Detection and enrichment of antigen-specific CD4+ and CD8+ T cells based on cytokine secretion

The Cytokine Secretion Assay is an innovative method for analysing and enriching live cytokine-secreting cells. In this assay, a cytokine affinity matrix is built on the cell plasma membrane, which traps cytokines produced by the cell in response to specific stimuli. The specifically bound cytokine is then detected, and the cells optionally enriched, using fluorochrome-conjugated cytokine-specific antibodies and magnetic microbeads. This method allows extremely detailed phenotyping of live cells and the detection of cytokine responses at very low frequencies. Here, the latest cell staining and separation procedures are reviewed, with particular reference to the best application of the technology and troubleshooting in a variety of different situations.     

Use of monoclonal antibodies to separate the enantiomers of abscisic acid

The resolution of racemates often requires difficult and time consuming purification procedures. McAb technology allows the production of specific antibodies in quantities suitable for the preparation of matrices for large scale affinity purification. Here we report the rapid separation of abscisic acid (ABA) enantiomers by affinity chromatography using McAb. This method appears to be far superior to previously published separations based on crystallization, chromatography, and affinity purification with conventional antisera. The approach here described will be particularly attractive in a wide variety of similar situations.     

Selective protein separations using Formed-In-Place anion exchange membranes.

Anion exchange membranes prepared by adsorption of polymers on Formed-In-Place microfiltration substrates were formed and ion-exchange separations of solutions containing two proteins were determined by ion exchange membrane sequential separation procedures, similar to affinity membrane separation procedures. Representative ion exchange separation processes utilizing adsorbed poly(ethylene imine) (PEI) as the ion exchange membrane for the separation of the components of solutions containing two proteins, bovine serum albumin (BSA) and lysozyme and ovalbumin and lysozyme, are described. The stability of the PEI adsorbed layer, binding characteristics of the BSA to the membrane and purification properties of the procedure were determined.     

Affinity purification of hexosaminidases

Hexosaminidases A and B were purified by affinity chromatography from normal gastric mucosa, after preliminary separation of isozymes by anion exchange chromatography. Heparin and mannosamine were coupled to Sepharose 4B and used as affinity matrices and the purified enzymes were found to be homogeneous when analysed by polyacrylamide slab gel electrophoresis. This combination of 2 novel affinity chromatographic procedures is superior to existing methods in that a final yield of over 70% could be achieved. Also, the number of steps required to obtain homogeneity are less in contrast to the conventional methods used previously.     

Endotoxin depletion of recombinant protein preparations through their preferential binding to histidine tags

The presence of endotoxins in preparations of recombinantly produced therapeutic proteins poses serious problems for patients. Endotoxins can cause fever, respiratory distress syndromes, intravascular coagulation, or endotoxic shock. A number of methods have been devised to remove endotoxins from protein preparations using separation procedures based on molecular mass or charge properties. Most of the methods are limited in their endotoxin removal capacities and lack general applicability. We are describing a biotechnological approach for endotoxin removal. This strategy exploits the observation that endotoxins form micelles that expose negative charges on their surface, leading to preferential binding of endotoxins to cationic surfaces, allowing the separation from their resident protein. Endotoxins exhibit high affinity to stretches of histidines, which are widely used tools to facilitate the purification of recombinant proteins. They bind to nickel ions and are the basis for protein purification from cellular extracts by immobilized metal affinity chromatography. We show that the thrombin-mediated cleavage of two histidine tags from the purified recombinant protein and the adsorption of these histidine tags and their associated endotoxins to a nickel affinity column result in an appreciable depletion of the endotoxins in the purified protein fraction.     

Protein fractionation using fast flow immobilized metal chelate affinity membranes

A new group-specific affinity membrane using metal chelates as ligands and inorganic glass hollow fiber microfiltration membranes as support matrices is developed and tested. The study focused on developing the optimum activation and coupling procedures to bind the chelating agent (iminodiacetic acid, IDA) to the surface of the microporous glass hollow fiber membrane and testing the resultant affinity membrane. Starting with three different glass surfaces, five modification reactions were evaluated. All the modified "active surfaces" were first tested for their protein adsorptive properties in batch mode with suspended microporous glass grains using model proteins with known binding characteristics with Cu-IDA systems. The metal loading capacities of the surfaces exhibiting favorable fractionation were then measured by atomic absorption spectroscopy.The results were compared with the results obtained with a commercial material used in immobilized metal affinity column chromatography. The protein binding characteristics of the hollow fiber affinity membranes were also evaluated under conditions of convective flow. This was performed by flowing single solute protein solutions through the microporous membrane at different flow rates. These results were then used to estimate the optimum loading and elution times for the process. A mathematical model incorporating radial diffusion was solved using a finite difference discretization method. Comparison between model predictions and experimental results was performed for four different proteins at one flow rate. These results suggested that the kinetics of adsorption was concentration dependent. Finally, the hollow fiber affinity membranes were challenged with two component mixtures to test their ability to fractionate mixed protein solutions. Efficient separation and good purity were obtained.The results presented here represent the development of a new fast flow affinity membrane process-immobilized metal affinity membranes (IMAM). (c) 1994 John Wiley & Sons, Inc.     

Synthesis of immobilized flavin derivatives and their use in purification of chicken egg-white ovoflavoprotein.

Affinity adsorbents for flavoproteins were prepared by the covalent attachment of polyacrylamide and agarose to flavin derivatives linked through position N(3) of the flavin nucleus. 3-Carboxymethyl-FMN covalently linked to aminoalkyl substituted agarose was successfully used for the separation and purification of the apo form of the ovoflavoprotein from chicken egg white. High yields and high purities were achieved by two different isolation procedures employing the affinity adsorbent.     

Determination of carbohydrate-deficient transferrin separated by lectin affinity chromatography for detecting chronic alcohol abuse.

Carbohydrate-deficient transferrin (CDT) has been established as a valuable biological marker for detecting chronic alcohol abuse. To improve the diagnostic efficiency, we studied new CDT determination procedures involving the use of lectin affinity chromatography with Allomyrina dichotoma agglutinin (allo A) and Trichosanthes japonica agglutinin I (TJA-I) to isolate the CDT isoforms CDT-allo A and CDT-TJA, respectively. These procedures, based on detection of the CDT-allo A and CDT-TJA isoforms in sera, showed high sensitivity (100% and 98%, respectively) and high specificity (93% and 85%, respectively). These results demonstrate that the new procedures involving the use of lectin affinity chromatography are more useful for isolating markers in the CDT test than the conventional charge-based separation method.     

Use of high-affinity cell wall-binding domains of bacteriophage endolysins for immobilization and separation of bacterial cells

Immobilization and magnetic separation for specific enrichment of microbial cells, such as the pathogen Listeria monocytogenes, depends on the availability of suitable affinity molecules. We report here a novel concept for the immobilization and separation of bacterial cells by replacing antibodies with cell wall-binding domains (CBDs) of bacteriophage-encoded peptidoglycan hydrolases (endolysins). These polypeptide modules very specifically recognize and bind to ligands on the gram-positive cell wall with high affinity. With paramagnetic beads coated with recombinant Listeria phage endolysin-derived CBD molecules, more than 90% of the viable L. monocytogenes cells could be immobilized and recovered from diluted suspensions within 20 to 40 min. Recovery rates were similar for different species and serovars of Listeria and were not affected by the presence of other microorganisms. The CBD-based magnetic separation (CBD-MS) procedure was evaluated for capture and detection of L. monocytogenes from artificially and naturally contaminated food samples. The CBD separation method was shown to be superior to the established standard procedures; it required less time (48 h versus 96 h) and was the more sensitive method. Furthermore, the generalizability of the CBD-MS approach was demonstrated by using specific phage-encoded CBDs specifically recognizing Bacillus cereus and Clostridium perfringens cells, respectively. Altogether, CBD polypeptides represent novel and innovative tools for the binding and capture of bacterial cells, with many possible applications in microbiology and diagnostics.     

Simple and rapid procedure for the purification of lipoprotein(a)

Lipoprotein(a) [Lp(a)] is a low-density lipoprotein-like particle displaying strong athero-thrombotic properties. Highly purified Lp(a) is increasingly requested for standardization of Lp(a) measurements and for biological studies. Several procedures have been described for Lp(a) separation and purification but none of them appear completely suitable. We present here a procedure for Lp(a) purification based on sequential elutions after lysine-Sepharose affinity chromatography. We were able to identify four distinct subspecies of Lp(a) showing different affinity to ε-amino groups of lysine-Sepharose, simply by modifying molarity and pH of the eluents; the fraction obtained in highly purified state represented the major form and could be eluted with 0.5 M sodium phosphate buffer (pH 4.4). Advantages of this procedure are represented by simplicity, rapidity and final yield.     

Affinity chromatography of endoglucanase of Trichoderma viride by concanavalin A-agarose.

Endoglucanase (C kappa cellulase) and cellobiase are often cross-contaminated in separation procedures by ion-exchange chromatography such as DEAE-cellulose. By using concanavalian A (Con A)-agarose chromatography, C kappa cellulase and cellobiase from Trichoderma virde can be separated. C kappa cellulase showed affinity toward Con A. indicating a glycoprotein containing alpha-D-mannopyransyl and alpha-D-glucopyranosyl end groups or internal 2-O-D-mannopyranosyl residues in sugar moieties. This method provides a way to estimate the quantities of C kappa enzyme produced by T. viride and possibly by other organisms.     

Cell separation by expanded bed adsorption: use of ion exchange chromatography for the separation of <Emphasis Type="Italic">E. coli</Emphasis> and <Emphasis Type="Italic">S. cerevisiae</Emphasis>

In a wide variety of biotechnological and medical applications it is necessary to separate different cell populations from one another. A promising approach to cell separations is demonstrated to be the adoption of chromatographic techniques conducted in expanded beds. The high voidage between the adsorbent beads in an expanded bed allows for the efficient capture of particulate entities such as cells together with washing and subsequent elution without entrapment and loss. In addition, the combination of a gentle hydrodynamic environment, a high surface area and low mixing within the expanded bed make this technique highly favourable. A model system for the separation of two types of microbial cells using STREAMLINE DEAE adsorbent in expanded bed procedures has been investigated. The use of a less selective ligand such as an ion exchange group, which is often characterised by gentle elution procedures, has been investigated as an alternative to affinity ligands whose strong binding characteristics can result in harsh elution procedures with consequent loss of yield and cell viability. Expanded bed experiments have demonstrated selective and high capacity capture of cells from feedstocks containing either a single type of cell or as a mixture of cells of Saccharomyces cerevisiae and Eschericia coli. The capture, washing and elution phases of the separation have been studied with respect to capacity, selectivity and yield of released cells. In these procedures, separation of cell types is achieved by the presence of multiple equilibrium stages within the expanded bed. The results show the potential for carrying out cell separations in expanded beds as an alternative to immunomagnetic cell separations. The combination of these recently developed technologies promises to be a powerful, but economic technique for cell separations involving simple equipment that can readily be scaled up.     

金属螯合亲和层析分离蛋白质的研究

金属螯合亲和层析是近20年发展起来的一项新型分离技术。它以配基简单、吸附量大、分离条件温和、通用性强等特点,逐渐成为分离纯化蛋白质等生物工程产品最有效的技术之一。本文从单组分蛋白质入手,考查了pH值、铵离子浓度、不同铵盐等对蛋白质洗脱的影响,并进行了分析。还对不同的金属螯合柱和不同性质蛋白质的洗脱性能进行了研究,比较了不同金属离子与蛋白质亲和力的区别,为实际体系的分离研究打下了基础。     

Facile preparation of the alpha-Gal-recognizing Griffonia simplicifolia I-B4 isolectin

The B4 isolectin from Griffonia simplicifolia is of great utility as a reagent for the identification of alpha-D-galactopyranosyl end groups. Its separation from isolectins containing A subunits has been greatly improved by a simple, rapid procedure using a column of N-acetylgalactosamine coupled to vinyl sulfone-activated Sepharose 4B to selectively retain the A subunit-containing isolectins. The procedure has the advantages over previous affinity procedures of speed (the isolation of B4 isolectin can be achieved in one day), simplicity, and high degree of resolution of the B4 isolectin.     

Affinity chromatography of tyrosine transfer RNAs.

Ricinus communis agglutinin, a lectin from castor beans has an affinity for β-d-galactose and tyrosine tRNAs of mammalian tissues have galactose in gal-Q base of their anticodons. We have studied interaction between tyrosine tRNAs and this lectin immobilized on solid supports using spacer arms of different lengths. Tyrosine tRNAs are separated from nineteen other tRNAs of bovine liver by affinity chromatography using the lectin immobilized to an agarose matrix. The results indicate that a spacer arm length of 10 Å between the agarose bead and the lectin gives the best separation. Two tyrosine tRNA isoacceptors are separated from each other and from other tRNAs in one step using this affinity column chromatography.     

Use of High-Affinity Cell Wall-Binding Domains of Bacteriophage Endolysins for Immobilization and Separation of Bacterial Cells

Immobilization and magnetic separation for specific enrichment of microbial cells, such as the pathogen Listeria monocytogenes, depends on the availability of suitable affinity molecules. We report here a novel concept for the immobilization and separation of bacterial cells by replacing antibodies with cell wall-binding domains (CBDs) of bacteriophage-encoded peptidoglycan hydrolases (endolysins). These polypeptide modules very specifically recognize and bind to ligands on the gram-positive cell wall with high affinity. With paramagnetic beads coated with recombinant Listeria phage endolysin-derived CBD molecules, more than 90% of the viable L. monocytogenes cells could be immobilized and recovered from diluted suspensions within 20 to 40 min. Recovery rates were similar for different species and serovars of Listeria and were not affected by the presence of other microorganisms. The CBD-based magnetic separation (CBD-MS) procedure was evaluated for capture and detection of L. monocytogenes from artificially and naturally contaminated food samples. The CBD separation method was shown to be superior to the established standard procedures; it required less time (48 h versus 96 h) and was the more sensitive method. Furthermore, the generalizability of the CBD-MS approach was demonstrated by using specific phage-encoded CBDs specifically recognizing Bacillus cereus and Clostridium perfringens cells, respectively. Altogether, CBD polypeptides represent novel and innovative tools for the binding and capture of bacterial cells, with many possible applications in microbiology and diagnostics.     

Affinity chromatography and capillary electrophoresis for analysis of the yeast ribosomal proteins

We present a top down separation platform for yeast ribosomal proteins using affinity chromatography and capillary electrophoresis which is designed to allow deposition of proteins onto a substrate. FLAG tagged ribosomes were affinity purified, and rRNA acid precipitation was performed on the ribosomes followed by capillary electrophoresis to separate the ribosomal proteins. Over 26 peaks were detected with excellent reproducibility (<0.5% RSD migration time). This is the first reported separation of eukaryotic ribosomal proteins using capillary electrophoresis. The two stages in this workflow, affinity chromatography and capillary electrophoresis, share the advantages that they are fast, flexible and have small sample requirements in comparison to more commonly used techniques. This method is a remarkably quick route from cell to separation that has the potential to be coupled to high throughput readout platforms for studies of the ribosomal proteome.     

High-performance liquid affinity chromatography with phenylboronic acid, benzamidine, tri-L-alanine, and concanavalin A immobilized on 3-isothiocyanatopropyltriethoxysilane-activated nonporous monodisperse silicas

Nonporous, microparticulate, monodisperse silicas with particle diameters between 0.7 and 2.1 microns are introduced as stationary phases in high-performance affinity chromatography. The immobilization of m-aminophenylboronic acid, p-aminobenzamidine, tri-L-alanine, and concanavalin A onto these silicas was successfully achieved using 3-isothiocyanatopropyl-triethoxysilane as an activation reagent. Immobilized phenylboronic acid was applied to the isolation of nucleosides, nucleotides, and glycoprotein hormones such as bovine follicotropin and human chorionic gonadotropin, while immobilized benzamidine was employed for the isolation of the serine proteases thrombin and trypsin, immobilized tri-L-alanine for the separation of pig pancreatic elastase and human leukocyte elastase, and immobilized concanavalin A for the isolation of horseradish peroxidase. In all affinity chromatographic systems studied, the nonporous monodisperse silicas showed improved chromatographic performance compared to results obtained with porous silica supports using identical activation and immobilization procedures. Furthermore, frontal analysis was used as a method to evaluate the influence of experimental parameters on biological activity and accessible ligand densities. Only minor changes in bioactivity were found with the nonporous affinity supports, where accessibilities were typically higher than ca. 60%. The immobilization of affinity ligands onto porous supports as used in this and associated papers thus represents a successful general procedure for the preparation of stable matrices with fast kinetics for use in high-performance affinity chromatography.     

5-Methylcytosine as a marker for the monitoring of DNA methylation

The extent of the DNA methylation of genomic DNA as well as the methylation pattern of many gene-regulatory areas are important aspects with regard to the state of genetic information, especially their expression. There is growing evidence that aberrant methylation is associated with many serious pathological consequences. As genetic research advances, many different approaches have been employed to determine the overall level of DNA methylation in a genome or to reveal the methylation state of particular nucleotide residues, starting from semiquantitative methods up to new and powerful techniques. In this paper, the currently employed techniques are reviewed both from the point of view of their relevance in genomic research and of their analytical application. The methods discussed include approaches based on chromatographic separation (thin-layer chromatography, high-performance liquid chromatography, affinity chromatography), separation in an electric field (capillary electrophoresis, gel electrophoresis in combination with methylation-sensitive restriction enzymes and/or specific sequencing protocols), and some other methodological procedures (mass spectrometry, methyl accepting capacity assay and immunoassays).     

Affinity membrane chromatography: relationship of dye-ligand type to surface polarity and their effect on lysozyme separation and purification

Two different dye-ligands, i.e. Procion Brown MX-5BR (RB-10) and Procion Green H-4G (RG-5) were immobilised onto poly(2-hydroxyethylmethacrylate) (pHEMA) membranes. The polarities of the affinity membranes were determined by contact angle measurements. Separation and purification of lysozyme from solution and egg white were investigated. The adsorption data was analysed using two adsorption kinetic models the first order and the second order to determine the best-fit equation for the separation of lysozyme using affinity membranes. The second-order equation for the adsorption of lysozyme on the RB-10 and RG-5 immobilised membranes systems is the most appropriate equation to predict the adsorption capacity for the affinity membranes. The reversible lysozyme adsorption on the RB-10 and RG-5 did not follow the Langmuir model, but obeyed the Temkin and Freundlich isotherm model. Separation and purification were monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purities of the eluted lysozyme, as determined by HPLC, were 76 and 92% with recovery 63 and 77% for RB-10 and RG-5 membranes, respectively. For the separation and purification of lysozyme the RG-5 immobilised membrane provided the best results. The affinity membranes are stable when subjected to sanitization with sodium hydroxide after repeated adsorption-elution cycles.