sepharose and 2-2-2-trifluoroethanesulfonyl-chloride

Immobilization of proteins, nucleic acids, and other "bioligands" requires selection of the proper bioligand for the specific application. This decision influences all others: the matrix is chosen and activated by the method that is most appropriate for this specific application and the ligand is coupled under conditions dictated by the activation method and the nature of the ligand (e.g., is it a labile protein or a sturdy enzyme cofactor?). There are many matrices and activation and coupling methods, and new ones are constantly being developed. This unit provides three protocols for activating and coupling proteins and other nucleophilic ligands to beaded agarose gel: cyanogen bromide, p-nitrophenyl chloroformate, and tresyl chloride.

Topics: Chromatography, Affinity; Cyanogen Bromide; Ligands; Nucleic Acids; Protein Binding; Proteins; Sepharose; Sulfones

During the use of chromatographic supports for the purification of proteins or the selective removal of substances by immunoaffinity, leakage of the antibodies immobilized on the matrix is systematically observed. When the cleansing of blood plasma by extracorporeal circulation is concerned, it is of prime importance that the immunoadsorbents exhibit an extensive chemical stability over the whole range of experimental conditions. To study and minimize this leakage, a matrix, Sepharose CL-4B, was activated by various chemical reagents and coupled to goat anti-apolipoprotein B polyclonal antibodies. Immunoadsorbents thus prepared were compared with those obtained earlier by cyanogen bromide activation. It turns out that divinyl sulphone- and tresyl chloride-activated supports lead to similar results in terms of coupling yield and adsorption capacity, but to a significant reduction in released antibodies.

Topics: Animals; Antibodies; Antibody Specificity; Apolipoproteins B; Butylene Glycols; Chromatography, Affinity; Cyanogen Bromide; Goats; Humans; Hydrogen-Ion Concentration; Immunosorbent Techniques; Immunosorbents; Sepharose; Sulfones

Study was made of controlled fabrication and operation of immunoadsorbents exploiting beaded composites of agarose or kieselguhr-agarose. Materials were activated by cyanogen bromide and tresyl chloride, derivatised with human IgG antigens, and utilised in direct, one-step purifications of anti-huIgG monoclonal antibodies produced in serum-based cultures of murine hybridomas. The influence of solid phase composition, degrees of activation, concentration of immobilised antigen, capping chemistries, and mode of product desorption was studied in respect of purification performance. Maximum concentrations of immobilised huIgG could be achieved following activation of 50% available hydroxyl groups in both materials. Specific adsorption and desorption of monoclonal antibodies, expressed per mole of immobilised ligand, declined with increasing ligand concentrations. Control of activation and derivatisation of agarose solid phases enhanced the overall specification and performance of both homogeneous and composite fabricates. The large particle size of composites (150-1000 microns) restricted efficient performance in fixed bed contactors operated under non-equilibrium conditions. However, their physical nature recommended adsorptive operations with particulate feedstocks in fixed or fluidised beds, batch suspension contactors, or fast flow regimes adopted for cleaning and equilibration operations.

Topics: Animals; Antibodies, Monoclonal; Cyanogen Bromide; Diatomaceous Earth; Electrophoresis, Polyacrylamide Gel; Evaluation Studies as Topic; Humans; Hybridomas; Immunosorbent Techniques; Ligands; Sepharose; Sulfones

Topics: Alcohol Dehydrogenase; Animals; Chymotrypsin; Enzymes, Immobilized; Horses; Indicators and Reagents; Liver; Methods; Peptide Hydrolases; Sepharose; Silicon Dioxide; Sulfones

A stable T-2 hydrazide gel is prepared by activating T-2 toxin with tresyl chloride followed by coupling to agarose-adipic acid hydrazide. Utilized as an affinity chromatography column, this T-2 hydrazide gel purifies a monoclonal antibody for T-2 in high yield directly from ascites fluid. Specific antibody trapped on the column is eluted either with excess T-2 or at pH 11.6. Much less successful are two other T-2 affinity columns that were prepared and evaluated: T-2 bovine serum albumin Affi-Gel 15 and T-2 hexylamine Sepharose.

Topics: Antibodies; Biotransformation; Chromatography, Affinity; Gels; Hydrazines; Ligands; Protein Binding; Sepharose; Sesquiterpenes; Sulfones; T-2 Toxin

Yeast alcohol dehydrogenase was successfully immobilized on tresyl-chloride-activated agarose; the optimized conditions allowed an enzyme activity recovery of over 90%. Comparison of free and immobilized enzyme properties showed an unchanged intrinsic activation energy of the reaction and a shift of optimum activity to a higher pH medium after immobilization. Comparison of the kinetic parameters for both substrates of the reaction showed that the Michaelis-Menten model could not take into consideration all the constraints induced by the immobilization on the enzyme properties but that the Theorell-Chance model was more appropriate. These results are discussed taking into consideration the factors affecting the immobilized enzyme. Finally, we discuss the possibilities of cofactor regeneration with this immobilized alcohol dehydrogenase.

Topics: Alanine Dehydrogenase; Alcohol Dehydrogenase; Amino Acid Oxidoreductases; Enzyme Activation; Enzymes, Immobilized; Kinetics; NAD; Oxidation-Reduction; Sepharose; Sulfones; Thermodynamics