IgG Solution Research Articles

Observation of protein-protein interaction by dielectric relaxation spectroscopy of protein solutions for biosensor application

Dielectric spectroscopy was used to measure the dielectric relaxation of bovine serum albumin, Protein A, and rabbit IgG in solution. Measurements were performed with an impedance analyzer and dielectric cell. Protein additions to bulk solution were measured by differential dielectric relaxation spectroscopy, presented as a method to measure the individual contributions of proteins to relaxation phenomena and reduce effects of electrode polarization. Relaxation characteristics of the proteins individually and as a complex were observed. Frequency shifts in the relaxation characteristics indicate binding. This illustrates the potential use of bulk solution dielectric relaxation as the principle of operation for a biosensor.

Multiple enzyme linked immunosorbent assay system on a capillary-assembled microchip integrating valving and immuno-reaction functions

Multiple enzyme linked immunosorbent assay (ELISA) chip is developed by using capillary-assembled microchip (CAs-CHIP) technique, which involves simple embedding of 2–3 mm length of square capillaries possessing valving and immuno-reaction functions into the microchannels fabricated on a PDMS substrate. In contrast to the previously reported ELISA chips, our system enables not only the flexible design of the multi-ELISA chip required for many different diagnostic purposes, but also the valving operation required for a reliable analysis. Here, a thermo-responsive polymer-immobilized capillary was used together with a small Peltier device, as a valving part, and different antibody-immobilized capillaries were used as immuno-reaction part. Sample solution and detecting reagent solutions were sequentially introduced through the valving capillary, and the valve is closed to completely stop the solution flow inside the immuno-reaction capillaries and detected using thermal lens microscope (TLM). Different anti-IgGs (human, goat, chicken) were immobilized and used as ELISA parts of CAs-CHIP. Sequential introductions of the mixed IgG solution, mixed enzyme–antibody solution and substrate solution facilitated the multiple determinations of 0.1 ng mL −1 IgGs (human, goat, chicken) with total analysis time of about 30 min. The valve-integrated multi-ELISA chip developed here can be applied for many different diagnostic purposes by using different immuno-reaction capillaries necessary for a specific clinical diagnostic application.

Fc function of a new intravenous immunoglobulin product: IGIV 10% triple virally inactivated solution

Baxter AG has developed a new liquid intravenous immunoglobulin product [Immune Globulin Intravenous (IGIV) 10%] using a new manufacturing procedure. A modified Cohn fractionation and ion exchange chromatography is used to produce an IgG solution with no alterations to the Fc region. Three dedicated virus reduction steps are included: solvent-detergent treatment, nanofiltration, and incubation at low pH and elevated temperature in final formulation. We applied the reference method of the European Pharmacopoeia (EP) together with a flow-cytometric binding assay for the evaluation of the Fc function of the new product. The EP reference method was done as described in the EP. The flow-cytometric method measured binding of IgG to Fc receptors of human monocytic THP-1 cells after exclusion of apoptotic cells. Sixteen lots of the new product expressed Fc functions between 84% and 110% when analysed with the EP reference method and Fc-binding activities between 82% and 121% when determined by the flow-cytometric method. All tested lots of the new product demonstrated a high level of Fc activity and met the requirements of the EP for Fc function.

G Protein-induced Trafficking of Voltage-dependent Calcium Channels

Calcium channels are well known targets for inhibition by G protein-coupled receptors, and multiple forms of inhibition have been described. Here we report a novel mechanism for G protein-mediated modulation of neuronal voltage-dependent calcium channels that involves the destabilization and subsequent removal of calcium channels from the plasma membrane. Imaging experiments in living sensory neurons show that, within seconds of receptor activation, calcium channels are cleared from the membrane and sequestered in clathrin-coated vesicles. Disruption of the L1-CAM-ankyrin B complex with the calcium channel mimics transmitter-induced trafficking of the channels, reduces calcium influx, and decreases exocytosis. Our results suggest that G protein-induced removal of plasma membrane calcium channels is a consequence of disrupting channel-cytoskeleton interactions and might represent a novel mechanism of presynaptic inhibition.

Removal of small viruses (parvovirus) from IgG solution by virus removal filter Planova ®20N

To clarify virus removability from IgG solutions by Planova ®20N, which is composed of cuprammonium regenerated cellulose hollow fiber membranes, we investigated the effects of virus spiking conditions as well as IgG concentration on the filtration performance (i.e. protein filterability and virus removability). The relationship between the performance and the membrane structure was also discussed. We used human IgG as the model protein and porcine parvovirus (PPV) as the model virus. The results revealed Planova ®20N's IgG permeability at 5 mg/ml IgG concentration was independent of PPV spiking as well as PPV purity, and its flux decay was small. The filter also maintained a high virus logarithmic rejection value (LRV) (>4) regardless of virus spiking conditions and filtration volume. For a wide range of IgG concentrations of 1–30 mg/ml, the flux decay was negligibly small keeping the high LRV. We consider these outcomes are due to the Planova ®20N's unique membrane structure (i.e. the multi-layered, capillary-void structure, having the thick effective layer with high porosity), which enables a high virus trapping capacity (volume) with a low plugging rate of protein. These results indicate that Planova ®20N has a well-balanced performance, eminently efficient viral removal capability and robust feature.

Reduction of the Nonspecific Binding of a Target Antibody and of Its Enzyme-Labeled Detection Probe Enabling Electrochemical Immunoassay of an Antibody through the 7 pg/mL−100 ng/mL (40 fM−400 pM) Range

We describe a simple, potentially low-cost, amperometric, enzyme-amplified, sandwich-type immunoassay, monitoring IgG at a concentration as low as approximately 7 pg/mL with a dynamic range of 10(4). The assay utilizes a screen-printed carbon electrode on which a redox hydrogel and avidin are co-electrodeposited. To neutralize nonspecifically binding positively charged microdomains of the avidin, two polyanions, poly(acrylic acid-co-maleic acid) and poly(acrylic acid), are applied. These polyanions bind to the film not only electrostatically but also by Michael addition reaction to cysteine, lysine, or arginine functions of the avidin. The electrode is then made specific for the analyte, for which rabbit IgG was chosen, by conjugating the film-bound avidin to biotin-labeled anti-rabbit IgG. After exposure to the tested solution and capture of rabbit IgG, the sandwich is completed by conjugation of horseradish-peroxidase (HRP)-labeled anti-rabbit IgG. Electrical contact between the HRP and the electrode-bound hydrogel results in the formation of an electrocatalyst for the electroreduction of H2O2 to water. The application of the poly(acrylic acid-co-maleic acid) and the poly(acrylic acid) reduces the nonspecific adsorption-associated noise, lowers the detection limit from 3 ng/mL (approximately 20 pM analyte antibody concentration) to approximately 7 pg/mL (approximately 40 fM analyte antibody concentration), and also expands the dynamic range to 10(4).

The effect of solvent environment on the conformation and stability of human polyclonal IgG in solution

Stability of therapeutic IgG preparations is an important issue as adequate efficacy and safety has to be ensured throughout a long shelf life. To this end, denaturation and aggregation have to be avoided. In many cases sugars are applied for stabilizing IgG in relatively high concentration (5–10%). However, certain sugars (sucrose, maltose) are responsible for adverse effects including renal failure. In this work we reassessed the effect of pH and stabilizers to optimize the solvent environment and minimize the amount of additives without endangering quality and stability. Since both biological function and aggregation depend on the conformational properties of individual IgG molecules, two sensitive and rapid physical methods were introduced to assess conformational changes and structural stability as a function of pH and addition of standard stabilizers. It was observed that the conformational stability decreases with decreasing pH, while the resistance against aggregation improves. The optimum pH range for storage is 5.0–6.0, as a compromise between conformational stability and the tendency for oligomerization. Intriguingly, additives in physiologically acceptable concentration have no effect on the thermal stability of IgG. On the other hand, glucose or sorbitol, even at a concentration as low as 1%, have significant effect on the tertiary structure as revealed by near-UV-CD spectroscopy, reflecting changes in the environment of aromatic side-chains. Although, 0.3% leucine does not increase conformational stability, it decreases the aggregation tendency even more efficiently than 1% glucose or sorbitol. Both pH and storage temperature are decisive factors for the long-term stability of IgG solutions. An increase in the dimer content was observed upon storage at 5 °C which was partly reverted upon incubation at 37 °C. Storage at temperatures higher than 5 °C may help to maintain an optimal proportion of dimers. Regarding the known side effects, and their limited stabilizing capacity at low concentration, it is advisable to omit sugars at intravenous immunoglobulin (IVIG) formulation. Hydrophobic amino acids give promising alternatives.

Design and evaluation of a coupled monolithic preconcentrator-capillary zone electrophoresis system for the extraction of immunoglobulin G from human serum

The analysis of proteins in biological fluids by capillary electrophoresis (CE) is of interest in clinical chemistry. However, due to low analyte concentrations and poor concentration limits of detection (CLOD), protein analysis by this technique is frequently challenging. Coupling preconcentration techniques with CE greatly improves the CLOD. An on-line preconcentration-CE method that can selectively preconcentrate any protein for which an antibody is available would be very useful for the analysis of low abundance proteins and would establish CE as a major tool in biomarker discovery. To accomplish this, the development of an on-line protein G monolithic preconcentrator-CE device is proposed. To generate active groups for protein immobilization, glycidyl methacrylate (GMA) was used to prepare polymer monoliths. A 1.5–2 cm monolith was cast inside a 75 μm I.D. fused silica capillary that had previously been coated with alternating layers of negatively (dextran) and positively (polybrene) charged polymers. Protein G was covalently bound to GMA. Monoliths from different formulations were prepared and evaluated for binding capacity to optimize the monolith formulation for protein preconcentration. The physical properties of the column considered best for preconcentration were determined by mercury intrusion porosimetry. The total pore area was 4.8 m 2/g, the average pore diameter was 3.3 μm and the porosity was 82%. The monolith had a low flow resistance and was macroscopically homogeneous. The effectiveness of the monolith to rapidly preconcentrate proteins at flow rates as high as 10 μL/min was demonstrated using a 1.8 μM IgG solution. This system proved effective for on-line sample extraction, clean-up, preconcentration, and CE of IgG in human serum. IgG from diluted (500 and 65,000 times) human serum samples was successfully analyzed using this system. The approach can be applied to the on-line preconcentration and analysis of any protein for which an antibody is available.

Mercaptoheterocyclic ligands grafted on a poly(ethylene vinyl alcohol) membrane for the purification of immunoglobulin G in a salt independent thiophilic chromatography

In this study, we attempted a limited combinatorial approach for designing affinity ligands based on mercaptoheterocyclic components. The template, divinyl sulfone structure (DVS), which was grafted on poly(ethylene vinyl alcohol) (PEVA) hollow fiber membrane, has served for the tethering of different heterocyclic compounds as pyridine, imidazole, purine and pyrimidine rings. Their ability to adsorb specifically IgG in a salt independent manner out of pure IgG solution, mixture of IgG/albumin and human plasma was demonstrated. Mercapto methyl imidazole (MMI) has shown the best adsorption of IgG in terms of binding capacity. No subclass discrimination was observed on all tested ligands except for mercapto methyl pyrimidine where the major IgG subclass adsorbed was IgG3. MMI gave an IgG binding capacity of 100 μg/cm 2 of hollow fiber membrane surface area.

In vitro glycation of human immunoglobulin G

Background: Glucose can covalently bind to the proteins by nonenzymatic process often termed glycation. Glycation of IgG is of special interest due to its possible influence on the functionality of immunoglobulins and overall immunocompetence. Methods: The glycation of IgG was studied using radioactive d-[U- 14C]-glucose. Results: The kinetics, concentration/temperature dependence and distribution of glycation binding sites of human IgG were studied under various conditions. (a) In average, 0.7 glucose molecules were found to be bound per IgG after 90 days of incubation at 37 °C with normal serum IgG and glucose concentrations, and 3.1 molecules after incubation with glucose in the concentration characteristic for diabetics. (b) Incubation of the same solutions for 270 days at 4–8 °C resulted in binding of 0.3 and 0.8 glucose molecules per IgG, respectively. (c) After 90 days at 37 °C in the presence of 0.56 mol/l of glucose, IgG was glycated with averagely 47 glucose molecules per IgG and with 9 after 270 days at 4–8 °C. (d) At very high glucose concentration (1.67 mol/l) in concentrated IgG solution (87 g/l), the molar ratio glucose/IgG reached 41 after incubation at 60 °C for 10 h. Conclusions: Glycation of IgG with glucose nearly resembled the first order reaction kinetics. No preferential glycation sites were found as bound glucose was equally distributed throughout F(ab) 2 and Fc fragments as well as on the light and heavy immunoglobulin chains.

Structure and Flexibility of Individual Immunoglobulin G Molecules in Solution

In contrast to averaging methods of determining structure, such as X-ray diffraction, NMR, and single-particle tomography, cryo-electron tomography allows three-dimensional imaging of an individual object in solution. The method has previously been used to study cells and very large macromolecules. We have used cryo-electron tomography to analyze a monoclonal IgG, with a molecular weight of only 150 kDa. Tomograms reveal y-shaped IgG molecules with three protruding subunits. Docking X-ray structures enabled us to recognize the three subunits as two ellipsoidal Fab arms and a heart-shaped Fc stem. Each subunit has a similar structure in the tomograms and in the X-ray map. Notably, the positions of the Fab arms relative to the Fc stem differed greatly from one molecule to another. The large flexibility of IgG in solution is most likely of functional significance in antigen recognition. This distribution of individual structures provides a qualitative insight into the system dynamics.

Colorimetric detection of protein microarrays based on nanogold probe coupled with silver enhancement.

This work presents a method for analyzing protein microarrays using a colorimetric nanogold probe coupled with silver enhancement (gold-silver detection). In this method, the gold nanoparticles were introduced to the microarray by the specific binding of the gold-conjugated antibodies or streptavidins and then coupled with silver enhancement to produce black image of microarray spots, which can be easily detected with a commercial CCD camera. The method showed high detection sensitivity (1 pg of IgG immobilized on slides or 2.75 ng/ml IgG in solution) and a good linear correlation between the signal intensity and the logarithm of the sample concentration. The examination of this method in analyzing a demonstrational ToRCH antigen microarray developed in our lab showed an identical result as in the fluorescent method. These results suggest the colorimetric gold-silver detection method has potential applications in proteomics research and clinical diagnosis.

Two generations of synthetic membranes for biological/medical applications

Two generations of amphiphilic membranes synthesized in our laboratories for biological/medical applications are highlighted. Recent results obtained with representative 2nd generation membranes designed specifically for immunoencapsulation of pancreatic islets and cells are summarized. The latter tri-component membranes comprise well-defined hydrophilic poly(ethylene glycol) and hydrophobic/oxyphilic polydimethylsiloxane segments crosslinked by oxyphilic polycyclosiloxane domains, which also provide reinforcement. Select membranes are demonstrated to allow the diffusion of glucose and insulin but prevent the permeation of immunoglobulin G. These membranes remain permeable despite long incubation in IgG solution.

Piezoelectric crystal immunosensors based on immobilized fullerene C60-antibodies

Fullerene C60-antibodies, e.g., C60-anti-human IgG and C60-anti-hemoglobin (Hb), coated piezoelectric crystals were prepared and applied in piezoelectric quartz crystal immunosensors for human IgG and hemoglobin, respectively. The immobilization of anti-IgG and anti-Hb onto fullerene C60 were studied with a C60-coated piezoelectric crystal detection system. The partially irreversible responses for these antibodies were observed by the desorption study, which implied that C60 could chemically react with these bio-species. Thus, immobilized C60-anti-IgG and C60-anti-Hb coating materials were successfully prepared and identified with FTIR spectrometry. The C60-anti-human IgG and C60-anti-Hb coated piezoelectric (PZ) quartz crystal immunosensors with homemade computer interfaces for signal acquisition and data processing were developed and applied for detection of human IgG and hemoglobin, respectively. The C60-antibody coated PZ Bio-Sensors for IgG and hemoglobin exhibited linear frequency responses to the concentrations of human IgG and hemoglobin with sensitivities of 1.25×10 2 Hz per decade (ΔHz/Δlog (mg/ml)) and 1.56×10 4 Hz/(mg/ml), respectively. Both C60-antibody coated PZ immunosensors showed detection limit of <10 −4 mg/ml for IgG and hemoglobin in aqueous solutions. Effects of pH and temperature on the frequency response of the IgG PZ immunosensor were also investigated. Optimum pH of the IgG solutions and optimum temperature for the IgG sensor were observed at pH=6.7 and 30 °C, respectively. The interference of various common species in the human blood, e.g., urea, uric acid, cysteine, tyrosine, ascorbic acid and metal ions, to the IgG PZ biosensor was also investigated. These species showed nearly no interference with the IgG PZ biosensor. The reproducibility and lifetime of these immobilized C60-antibody coated PZ immunosensors were also investigated and discussed.

Ligand-Receptor Kinetics Measured by Total Internal Reflection with Fluorescence Correlation Spectroscopy

Total internal reflection excitation used in combination with fluorescence correlation spectroscopy (TIR-FCS) is a method for characterizing the dynamic behavior and absolute concentrations of fluorescent molecules near or at the interface of a planar substrate and a solution. In this work, we demonstrate for the first time the use of TIR-FCS for examining the interaction kinetics of fluorescent ligands in solution which specifically and reversibly associate with receptors in substrate-supported planar membranes. Fluorescence fluctuation autocorrelation functions were obtained for a fluorescently labeled IgG reversibly associating with the mouse receptor Fc γRII, which was purified and reconstituted into substrate-supported planar membranes. Data were obtained as a function of the IgG solution concentration, the Fc receptor surface density, the observation area size, and the incident intensity. Best fits of the autocorrelation functions to appropriate theoretical forms gave measures of the average surface density of bound IgG, the local solution concentration of IgG, the kinetic rate constant for surface dissociation, and the rate of diffusion through the depth of the evanescent field. The average number of observed fluorescent molecules, both in solution and bound to the surface, scaled with the solution concentration of IgG, observation area size, and Fc receptor surface density as expected. The dissociation rate constant and rate of diffusion through the evanescent field agree with previous results, and all measured parameters were independent of the incident intensity.

Direct-write dip-pen nanolithography of proteins on modified silicon oxide surfaces.

Direct-write dip-pen nanolithography of proteins on modified silicon oxide surfaces.

Removal of neutralized model parvoviruses and enteroviruses in human IgG solutions by nanofiltration.

Nanofiltration has proven to be an effective technology for virus removal. The small nonenveloped viruses, such as HAV and parvovirus B19 (B19), are evidently more difficult to remove than the larger enveloped viruses. However, since antibodies against these viruses, HAV and B19, are constantly present in large plasma pools, it is conceivable that antibody binding will lead to an increase in size of the viruses due to a corona of antibodies on the virus surfaces. These enlarged viruses should be easier to remove by nanofiltration than naked viruses. In this study, antibody-coated viruses and free virions were subjected to nanofiltration to determine whether the antibody-mediated increase in size contributes to the elimination of viruses which are otherwise too small to be retained by filters of a given nominal pore diameter. One-percent IgG or 1-percent albumin solutions were spiked with bovine enterovirus (BEV) and filtered through 50-nm filters. This virus cross-reacts with antibodies in pooled human IgG solutions but persists as free virions in albumin solutions. Similarly, two parvoviruses, bovine parvovirus (BPV) and minute virus of mice (MVM), in 1-percent IgG solutions were filtered through 20-nm filters. BPV cross-reacts with antibodies in human IgG, whereas MVM remains unaffected. Virus removal was assessed by in vitro infectivity assays. Two methods were developed to displace the antibodies from BEV and BPV and thereby render them accessible for titration. Antibody-coated BEV was eliminated to below detection limit by the 50-nm filters (logarithmic reduction factors [LRF] > or = 5), while free BEV virions passed without restraint through the same filters. Similarly, antibody-coated BPV was completely retained by the 20-nm filters (LRF > or = 5). MVM virions were strongly retained by the 20-nm filters; however, the virus was still detectable in the filtrate. These results indicate that viruses with bound antibodies are efficiently eliminated by nanofiltration by use of filters having nominal pore sizes larger than the diameter of the respective free virions.

Detection of Pyrogens in Intravenous IgG Preparations

Five different intravenous IgG (i.v. IgG) preparations were assessed for their capacity to modify the pyrogenic response to bacterial lipopolysaccharide (LPS) of rabbits under the conditions of a pharmacopoeal test. Four of the five preparations were found to mitigate the reaction rendering the result “non-pyrogenic” with an LPS dose proved pyrogenic when administered in saline or in albumin. Bacterial LPS was found readily detectable by a simple Limulus amoebocyte lysate (LAL) gelation test. Four of six brands of i.v. IgG were found reactive in the test under conditions adjusted to detect the FDA limit. The reaction obtained upon addition of standard LPS to the negative preparations supported the validity of the assay. The LAL reactivity of two of the reactive preparations was inhibited by laminarin, a compound known to inhibit Limulus lysate gelation by β-D-glucan, but not by Polymyxin B. Specific detection of bacterial endotoxins in i.v. IgG solutions requires inhibition of the β-D-glucan pathway of the Limulus lysate coagulation. Using an appropriate inhibitor, the LAL gelation test is suitable to detect a potential endotoxin contamination in i.v. IgG which might have not been unravelled by the in vivo test for pyrogens. Copyright 2002 Published by Elsevier Science Ltd on behalf of The International Association for Biologicals

Synthesis of porous macrospheres from amino-polymers

Porous macrospheres with controlled diameter were produced using N-methylolacrylamide and urea–formaldehyde resins synthesized in our laboratories. Hot silicon oil was used to form the porous macrospheres which were characterized by using infrared spectroscopy (FTIR), scanning electron microscopy, mercury porosimetry and surface area determination (BET). Porous macrospheres of amino-polymers were used as support in immunoassays type ELISA (enzyme linked immunosorbent assay) in which mouse immunoglobulin (IgG) was immobilized, and detected by rabbit anti-mouse immunoglobulin (a-IgG) labeled with horseradish peroxidase. Responses were obtained up to a dilution ratio of 1:4000 of the conjugate standard (1 mg/ml) and 10 μ g/ml of IgG solution to recover the spheres in pH=4.5.

A General Strategy for Epitope Mapping by Direct MALDI-TOF Mass Spectrometry Using Secondary Antibodies and Cross-Linking

The combination of limited proteolysis and MALDI-TOF mass spectrometry has become an important tool for the determination of epitopes but works best with highly purified antibodies. Here we report the use of capture antibodies to reduce the need for purification of the antibody in the mass spectrometric determination of the epitope. In this new method, a secondary Fc-specific antibody, covalently bound to Sepharose beads, is used to capture the primary antibody (the antibody of interest). After capture, the two antibodies are cross-linked. The antigen is then bound to the immobilized antibodies and subjected to proteolysis using several successive proteinases. In this study, this strategy is demonstrated with a crude mouse anti-ACTH IgG solution and adrenocorticotropin (ACTH). Comparing this strategy with previous methods where the antibody is bound directly to activated beads, the new method (1) results in a higher binding capacity of the bound antibody to ACTH, (2) does not require purification of the antibody of interest, and (3) dramatically reduces the chemical background in the MALDI mass spectra.