Development of a DNA immunoadsorbent: coupling DNA on sepharose 4FF by an efficient activation method.

To remove anti-DNA antibodies from a patient's plasma with systemic lupus erythematosus (SLE), a DNA immunoadsorbent was developed by covalently coupling calf thymus DNA on activated Sepharose 4FF. Sepharose 4FF was activated with 5-norbornene-2,3-dicarboximido carbonochloridate (Cl-CO-ONB), which was proven to be a very effective method for preparation of affinity chromatographic adsorbents. The activation was carried out in dry acetone using 4-(dimethylamine)pyridine (DMAP) and triethylamine (TEA) as catalysts at 4 degrees C or at room temperature. The coupling of DNA to the activated support was investigated as a function of pH, temperature, time, concentration of DNA, and activation level. It was found that the pH for optimal coupling is 3.0, and the amount of coupled DNA increases with an increase either in the concentration of DNA or the activation level. The maximum amount of coupled DNA could reach 1.0 mg DNA/ml support. The incubation of 5 to 20 ml of SLE plasma with 1.0 ml of adsorbent resulted in an 80 to 90% decline in the anti-DNA antibody level. Nonspecific adsorption for normal IgG and total protein is less than 15%.

Bead cellulose derivatives as supports for immobilization and chromatographic purification of proteins.

Characteristic data are presented for Divicell, a macroporous bead cellulose with excellent flow parameters. The preparation of Divicell derivatives and their properties are described with respect to their application as chromatographic supports. The ion exchangers Divicell DEAE and Divicell CM were manufactured in two types with different exclusion limits and an available capacity for proteins of up to 100 mg/ml gel. Divicell Blue is a bead cellulose with covalently bound Cibacron Blue F3G-A and was found to be a very suitable adsorbent for the selective separation and purification of human serum albumin. Activation of Divicell with sodium periodate, epichlorohydrin and 5-norbornene-2,3-dicarboximido carbonochloridate provided activated supports used for immobilization of ligands in organic solvents and in aqueous solutions. Coupling of amines, diamines, amino acids, carbohydrates and proteins is described. The immobilized ligands retained their biological activity as determined by their specific adsorption of proteins. Divicell alkyl derivatives were tested in hydrophobic interaction chromatography with bovine serum albumin as a model. Examples are presented of the application of Divicell derivatives to the purification of biomacromolecules such as immunoglobulins and lectins by affinity chromatography. The results were comparable to those obtained using the corresponding Sepharose-derived absorbents.

Biospecific adsorbents on the basis of chloroformate-activated bead cellulose.

Carbohydrate-derived polymers are activated by the chloroformate N-chlorocarbonyloxy-5-norbornene-2.3-dicarboximide (ClCOONB). The advantages of this activation method are presented. The application of bead cellulose as adsorbent for biomedical and biotechnological purposes is demonstrated. Examples for immunoglobulin purification, streptavidin isolation, and biotransformation of porcine insulin are given.

A novel carbonochloridate for activation of supports containing hydroxyl groups.

The conditions for the introduction of active carbonate groups into supports containing hydroxyl groups by reaction with 5-norbornene-2.3-dicarboximido carbonochloridate are described. Up to 1.5 mmol carbonate groups/g dry Sepharose 4B could be bound. In the case of glycine the reaction of the activated supports with the amino groups takes place with a 10-fold higher rate than the hydrolysis of the carbonate groups, and high coupling yields can be reached. It is shown that the activated supports are well suitable for the preparation of carriers for affinity chromatography or the immobilization of enzymes.