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

Abstract

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.