Evaluation of activation methods with cellulose beads for immunosorbent purification of immunoglobulins

Attempts were made to evaluate the chemical properties of cross-linked cellulose beads in order to utilize them as a support material for the large scale purification of specific immunoglobulins via immunosorbent chromatography with goat anti-human IgG serving as the model affinity ligand. Since these cellulose beads have sufficient mechanical strength to sustain a high flow rate of viscous fluids, they are ideal for rapid purification of large fluid volumes. The beads were activated with cyanogen bromide, tosyl chloride, cyanuric chloride or oxidation reagents such as chromium trioxide, sodium periodate and dimethylsulfoxide-carbodiimide before the antibodies were immobilized under mild conditions. The inert hydroxyl groups were thus converted into more active cyanate ester, tosylate, reactive acyl-like chlorines, and carbonyl groups which readily react with amino groups of antibodies. Antibodies were immobilized on the activated cellulose beads under mild conditions with an average yield of 42.3%. Every immobilization method had disadvantages. The binding activity of the immobilized antibody depended on its concentration. Very high binding efficiency was achieved when the concentration was less than 0.2 mg/ml; however, the efficiency was only about 5% when the concentration was greater than 2 mg/ml. The binding activity of immobilized antibodies was affected by the steric factors imposed by the support material but not affected by the immobilization methods. Although some non-specific interaction between plasma components and the cellulose bead immunosorbent occurred, specific immunoglobulin could be purified from plasma in a single step.     

Use of thiol sorbents for obtaining human recombinant proinsulin

A convenient route of obtaining recombinant human proinsulin from the hybrid protein produced by the bacteria was developed. Chimeric protein was prepared by ultra- or gel-filtration, immobilized on thiol-support at cysteine residues and cleaved by cyanogen bromide to liberate purified proinsulin. Conditions of treatment hybrid protein with cyanogen bromide at methionine residues without affecting disulfide bonds between proinsulin and support are described. Proinsulin with correct disulfide bonds, directly obtained from polymer--attached polypeptide, followed was converted into insulin.     

Quantification and specific detection of collagenous proteins using an enzyme-linked immunosorbent assay and an immunoblotting for cyanogen bromide peptides

A method for the detection of collagenous proteins within cyanogen bromide digests of tissues has been devised. The peptides produced by digestion with cyanogen bromide were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose filter. They were stained on the filter by incubation first with antibodies to collagen and then with a second antibody covalently linked to horseradish peroxidase, 4-chloro-1-naphthol was added, and the bound enzyme was assayed. This procedure is useful for the identification and characterization of collagens of types I, III, IV, and V in tissues. In addition, we have developed a sensitive and specific competitive enzyme-linked immunosorbent assay (ELISA) which is convenient for quantifying collagens (types I, III, and IV) in tissues. In this kind of assay, soluble cyanogen bromide peptides compete with cyanogen bromide peptides adsorbed onto a solid-phase support for rabbit anti-collagen antibodies. We determined the amount of bound antibody by using goat anti-rabbit immunoglobulin G covalently conjugated to horseradish peroxidase and then provided a substrate for the enzymatic reaction. The sensitivity range of the ELISA is 0.09 micrograms/ml in the region of 90 to 10% binding.     

Cellulase immobilized on a soluble polymer

Summary Aspergillus niger cellulase was imobilized on cyanogen bromide activated dextran of varying molecular weights. The effect of different concentrations of cyanogen bromide used for the activation process was also studied. About 50% conjugation and 70% retention activity was achieved in the immobilized cellulase. The pH activity of immobilized enzyme was unchanged, but exhibited more stable activity at acidic pH than the free enzyme. Higher resistance to heat inactivation was also observed.     

Quantitation of types I and III collagens in human tissue samples and cell culture by cyanogen bromide peptide analysis

A procedure for the quantitation of types I and III collagens by cyanogen bromide peptide analysis was developed with the aim of eliminating certain problems associated with this method. Ion-exchange chromatography reduced high background levels on gel scans used to quantitate the peptides; reduction with beta-mercaptoethanol substantially increased the efficiency of the cyanogen bromide cleavage; use of a concave gradient in acrylamide from 8 to 20% improved the resolution of cyanogen bromide peptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; and a normalization procedure eliminated variations due to differences in the amount of material loaded on the gel system. This method of quantitation was applied to human aorta samples and to collagen secreted by human skin fibroblasts. Metachromasy of type I and type III collagen cyanogen bromide peptides stained with Coomassie blue R-250 was established and this was used as an index of the purity of the cyanogen bromide peptide preparations. Type I and III collagens were prepared from human placental tissue, and these purified collagens were used to construct calibration curves to determine the relationship between the quantity of diagnostic cyanogen bromide peptides present and the composition of the sample in terms of types I and III collagens.     

Activity of synchronized cells of a steady-state biofilm recirculated reactor during xenobiotic biodegradation.

The maintenance of a steady-state biofilm in a continuous-flow fixed-bed reactor, as a consequence of the reproduction-detachment of cells (an interfacial cell physiology phenomenon of steady-state biofilm) during the biodegradation of 2,4,6-trichlorophenol by Pseudomonas cells, was determined. After cell adhesion on an open-pore glass support, the biofilm was formed in a packed-bed recirculated reactor. After the steady-state biofilm was reached, the mechanisms of the interfacial cell detachment (at the biofilm-liquid interface) were determined. It was established that (i) the hydrophobicity of immobilized sessile cells (parent cells) increased (from 50 to 80%) as the dilution rate increased, while the hydrophobicity of detached suspended cells (daughter cells) remained constant (about 45%); and (ii) the immediately detached suspended cells showed a synchronized growth in about three generations. These results indicate that (i) the immobilized sessile and suspended detached cells grew synchronically at the end and at the beginning of the cell cycle, respectively; and (ii) the hydrophobicity difference of immobilized sessile and suspended detached cells permitted the cells detachment. Therefore, it is probable that independent of shear stress (due to recirculated flow), the synchronized growth and hydrophobicity of cells (which vary during the cell cycle) are the main factors permitting the maintenance of a steady-state xenobiotic-degrading biofilm reactor (in which the overall accumulation of biofilm is determined by the average growth rate of the biofilm cells minus the rate of detachment of cells from the biofilm).     

Cooperative elution of oligoadenylic acid in polyU immobilized chromatography.

Characterization of polyU immobilized chromatography was performed in order to use this technique as an analytical device. A method of analysis of the elution profile related to thermodynamic parameters was also developed. Sites of attachment of polyU to agarose gel activated by cyanogen bromide were studied using uridine diphosphate and adenine. Independent equilibrium dialysis of ApA for different states of polyU, in solution and immobilized in gel, were carried out. These results show that the immobilized polyU is attached to agarose only at the 5′-terminal phosphate groups and behaves as it does in solution. Column chromatography of ApA with the immobilized polyU was performed at several temperatures and concentrations. To analyze the elution profile, the theory of cooperative binding of oligonucleotides to polynucleotides was extended to the frame-work of plate theory. A computer simulation for the elution profiles was performed using thermodynamic parameters obtained by equilibrium dialysis. This simulation duplicated the experimental results. This fact shows that the peculiar leading form of elution profile is due to the cooperative binding. The thermodynamic parameters of the polyU–ApA system were obtained from the “peak trajectory.”     

Evaluation of CNBr, FMP and hydrazide resins for immunoaffinity purification of factor IX.

The American Red Cross has developed an immunoaffinity chromatography method to purify human coagulation Factor IX to high levels of purity for therapeutic treatment of hemophilia B. The resin currently used in this process is Sepharose CL2B, a cross-linked 2% agarose, which is activated with cyanogen bromide to immobilize an anti-Factor IX monoclonal antibody. This study evaluated two alternative resins and coupling chemistries, a synthetic polymer bead activated by 2-fluoro-1-methyl-pyridinium toluene 4-sulfonate (FMP) and a cross-linked 2% agarose bead with free hydrazide groups for site-specific coupling. The cyanogen bromide and FMP chemistries immobilize the monoclonal antibody in a random orientation. In hydrazide coupling, the monoclonal antibody is immobilized by the non-antigen-binding part of the molecule which, theoretically, should increase the amount of immobilized monoclonal antibody able to bind antigen. To examine this, the capacity of the resins to bind Factor IX and the purity and recovery of Factor IX eluted from the resins were measured. The FMP-activated resin exhibited the lowest capacity, binding only 2% of the Factor IX feed. Sepharose CL2B bound 87% of the loaded protein, while the hydrazide resin bound 43%. These results suggest that (a) hydrazide activation may be insufficient to orient monoclonal antibody and (b) other factors such as steric hindrances and diffusional resistances during immobilization may be important. Neither of the other resins tested demonstrated improved performance compared with cyanogen bromide-activated Sepharose CL2B for the immunoaffinity purification of Factor IX.     

Quantification of Dehalospirillum multivorans in Mixed-Culture Biofilms with an Enzyme-Linked Immunosorbent Assay

A fast, highly selective and sensitive method to quantify specific biomasses in mixed-culture biofilms is described. It consists of detachment of a biofilm from its support material, resolution of the detached biofilm flocs in order to separate the enclosed cells and antigens, and quantification of specific biomass by an enzyme-linked immunosorbent assay.     

Structural organization of glutamate dehydrogenase hexamer. 1. Immobilization on sepharose as a model for analysis of structural-functional characteristics of the enzyme

Bovine liver glutamate dehydrogenase (L-glutamate-NAD(P)-oxidoreductase, EC 1.4.1.3) and its radioactive phosphopyridoxyl derivative were covalently immobilized on Sepharose CL-4B with different degrees of cyanogen bromide activation. The catalytical and regulatory properties of the immobilized samples of the enzymes were studied. It was shown that the enzymes were immobilized through a single subunit of hexamer when sepharose was activated by small amounts of cyanogen bromide (less than 5 mg per 1 ml of gel). In this case, the immobilization did not alter the catalytical and regulatory properties of glutamate dehydrogenase. The immobilized radioactive phosphopyridoxyl derivative of glutamate dehydrogenase completely imitated the immobilized native enzyme and can be used as a convenient model for structural and functional investigation of catalytically active hexamer of glutamate dehydrogenase.     

Immunoaffinity chromatography

Immunoaffinity chromatography is a process in which the binding affinity of an antigen to a parent antibody is utilized as a basis of separation. Owing to the customized avidity and specificity, monoclonal antibodies (Mabs) have become indispensable for both protein characterization and purification. The immunosorbent performance is dependent on the support matrix upon which the antibody is immobilized and on the activation chemistry used couple the antibody to the matrix. This report details, protocols to immobilize Mabs on commercially available supports, and a method to compute immunosorbent efficiency.     

Preparation of inert magnetic nano-particles for the directed immobilization of antibodies

Various activated supports (cyanogen bromide, glutaraldehyde, epoxy-chelates, primary amino) were evaluated for the immobilization of IgG anti-horseradish peroxidase. Cyanogen bromide and glutaraldehyde supports greatly reduced the recognition capacity of the antigen, probably due to the incorrect orientation of the antibody on the support. Hetero-functional epoxy-chelate and immobilization by the sugar chain on primary amino groups had little effect on high recognition of the antigen (near to the theoretically expected value). However, the immobilization by the sugar chain resulted in a higher adsorption rate of horseradish peroxidase, possibly due to a favourable orientation on a flexible spacer arm). Antibodies immobilized on aminated surfaces showed two major drawbacks. Firstly, the biological activity of the immobilized antibody sharply decreased over several days when stored at low ionic strength, although this effect could be partially reversed by incubation at high ionic strength. Secondly, a high level of non-specific proteins adsorption on the support surface was observed. Both problems could be successfully resolved by controlling the coating of the support with aldehyde-aspartic-dextran. We propose that the loss of biological activity was related to the ionic adsorption of the immobilized antibody on the support surface, leading to a blocking of the recognition areas. This optimized protocol was applied to the immobilization of IgG anti-horseradish peroxidase from rabbit on magnetic nano-particles. A 10 microg preparation of nano-particles was able to capture more than 75% of the 0.1 microgram of recombinant horseradish peroxidase present in 10 L of crude protein extract (1g/L) from Escherichia coli.     

Stability of nicotinamide adenine dinucleotide immobilized to cyanogen bromide activated agarose

The stability of NAD(H) immobilized to a crosslinked agarose support (Sepharose(R)-4B) was examined in buffer solutions at a pH of 7.0 and 8.5. Specifically, this study investigated particle attrition and ligand leakage rates from a cyanogen bromide activated agarose support. Particle attrition did not occur under the experimental conditions. Ligand leakage rates were found to be first order in immobilized ligand concentration with two labile populations of ligand. The two-population model is consistent with the cyanogen bromide coupling chemistry, which results in both an isourea and imidocarbonate ligand linkage. The rate of ligand leakage was found to occur over a time scale of days, with first order rate constants ranging from 0.007 to 0.15 d(-1), depending on solution pH. (c) 1997 John Wiley & Sons, Inc.     

Micro-quantitative determination of ciprostene in plasma by gas chromatography—mass spectrometry coupled with an antibody extraction

A simple and highly sensitive method has been developed for the determination in plasma of ciprostene, 9β-methyl-6α-carbaprostaglandin I₂, using gas chromatography—mass spectrometry following solid-phase extraction on an immobilized antibody column. The anti-ciprostene antibody obtained from rabbit serum was coupled to an agarose support matrix, and the immobilized antibody thus prepared was used as an extraction phase for sample clean-up. The extracted drug was treated with pentafluorobenzyl bromide followed by bis(trimethylsilyl)trifluoroacetamide. The derivative was quantitatively analysed by negative-ion chemical ionization gas chromatography—mass spectrometry. The lower limit of quantitation was 50 pg/ml when 1 ml of human plasma was used. The plasma concentration of ciprostene in a dog treated with ciprostene at 2.5 μg/kg was determined successfully by this method.     

Coimmobilization of Lactate Dehydrogenase and Low-Molecular-Weight Thiols on Sepharose

Lactate dehydrogenase (EC 1.1.1.27) and dithiothreitol (DTT) were coimmobilized on Sepharose activated with cyanogen bromide. It was demonstrated that the addition of 10 mM DTT (but not 2-mercaptoethanol) during immobilization increased the enzyme specific activity 1.5–5-fold depending on the initial extent of Sepharose activation by cyanogen bromide. The total activity increased two- to threefold. The lactate dehydrogenase preparations were rich in matrix-immobilized sulfhydryl groups (1.8–13.0 nmol per ml gel). The presence of DTT increased the stability of immobilized lactate dehydrogenase.     

An immunochemical aid to sequence determination of proteins.

A specific radioimmunoassay for peptides has been developed using ¹²⁵I-labeled peptides and a double-antibody precipitation. Cross-reacting peptides are measured by inhibition of the binding of the labeled cyanogen bromide peptide to its antibody. The assay, which allows detection of picomole quantities, was used to monitor the purification of two overlapping tryptic peptides from a complex mixture of peptides. These were shown to contain a portion of the sequence of the radio-labeled cyanogen bromide peptide and a portion of the sequence of a cyanogen bromide peptide which follows in the polypeptide chain. The need to analyze many fractions in a digest in order to locate a desired peptide is thus avoided. The general suitability of this method for the purification of specific peptides from digestion mixtures of other large proteins is discussed.     

A micromethod for the quantitation of sialoglycoconjugate immobilization on insoluble supports: Its use in the preparation of supports with varying ligand concentration

A micromethod is described for the evaluation of immobilization of sialoglycoconjugates on insoluble supports. Ligands were radioactively labeled in their sialic acid moieties after mild periodate oxidation and borotritide reduction, or in the glycosylamino residue after borotritide reduction of the Schiff's base formed between reducing sialooligosaccharides and β-(p-aminophenyl)-ethylamine. Sephadex G-25, Sepharose 4B, and Cellulose MN 2100 were activated by CNBr or periodate oxidation. The hydrazido derivatives of these supports were prepared using both activation methods, and activated to azido-supports using nitrous acid. Controlled Pore Glass-glycophase activated by periodate oxidation was also studied. The investigation of conditions for the binding of the radioactive ligands was carried out in the microassay using 0.5-ml aliquots of the activated supports. The stability of the bound ligands in dependence on various parameters was investigated using the immobilized radioactive ligands. Multivalent linkages formed between ligand and support gave increased stability to release compared to monovalent attachment, for cyanogen bromide activation. The use of periodate activation yielded ligands with much greater stability even for monovalent linkages.The microassay was used successfully to predict conditions for the batchwise preparation of immobilized ligands.     

Immobilization of luciferase from the firefly Luciola mingrelica — Catalytic properties and stability of the immobilized enzyme

Firefly (Luciola mingrelica) luciferase [Photinus luciferin 4-monooxygenase (ATP-hydrolysing); Photinus luciferin: oxygen 4-oxidoreductase (decarboxylating, ATP-hydrolysing), EC 1.13.12.7] has been immobilized on albumin and polyacrylamide gel, on AH-, CH- and CNBr-Sepharose 4B as well as on Ultragel, Ultradex and cellophane film activated by cyanogen bromide. Only immobilization on cyanogen bromide-activated polysaccharide carriers resulted in highly active immobilized luciferase. Kinetic properties of immobilized luciferase hardly differed from those of the soluble enzyme. The inactivation rate constants of soluble and immobilized luciferase were measured at pH 5.5–9.0 and 25°C as well as at pH 7.8 and 20–40°C. The ΔH^≠ and ΔS^≠ values for inactivation of soluble and immobilized luciferases were obtained. A 1000-fold stabilization effect was noted for the luciferase immobilized on CNBr-Sepharose 4B at pH 7.5 and 25°C. A stabilization mechanism for the immobilized luciferase is discussed.     

Isolation of a tubulin-like protein from Phaseolus

Colchicine binds to a protein fraction isolated from Phaseolus aureus. A protein with characteristics similar to calf brain tubulin, in terms of its MW, elution properties from DEAE cellulose, precipitation by Ca²⁺ ions and Chlorpromazine was detected in whole cell supernatants. This protein consisted of two monomeric subunits with MWs of 56000 and 53000. This protein, tentatively identified as tubulin, was compared by cyanogen bromide peptide mapping with calf brain tubulin.     

Protection of cellulose synthesis in detached cotton fibers by polyethylene glycol

Detachment of the cotton fiber cell from the ovule results in loss of over 90% of the in vivo capacity for synthesis of [¹⁴C]cellulose from [¹⁴C]glucose. However, over 50% of the capacity for cellulose synthesis in the detached fiber population is protected when polyethylene glycol 4000 is present during detachment and incubation. Radioautography shows that approximately full capacity is restored in about half the fibers, whereas the other half of the population are incapable of cellulose synthesis from supplied glucose. The rate of cellulose synthesis in such fibers has a pH optimum of 6 and the optimum polyethylene glycol 4000 concentration is 0.06 molal (−9 bars). Cellulose synthesis in such detached fibers is synergistically stimulated by Ca²⁺ and Mg²⁺ and inhibited by K⁺.