Combining Low Pressure Liquid Affinity Chromatography and Flow Injection Analysis for the Quantitation of Immunoglobulins

Abstract

A simple, accurate method combining low pressure liquid affinity chromatography and flow injection analysis is described for the quantitation of immunoglobulins in biologicals fluids. The affinity matrix consists of Protein A covalently immobilized to a 2-fluoro-1 methylpyridinium salt activated Fractogel support. Utilizing a 1 cm³ affinity column, optimized binding and eluting buffer flow rates of 0.7 and 1.5 mL min^?1, respectively, and a sample loop size of 100 μL, IgG's can be eluted from the affinity column in about 9 min. Linear standard curves (r > 0.99) were obtained at concentrations up to at least 4.0 and 8.0 mg mL^?1 respectively for human and bovine IgG. Recovery yields are good ranging from 96–100%. The within day CV for human and bovine IgG was found to be less than 3.0% whereas the day to day CV was less than 3.4% (n=10). IgG concentrations of spiked and unspiked bovine plasma samples obtained by the low pressure affinity/flow injection method when compared to those obtained by the radial immunodiffusion agreed to within 4%.     

Enzyme kinetics and glycan structural characterization of secreted alkaline phosphatase prepared using the baculovirus expression vector system

Secreted human alkaline phosphatase (SEAP, a model protein containing a single N-glycan chain) was expressed in Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines infected with recombinant Autographa californica multiple nuclear polyhedrovirus expressing SEAP under control of the polyhedrin promoter. SDS-PAGE showed that both systems expressed fairly pure rSEAP products. The rSEAP expression level was 7.0 U/mL in Tn-5B1-4, higher than the 4.1 U/mL produced by Sf-9. Kinetic analysis showed that V _max and K _m of human placental SEAP were approx 10-fold higher than that of rSEAP, whereas the V _max and K _m of rSEAP prepared using both insect cell lines were comparable. To characterize the recombinant SEAP (rSEAP) glycosylation, the purified rSEAP was digested with PNGase F to release the N-glycan chains. Glycan analysis showed the presence of oligomannose-type N-linked glycans (i.e., Man_2–8 GlcNAc₂ and FucMan_{3 or 4}GlcNAc₂) in rSEAP from Sf9 and Tn-5B1-4 cell lines. The proportions of these oligosaccharide structures were different in the two cell lines. Man₄GlcNAc₂ and FucMan₄GlcNAc₂ were the major rSEAP N-glycans produced in Sf-9 cells, while Man₂GlcNAc₂ was the major rSEAP N-glycan produced in Tn-5B1-4 cells.     

Two‐step chromatography purification of IgGs possessing sialidase activity from human blood serum

Sialation of cell surface is known to be tightly connected with tumorigenicity, invasiveness, metastatic potential and clearance of aged cells, while sialation of immunoglobulin G (IgG) molecules determines their anti‐inflammatory properties. Recently, we have found for the first time IgG‐antibodies possessing sialidase‐like activity (sialylic abzyme) in blood serum of multiple myeloma and systemic lupus erythematosis patients. This abzyme was detected in a pool of IgGs purified by a typical procedure including immunoglobulin's precipitation with ammonium sulfate and following chromatography on protein G–Sepharose column. Here we describe a novel matrix for affinity purification of sialylic abzyme that is based on using bovine submandibular gland mucin conjugated to Sepharose matrix (mucin–Sepharose). This matrix preferentially binds sialidase‐like IgGs from a pool of sialidase‐active fraction of proteins precipitated with 50% ammonium sulfate from blood serum of the systemic lupus erythematosis patients. That allowed us to develop a new scheme of double‐step chromatography purification of sialidase‐like IgGs from human blood serum. Copyright © 2014 John Wiley & Sons, Ltd.     

An integrated coupled column liquid chromatography system for the determination of leukotriene metabolites in biological samples

Summary A fully integrated chromatographic system was developed for the determination of leukotrienes in biological samples using photodiode-array detection (PDAD), which eliminates time consuming manual sample handling steps. A special solid phase extraction, (SPE) methodology for leucotriene metabolite stability was developed which increased the recoveries and eliminates the contamination risk of biological samples. The inherent instability (autooxidation) of many of the leukotriene mediators, and the adsorption effects onto exposed surfaces in vials and in the chromatographic system were found to be very important parameters to control in order to circumvent high loss of sample analytes. By binding the cell supernatants to the functionalities of the SPE support stabilised these mediators. Cell culture samples were eluted through a disposable C₁₈ SPE column. The SPE columns were allowed to thaw and deposited in an automated sample handling unit (ASPEC XL). Desorption of the analytes was followed by a second on-line SPE step, to eliminate remaining interfering matrix compounds. Typical recoveries when stored at −70°C were in-between 55–97% except for (LTE₄) which was found to be around 40% after 72 days of storage. Seven reversed-phase packings were studied. Selectivity factors, as well as the separation efficiencies, were found to differ for the various C₁₈ bonded silica stationary phases. This integrated on-line column liquid chromatographic system was applied to the determination of leukotriene B₄, leukotriene C₄, leukotriene D₄, leukotriene and E₄ in human cell extracts using prostaglandin B₂ as the internal standard. More than 1500 biological samples were analysed. Some validation data are presented for unattended operations.     

Evaluation of Relative Aptamer Binding to Campylobacter jejuni Bacteria Using Affinity Probe Capillary Electrophoresis

Abstract

A qualitative capillary electrophoresis immunoassay was developed for the first-time to evaluate aptamer binding to bacterial cells. Binding affinity of aptamers developed against a Campylobacter jejuni bacterial cell target, relative to other common food-borne pathogens was investigated and specific binding affinity was evidenced by pronounced mobility shift and peak broadening with increasing bacteria concentration for both aptamers. Little to no mobility shift was observed for food-borne pathogens, Salmonella typhirium and Escherichia coli, even when increasing concentrations 10-fold over target. These results suggest that affinity probe capillary electrophoresis could be useful for qualitative screening of aptamer candidates for bacterial cell targets.     

Evaluation of Fumonitest Immunoaffinity Columns

Abstract

A method for the determination of fumonisins B1 and B2 in corn was developed. The method involves sample extraction with methanol:water (80:20) and the use of a commercially available Fumonitest column for sample cleanup. The capacity, selectivity, column-to-column and lot-to-lot reproducibility of the Fumonitest columns were evaluated. The total capacity of the column was found to be 1.2 μg fumonisin. Both fumonisins B1 and B2 had an equal affinity toward the Fumonitest column, with the sample matrix demonstrating little effect on the column performance. The maximum sample size was 0.5 g for samples containing total fumonisins of less than 2 ppm. After elution from the immunoaffinity column, fumonisins B1 and B2 were reacted with naphthalene-2, 3-dicarboxaldehyde (NDA) to produce a highly fluorescent derivative, 1-cyano-2-alkyl-benz[f]isoindole (CBI). The derivatives were then separated from the sample matrix on a reverse phase C-18 column with a mobile phase consisting of acetonitrile:water:acetic acid (55:45:1) Average recoveries of fumonisins B1 and B2 from corn samples spiked at a level of 1000 ng (500ng B1 + 500ng B2)/g were 85.4 and 87.1%, respectively. The detection limit for B1 and B2 was estimated to be 10 and 4 ppb, respectively. The coefficient of variations for fumonisins B1 and B2 were determined to be 10.2% and 10.6%, respectively.     

Novel affinity monolithic column modified with cuprous sulfide nanoparticles for the selective enrichment of low‐molecular‐weight electron‐rich analytes

A novel monolithic column modified with cuprous sulfide nanoparticles was developed and its affinity characteristics towards low‐molecular‐weight electron‐rich analytes were investigated. In the synthesis process, home‐made cuprous oxide nanocubes were immobilized on the surface of monolithic skeleton with the moderate thickness based on the strong interaction between imidazole groups and cuprous oxide, then the cuprous oxide layer was transformed into the more stable cuprous sulfide layer through the treatment by sodium sulfide. The resulting cuprous sulfide modified monolithic column presented good permeability and stability in a wide pH range from 2 to 10. Two kinds of typical electron‐rich analytes, kanamycin A and purine, were chosen to assess its affinity characteristics. Compared with the commercial Cu²⁺‐ and Ni²⁺‐based affinity sorbents, a larger binding capacity of cuprous sulfide modified column toward kanamycin A was obtained under basic condition and the recovery of kanamycin A in a milk sample was over 70%. Moreover, the binding capacity of cuprous sulfide modified column for purine was up to 5.57 mg/mL in frontal elution mode. These results suggested that the Cu₂S column has a promising application for the enrichment of electron‐rich analytes.     

Foot and mouth disease virus concentration and purification by affinity chromatography

Foot and mouth disease virus, (FMDV) from a crude cell lysate was purified in a single step by affinity chromatography with heparin as a ligand. The virus eluted from an Heparin-Ultrogel A₄R column at 1M sodium chloride in 10 mM sodium phosphate buffer, pH 7.0, while most cell protein and albumin did so at lower concentrations of sodium chloride in the same buffer. Purity of the eluted fraction containing the virus was assessed by SDS-PAGE, HPLC, ultracentrifugation, and UV absorption spectrum. With this method, intact viral particles are recovered in high yield (over 90%) and specific virus purity increases nearly 1000-fold. The capacity of the Chromatographic matrix for the virus was found to be 1.1 mg viral mass per mL of hydrated gel.     

Recording of the streaming potential over an affinity column in a continuous flow system as a means of quantifying proteins biospecifically

Measurement of the streaming potential over an affinity column is used as a means of following affinity binding of proteins to immobilized ligands. The system designed operates with one sample column and one reference column and the differential signal is a good measure of the amount of affinity-bound material. The system is used for the determination of albumin in discrete samples as well as for continuous measurements. Assays of rabbit anti-albumin antibodies are also reported; and various glycoproteins which bind to immobilized concanavalin A can be quantified. The limit of detection for proteins (down to 10^?9 moles l^?1) seems to be lower than that which is required for applications to process control.     

Evaluation of interactions between RAW264.7 macrophages and small molecules by capillary electrophoresis

In this study, the affinity interactions between RAW 264.7 macrophages and three small molecules including naringin, oleuropein and paeoniflorin were evaluated by affinity capillary electrophoresis (ACE), partial filling affinity capillary electrophoresis (PFACE) and frontal analysis capillary electrophoresis (FACE), respectively. The result indicated that ACE (varying concentrations of cell suspension were filled in the capillary as receptor) may not be suitable for the evaluation of interactions between cell and small molecules due to the high viscosity of cell suspension; PFACE can qualitatively evaluate the interaction, but the difference in viscosity between RAW264.7 suspension and buffer effects on the liner relationship between filling length and injection time, which makes the calculation of binding constant difficult. Furthermore, based on the PFACE results, naringin showed stronger interaction with macrophages than the other two molecules; taking advantage of the aggregation phenomenon of cell induced by electric field, FACE was successfully used to determine the stoichiometry (n = 5×10⁹) and binding constant (K_b = 1×10⁴ L/mol) of the interaction between RAW264.7 and naringin.     

Determination of Hyaluronic Acid in a Chitosan-Based Formulation by RP C18 and HILIC LC–ESI-MS: an Evaluation of Matrix Effect

Two simple and fast C18 and HILIC liquid chromatography–electrospray mass spectrometry methods for the determination of hyaluronic acid (HA) in a mucoadhesive chitosan-based formulation were developed and validated. The performances of both methods were compared in terms of validation parameters and matrix effect. A simple sample preparation method based on sulphuric acid-based degradation was optimized for the detection of HA fragments (i.e. m/z 380 2-mer, m/z 759 4-mer, m/z 1,138 8-mer and m/z 1,518 16-mer). By operating under selected ion-monitoring mode, excellent selectivity towards chitosan fragments was obtained. For validation, good linearity, detection limits (<4 μg mL⁻¹) and precision (RSD % < 16 %) were generally obtained on matrix with both columns. However, HILIC column exhibited improved performances in terms of HA fragment separation and selectivity. By analyzing on the C18 column the chitosan-based formulation and sample extracts from pig mucosa treated with the formulation, matrix effects exhibited a dependence of signal suppression degree (ranging from 37 to 83 %) as a function of the HA fragment dimension. The HILIC column afforded instead a significantly reduced suppression degree (ranging from 1 to 16 %) and a better separation. These findings demonstrated the improved performances of the HILIC column with respect to conventional C18 mechanism for the analysis of HA fragments in complex matrices.

     

Preparation of a high-performance multi-lectin affinity chromatography (HP-M-LAC) adsorbent for the analysis of human plasma glycoproteins

We report on the preparation of an improved multi-lectin affinity support for HPLC separations. We combined the selectivity of three different lectins: concanavalin A (ConA), wheat germ agglutinin (WGA), and jacalin (JAC). Each lectin was first covalently immobilized onto a polymeric matrix and then the three lectin media were combined in equal ratios. The beads were packed into a column to produce a mixed-bed multi-lectin HPLC column (high-performance multi-lectin affinity chromatography (HP-M-LAC)) for fast chromatographic affinity separations. The support was characterized with respect to kinetics of immobilization, ligand density, and binding capacity for human plasma glycoproteins. A high lectin density (15 mg/mL of beads) was found to be optimal for the binding of glycoproteins from human plasma. A single clinical sample can be fractionated in less than 10 min per run, making this a useful sample preparation tool for proteomics/glycoproteomics studies associated with disease abnormalities.     

Biointeraction analysis of carbamazepine binding to α1‐acid glycoprotein by high‐performance affinity chromatography

Interactions of the drug carbamazepine with the serum protein α₁‐acid glycoprotein (AGP) were examined by high‐performance affinity chromatography. Frontal analysis studies with an immobilized AGP column and control column indicated carbamazepine had both low‐affinity interactions with the support and high‐affinity interactions with AGP. When a correction was made for binding to the support, the association equilibrium constant measured at pH 7.4 and 37°C for carbamazepine with AGP was 1.0 (±0.1)×10⁵ M^?1, with values that ranged from 5.1 to 0.58×10⁵ M^?1 in going from 5 to 45°C. It was found in competition studies that these interactions were occurring at the same site that binds propranolol on AGP. Temperature studies indicated that the change in enthalpy was the main driving force for the binding of carbamazepine to AGP. These results provide a more complete picture of how carbamazepine binds to AGP in serum. This report also illustrates how high‐performance affinity chromatography can be used to examine biological interactions and drug–protein binding in situations in which significant interactions for an analyte are present with both the chromatographic support and an immobilized ligand.     

Electrochemical detection for trace analysis by capillary HPLC

An amperometric-type electrochemical detector is proposed for use in capillary HPLC. Its cell (volume 60 nl) contains a cylindrical working electrode for detecting the presence of electroactive compounds. The noise level is 3–5 pA depending on the potential. The minimum detectable concentrations of the o-, m-, and p- isomers of nitrophenol (after separation of a 20 μl sample on a fused silica column) lies between 4 and 18 ng/l. The relative standard deviation of the height of the chromatographic peaks of phenols present at concentrations of 10 ppb does not exceed 0. 08 (n = 5).     

Synthesis of affinity column of phosphatidylinositol-3,4-diphosphate

Phosphatidylinositol polyphosphates (PIPx) are related with tyrosine kinase activation, cell proliferation and carcinogenesis. In order to investigate the action mechanism of PIPx, it is desirable to synthesize affinity column of PI-3,4-P2, which is expected to be able to isolate the binding proteins of PI-3,4-P2. Thyramine reacted with CH-Sepharose 4B giving column 13. The p-amino group of 3'-(1',2'-distearoyl-glyceryl)-1-(2-p-aminobenzyl)-3,4-di-O-phosphoryl-myo-inosityl phosphate (12) was diazotized, then diazo-coupled with column 13 to give PI-3,4-P2 affinity column 14. This PI-3,4-P2 affinity column is an effective tool to pick up binding proteins of PI-3,4-P2.     

Supermacroporous cryogel matrix for integrated protein isolation. Immobilized metal affinity chromatographic purification of urokinase from cell culture broth of a human kidney cell line

A new type of supermacroporous, monolithic, cryogel affinity adsorbent was developed, allowing the specific capture of urokinase from conditioned media of human fibrosarcoma cell line HT1080. The affinity adsorbent was designed with the objective of using it as a capture column in an integrated perfusion/protein separation bioreactor setup. A comparative study between the utility of this novel cryogel based matrix and the conventional Sepharose based affinity matrix for the continuous capture of urokinase in an integrated bioreactor system was performed. Cu(II)-ion was coupled to epoxy activated polyacrylamide cryogel and Sepharose using iminodiacetic acid (IDA) as the chelating ligand. About 27-fold purification of urokinase from the conditioned culture media was achieved with Cu(II)-IDA-polyacrylamide cryogel column giving specific activity of about 814 Plough units (PU)/mg protein and enzyme yields of about 80%. High yields (95%) were obtained with Cu(II)-IDA-Sepharose column by virtue of its high binding capacity. However, the adsorbent showed lower selectivity as compared to cryogel matrix giving specific activity of 161 PU/mg protein and purification factor of 5.3. The high porosity, selectivity and reasonably good binding capacity of Cu(II)-IDA-polyacrylamide cryogel column make it a promising option for use as a protein capture column in integrated perfusion/separation processes. The urokinase peak pool from Cu(II)-IDA-polyacrylamide cryogel column could be further resolved into separate fractions for high and low molecular weight forms of urokinase by gel filtration chromatography on Sephacryl S-200. The selectivity of the cryogel based IMAC matrix for urokinase was found to be higher as compared to that of Cu(II)-IDA-Sepharose column.     

Preparation and Evaluation of Uniform-Sized Sol–Gel Theophylline Imprinted Microspheres as Solid Phase Extraction Sorbent

A highly selective imprinted amino-functionalized silica gel microsphere was prepared by a simple molecular imprinting technique with a sol–gel process. Theophylline was used as template; 3-aminopropyltrimethoxysilane (APTMS) as functional monomer and tetraethylorthosilicate (TEOS) as reticulating agent. These theophylline imprinted silica microspheres were characterized by Fourier transform infrared spectroscopy (FT-IR), scanning electron micrographs (SEM), thermogravimetric analysis (TGA), and high performance liquid chromatography (HPLC) in detail. The results showed that monodispersive APTMS-silica imprinted microspheres with size of 250 nm were synthesized successfully. Two kind of binding sites exist between the sol–gel imprinted microspheres and theophylline with K _D and Q _max for the higher affinity binding parameters was 1.89 μg/mL and 7.47 μg/mg dry polymers, and for the lower affinity binding sites were found to be 0.18 μg/mL and 3.47 μg/mg dry polymers, respectively. Packed in column, the imprinted microspheres were employed to extract theophylline from green tea successfully.     

Micro-Scale Frontal Affinity Chromatography with Mass Spectrometric Detection: A New Method for the Screening of Compound Libraries

In one run the binding constants K_d for all the active components of a ligand library at sub-microgram quantities can be determined. A mixture of ligands is continuously infused through a column of immobilized receptor, and the eluent analyzed by electrospray mass spectrometry. From the affinity chromatogram produced (see picture) the breakthrough volume of a single compound and hence its K_d value can be determined.     

Dependence of the Formation Function of a Complex with an Oligomer on the Number of Monomers in Its Chain

Isotherms of ligand adsorption by short oligomers were calculated in the form of Scatcharddependences by the matrix method, were used to calculate ligand-polymer binding constants by theMcGhee-Hippel method; the resulting binding parameters are close to theoretical. The complex formationwith a polymer can be distinguished from the complex formation with an oligomer statistically, when a precise formation function and physical methods sensitive to the concentrations of monomer forms are available. Parameters of binding 5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H-porphine with the DNA [d(T₄4G₄4)]₄4 tetraplex at 25°C were calculated on the basis of the matrix and statistical approaches.     

MALDI‐based intact spore mass spectrometry of downy and powdery mildews

Fast and easy identification of fungal phytopathogens is of great importance in agriculture. In this context, matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) has emerged as a powerful tool for analyzing microorganisms. This study deals with a methodology for MALDI‐TOF MS‐based identification of downy and powdery mildews representing obligate biotrophic parasites of crop plants. Experimental approaches for the MS analyses were optimized using Bremia lactucae, cause of lettuce downy mildew, and Oidium neolycopersici, cause of tomato powdery mildew. This involved determining a suitable concentration of spores in the sample, selection of a proper MALDI matrix, looking for the optimal solvent composition, and evaluation of different sample preparation methods. Furthermore, using different MALDI target materials and surfaces (stainless steel vs polymer‐based) and applying various conditions for sample exposure to the acidic MALDI matrix system were investigated. The dried droplet method involving solvent evaporation at room temperature was found to be the most suitable for the deposition of spores and MALDI matrix on the target and the subsequent crystallization. The concentration of spore suspension was optimal between 2 and 5 × 10⁹ spores per ml. The best peptide/protein profiles (in terms of signal‐to‐noise ratio and number of peaks) were obtained by combining ferulic and sinapinic acids as a mixed MALDI matrix. A pretreatment of the spore cell wall with hydrolases was successfully introduced prior to MS measurements to obtain more pronounced signals. Finally, a novel procedure was developed for direct mass spectra acquisition from infected plant leaves. Copyright © 2012 John Wiley & Sons, Ltd.