Strategies for the reversible immobilization of enzymes by use of biotin-bound anti-enzyme antibodies

Glucose oxidase (E.C. 1.1.3.4) is reversibly immobilized in a reactor coupled to a flow-injection analysis system using an immunological reaction. The antibody used is irreversibly immobilized on the reactor support by an avidin—biotin linkage. The bond between avidin and biotin is nearly irreversible under normal elution conditions for antibody—antigen reactions. The reactor is packed with a support on which avidin is covalently attached and a biotin-bound second antibody is passed over the reactor packing, which immobilizes this antibody. An immune complex of the enzyme, or first anti-enzyme antibody followed separately by enzyme, is introduced into the flow system, resulting in enzyme immobilization. The reactor produced can be used in the determination of 1 × 10 −11 −1 × 10 −6 mole of glucose with a sample size of 20 μl and a sample throughput of 20–30/hr. These results are comparable to or better than those obtained with glucose oxidase directly immobilized on the same support. The enzyme can be removed by elution with low-pH buffers, and the reactor regenerated by injection of the anti-enzyme antibody and the enzyme.