Immunoglobulin G Solution Research Articles

Recombinant streptococcal protein G-modified metal-organic framework ZIF-8 for the highly selective purification of immunoglobulin G from human serum

A novel solid phase extractant His-rSPG@ZIF-8 was prepared by covalently coupling recombinant streptococcal protein G (His-rSPG) with ZIF-8. The His-rSPG@ZIF-8 composite was characterized by Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy (Raman), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Due to the specific binding between the immunoglobulin binding region of His-rSPG and the Fc region of immunoglobulin G (IgG), the His-rSPG@ZIF-8 composite demonstrated exceptional selectivity in adsorbing IgG. In Britton-Robinson buffer (BR buffer) with a salt concentration of 500 mmol L−1 (0.04 mol L−1, pH 8.0), the His-rSPG@ZIF-8 composite exhibited a remarkable adsorption efficiency of 99.8 % for 0.05 mg of the composite on 200 μL of IgG solution (100 μg mL−1). The adsorption behavior of the His-rSPG@ZIF-8 composite aligns with the Langmuir adsorption model, and the theoretical maximum adsorption capacity is 1428.6 mg g−1. The adsorbed IgG molecules were successfully eluted using a SDS solution (0.5 %, m/m), resulting in a recovery rate of 91.2 %. Indeed, the His-rSPG@ZIF-8 composite was successfully utilized for the isolation and purification of IgG from human serum samples. The obtained IgG exhibited high purity, as confirmed by SDS-PAGE analysis. Additionally, LC-MS/MS analysis was employed to identify the human serum proteins following the adsorption and elution process using the His-rSPG@ZIF-8 composite material. The results revealed that the recovered solution contained an impressive content of immunoglobulin, accounting for 62.4 % of the total protein content. Furthermore, this process also led to the significant enrichment of low abundance proteins such as Serpin B4 and Cofilin-1. Consequently, the His-rSPG@ZIF-8 composite holds great promise for applications such as IgG purification and immunoassays. At the same time, it expands the application of metal-organic frameworks in the field of proteomics.

Subcutaneous Immunoglobulin in Chronic Inflammatory Demyelinating Polyneuropathy: A Historical Perspective.

The therapeutic administration of subcutaneous immunoglobulin (SCIg) offers various advantages over intravenous immunoglobulin (IVIg). This narrative review examines and compares SCIg versus IVIg in chronic inflammatory demyelinating polyneuropathy (CIDP). SCIg is as effective as IVIg but is better tolerated and easier to administer, as intravenous access is not required. Furthermore, SCIg administration is more convenient and cost-effective than IVIg, enabling flexible treatment scheduling at home and improving patients' overall quality of life. The availability of highly concentrated immunoglobulin G (IgG) subcutaneous solutions, such as IgPro20, a 20% IgG solution stabilized with L-proline, allows for the administration of larger volumes in a single session, while the parallel development of new technological devices enables the delivery of higher doses over a shorter time. Based on the results of the PATH study, SCIg has become a well-established therapy in CIDP. In addition to discussing the advantages of SCIg, this review summarizes the evolution of SCIg by discussing all the relevant clinical studies which have considered its use in the treatment of CIDP.

Aerosolization Performance of Immunoglobulin G by Jet and Mesh Nebulizers

Recently, many preclinical and clinical studies have been conducted on the delivery of therapeutic antibodies to the lungs using nebulizers, but standard treatment guidelines have not yet been established. Our objective was to compare nebulization performance according to the low temperature and concentration of immunoglobulin G (IgG) solutions in different types of nebulizers, and to evaluate the stability of IgG aerosols and the amount delivered to the lungs. The output rate of the mesh nebulizers decreased according to the low temperature and high concentration of IgG solution, whereas the jet nebulizer was unaffected by the temperature and concentration of IgG. An impedance change of the piezoelectric vibrating element in the mesh nebulizers was observed because of the lower temperature and higher viscosity of IgG solution. This affected the resonance frequency of the piezoelectric element and lowered the output rate of the mesh nebulizers. Aggregation assays using a fluorescent probe revealed aggregates in IgG aerosols from all nebulizers. The delivered dose of IgG to the lungs in mice was highest at 95 ng/mL in the jet nebulizer with the smallest droplet size. Evaluation of the performance of IgG solution delivered to the lungs by three types of nebulizers could provide valuable parameter information for determination on dose of therapeutic antibody by nebulizers.Graphical abstract

Arginine and its Derivatives Suppress the Opalescence of an Antibody Solution

Opalescence is a problem concerned with the stability of an antibody solution. It occurs when a high concentration of a protein is present. Arginine (Arg) is a versatile aggregation suppressor of proteins, which is among the candidates that suppress opalescence in antibody solutions. Here, we investigated the effect of various types of small molecular additives on opalescence to reveal the mechanism of Arg in preventing opalescence in antibody solution. As expected, Arg suppressed the opalescence of the immunoglobulin G (IgG) solution. Arg also concentration dependently inhibited the formation of microstructures in IgG molecules. Interestingly, the intrinsic fluorescence spectra of highly concentrated IgG solutions differed from those having low concentrations, even though IgG retained a distinct tertiary structure. Arginine ethylester was more effective in suppressing the opalescence of IgG solutions than Arg, whereas lysine and γ-guanidinobutyric acid were less effective. These results indicated that positively charged groups of both α-amine and guanidinium actively influence Arg as an additive for suppressing opalescence. Diols, which are the suppressors of the liquid–liquid phase separation of proteins were also effective in suppressing the opalescence. These results therefore provide insight into the control of opalescence of antibody solutions at high concentrations using solution additives.

Performance of phospho-L-tyrosine immobilized onto alginate/polyacrylamide-based cryogels: Effect of ligand coupling on human IgG adsorption and Fab fragments separation

The ortho-phospho-tyrosine (P-Tyr) pseudoaffinity ligand was immobilized via ether linkage onto polyacrylamide-alginate (PAAm-Alg)-epoxy cryogels prepared according to two different approaches in order to explore their performance in the immunoglobulin G (IgG) purification from human serum. In the first approach, the P-Tyr was attached to cryogel prepared by cryocopolymerization of acrylamide and alginate with allyl glycidyl ether (AGE) as functional comonomer, and methylenebisacrylamide and Ca(II) as crosslinkers, obtaining the PAAm-Alg-AGE-P-Tyr. In the second approach, the PAAm-Alg was synthesized under the same conditions, but without AGE, and the P-Tyr was coupled to epichlorohydrin (ECH)-activated cryogel, obtaining the PAAm-Alg-ECH-P-Tyr. Both pseudoaffinity cryogels were characterized by scanning electron microscopy, swelling tests, porosity, ligand density, and flow dynamics. The human IgG differently interacted with the PAAm-Alg-ECH-P-Tyr and PAAm-Alg-AGE-P-Tyr cryogels, depending on the pH and adsorption buffer system used. The selectivity analyzed by electrophoretic profiles was similar for both cryogels, but PAAm-Alg-ECH-P-Tyr achieved higher IgG adsorption capacity (dynamic capacity of 12.62 mg of IgG/mL of cryogel). The IgG purity assayed by ELISA was 95%. The maximum IgG adsorption capacity and dissociation constant of the PAAm-Alg-ECH-P-Tyr, determined by Langmuir isotherm, were found to be 91.75 mg IgG/g of dry cryogel and 4.60 × 10−6 mol/L at pH 6.0 from aqueous solutions. The PAAm-Alg-AGE-P-Tyr showed potential to purify the Fab fragments from papain-digested human IgG solution at pH 7.0. Fab fragments were separated from Fc fragments (but with uncleaved IgG) in eluted fractions (analyzed by the Western blot technique), with yield of 82% and purity of 95% (determined by radial immunodiffusion).

Long-term human IgG treatment improves heart and muscle function in a mouse model of Duchenne muscular dystrophy.

BackgroundDuchenne muscular dystrophy (DMD) is a progressive muscle‐wasting disease caused by mutations in the dystrophin gene, which leads to structural instability of the dystrophin–glycoprotein‐complex with subsequent muscle degeneration. In addition, muscle inflammation has been implicated in disease progression and therapeutically addressed with glucocorticosteroids. These have numerous adverse effects. Treatment with human immunoglobulin G (IgG) improved clinical and para‐clinical parameters in the early disease phase in the well‐established mdx mouse model. The aim of the present study was to confirm the efficacy of IgG in a long‐term pre‐clinical study in mdx mice.MethodsIgG (2 g/kg body weight) or NaCl solution as control was administered monthly over 18 months by intraperitoneal injection in mdx mice beginning at 3 weeks of age. Several clinical outcome measures including endurance, muscle strength, and echocardiography were assessed. After 18 months, the animals were sacrificed, blood was collected for analysis, and muscle samples were obtained for ex vivo muscle contraction tests, quantitative PCR, and histology.ResultsIgG significantly improved the daily voluntary running performance (1.9 m more total daily running distance, P < 0.0001) and slowed the decrease in grip strength by 0.1 mN, (P = 0.018). IgG reduced fatigability of the diaphragm (improved ratio to maximum force by 0.09 ± 0.04, P = 0.044), but specific tetanic force remained unchanged in the ex vivo muscle contraction test. Cardiac function was significantly better after IgG, especially fractional area shortening (P = 0.012). These results were accompanied by a reduction in cardiac fibrosis and the infiltration of T cells (P = 0.0002) and macrophages (P = 0.0027). In addition, treatment with IgG resulted in a significant reduction of the infiltration of T cells (P ≤ 0.036) in the diaphragm, gastrocnemius, quadriceps, and a similar trend in tibialis anterior and macrophages (P ≤ 0.045) in gastrocnemius, quadriceps, tibialis anterior, and a similar trend in the diaphragm, as well as a decrease in myopathic changes as reflected by a reduced central nuclear index in the diaphragm, tibialis anterior, and quadriceps (P ≤ 0.002 in all).ConclusionsThe present study underscores the importance of an inflammatory contribution to the disease progression of DMD. The data demonstrate the long‐term efficacy of IgG in the mdx mouse. IgG is well tolerated by humans and could preferentially complement gene therapy in DMD. The data call for a clinical trial with IgG in DMD.

DMSA-Functionalized Mesoporous Alumina with a High Capacity for Selective Isolation of Immunoglobulin G.

A novel dimercaptosuccinic acid-functionalized mesoporous alumina (DMSA-MA) is synthesized by the dicarboxylic acid groups of dimercaptosuccinic acid molecules coordinating to the Al3+ ions located in the mesostructure. The as-prepared DMSA-MA composites possess a large surface area of 91.17 m2/g as well as a uniform pore size and a high pore volume of 17.22 nm and 0.23 cm3/g, respectively. DMSA coating of mesostructures significantly enhanced their selectivity for glycoprotein adsorption through a powerful hydrophilic binding force, and the maximum adsorption capacity of immunoglobulin G (IgG) can reach 2298.6 mg g-1. The captured IgG could be lightly stripped from the DMSA-MA composites with an elution rate of 98.3% by using 0.5 wt % CTAB solution as the elution reagent. DMSA-MA is further employed as a sorbent for the enrichment of IgG heavy chain and light chain from human serum sample. SDS-PAGE assay results showed the obtained IgG with high purity compared to that of the standard solution of IgG.

Highly Sensitive SPR Biosensor Based on Graphene Oxide and Staphylococcal Protein A Co-Modified TFBG for Human IgG Detection

A highly sensitive optical fiber surface plasmon resonance (SPR) biosensor based on graphene oxide (GO) and staphylococcal protein A (SPA) co-modified tilted fiber Bragg grating (TFBG) is proposed and demonstrated for the detection of human immunoglobulin G (IgG) for the first time. The gold film on the surface of the sensor was first fixed with GO and then modified with an SPA to improve the sensitivity of the sensor. Large specific surface area and abundant functional groups of GO can adsorb more antibodies. The combination of SPA and the antibody molecule Fc region makes the Fab area with antigen-binding sites extend outward, resulting in highly oriented antibody immobilization on the sensor surface and high antigen–antibody binding efficiency. The experimental results show that the sensitivity as well as the limit of detection of GO-SPA-modified TFBG-SPR biosensor is around 0.096 dB/( $\mu \text{g}$ /mL) and $0.5~\mu \text{g}$ /mL, showing better responses to human IgG solutions with a concentration range of 30– $100~\mu \text{g}$ /mL compared with the TFBG-SPR biosensors modified singly with GO or SPA. The biosensor exhibits the advantages of small size, ease of fabrication, high sensitivity, label-free, and rapid response, and provides a new solution for detecting low concentration of biological solution, presenting great application potential in the biochemistry field.

Selective separation method of aggregates from IgG solution by aqueous two-phase system

Aggregation of immunoglobulin G (IgG) is a serious concern that results in immunogenicity in pharmaceutical applications. Removal of the small and soluble aggregates in protein solutions through a simple method remains challenging. Here we show that an aqueous two-phase system (ATPS) can be used for the elimination of soluble aggregates from IgG solution. Polyethylene glycol (PEG) and dextran (DEX) were selected as components of the ATPS. As expected, IgG monomers were partitioned into the top or bottom phases of ATPS. Interestingly, almost all the small and soluble aggregates of IgG were extracted to the interface between top and bottom phases, rather than in the liquid phases. The partitioning of monomers and aggregates of IgG can be attributed to the solubility of these protein states in PEG and DEX. Thus, ATPS using PEG and DEX can be employed for the simple removal method of soluble aggregates from IgG solution.

MWCNT interactions with protein: surface-induced changes in protein adsorption and the impact of protein corona on cellular uptake and cytotoxicity.

PurposeProtein adsorption onto nanoparticles in the form of protein corona, affects properties of nanomaterials and their behavior in the biological milieu. This study aims at exploring the effects of multiwalled carbon nanotubes (MWCNTs) surface chemistry on bovine serum albumin (BSA) and immunoglobulin G (IgG), including their adsorption behavior and spatial configurations, as well as the impact on cellular uptake, cytotoxicity, and cellular responses.MethodsThree types of MWCNTs (pristine MWCNTs, MWCNTs-COOH, and MWCNTs-PEG) were synthesized by classical chemical reduction. The size, morphology, hydrodynamic size, and zeta potential were characterized using transmission electron microscopy and dynamic light scattering. MWCNTs were exposed to BSA and IgG solutions, then the amount of MWCNT absorption was performed by bicinchoninic acid assay, and the effects were assessed by utilizing fluorescence spectroscopy, circular dichroism (CD) spectroscopy. Quantitative measurement of MWCNTs uptake with or without protein corona was performed as turbidity method. CCK assay and a microdilution method were performed to evaluate the effects of protein corona on cytotoxicity and pro-inflammatory cytokines release.ResultsThe BSA and IgG adsorption capacities of MWCNTs followed the order pristine MWCNTs>MWCNTs-COOH and MWCNTs-PEG. MWCNT binding can cause fluorescence quenching and conformational changes in BSA and IgG, indicating that both the physicochemical properties of MWCNTs and protein properties play critical roles in determining their adsorption behavior. Further study showed time-dependent increases in MWCNT cellular uptake and internalization. Hydrophobicity is the major factor increasing cellular uptake of pristine MWCNTs, but a protein corona enriched with dysoposnins is the main factor reducing uptake of MWCNT-COOH by RAW264.7 cells. The cytotoxicity and pro-inflammatory response related to physicochemical properties of MWCNTs, and frustrated phagocytosis is a key initiating event in the pro-inflammatory response of MWCNT-exposed macrophages.ConclusionThese findings shed light on how functionalized MWCNTs interact with protein coronas and provide useful insight into the dramatic effect of protein coronas on different functionalized MWCNTs. These events affect cellular uptake and cytotoxicity, which could inform how to enhance MWCNT biocompatibility and develop approaches for managing MWCNT hazards.

Separation of human Fab fragments by negative chromatography on ω-aminohexyl- and poly(ethyleneimine)-agarose

Polyamines ω-aminohexyl and poly(ethyleneimine) (PEI) immobilized on agarose gel were evaluated as ligands for the purification of Fab fragments from papain-digested human immunoglobulin G (IgG) solution by negative chromatography. The adsorption of proteolytic fragments was investigated using different buffer systems and pH values. The results showed that the buffer system and pH had stronger effects on the adsorption of the proteolytic fragments onto ω-aminohexyl-agarose than onto PEI-agarose. Tris-HCl buffer pH 8.0 could be used to purify Fab fragments by negative chromatography using both adsorbents, achieving purity and recovery above 96% and 93%, respectively. Static binding experiments were conducted to determine the maximum binding capacity of ω-aminohexyl-agarose adsorbent (Qm). The experimental isotherm was best described by the Langmuir–Freundlich model, which indicated the positive cooperativity of adsorption (n = 1.68) and a Qm value of 44.32 mg mL−1 of gel. The results suggest that negative chromatography on ω-aminohexyl- and PEI-agarose is a potential technique for the purification of Fab fragments from papain-digested human IgG solution.

Biosensing Probe for Quality Control Monitoring of the Structural Integrity of Therapeutic Antibodies.

Ideal quality control of therapeutic antibodies involves analytical techniques with high-sensitivity, high-resolution, and high-throughput performance. Few technologies that assess the physicochemical heterogeneity of antibodies, however, meet all the required demands. We developed a biosensing method for the quality control of therapeutic antibodies based on an artificial protein, AF.2A1, which discriminates between the native and the non-native three-dimensional structures of immunoglobulin G (IgG). AF.2A1 specifically recognized non-native IgG spiked into a solution of native IgG, thereby making it possible to detect contamination by a small amount of non-native IgG, which is difficult using conventional size-based separation or spectroscopic techniques. Using AF.2A1 as an analytical probe, we determined the concentration of non-native IgG formed under various pH conditions. The probe was also applicable to accelerated tests of the long-term stability of a therapeutic antibody, allowing monitoring of the formation of non-native IgG at elevated temperatures and extended periods of storage. AF.2A1, a proteinous probe, can be combined with established methods or devices to achieve high-throughput assays and provides the potential for probe-based biosensing for the quality control of therapeutic antibodies.

PHEMA/PEI]–Cu(II) based immobilized metal affinity chromatography cryogels: Application on the separation of IgG from human plasma

The immobilized metal-affinity chromatography (IMAC) has gained significant interest as a widespread separation and purification tool for therapeutic proteins, nucleic acids and other biological molecules. The enormous potential of IMAC for proteins with natural surface exposed-histidine residues and for recombinant proteins with histidine clusters. Cryogels as monolithic materials have recently been proposed as promising chromatographic adsorbents for the separation of biomolecules in downstream processing. In the present study, IMAC cryogels have been synthesized and utilized for the adsorption and separation of immunoglobulin G (IgG) from IgG solution and whole human plasma. For this purpose, Cu(II)-ions were coupled to poly(hydroxyethyl methacrylate) PHEMA using poly(ethylene imine) (PEI) as the chelating ligand. In this study the cryogels formation optimized by the varied proportion of PEI from 1% to 15% along with different amounts of Cu (II) as chelating metal. The prepared cryogels were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The [PHEMA/PEI]–Cu(II) cryogels were assayed for their capability to bind the human IgG from aqueous solutions. The IMAC cryogels were found to have high affinity toward human IgG. The adsorption of human IgG was investigated onto the PHEMA/PEI cryogels with (10% PEI) and the concentration of Cu (II) varied as 10, 50, 100 and 150mg/L. The separation of human IgG was achieved in one purification step at pH7.4. The maximum adsorption capacity was observed at the [PHEMA/PEI]–Cu(II) (10% PEI) with 72.28mg/g of human IgG. The purification efficiency and human IgG purity were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Linking the Solution Viscosity of an IgG2 Monoclonal Antibody to Its Structure as a Function of pH and Temperature

Although the viscosity of concentrated antibody solutions has been the focus of many recent studies, less attention has been concentrated on how changes in protein structure impact viscosity. This study examines viscosity profiles of an immunoglobulin G (IgG) 2 monoclonal antibody at 150 mg/mL as a function of temperature and pH. Although the structure of the antibody at pH 4.0-7.0 was comparable at lower temperatures as measured by second derivative UV absorbance and Fourier transform infrared spectroscopy, differences in 8-anilino-1-naphthalene sulfonate (ANS) fluorescence intensity indicated small structural alterations as a function of pH. Below the structural transition onset temperature, the viscosity profiles were pH dependent and linearly correlated with fluorescence intensity, and followed semilogarithmic behavior as a function of temperature. The transitions of the viscosity profiles correlated well with the major structure transitions at a protein concentration of 150 mg/mL. The viscosity correlated particularly well with ANS fluorescence intensity at 0.2 mg/mL below and above the structural transition temperatures. These results suggest: (1) ANS can be an important measure of the overall structure and (2) hydrophobic interactions and charge-charge interactions are the two major physical factors that contribute collectively to the high viscosity of concentrated IgG solutions.

Integrated dual‐grating inteferometric detection with polymer microbeams for bio‐chemical sensing in liquid environment

An interferometric displacement detection method of polymer microbeams based on integrated dual-grating was developed for bio-chemical sensing in a liquid environment. Sensing microstructures made of polyimide (PI) with four symmetrically arranged L-shaped beams suspending a central plate were designed and fabricated on a glass substrate using a surface micromachining process. With these structures, deflection changes of the microbeams were transduced to out-of-plane movements of the central plate, which were then measured from the interference of light spots diffracted from the integrated gratings using a charge coupled device image measurement setup. To extend the displacement detection range, two adjacent metal gratings corresponding to a microbeam were formed inside and outside a groove with a depth of 55 nm, respectively, to achieve a phase shift of about π/2 in liquid. The detected noise of aluminium (Al) microcantilevers in ethanol slightly increased compared with that in air, and the noise of PI microbeams in phosphate-buffered saline was about three times larger than that of Al cantilevers in ethanol. The preliminary experimental results in ethanol show that the detection method based on polymer microbeams integrated with dual-gratings is feasible in a liquid environment. Antibody–antigen binding detection was also conducted with a rabbit immunoglobulin G (IgG) immobilised chip in 100 μg/ml goat anti-rabbit IgG solution.

Simultaneous depletion of immunoglobulin G and albumin from human plasma using novel monolithic cryogel columns

In this study, we aimed to develop an alternative matrix able to deplete the albumin (Alb) and immunoglobulin G (IgG) from blood plasma simultaneously to prepare plasma samples for large-scale applications of blood-related proteomics. As a first step, nano-protein A nanoparticles (nanoProA) were prepared and characterized. Subsequently, cibacron blue F3GA (CB) was immobilized onto the nanoProA's to enhance their specific affinity for Alb molecules. Finally, both nanoparticles, specifically, nanoProA and CB-nanoProA, were separately embedded into cryogel structures to combine advantages of the nanoparticles with those of the cryogels. The protein adsorption was optimized using aqueous Alb and IgG solutions separately. Subsequently, competitive protein adsorption was performed using a protein mixture prepared with Alb and IgG adhering to their plasma protein ratios. Because of the CB-immobilization, the Alb depletion performance of the cryogels increased whereas the IgG depleting performance decreased. Using the nanoProA, embedded cryogel removed 99.3% of the IgG, while using the CB-nanoProA embedded cryogel removed 97.5% of the Alb content. The simultaneous depletion performances of the cryogels for Alb and IgG were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In this study, the monolithic cryogel-based adsorbents were classified as an alternative matrix to prepare plasma samples for proteomics applications at the preparative scale.

The fabrication of nanosensor-based surface plasmon resonance for IgG detection

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

Fab fragments imprinted SPR biosensor for real-time human immunoglobulin G detection

Fab fragments imprinted surface plasmon resonance (SPR) chip was prepared for the real-time detection of human immunoglobulin G (IgG). In order to attach polymerization precursor on SPR chip, the SPR chip surface was modified with allyl mercaptan. Fab fragments of the IgG molecules were prepared by papain digestion procedure and collected by fast protein liquid chromatography (FPLC) system using Hi-Trap_r Protein A FF column. The collected Fab fragments were complexed with histidine containing specific monomer, N-methacryloyl-l-histidine methyl ester (MAH). Molecular imprinted polymeric nanofilm was prepared on SPR chip in the presence of ethylene glycol dimethacrylate and 2-hydroxyethylmethacrylate. The template molecules, Fab fragments, were removed from the polymeric nanofilm using 1M NaCl solution (pH: 7.4, phosphate buffer system). The molecular imprinted SPR chip was characterized by contact angle, atomic force microscopy and Fourier transform infrared spectroscopy. By the real-time IgG detection studies carried out using aqueous IgG solutions in different concentrations, the kinetics and isotherm parameters of the molecular imprinted SPR chip–IgG system were calculated. To show selectivity and specificity of the molecular imprinted SPR chip, competitive kinetic analyses were performed using bovine serum albumin (BSA), IgG, Fab and Fc fragments in singular and competitive manner. As last step, IgG detection studies from human plasma were performed and the measured IgG concentrations were well matched with the results determined by enzyme-linked immunosorbent assay (ELISA). The results obtained with the molecular imprinted SPR chip were well fitted to Langmuir isotherm and the detection limit was found as 56ng/mL. In the light of the results, we can conclude that the proposed molecular imprinted SPR chip can detect IgG molecules from both aqueous solutions and complex natural samples.

Influence of protein adsorption kinetics on breakthrough broadening in membrane affinity chromatography

Existing mathematical models developed to describe membrane affinity chromatography are unable to match the complete breakthrough curve when a single Langmuir adsorption isotherm is used, because important deviations from the observed behavior are systematically encountered in the simulation of breakthrough broadening near saturation. The relevant information required to overcome that limitation has been obtained by considering simultaneously both loading and washing curves, thus evaluating the adsorption data at equilibrium and recognizing what are the appropriate adsorption mechanisms affecting the observed behavior. The analysis indicates that a bi-Langmuir binding kinetics is essential for a correct process description up to the saturation of the stationary phase, together with the use of the relevant transport phenomena already identified for the experimental system investigated. The input parameters used to generate the resulting simulations are evaluated from separate experiments, independent from the chromatographic process. Model calibration and validation is accomplished comparing model simulations with experimental data measured by feeding pure human immunoglobulin G (IgG) solutions as well as a cell culture supernatant containing human monoclonal IgG 1 to B14-TRZ-Epoxy2 bio-mimetic affinity membranes. The simulations obtained are in good agreement with the experimental data over the entire adsorption and washing stages, and breakthrough tailing appears to be associated to the reversible binding sites of the bi-Langmuir mechanism. Remarkably, the model proposed is able to predict with good accuracy the purification of IgG from a complex mixture simply on the basis of the results obtained from pure IgG solutions.

Label-Free Raman Mapping of Surface Distribution of Protein A and IgG Biomolecules

We have demonstrated a nanoengineered substrate composed of micropatterned silver nanoparticles to be used for the label-free mapping of adsorbed biomolecules. We utilized surface-enhanced Raman scattering (SERS) phenomenon to monitor the known bioanalytes, protein A and human immunoglobulin G (IgG). The SERS substrate was composed of a poly(alylamine hydrochloride) (PAH)/poly(styrenesulfonate) (PSS) layer-by-layer (LbL) nanocoating micropatterned with silver nanoparticles confined to microscopic stripes. Selective adsorption of biomacromolecules is facilitated by the amine-terminated LbL nanocoating, which prevents the surface adsorption of positively charged protein A across the surface except on the patterned regions containing negatively charged silver nanoparticles. Furthermore, adsorption of IgG on predetermined regions is facilitated by the selective binding of the Fc region of IgG to protein A. This label-free SERS approach provides accurate, selective, and fast detection of protein A and IgG solutions with a nanomolar concentration, down to below 1 nM for IgG in solution. This method could also be utilized for the facile detection of proteins in field conditions as well as in clinical, forensic, industrial, and environmental laboratories.