Immunoaffinity Membrane Research Articles

Assessment of thermally stabilized electrospun poly(vinyl alcohol) materials as cell permeable membranes for a novel blood salvage device

The use of Intraoperative Cell Salvage (ICS) is currently limited in oncological surgeries, due to safety concerns associated with the ability of existing devices to successfully remove circulating tumour cells. In this work, we present the first stages towards the creation of an alternative platform to current cell savers, based on the extremely selective immunoaffinity membrane chromatography principle. Non-woven membranes were produced via electrospinning using poly(vinyl alcohol) (PVA), and further heat treated at 180 °C to prevent their dissolution in aqueous environments and preserve their fibrous morphology. The effects of the PVA degree of hydrolysis (DH) (98 % vs 99 %), method of electrospinning (needleless DC vs AC), and heat treatment duration (1–8 h) were investigated. All heat treated supports maintained their cytocompatibility, whilst tensile tests indicated that the 99 % hydrolysed DC electrospun mats were stronger compared to their 98 % DH counterparts. Although, and at the described conditions, AC electrospinning produced fibres with more than double the diameter compared to those from DC electrospinning, it was not chosen for subsequent experiments because it is still under development. Evidence of unimpeded passage of SY5Y neuroblastoma cells and undiluted defibrinated sheep's blood in flow-through filtration experiments confirmed the successful creation of 3D networks with minimum resistance to mass transfer and lack of non-specific cell binding to the base material, paving the way for the development of novel, highly selective ICS devices for tumour surgeries.

Development of biohybrid immuno-selective membranes for target antigen recognition

Membranes are gaining increasing interest in solid-phase analytical assay and biosensors applications, in particular as functional surface for bioreceptors immobilization and stabilization as well as for the concentration of target molecules in microsystems.In this work, regenerated cellulose immuno-affinity membranes were developed and they were used for the selective capture of interleukin-6 (IL-6) as targeted antigen. Protein G was covalently linked on the membrane surface and it was successfully used for the oriented site-specific antibody immobilization. The antibody binding capacity of the protein G-coupled membrane was evaluated. The specific anti IL-6 antibody was immobilized and a quantitative analysis of the amount of IL-6 captured by the immuno-affinity membrane was performed. The immobilization procedure was optimized to eliminate the non-specific binding of antigen on the membrane surface. Additionally, the interaction between anti IL-6 antibody and protein G was stabilized by chemical cross-linking with glutaraldehyde and the capture ability of immuno-affinity membranes, with and without the cross-linker, was compared. The maximum binding capacity of the protein G-coupled membrane was 43.8µg/cm2 and the binding efficiency was 88%. The immuno-affinity membranes showed a high IL-6 capture efficiency at very low antigen concentration, up to a maximum of 91%, the amount of captured IL-6 increased linearly as increasing the initial concentration. The cross-linked surface retained the antigen binding capacity demonstrating its robustness in being reused, without antibody leakage or reduction in antibody binding capacity. The overall results demonstrated the possibility of a reliable application of the immuno-affinity membrane developed for biosensors and bioassays also in multiple use.

Inhibition of Amyloid β Protein Fibrillation via Carboxypeptidase Y after Protein Trapping Using Immunoaffinity Membranes

In this study, we firstly report inhibition of amyloid β42 (Aβ42) fibrillation, which is related to the progression of Alzheimer’s disease, by digestion using proteases, combined with Aβ42 isolation using an immunoaffinity membrane. Especially, digestion of Aβ42 with carboxypeptidase Y (CPY) is effective for the inhibition of Aβ42 fibrillation, indicating that carboxy-terminal digestion of Aβ42 is efficient for the prevention of its fibrillation. The combinational method of the immunoaffinity membrane with CPY digestion is applicable to suppress Aβ42 fibrillation.

Retaining activity of enzymes after capture and extraction within a single-drop of biological fluid using immunoaffinity membranes.

The purpose of this study was the measurement of enzyme activity within a single-drop of biological fluid after micropurification. Esterase and lactate dehydrogenase (LDH) retained their enzymatic activities after being captured by membrane-immobilized antibodies, which were prepared by non-denaturing two-dimensional electrophoresis, transferred to polyvinylidene difluoride and then stained by Ponceau S. The activities of both enzymes were also measured after being captured by antibodies and biotinylated antibodies bound to membrane-immobilized protein A or avidin, respectively. After esterase and LDH were captured from biological samples by membrane-immobilized protein A or avidin, their activities were semi-quantitatively measured on the surface of the membrane using fluorescence determination. More than 51% of enzyme activities were retained even after the enzymes were captured by biotinylated antibody bound to membrane-immobilized avidin and eluted by rinsing with 5μL of 1% Triton X-100, compared with the activities of the enzyme on the immunoaffinity membrane.

Combined method of immunoaffinity membrane within tubes and MALDI-TOF MS for capturing and analyzing amyloid beta.

Amyloid beta 1-40 peptide was specifically isolated and analyzed from human plasma spiked with amyloid beta using a combined method of biotinylated anti-amyloid beta antibody binding to membrane-immobilized avidin (immunoaffinity membrane) and matrix-assisted laser desorption /ionization time-of-flight mass spectrometry (MALDI-TOF MS). A solution of 10μL containing 13.6ng to 2.9μg of amyloid beta peptide was examined in this method. After the isolated amyloid beta peptide from the spiked human plasma containing 2.9μg of amyloid beta peptide was incubated in the presence of trifluoroacetic acid, fibrillization of the peptides was observed using a thioflavin T assay. Furthermore, an immunoaffinity membrane present on the inner wall of a tube (diameter 2mm) captured the amyloid beta peptide from the spiked human plasma. Our results indicate that the combination of the immunoaffinity membrane procedure and MALDI-TOF MS can be used to capture and analyze the target antigens such as amyloid beta in micro-spaces.

Isolation and analysis of amyloid-β 1-42 monomer and oligomers in liquid droplets using an immunoaffinity membrane.

Monomeric molecules such as amyloid-β can aggregate and transform into oligomeric and fibrous forms, which are implicated in the development and progression of Alzheimer's disease. Novel analytical techniques for the formation of oligomers are required to examine the neurotoxic amyloid-β oligomers involving fibrils. After isolating amyloid-β monomer 1-42 using a biotinylated antibody bound to membrane-immobilized avidin (immunoaffinity membrane), their masses were determined by MALDI-TOF MS. Fluorometric determination of more than 0.5μM of aggregated amyloid-β in pipette droplets was performed after aggregation and dilution of 1mM amyloid-β. Thus, large (>105nm) amyloid-β oligomers in microliter volumes of fluids were isolated using the immunoaffinity membrane and quantitatively analyzed after removal of amyloid-β monomers and small oligomers by non-denaturing electrophoresis. In addition, amyloid-β oligomers were specifically isolated from a mixture of human plasma and aggregated amyloid-β and then fluorometrically analyzed. Our results show that the combination of immunoaffinity membrane-binding and fluorescence determinations together with one drop analysis could be used to isolate and detect huge neurotoxic amyloid-β oligomers such as fibrils in plasma samples.

Micro-Scale Extraction and Analysis of Intact Carboxylesterase after Trapping on an Immunoaffinity Membrane Surface

Porcine liver carboxylesterase was captured using an immunoaffinity membrane, which was prepared by separating an anti-porcine esterase antibody using non-denaturing two-dimensional electrophoresis, followed by transfer to a polyvinylidene difluoride membrane and staining. The activity of this esterase was 0.008 units after it was captured in the tiny spaces (4 mm(2)) of this membrane and eluted by rinsing with 5 μL of aspartic acid solution. The molecular mass of the eluted esterase was m/z 61,885 according to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after the purification of this enzyme from the porcine liver cytosol. The purified enzyme's activity was inhibited by 6,9-diamino-2-ethoxyacridine, and this inhibition was retained even after extracting the enzyme from the immunoaffinity membrane. These results indicate that micro-scale extraction and analysis of a carboxylesterase are possible when the enzyme is trapped using an immunoaffinity membrane and eluted with aspartic acid.

Antigen digestion on the target plate of MALDI-TOF MS after isolation using an immunoaffinity membrane

A combination of methods is required to achieve separation of intact proteins and subsequently perform structure analysis to examine their unstable or external structures. The aim of this study was to develop a method of structure analysis in intact proteins after purification. Transferrin from human plasma was trapped by membrane-immobilized anti-transferrin antibody, which was produced by non-denaturing two-dimensional electrophoresis (2-DE), and transferred to a polyvinylidene fluoride (PVDF) membrane and stained with Ponceau S. The antigen transferrin was eluted by rinsing the membrane with trifluoroacetic acid (TFA) or aspartic acid. In addition, a method was established by which the purified human transferrin was enzymatically digested on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) target plate. Thus, after purification of the human transferrin antigen from tens of microlitres of human plasma using an immunoaffinity membrane, transferrin polypeptide fragments were obtained on the plate following digestion with pepsin in the presence of 0.1% TFA or endoproteinase Lys-C or Lys-C/trypsin with 0.001% sodium dodecyl sulphate (SDS). The results indicated that the combined methods of isolation using an immunoaffinity membrane and enzymatic digestion on a MALDI-TOF MS plate could be applied to the purification and microanalysis of antigens. This approach would be particularly applicable to the analysis of the primary structure and the less stable and highly accessible regions of antigens from limited sample volumes.

Analysis of activity of esterase captured onto an immunoaffinity membrane

BackgroundSpecific proteins in biological fluids can be captured on an immunoaffinity membrane after polyclonal anti-porcine liver esterase antibodies are separated by non-denaturing 2-dimensional electrophoresis (2-DE) and transferred onto the membrane. The enzymatic activities of these captured proteins can then be monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). MethodsPolyclonal anti-porcine liver esterase antibody was separated by non-denaturing 2-DE, transferred onto a polyvinylidene difluoride membrane and stained with Ponceau S. Esterase activity was examined by enzyme activity staining and MALDI-TOF MS after antigens, including purified carboxylesterase from porcine liver and cytosolic esterase from porcine retina, were captured on the immunoaffinity membrane. ResultsEsterase activity was detected on the immunoaffinity membrane after the enzyme was captured. Phosphatidylcholine hydrolysis by the esterase was monitored after the esterase was captured onto the membrane and attached to the target plate for MALDI-TOF MS. ConclusionsThis method could be used to analyze changes in enzymatic activity under biological conditions such as health and disease conditions using immunoaffinity membranes and MALDI-TOF MS.

Preparation of nitrocellulose (NC) immuno‐affinity membrane for purification of rAPC antibody

In this study, recombinant allophycocyanin (rAPC) with a purity of 98% was transferred from a gel to a nitrocellulose (NC) membrane to develop a simple and efficient immuno-affinity membrane. Atomic force microscopy (AFM) was used to investigate the surface topography of the affinity membrane and its characterization indicated that rAPC easily forms trimers or hexamers on the membrane surface on use of the given transfer method. The hydrodynamic radius (R(h)) of the rAPC aggregation was equal to 103 nm or 365 nm according to dynamic light scattering (DLS), which was in agreement with the result obtained by AFM. Based on the specific immunological reaction of antigen and antibody, anti-APC antibodies were purified from rabbit polyclonal serum in a single step. The amount of absorbed antibody was 5.79 mg/g membrane according to analysis by ELISA methods. The purity of antibodies was up to 98% according to SDS-PAGE. The adsorption-desorption cycle of rAPC was repeated six times using the same immuno-affinity membrane, and there was no significant loss in adsorption capacity. The method provides a novel and efficient immunological affinity membrane for the purification of antibodies.

High-performance immunoaffinity chromatography, an immunoaffinity membrane for selective removal of plasma components, and safety evaluation of the latter system

This article reviews our studies on evaluating the suitability of high-performance immunoaffinity chromatography (HPIAC), an immunoaffinity membrane (IAM) for removing unwanted plasma component, and the safety of the IAM. In an attempt to resolve the problem of amyloid deposition in dialysis patients, we used an HPIAC column, bearing anti-β2-microglobulin to remove specifically β2-MG from human plasma. The use of a membrane as an affinity ligand support was also studied. A specific antibody immobilized on the membrane was highly effective for the removal of rat immunoglobulin E and of a human serum amyloid P component passed through an extracorporeal circulation (EC) system. Biocompatibility of the specific antibody-bearing IAM was also examined. These techniques should prove useful for medical applications and may have broad applicability to the elimination of any unwanted plasma component. The IAM exerts two functions simultaneously, usual dialysis and elimination by immunoaffinity binding. The rat EC model has been applied as an evaluation system for the safety of medical devices in contact with the blood stream. Combining commonly used hemodialysis (HD) membranes with rat EC, we evaluated the elicitation of immunological responses, as well as the effect of repeated EC. The data suggest that this EC model can reproduce similar immunological responses in HD patients, and can be employed to evaluate medical devices and materials for their delayed, systemic, and repeated exposure effects. The EC system described here can reproduce human HD treatment, remove unwanted substances, and evaluate medical devices and materials for toxicological responses.

Quantitative fast fractionation of a pool of polyclonal antibodies by immunoaffinity membrane chromatography

A new affinity method for the direct quantitative analysis of monospecific anti-peptide immunoglobulins (antibodies) and, simultaneously, their semi-preparative isolation from blood serum of the immunized animals has been developed. Immunoaffinity discs based on macroporous poly(glycidyl methacrylate–co-ethylene dimethacrylate) were used as the supporting stationary phase. The specifically prepared synthetic peptides with biological activity imitating that of the immunoglobulin binding sites of various proteins were used as the selective ligands instead of native proteins. These ligands were immobilized by a single-step reaction that involves epoxy groups located on the pore surface of the porous polymer disc with amine groups of the peptide molecules. A spacer between biospecific ligands and the linking site was not required to achieve good separation. These novel immunosorbents characterized by large binding capacity are well suited for high throughput screening. Dissociation constants of the peptide–antibody complexes calculated from the experimental adsorption isotherms confirm the excellent selectivity of the proposed separation method. The discs were used in a single step enrichment of antibodies both from precipitated blood fraction and crude blood serum of immunized animals. The quantitative data of the immunoaffinity disc chromatography were compared to those obtained by an enzyme-linked immunosorbent assay. Gel electrophoresis was also used to demonstrate the high degree of purity of the final product. In contrast to typical techniques that involve proteins, this immunoaffinity approach allows for the first time direct determination of concentration of specific antibodies using the immunosorbent prepared from the short peptide molecules immobilized on the internal surface of reactive porous polymer discs.

In vitro Characterization of a Membrane-Based Low-Density Lipoprotein Affinity Adsorption Device

The objective of this study was to explore the use of microporous membranes as an alternative substrate to porous beads in affinity adsorption of low-density lipoprotein (LDL) for therapeutic purposes. Flat sheet immunoaffinity membranes containing a polyclonal antibody preparation were utilized as the affinity substrate. The antibody was covalently immobilized to the surface through a poly(ethylene glycol) (PEG) spacer. Equilibrium adsorption of LDL from plasma was measured. Adsorption from plasma and elution of bound LDL using citrate buffer were studied as a function of flow rate.Specific capacity was as great as 2 mg apolipoprotein B per milliliter membrane volume. The superior transport properties of the membrane allowed rapid adsorption and regeneration, which translated to a large number of adsorptive cycles that can be performed within a given treatment time. On the basis of in vitro performance characteristics, it is estimated that an immunoaffinity membrane device can provide a reduction in patient plasma LDL concentration comparable to that provided by packed columns, but with almost an 80% reduction in the device volume.

Selective Removal of Plasma Components by High‐Performance Immunoaffinity Chromatography

Affinity chromatography employing immobilized antibody is a rapid and specific technique for isolating biologically active materials from different sources, and high-performance immunoaffinity chromatography (HPIAC) has been used to isolate antibodies and antigens for medical application. In an attempt to resolve the problem of amyloid deposition in dialysis patients, we used HPIAC to specifically remove beta2-microglobulin from human plasma. The use of a membrane as an affinity ligand support was also studied. The specific antibody immobilized on a membrane was highly effective for removal of rat immunoglobulin E passed through an extracorporeal circulation system (ECS). Then we investigated the removal of injected human serum amyloid P component from the blood of rats by means of the ECS. Biocompatibility of the specific antibody-bearing immunoaffinity membrane was also examined in terms of nonspecific binding of other plasma components. These techniques should prove useful for medical application and may have broad applicability for the elimination of any unwanted plasma component.

Selective removal of human serum amyloid P component from rat blood by use of an immunoaffinity membrane in an extracorporeal circulation system

We examined the suitability of an immunoaffinity membrane (IAM) bearing specific antibody as a ligand for removing human serum amyloid P component (hSAP) from blood passed through a simple extracorporeal circulation system established in rats. The specific antibody was the most effective of the various ligands tested for removing hSAP from human blood. To determine the value of the hSAP in human or rat plasma, we also developed a simple ELISA. In the rat extracorporeal circulation system, the hSAP level in the inlet blood to the IAM module decreased to 49% of the initial concentration within 60 min. In contrast, the hSAP remained at the initial concentration throughout the study in the module without the IAM. The use of this extracorporeal circulation system in this case allows preclinical evaluation of the ex vivo removal of a human plasma component in an animal model. Biocompatibility of the IAM was also examined. No change in blood cell counts or activation of the coagulation system occurred after contact with the IAM. Non-specific adsorption was not observed, since there was no statistically significant difference in IgG, complement C3, or albumin level between the pre- and post-treatment with this module. The immunological effects of the IAM were also examined using this system. Four weeks after the termination of the extracorporeal circulation, the rats examined showed no detectable antibody titer to the ligand.

Selective removal of immunoglobulin E from rat blood by membrane-immobilized antibody.

We examined the suitability of an immunoaffinity membrane [rabbit IgG specific for rat immunoglobulin E (IgE) immobilized on a cellulose membrane] for removing IgE from rat blood passed through a simple extracorporeal circulatory system. To determine the concentration of IgE in the blood, we also developed a highly sensitive chemiluminescence enzyme immunoassay for rat IgE. The IgE levels in the outlet blood from the immunoaffinity membrane module decreased to 30% of the initial concentration within 30 min.

Mediatorless horseradish peroxidase enzyme electrodes based on activated carbon: potential application to specific binding assay

Horseradish peroxidase (HRP) enzyme electrodes based on activated carbon containing various amounts of platinum have been constructed by simple passive adsorption. Direct electron transfer between the electrodes and HRP resulted in the electroenzymic reduction of H 2O 2 at potentials of less than +480 mV (vs. Ag/AgCl). The highest cathodic current was obtained using HRP adsorbed to non-platinized activated carbon (NPAC), which gave a current density of 637 nA dm 3 μmol −1 cm −2 at +50 mV. A linear calibration curve for H 2O 2 measurement was obtained over the range 0.2–150 μmol dm −3. Kinetic analyses of these data gave a heterogeneous rate constant k′ ME of 5.3 × 10 −3 cm s −1 and an enzyme turnover number k′ cat,E of 2.8 × 10 −2 cm s −1. The HRP-NPAC electrode showed good storage stability in phosphate-buffered saline solution (pH 7.4) at 4°C, with a calculated half-life of 235 days. The potential for HRP-NPAC electrodes to be applied in specific binding assays, such as immunoassays, was assessed using a biotin binding procedure based on competition with glucose-oxidase-labelled biotin for available avidin binding sites on an immunoaffinity membrane. The detection of H 2O 2 generated from a specifically bound glucose oxidase label, on the addition of β- d-glucose, resulted in a cathodic current which was inversely proportional to the biotin concentration over the range 0.1–300 μg dm −3.