Antigen-antibody dissociation in Alzheimer disease: a novel approach to diagnosis

With the ever-increasing population of aged individuals at risk of developing Alzheimer's disease (AD), there is an urgent need for a sensitive, specific, non-invasive, and diagnostic standard. The majority of efforts have focused on auto-antibodies against amyloid-β (Aβ) protein, both as a potential treatment, and a reliable biomarker of AD pathology. Naturally occurring antibodies against Aβ are found in the CSF and plasma of patients with AD as well as healthy control subjects. To date, differences between diseased and control subjects have been highly variable. However, some of the antibody will be in preformed antigen–antibody complexes and the extent and nature of such complexes may provide a potential explanation for the variable results reported in human studies. Thus, measuring total amounts of antigen or antibody following unmasking is critical. Here, using a technique for dissociating antibody–antigen complexes, we found significant differences in serum antibodies to Aβ between AD and aged-matched control subjects. While the current study demonstrates the relevance of measuring total antibody, bound and unbound, against Aβ in AD, this technique may be applicable to diseases such as acquired immune deficiency syndrome and hepatitis B where determination of antigen and antibody levels are important for disease diagnosis and assessing disease progression.     

Double-ternary complex affinity chromatography: preparation of alcohol dehydrogenases.

A general affinity chromatographic method for alcohol dehydrogenase purification has been developed by employing immobilized 4-substituted pyrazole derivatives that isolate the enzyme through formation of a specific ternary complex. Sepharose 4B is activated with 300 mg of cyanogen bromide/ml of packed gel and coupled to 4-[3-(N-6-aminocaproyl)aminopropyl]pyrazole. From crude liver extracts in 50 mM phosphate-0.37 mM nicotinamide adenine dinucleotide, pH 7.5, alcohol dehydrogenase is optimally bound at a capacity of 4-5 mg of enzyme/ml of gel. Addition of ethanol, propanol, or butanol, 500 mM, results in the formation of a second ternary complex, which allows the elution of bound enzyme in high yield and purity. This double-ternary complex affinity chromatography has been applied successfully to human, horse, rat, and rabbit liver extracts to isolate the respective homogeneous alcohol dehydrogenases.     

Preparation of a stable folate-sepharose complex for affinity chromatography.

A stable folic acid affinity gel has been developed for the purification of nanograms of protein that bind folic acid or its derivatives. The affinity gel was prepared by first coupling folic acid covalently to bovine serum albumin, followed by covalent coupling of the albumin to p-benzoquinone-activated Sepharose. After the albumin-folic acid complex was formed, it was treated with charcoal to remove ionically bound folate which would otherwise elute from the gel and decrease the recovery of the binding protein. The p-benzoquinone activation resulted in a more stable binding of the albumin to the Sepharose.     

Antigen-antibody complex binding and cell interaction in stimulating normal rabbit lymphocytes

Complexes of antigen with specific antibody have been shown to enhance or suppress the specific antibody response in vivo. In vitro, antigen-antibody (Ag-Ab) complexes prepared in a slight antigen excess with rabbit antibody induced proliferation of unprimed rabbit lymphocytes. The Ag-Ab stimulated cells from a number of different normal lymphoid organs, including peripheral blood, bone marrow, spleen, and lymph node, but not thymus. Cells exposed to Ag-Ab for 1 hr and washed, bound Ag-Ab through Fc and complement receptors (CR), but were not induced to proliferate unless additional Ag-Ab was added. Specific antigen, which was otherwise unstimulatory, interacted with Ag-Ab-coated cells to activate them, probably through the cross-linking of membrane-bound ligands. Proliferation stimulated by Ag-Ab involved the interaction of three bone marrow cell subpopulations; a macrophage-enriched, a B-cell-enriched, and an mIgM- cell-enriched population. The separated subpopulations were poorly responsive to Ag-Ab stimulation, even though Ag-Ab bound to cells in each of the populations. Low levels of responsiveness to Ag-Ab also resulted when any two of the three subpopulations were combined. Only when all three subpopulations were mixed, was stimulation equivalent to the levels of stimulation reached by unseparated cells.     

Lectin affinity chromatography

A protocol is described for the preparation of lectin affinity chromatography columns using purified lectins and preactivated matrices. A general method is given for the purification of glycoproteins on immobilized Con A. Methods for immobilizing Con A on CDI agarose, Affi-Gel 15, and carbonyl-diimidazole-activated agarose are described.     

New developments in affinity chromatography

The design, synthesis and chromatographic operation of a new range of stable and selective immobilized dye affinity adsorbents for potential application in the purification of pharmaceutical proteins is described. Computer aided molecular design has been exploited to design novel dye ligands which show a predictable selectivity for the target protein and which, when coupled to stable perfluoropolymer supports, yield high capacity, low leakage adsorbents for affinity chromatography. It is anticipated that these new materials will withstand the rigorous conditions required for sanitization and cleaning in situ of industrial scale processes.     

Fractionation of the naringinase complex from Aspergillus terreus by dye affinity chromatography

Affinity chromatography with immobilised triazine dyes was used to separate the main enzymes present in the naringinase complex produced by Aspergillus terreus CECT 2663. One alpha-L-rhamnosidase and two beta-D-glucosidases (beta G1 and beta G2) were separated by a simple two-step procedure involving chromatography with Red HE-3B immobilised on Sepharose 4B first at pH 5.5 and then at pH 4.7. Maximum activity of the beta-D-glucosidases was from pH 4 to 6 and at 65 degrees C. Both glucosidases were active on p -nitrophenol glucoside and prunin with respective Km values of 1.9 mm and 1.6 mm for beta G1 and 2.1 mm and 0.25 mm for beta G2. Only beta G1 hydrolysed cellobiose (Km = 5.7 mm).     

Quantification of monoclonal antibodies in complex mixtures by protein G high-performance liquid affinity chromatography

High-performance liquid affinity chromatography (HPLAC) utilizing Protein G as a ligand has been evaluated for rapid quantification of monoclonal antibodies (MAbs) in various solutions. The results obtained by HPLAC agreed to within 10% of a standard enzyme-linked immunospecific assay (ELISA). A standard curve was prepared by injection of known amounts of a purified murine IgG1 with the elution peak area analyzed by computer integration software. Accuracy of quantification was independent of the injection volume, solution compositions, or mouse IgG subclass. A method is described for using Protein G HPLAC to determine murine IgG levels in various complex mixtures within 15 min, compared to the ELISA which required 5 h.     

Semicontinuous affinity chromatography separations

The semicontinuous operation of affinity chromatography columns charged and eluted alternately is studied mathematically. A strongly nonlinear adsorption equilibrium relation is utilized to examine the periodic mode of adsorption and desorption. The governing differential equations are solved by the method of characteristics. The time interval between desorption and adsorption periods is estimated, and implications for separations are discussed to show the advantages of semicontinuous cyclic operation.     

Quantitative affinity chromatography.

The objective of this review is to summarize developments in the use of quantitative affinity chromatography to determine equilibrium constants for solute interactions of biological interest. Affinity chromatography is an extremely versatile method for characterizing interactions between dissimilar reactants because the biospecificity incorporated into the design of the affinity matrix ensures applicability of the method regardless of the relative sizes of the two reacting solutes. Adoption of different experimental strategies, such as column chromatography, simple partition equilibrium experiments, solid-phase immunoassay, and biosensor technology, has led to a situation whereby affinity chromatography affords a means of characterizing interactions governed by an extremely broad range of binding affinities--relatively weak interactions (binding constants below 10(3) M(-1)) through to interactions with binding constants in excess of 10(9) M(-1). In addition to its important role in solute separation and purification, affinity chromatography thus also possesses considerable potential for investigating the functional roles of the reactants thereby purified.     

Histidine ligand affinity chromatography

The sorbents with immobilized histidine as a pseudo affinity ligand with a wide specificity is described. The possibilities and relevant chemistries to use both particulate and flat or hollow fiber membranes as support matrices are discussed. The usefulness of such adsorbents for the purification of a wide variety of proteins, with relevant interaction mechanism are described. Practical protocols of sample quality, capacity and scaled up and scaled down operations are discussed. Possibilities of pyrogen removal from high value blood proteins and their simultaneous recovery in the pure form, using histidine immobilized sorbents are described.     

Enrichment and analysis of nonenzymatically glycated peptides: boronate affinity chromatography coupled with electron-transfer dissociation mass spectrometry

Nonenzymatic glycation of peptides and proteins by d-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low-abundance post-translational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron-transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. ETD fragmentation mode permitted identification of a significantly higher number of glycated peptides (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing enrichment on first the protein and then the peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS and using ETD as the fragmentation mode is an efficient approach for analysis of glycated proteins and may have broad application in studies of diabetes mellitus.     

Thermostable carbohydrate-binding modules in affinity chromatography

Affinity chromatography is routinely used mostly on a preparative scale to isolate different biomolecules such as proteins and carbohydrates. To this end a variety of proteins is in common use as ligands. To extend the arsenal of binders intended for separation of carbohydrates, we have explored the use of carbohydrate-binding modules (CBM) in affinity chromatography. The thermostable protein CBM4-2 and two variants (X-6 and A-6) thereof, selected from a newly constructed combinatorial library, were chosen for this study. The CBM4-2 predominantly binds to xylans but also crossreacts with glucose-based oligomers. The two CBM-variants X-6 and A-6 had been selected for binding to xylan and Avicel (a mixture of amorphous and microcrystalline cellulose), respectively. To assess the ability of these proteins to separate carbohydrates, they were immobilized to macroporous microparticulate silica and analyses were conducted at temperatures ranging from 25 to 65 degrees C. With the given set of CBM-variants, we were able to separate cello- and xylo-oligomers under isocratic conditions. The affinities of the CBMs for their targets were weak (in the mM-microM range) and by adjusting the column temperature we could optimize peak resolution and chromatographic retention times. The access to thermostable CBM-variants with diverse affinities and selectivities holds promise to be an efficient tool in the field of affinity chromatography for the separation of carbohydrates.     

Applications of affinity chromatography in proteomics

Affinity chromatography is a powerful protein separation method that is based on the specific interaction between immobilized ligands and target proteins. Peptides can also be separated effectively by affinity chromatography through the use of peptide-specific ligands. Both two-dimensional electrophoresis (2-DE)- and non-2-DE-based proteomic approaches benefit from the application of affinity chromatography. Before protein separation by 2-DE, affinity separation is used primarily for preconcentration and pretreatment of samples. Those applications entail the removal of one protein or a class of proteins that might interfere with 2-DE resolution, the concentration of low-abundance proteins to enable them to be visualized in the gel, and the classification of total protein into two or more groups for further separation by gel electrophoresis. Non-2-DE-based approaches have extensively employed affinity chromatography to reduce the complexity of protein and peptide mixtures. Prior to mass spectrometry (MS), preconcentration and capture of specific proteins or peptides to enhance sensitivity can be accomplished by using affinity adsorption. Affinity purification of protein complexes followed by identification of proteins by MS serves as a powerful tool for generating a map of protein-protein interactions and cellular locations of complexes. Affinity chromatography of peptide mixtures, coupled with mass spectrometry, provides a tool for the study of protein posttranslational modification (PTM) sites and quantitative proteomics. Quantitation of proteomes is possible via the use of isotope-coded affinity tags and isolation of proteolytic peptides by affinity chromatography. An emerging area of proteomics technology development is miniaturization. Affinity chromatography is becoming more widely used for exploring PTM and protein-protein interactions, especially with a view toward developing new general tag systems and strategies of chemical derivatization on peptides for affinity selection. More applications of affinity-based purification can be expected, including increasing the resolution in 2-DE, improving the sensitivity of MS quantification, and incorporating purification as part of multidimensional liquid chromatography experiments.     

The separation of subunits of phenylalanyl-tRNA-synthetase from Escherichia coli MRE-600 by means of affinity chromatography in dissociation conditions

The method of affinity chromatography on sepharose with immobilized tRNA in the presence of urea was developed for separating the subunits of phenylalanyl-tRNA synthetase from E. coli MRE-600 (subunit structure alpha 2 beta 2). Specific binding of large beta-subunits of the enzyme on immobilized tRNA testifies the localization of the tRNA-binding center on the beta-subunit of phenylalanyl-tRNA synthetase. Separately alpha- and beta-subunits of the enzyme exhibit no catalytic activity. Incubation of the mixture of alpha- and beta-subunits in conditions leading to reassociation of the oligomeric structure results in restoration of catalytic activity of the enzyme. In the presence of urea resin with immobilised analogs of ATP binds alpha- and beta-subunits of the enzyme. This testifies the presence of nucleotide-binding sites on both subunits. The possibility of using the affinity chromatography method to separate non-identical subunits of different enzymes is discussed.