 1000 µg/ml for three of four generic peptides. Overall, the SMART Digest™ kit resulted in similar quantitative data (linearity, sensitivity, accuracy, and precision) compared with the pellet digestion protocol. However, the SMART Digest™ required only 2 h of sample preparation with fewer reagents.

Poly(glycidyl methacrylate)-grafted hydrophobic charge-induction agarose resins with 5-aminobenzimidazole as a functional ligand.

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.

The solution structures of native and patient monomeric human IgA1 reveal asymmetric extended structures: implications for function and IgAN disease.

Native IgA1, for which no crystal structure is known, contains an O-galactosylated 23-residue hinge region that joins its Fab and Fc regions. IgA nephropathy (IgAN) is a leading cause of chronic kidney disease in developed countries. Because IgA1 in IgAN often has a poorly O-galactosylated hinge region, the solution structures of monomeric IgA1 from a healthy subject and three IgAN patients with four different O-galactosylation levels were studied. Analytical ultracentrifugation showed that all four IgA1 samples were monomeric with similar sedimentation coefficients, s(0)20,w. X-ray scattering showed that the radius of gyration (Rg) slightly increased with IgA1 concentration, indicating self-association, although their distance distribution curves, P(r), were unchanged with concentration. Neutron scattering indicated similar Rg values and P(r) curves, although IgA1 showed a propensity to aggregate in heavy water buffer. A new atomistic modelling procedure based on comparisons with 177000 conformationally-randomized IgA1 structures with the individual experimental scattering curves revealed similar extended Y-shaped solution structures for all four differentially-glycosylated IgA1 molecules. The final models indicated that the N-glycans at Asn(263) were folded back against the Fc surface, the C-terminal tailpiece conformations were undefined and hinge O-galactosylation had little effect on the solution structure. The solution structures for full-length IgA1 showed extended hinges and the Fab and Fc regions were positioned asymmetrically to provide ample space for the functionally-important binding of two FcαR receptors to its Fc region. Whereas no link between O-galactosylation and the IgA1 solution structure was detected, an increase in IgA1 aggregation with reduced O-galactosylation may relate to IgAN.

Molecular modeling and multi-spectroscopic approaches to study the interaction between antibacterial drug and human immunoglobulin G.

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 ΔH(0) and entropy ΔS(0), 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.

Aptamer-based detection and quantitative analysis of human immunoglobulin E in capillary electrophoresis with chemiluminescence detection.

A novel aptamer-based CE with chemiluminescence (CL) assay was developed for highly sensitive detection of human immunoglobulin E (IgE). The IgE aptamer was conjugated with gold nanoparticles (AuNPs) to form AuNPs-aptamer that could specifically recognize the IgE to produce an AuNPs-aptamer-IgE complex. The mixture of the AuNPs-aptamer-IgE complex and the unbounded AuNPs-aptamer could be effectively separated by CE and sensitively detected with luminol-H2 O2 CL system. By taking the advantage of the excellent catalytic behavior of AuNPs on luminol-H2 O2 CL system, the ultrasensitive detection of IgE was achieved. The detection limit of IgE is 7.6 fM (S/N = 3) with a linear range from 0.025 to 250 pM. Successful detection of IgE in human serum samples was demonstrated and the recoveries of 94.9-103.2% were obtained. The excellent assay features of the developed approach are its specificity, sensitivity, adaptability, and very small sample consumption. Our design provides a methodology model for determination of rare proteins in biological samples.

Direct immobilization of antibodies on a new polymer film for fabricating an electrochemical impedance immunosensor.

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.

Hydrophobic charge-induction resin with 5-aminobenzimidazol as the functional ligand: preparation, protein adsorption and immunoglobulin G purification.

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.